Campbell, Graham F. (Graham Findlay)
Dissertation (PhD(Agric)) -- University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Net blotch of barley, caused by Pyrenophora teres, is one of the most important diseases of this cereal in the south Western Cape Province of South Africa. This fungus exists as two different types (forms), namely a nettype and a spot-type that are distinguished by differential symptom expression on barley leaves. Based on this specific plant pathological difference a series of studies of agricultural importance were executed to investigate the effects of sexual recombination between these two types. In addition, studies were done to determine the difference between local net- and spot-type populations with regards to population structure and fungicide sensitivity. This dissertation therefore, consists of a collection of separate publications and as a result a certain degree of redundancy has been unavoidable. Recombination is one of the most important evolutionary forces involved with sexual reproduction. In plant-fungal agricultural ecosystems this may result in pathogenic fungal populations adapting more rapidly to control programs such as fungicide applications. The first section of the review in part 1 of this dissertation covers different aspects of sexual reproduction in ascomycetes, specifically focussing on mating-type genes, vegetative incompatibility and recombination. The major part of the review is then dedicated to various plant pathological aspects of P.teres, specifically addressing the differences between the two types, and in various cases highlighting the significance of sexual recombination within and between the net- and spot-type. Using morphological criteria for identification purposes there have been many conflicting reports concerning the identity of leaf spot isolates in the Western Cape Province of South Africa. In part 2, the correct identity was eventually achieved employing mating studies and molecular markers .: This was accomplished after single ascospores were obtained from pseudothecia after in vitro mating had occurred between a verified P. teres net-blotch isolate from Denmark and a representative Pyrenophora leaf spot isolate from South Africa. Using amplified fragment length polymorphism (AFLP) and RAPD markers, recombination was demonstrated in the progeny that had DNA banding patterns different from the two parental isolates. Pathogenicity trials also confirmed that recombination had taken place during mating. Inoculations were conducted on the differential cultivars susceptible to the net-blotch and leaf spot forms. The two parents induced typical net-blotch or leaf spot symptoms whereas the progeny mostly induced a jagged spot symptom on each cultivar. Fungicide sensitivity tests using the ergosterol biosynthesis inhibitors showed that, due to recombination, some progeny could have increased resistance to these fungicides. Due to mating and subsequent recombination between a net blotch isolate of P. teres and a representative leaf spot isolate, it was concluded that the latter was P. teres f. maculata. Fifteen of the net-spot hybrid progeny (F1) produced from the mating study in Part 2 were screened in Part 3 to assess their viability and genetic stability. Hybrid progeny (F1) inoculated onto barley seedlings consisting of the cultivars Stirling (differentially susceptible to net-type isolates), B87/14 and Clipper (both differentially susceptible to spot-type isolates) produced intermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated from foliar lesions, followed by repeated inoculation and isolation (F1-2) onto a healthy set of seedlings produced similar intermediate symptoms. RAPDs conducted with two 1Q-mer primers on all isolates of F1-1and F1-2progeny revealed profiles similar to those obtained for F1 isolates. RAPD molecular data, therefore, indicated that hybrid progeny of this net x spot mating were genetically stable after having been subjected to two repetitive inoculation and reisolation cycles. Phylogenetic analysis of DNA sequences of the internal transcribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNA gene and the 5' end partial histone-3 gene confirmed the genetic stability of the hybrid progeny. These results also indicated that the hybrid progeny produced consistent symptoms throughout the series of experiments, and maintained their virulence to the differential cultivars screened. Both types of P. teres are prevalent in the south Western Cape Province of South Africa, found on susceptible cultivars often grown within close proximity of each other. In Part 4, a net- and spot-type population were characterised in terms of their population structure using RAPD markers. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. A total of 65 loci were produced of which 54 were polymorphic. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spottype populations. A coefficient of genetic differentiation (Gs) of 0.0149 was obtained between sites within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis done on the two populations together with six progeny from the mating between a netand spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the nettype population also contained a unique spot-type RAPD fragment. This suggested that sexual recombination may be taking place between isolates of the net- and spot-type under field conditions. Fungicide application is the most important method used in the control of net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values) of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to sterol demethylation inhibiting fungicides were determined, based on the inhibitory effect on radial mycelial growth. The fungicides evaluated were triadimenol, bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- and spot-type isolates revealed strong resistance to triadimenol while flusilazole was shown to be the strongest inhibitor of fungal growth. Spot-type isolates showed a higher resistance than net-type isolates to all five fungicides screened. The ICsD values indicated significant differences between four of the fungicides (triadimenol, tebuconazole, flusilazole and propiconazole). The ICsD values between propiconazole and bromuconazole were not significant. This study suggested that spot-type isolates showed a higher degree of resistance to commercially used fungicides than net-type isolates. The overall conclusion of this study is that the spot-type of P. teres is the pathogen associated with leaf spots of barley in the south western Cape province of South Africa and not P. japonica as earlier reported. Together with the net-type, both types exist as genetically variable populations in this barley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent to one another in which either net- or spot-type susceptible cultivars are being cultivated may lead to sexual progeny being produced. This in turn may lead to an increased rate at which fungal populations may become resistant to commercially used fungicides. It is furthermore suggested that an alternative fungicide seed treatment is used instead of triadimenol due to high resistance of P. teres to this fungicide. / AFRIKAANSE OPSOMMING: Netvlek op gars is een van die belangrikste siektes van hierdie graansoort in die suidelike deel van die Westelike Kaapprovinsie. Dié siekte word veroorsaak deur die swam Pyrenophora teres. Hierdie swam kom voor as twee verskillende tipes, naamlik 'n net-tipe en 'n kol-tipe wat onderskei word op grand van die voorkoms van hulle simptome op garsblare. Hierdie planpatologiese verskil in ag genome, is 'n reeks studies van landboukundige waarde uitgevoer om die effek van geslagtelike rekombinasie tussen die twee tipes te ondersoek. Daarbenewens is ook studies uitgevoer om om die verskil te bepaal tussen plaaslike net- en koltipe populasies ten opsigte van populasiestruktuur en fungisiedsensitiwiteit. Hierdie verhandeling bestaan dus uit 'n versameling afsonderlike publikasies en as gevolg daarvan is daar onvermydelik'n mate van oorvleueling. Rekombinasie is een van die belangrikste evolusionêre kragte betrokke by geslagtelike voortplanting. In plant-swam landboukundige ekostelsels kan dit veroorsaak dat patogene swampopulasies vinniger aanpas by beheerpragramme soos fungisiedtoediening. Die eerste gedeelte in deel 1 van hierdie verhandeling dek die verskillende aspekte van geslagtelike voortplanting van ascomycetes, met spesifieke verwysing na paringstipe gene, vegetatiewe onverenigbaarheid en rekombinasie. Die grootste gedeelte van die oorsig word gewyaan verskeie plantpatologiese aspekte van P. teres,en wys veralop die verskille tussen die twee tipes. In verskeie gevalle word die betekenis van geslagsrekombinasie binne en tussen die net- en koltipe uitgelig. Deur morfologiese kenmerke vir identifikasiedoeleindes te gebruik, is daar baie teenstrydige verslae rakende die identifikasie van blaarvlekisolate in die Westlike Kaapprovinsie van Suid-Afrika. In deel 2 is die korrekte identifikasie eventueel verkry deur gebruik te maak van paringstudies en molekulêre merkers. Dit is bereik nadat enkel ascospore verkry is uit pseudothecia gevorm na in vitro paring plaasgevind het tussen 'n bevestigde P. teres netvlek isolaat uit Denemarke en 'n verteenwoordigende Pyrenophora blaarvlekisolaat van Suid- Afrika. Deur gebruik te maak van versterkte fragmentlengte polimorfisme [AFLP] en RAPD merkers, is rekombinasie gedemonstreer in die nasate wat DNA bandpatrone gehad het wat verskil het van dié van die "ouer" isolate. Patogenisiteitstoetse het ook bevestig dat rekombinasie tydens paring plaasgevind het. Inokulasies is uitgevoer op die verskillende cultivars wat vatbaar is vir die netvlek en blaarvlek vorme. Die twee ouers het tipiese netvlek of blaarvlek simptome veroorsaak, terwyl die nasate hoekige vlekke veroorsaak het op elke cultivar. Toetse vir fungisiedsensitiwiteit deur gebruik van die ergosterol biosintese inhibeerders het gewys dat a.g.v. rekombinasie sekere nasate verhoogde weerstand teen hierdie fungisiedes het. As gevolg van paring en daaropvolgende rekombinasie tussen 'n netvlek isolaat van P. teres en 'n verteenwoordigende blaarvlek isolaat is afgelei dat laasgenoemde P. teres f. maculata is. Vyftien van die netvlek hibried nakomelinge (F1) verkry van die paringstudie in deel 2 is ondersoek in deel 3 om hul lewensvatbaarheid en genetiese stabiliteit te bepaal. Hibried nasate (F1) geïnokuleer op garssaailinge bestaande uit die volgende cultivars: Stirling (soms vatbaar vir net-tipe isolate) , B87/14 en Clipper (albei soms vatbaar vir kol-tipe isolate) het intermediêre simptome op al die cultivars veroorsaak. Akseniese kulture (F1-1) geïsoleer uit blaarletsels gevolg deur herhaalde inokulasie en isolasie (F1-2) op 'n gesonde stel saailinge het dieselfde intermediêre simptome veroorsaak. RAPDs uitgevoer met twee 10-mer inleiers op al die isolate van F1-1 en F1-2 nasate het profiele opgelewer soortgelyk aan dié wat vir F1 isolate verkry is. RAPD molekulêre data het dus gewys dat die hibried nasate van hierdie net x kol paring geneties stabiel was nadat dit onderwerp is aan twee inokulasie en reïsolasie siklusse. Genetiese stabiliteit van die hibried nageslag is bevestig deur filogenetiese analise van die DNA volgorde van die interne getranskribeerde spasieerders (ITS1 en ITS2) reg langs die 5.8S nukluêre ribosomale RNA geen en die 5' end gedeeltelike histoon-3 geen. Hierdie resultate het ook gewys dat die hibried nasate konstante simptome getoon het tydens die hele reeks eksperimente en hulle virulensie behou het vir die kultivars wat getoets is. Beide tipes van P. teres kom algemeen voor in die suidelike deel van die Westelike Kaapprovinsie en word gevind op vatbare cultivars wat dikwels naby mekaar groei. In deel 4 is 'n net- en kol-tipe populasie gekarakteriseer in terme van hulle populasiestruktuur deur gebruik van RAPD merkers. Monsters is versamel van geïnfekteerde garsblare van twee aparte kwadrante in elke saailand. Die twee kwadrante is geplaas in die hoeke van die saailand, diagonaal tot mekaar. 'n Totaal van 65 lokusse is gevorm, waarvan 54 polimorfies was. Die algehele genetiese verskeidenheid bepaal vir alle lokusse, het gelei tot gemiddelde indekse van 0.063 en 0.082 soos gevind vir die net- en kol-tipe populasies. 'n Koëffisiënt van genetiese differensiasie (Gs ) van 0.0149 is gevind tussen gebiede tussen populasies, terwyl 'n koëffisiënt (GT) van 0.63 gevind is tussen die twee populasies. Genotipiese variasie het 13 duidelike multilokus genotipes (haplotipes) getoon in die net-tipe populasie, terwyl daar twaalf was in die kol-tipe populasie. UPGMA groeperingsanalises wat gedoen is op die twee populasies tesame met ses nasate van die paring van 'n net- en koltipe isolaat het tot gevolg gehad dat drie hoof groepe gevorm is, een vir elke populasie en een vir die nasate. Een isolaat wat versamel is, van die net-tipe populasie het 'n unieke kol-tipe RAPD fragment bevat. Dit wys daarop dat geslagtelike rekombinasie in veldomstandighede mag voorkom tussen isolate van die net- en kol-tipe. Fungisiedtoediening is die belangrikste metode wat gebruik word om netvlek in Suid-Afrika te beheer. In deel 5 is die fungisiedsensitiwteit (Ieso waardes) van 89 enkelkonidiale isolate (46 net-tipe en 43 kol-tipe) van P. teres teen sterol demetielasie inhiberende fungisiedes bepaal, op die basis van die onderdrukkende effek op die radiale groei van die miselium. Die volgende fungisiedes is geëvalueer: triadimenol, bromuconazole, flusilazole, propiconazole en tebuconazole. Beide net- en kol-tipe isolate het 'n sterk weerstand teen triadimenol openbaar, terwyl flusilazole gevind is as die sterkste onderdrukker van swamgroei. Kol-tipe isolate het 'n hoër weerstand as die net-tipe isolate teen al vyf fungisiedes wat getoets is, gehad. Die lesowaardes het aangedui dat daar beduidende verskille tussen vier van die fungisiedes IS (triadimenol, tebuconazole, flusilazole en propiconazole). Die leso waardes tussen propiconazole en bromuconazole was nie beduidend nie. Die gevolgtrekking van hierdie studie is dus dat die kol-tipe isolate 'n hoër graad van weerstand teen kommersiëel gebruikte fungisiedes as die net-tipe isolate gehad het. Die algehele gevolgtrekking van hierdie studie is dat die kol-tipe van P. teres, die patogeen is wat geassosieer word met blaarvlekke op gars in die suidwestelike Kaapprovinsie van Suid-Afrika, en nie P. japonica soos voorheen gerapporteer nie. Tesame met die net-tipe, kom altwee tipes voor as geneties veranderlike populasies in hierdie gars verbouingstreek. Paring tussen die twee tipes lei tot geslagtelike nasate wat geneties stabiel is. Dit impliseer dat aangrensende garsvelde waarop net- óf kol-tipe vatbare kultivars verbou word, mag lei tot die produksie van geslagtelike nasate. Dit kan weer lei tot 'n verhoogde tempo waarteen swampopulasies weerstandbiedend teenoor kommersiële fungisiedes raak. Daar word verder ook voorgestel dat alternatiewe fungisied saadbehandelings gebruik word in plaas van triadimenol as gevolg van verhoogde weerstand van P. teres teenoor laasgenoemde.
Thesis (PhD(Agric))--Stellenbosch University, 2014. / ENGLISH ABSTRACT: South Africa is the largest exporter of shipped fresh citrus fruit worldwide. One of the major factors that can lead to substantial losses is postharvest decay. Penicillium digitatum (PD) and P. italicum (PI) are the main wound pathogens, respectively causing green and blue mould decay. PD is more prevalent than PI and therefore also the focus in the majority of research in this field. Imazalil (IMZ) is applied by the majority of citrus packhouses through an aqueous dip treatment, and provides good curative and protective control, as well as sporulation inhibition activity. Two IMZ formulations are in use: the sulphate salt applied in aqueous treatments and the emulsifiable concentrate (EC) applied with wax coatings. The majority of research on IMZ has been done using the EC formulation. The maximum residue limit (MRL) for IMZ on citrus fruit is 5 μg.g-1, whereas 2-3 μg.g-1 is regarded as a biologically effective residue level that should at least inhibit green mould sporulation. A study was conducted to assess the current status of IMZ application in South African packhouses, to determine the adequate residue levels needed to control green mould and inhibit sporulation using IMZ sensitive and resistant isolates, and to study optimisation of modes of IMZ application in citrus packhouses. Factors studied were IMZ concentration, application type (spray vs. dip and drench), exposure time, solution temperature and pH, as well as curative and protective control of PD. The packhouse survey showed that the majority of packhouses applied IMZ in a sulphate salt formulation through a fungicide dip tank, and loaded an IMZ residue of ≈1 μg.g-1. In dip applications, IMZ had excellent curative and protective activity against Penicillium isolates sensitive to IMZ. However, curative control of IMZ resistant isolates was substantially reduced and protective control was lost, even at twice the recommended concentration, nor was sporulation inhibited. The use of sodium bicarbonate (2%) buffered imazalil sulphate solutions at pH ±8, compared with pH ±3 of the unbuffered solutions, markedly increased IMZ residue loading on navel and Valencia oranges and improved curative and protective control of IMZ resistant isolates. Exposure time did not affect IMZ residue loading in IMZ sulphate solutions at pH 3, although the MRL was exceeded after 45 s exposure in pH 8 solutions. Imazalil applied through spray or drench application improved residue loading, but green mould control was less effective than after dip application. IMZ formulation (IMZ sulphate and EC), solution pH (IMZ sulphate at 500 μg.mL-1 buffered with NaHCO3 or NaOH to pH 6 and 8) and exposure time (15 to 540 s) were subsequently investigated in order to improve IMZ residue loading and green mould control on Clementine mandarin, lemon, and navel and Valencia orange fruit. As seen previously, exposure time had no significant effect on residue loading in the unbuffered IMZ sulphate solution (pH 3). No differences were observed between the pH buffers used, but residue loading improved with increase in pH. The MRL was exceeded following dip treatment in IMZ EC (after 75 s exposure time), and IMZ sulphate at pH 8 using NaHCO3 (77 s) or NaOH (89 s) as buffer. The MRL was exceeded after 161 s in IMZ sulphate solutions buffered at pH 6 with either NaHCO3 or NaOH. Green mould control as influenced by residue data was modelled to predict control of IMZ-sensitive and IMZ-resistant PD isolates. From this model the effective residue levels for 95% control of an IMZ-sensitive isolate and of an IMZ-resistant isolate were predicted to be 0.81 and 2.64 ug g-1, respectively. The effects of incubation time (infection age), exposure time, solution pH, wounds size and fruit brushing after dip treatments on residue loading and curative green mould control were also investigated. Exposure time did not have a significant effect on residue loading on fruit dipped in pH 3 solutions of IMZ (< 2.00 μg.g-1). Increasing the pH to 6 resulted in significantly increased residue loading, which increased with longer exposure time, but mostly to levels below the MRL after 180 s. Post-dip treatment brushing reduced residue levels obtained in IMZ pH 3 solutions by up to 90% to levels < 0.5 μg.g-1; however, curative control of the IMZ sensitive isolate was mostly unaffected, but with poor sporulation inhibition. At pH 6, post-dip brushing reduced residues to ≈ 60%; again curative control of the sensitive isolate was unaffected, but with improved sporulation inhibition. Wounded rind sections loaded higher residue levels compared to intact rind sections and large wounds loaded higher levels than small wounds (≈ 10.19, ≈ 9.06 and ≈ 7.91 μg.g-1 for large, small and no wound, respectively). Curative control of infections originating from large wounds was significantly better than those from small wounds. The ability of IMZ to control sensitive green mould infections declined from 6 and 12 h after inoculation on Clementine mandarin fruit of infections induced by small and large wounds, respectively; on navel orange fruit, curative control declined 18 and 36 h after inoculation for the respective wound size treatments. Effective IMZ concentrations that inhibit 50% (EC50) growth of nine PD and five PI isolates were determined in vitro and the IMZ sensitivity of the various isolates categorized according to their EC50 values and resistance (R) factors. Effective residue levels that predicted 50% curative (ER50C) and protective (ER50P) control of these isolates were determined in vivo. All the PI isolates had sensitive EC50 values of 0.005 - 0.050 μg.mL-1. Three PD isolates were sensitive (0.027 – 0.038 μg.mL-1), while one resistant isolate was categorized as low resistant (R-factor of 19), one as moderately resistant (R-factor of 33.2), three as resistant (R-factor of 50 - 57.6) and one as highly resistant (R-factor of 70.7). Sensitive PD isolates had mean ER50C and ER50P values on Valencia orange fruit of 0.29 and 0.20 μg.g-1, and 0.33 and 0.32 μg.g-1 on navel fruit, respectively. ER50 values for resistant isolates did not always correlate with EC50 values and ranged from 1.22 – 4.56 μg.g-1 for ER50C and 1.00 – 6.62 μg.g-1 for ER50P values. ER50P values for resistant isolates could not be obtained on navel orange fruit, but ER50C values (1.42 – 1.65 μg.g-1) were similar to those obtained on Valencia fruit. The PI isolates all behaved similar to the sensitive PD isolates with ER50C and ER50P values on navel and Valencia fruit < 0.38 μg.g-1. Alternative fungicides were assessed for the control of an IMZ sensitive, resistant and highly resistant PD isolates; these included sodium ortho-phenylpenate (SOPP), thiabendazole (TBZ), guazatine (GZT), imazalil (IMZ), pyrimethanil (PYR) and Philabuster® (PLB; a combination of IMZ and PYR), fludioxonil (FLU), azoxystrobin (AZO), Graduate®A+ (a combination of FLU and AZO) and propiconazole (PPZ). Multiple resistance was shown against IMZ, GZT, TBZ and PPZ in both resistant PD isolates. For the sensitive isolates, IMZ, SOPP, TBZ, GZT and PLB provided best curative control, while IMZ, GZT and PLB provided best protective control. For the IMZ-resistant isolates, SOPP, PYR and PLB gave the best curative control, while none of the fungicides provided adequate protective control. Globally, this is the first in-depth study of green and blue mould control with the sulphate formulation of IMZ. Findings from this study are already being implemented by industry. Solution pH is monitored, exposure time is measured and residue loading specific to application method is assessed and interpreted by means of the ER50 values. Aqueous dip applications performed best in terms of curative control, and IMZ residue loading in wound sites was most important for curative control. Other studies confirmed this and showed that IMZ is better protectively applied with wax coatings. The practical impact of IMZ resistance has been highlighted as resistant isolates infections could never be adequately controlled. IMZ alternative fungicides were assessed and SOPP, TBZ, GZT, PYR and/or PLB could be used to reduce the development and impact of IMZ resistance. / AFRIKAANSE OPSOMMING: Suid-Afrika is die grootste uitvoerder van verskeepde vars sitrusvrugte wêreldwyd. Een van die vernaamste faktore wat tot substansiële verliese kan lei, is na-oesverrotting. Penicillium digitatum (PD) en P. italicum (PI) is die hoof wondpatogene, en veroorsaak onderskeidelik groenskimmel- en blouskimmelverval. PD is meer algemeen as PI en daarom ook die fokus in die meerderheid van navorsing in hierdie veld. Imazalil (IMZ) word deur die meerderheid van sitruspakhuise in ‘n waterige doopbehandeling toegedien, en verskaf goeie genesende en beskermende beheer, sowel as sporulasie-inhibisie aktiwiteit. Twee IMZ-formulasies word gebruik: die sulfaatsout wat in waterige behandelings toegedien word, en die emulsifiseerbare konsentraat (EK) wat in wakslaagbehandelings toegedien word. Die meerderheid van navorsing op IMZ is gedoen deur die gebruik van die EK-formulasie. Die maksimum residu limiet (MRL) vir IMZ op sitrusvrugte is 5 μg.g-1, terwyl 2-3 μg.g-1 as ‘n biologies effektiewe residuvlak beskou word wat ten minste groenskimmelsporulasie moet inhibeer. ‘n Studie is uitgevoer ten einde die huidige status van IMZ-toediening in Suid-Afrikaanse pakhuise vas te stel, om die voldoende residuvlakke vas te stel wat nodig is om groenskimmel te beheer en sporulasie te inhibeer deur die gebruik van IMZ-sensitiewe en -weerstandbiedende isolate, en om optimisering van metodes van IMZ-toediening in sitruspakhuise te bestudeer. Faktore wat bestudeer is, was IMZ-konsentrasie, toedieningstipe (spuit vs. doop en stort), blootstellingsperiode, oplossingstemperatuur en pH, asook genesende en beskermende beheer van PD. Die pakhuis-opname het aangedui dat die meerderheid van pakhuise IMZ in ‘n sulfaatsoutformulasie deur ‘n fungisieddooptenk toegedien het, en ‘n IMZ-residu van ≈1 μg.g-1 gelaai het. In dooptoedienings het IMZ uitstekende genesende en beskermende aktiwiteit teen ‘n Penicillium IMZ-sensitiewe isolaat gehad. Genesende beheer van ‘n IMZ-weerstandbiedende isolaat was egter substansiëel minder, en beskermende beheer was verlore, selfs teen twee keer die aanbevole konsentrasie. Sporulasie is ook nie geïnhibeer nie. Die gebruik van natriumbikarbonaat (2%) gebufferde imazalil sulfaat-oplossings by pH ±8, in vergelyking met pH ±3 van die ongebufferde oplossings, het IMZ-residulading op nawel en Valencia lemoene merkbaar verhoog, en genesende en beskermende beheer van IMZ-weerstandbiedende isolaat verbeter. Blootstellingsperiode het nie IMZ-residulading in IMZ-sulfaat-oplossings by pH 3 geaffekteer nie, hoewel die MRL ná 45 s blootstelling in pH 8 oplossings oorskry is. Imazalil wat deur spuit- of drenkhandeling toegedien is, het residulading verbeter, maar groenskimmelbeheer was minder effektief as ná dooptoediening. IMZ-formulasie (IMZ-sulfaat en EK), oplossing pH (IMZ-sulfaat teen 500 μg.mL-1 gebuffer met NaHCO3 of NaOH na pH 6 en 8) en blootstellingsperiode (15 tot 540 s) is daaropvolgend ondersoek ten einde IMZ-residulading en groenskimmelbeheer op Clementine mandaryn, suurlemoen, en nawel en Valencia lemoen vrugte te verbeter. Soos voorheen opgelet, het blootstellingsperiode geen betekenisvolle effek op residulading in die ongebufferde IMZ-sulfaat-oplossing (pH 3) gehad nie. Geen verskille is tussen die pH buffers wat gebruik is, waargeneem nie, maar residulading het met verhoogde pH verbeter. Die MRL is ná die doopbehandeling in IMZ EK (ná 75 s blootstellingsperiode), en IMZ-sulfaat by pH 8 en gebruik van NaHCO3 (77 s) of NaOH (89 s) as buffer, oorskry. Die MRL is ná 161 s in IMZ-sulfaat-oplossings gebuffer by pH 6 met óf NaHCO3 óf NaOH oorskry. Groenskimmelbeheer, soos beïnvloed deur residulading, is gemodelleer ten einde beheer van IMZ-sensitiewe en IMZ-weerstandbiedende PD isolate te voorspel. Vanaf hierdie model is die effektiewe residuvlakke vir 95% beheer van ‘n IMZ-sensitiewe isolaat en van ‘n IMZ-weerstandbiedende isolaat as onderskeidelik 0.81 en 2.64 ug.g-1 voorspel. Die effekte van inkubasieperiode (infeksie-ouderdom), blootstellingsperiode, oplossing pH, wondgrootte en borsel van vrugte ná doopbehandelings, op residulading en genesende groenskimmelbeheer, is ook ondersoek. Blootstellingsperiode het geen betekenisvolle effek op residulading op vrugte wat in pH 3 oplossings van IMZ (< 2.00 μg.g-1) gedoop is, gehad nie. Verhoging van pH tot 6 het tot betekenisvolle verhoogde residulading gelei, wat met verlengde blootstellingsperiode toegeneem het, maar meestal tot vlakke onder die MRL ná 180 s. Ná-doop borsel van vrugte het residuvlakke wat in IMZ pH 3 oplossings verkry is, met tot 90% verminder na vlakke < 0.5 μg.g-1; genesende beheer van die IMZ-sensitiewe isolaat was egter meestal ongeaffekteer, maar met swak sporulasie-inhibisie. By pH 6, het ná-doop borsel van vrugte residue tot ≈ 60% verminder; genesende beheer van die sensitiewe isolaat is weer nie geaffekteer nie, maar met verbeterde sporulasie-inhibisie. Gewonde skilsegmente het hoër residuvlakke gelaai in vergelyking met heel skilsegmente, en groot wonde het hoër vlakke gelaai in vergelyking met klein wonde (≈ 10.19, ≈ 9.06 en ≈ 7.91 μg.g-1 vir groot, klein en geen wond, onderskeidelik). Genesende beheer van infeksies wat vanaf groot wonde ontstaan het, was betekenisvol beter as dié vanaf klein wonde. Die vermoë van IMZ om sensitiewe groenskimmel-infeksies te beheer, het vanaf 6 en 12 h ná inokulasie op Clementine mandaryn vrugte van infeksies wat deur klein en groot wonde onderskeidelik geïnduseer is, afgeneem; op nawel lemoen vrugte, het genesende beheer 18 en 36 h ná inokulasie vir die onderskeie wondgrootte behandelings, afgeneem. Effektiewe IMZ-konsentrasies wat 50% (EK50) groei van nege PD en vyf PI isolate inhibeer, is in vitro vasgestel en die IMZ-sensitiwiteit van die verskillende isolate is volgens hul EK50 waardes en weerstandsfaktore (R) gekatogeriseer. Effektiewe residuvlakke wat 50% genesende (ER50C) en beskermende (ER50P) beheer van hierdie isolate voorspel, is in vivo vasgestel. Al die PI isolate het sensitiewe EK50 waardes van 0.005 - 0.050 μg.mL-1 gehad. Drie PD isolate was sensitief (0.027 – 0.038 μg.mL-1), terwyl een weerstandbiedende isolaat as laag weerstandbiedend (R-faktor van 19) gekatogeriseer is, een as matig weerstandbiedend (R-faktor van 33.2), drie as weerstandbiedend (R-faktor van 50 - 57.6) en een as hoogs weerstandbiedend (R-faktor van 70.7). Sensitiewe PD isolate het gemiddelde ER50C en ER50P waardes op Valencia lemoen vrugte van 0.29 en 0.20 μg.g-1 gehad, en 0.33 en 0.32 μg.g-1 op nawel vrugte, onderskeidelik. ER50 waardes vir weerstandbiedende isolate het nie altyd met EK50 waardes gekorreleer nie en het van 1.22 – 4.56 μg.g-1 vir ER50C en 1.00 – 6.62 μg.g-1 vir ER50P waardes gevariëer. ER50P waardes vir weerstandbiedende isolate kon nie op nawel lemoen vrugte verkry word nie, maar ER50C waardes (1.42 – 1.65 μg.g-1) was soortgelyk aan dié verkry op Valencia vrugte. Die PI isolate het almal soortgelyk aan die sensitiewe PD isolate opgetree, met ER50C en ER50P waardes op nawel en Valencia vrugte < 0.38 μg.g-1. Alternatiewe swamdoders is vir die beheer van ‘n IMZ-sensitiewe, -weerstandbiedende en -hoogs weerstandbiedende PD isolate getoets; hierdie het ingesluit: “sodium ortho-phenylpenate” (SOPP), thiabendazole (TBZ), guazatine (GZT), imazalil (IMZ), pyrimethanil (PYR) en Philabuster® (PLB; ‘n kombinasie van IMZ en PYR), fludioxonil (FLU), azoxystrobin (AZO), Graduate®A+ (‘n kombinasie van FLU en AZO) en propiconazole (PPZ). Veelvoudige weerstand is teen IMZ, GZT, TBZ en PPZ in beide weerstandbiedende PD isolate aangetoon. Vir die sensitiewe isolate, het IMZ, SOPP, TBZ, GZT en PLB die beste genesende beheer verskaf, terwyl IMZ, GZT en PLB die beste beskermende beheer verskaf het. Vir die IMZ-weerstandbiedende isolate, het SOPP, PYR en PLB die beste genesende beheer verskaf, terwyl geen van die swamdoders voldoende beskermende beheer verskaf het nie. Hierdie studie is wêreldwyd die eerste in-diepte studie van groenskimmel- en blouskimmelbeheer met die sulfaatformulasie van IMZ. Bevindinge vanuit hierdie studie word alreeds in die industrie geïmplementeer. Oplossing pH word gemonitor, blootstellingsperiode word gemeet en residulading spesifiek tot toedieningsmetode word bepaal en volgens die ER50 waardes geïnterpreteer. Waterige dooptoedienings het die beste ten opsigte van genesende beheer gevaar, en IMZ-residulading in wond-areas was die belangrikste vir genesende beheer. Ander studies het dit bevestig en getoon dat IMZ beter beskermend is wanneer in ‘n wakslaag toegedien word. Die praktiese impak van IMZ-weerstand is uitgelig aangesien weerstandbiedende isolaat-infeksies nooit voldoende beheer kon word nie. IMZ alternatiewe swamdoders is getoets en SOPP, TBZ, GZT, PYR en/of PLB kon gebruik word om die ontwikkeling en impak van IMZ-weerstand te verminder.
Marker-assisted selection for maize streak virus resistance and concomitant conventional selection for Downy Mildew resistance in a maize population.Mafu, Nothando Fowiza. January 2013 (has links)
Maize streak virus (MSV) disease, transmitted by leafhoppers (Cicadulina mbila, Naude), and maize downy mildew (DM) disease caused by Peronosclerospora sorghi (Weston and Uppal) Shaw, are major contributing factors to low maize yields in Africa. These two diseases threaten maize production in Mozambique, thus the importance of breeding Mozambican maize varieties that carry resistance to these diseases. Marker-assisted selection (MAS) was employed to pyramid MSV and DM disease resistant genes into a single genetic background through simultaneous selection. Firstly, it was essential to determine the genetic diversity of MSV disease resistance in 25 elite maize inbred lines to aid in the selection of suitable lines for the introgression of the msv1 gene; and subsequently, to introduce the msv1 resistance gene cluster from two inbred lines, CM505 and CML509, which were identified as the ideal parental lines for the introgression of MSV disease resistance into a locally adapted Mozambican inbred line LP23 that had DM background resistance. Pyramiding the resistance genes by the use of simple sequence repeat (SSR) molecular markers to track the MSV gene cluster was investigated in 118 F3 progeny derived from crosses of CML505 x LP23 and CML509 x LP23. High resolution melt (HRM) analysis using the markers umc2228 and bnlg1811 detected 29 MSV resistant lines. At the International Maize and Wheat Improvement Centre (CIMMYT) in Zimbabwe, MSV disease expression of the 118 F3 progeny lines was assessed under artificial inoculation conditions with viruliferous leafhoppers and the effect of the MSV disease on plant height was measured. Thirty-seven family lines exhibited MSV and DM (DM incidence ≤50) disease resistance. Individual plants from a total of 41 progeny lines, that exhibited MSV disease severity ratings of 2.5 or less in both locations within each of the F3 family lines, were selected based on the presence of the msv1 gene based on SSR data, or field DM disease resistance, and were then advanced to the F4 generation to be fixed for use to improve maize hybrids in Mozambique for MSV resistance. Simultaneous trials were run at Chokwe Research Station in Mozambique for MSV and DM disease assessment, under natural and artificial disease infestation, respectively. Thus the MSV and DM genes were effectively pyramided. Lines with both MSV and DM resistance were advanced to the F4 generation and will be fixed for use to improve maize hybrids in Mozambique for MSV and DM resistance, which will have positive implications on food security in Mozambique. This research discusses the results of combined selection with both artificial inoculation and the three selected SSR markers. It was concluded that a conventional maize breeder can successfully use molecular markers to improve selection intensity and maximise genetic gain. / Thesis (M.Sc.Agric)-University of KwaZulu-Natal, Pietermaritzburg, 2013.
Response of the endangered medicinal plant : Siphonochilus aethiopicus (Schweif) B.L. Burt. to agronomic practices.Hartzell, James Francis. January 2011 (has links)
This study examines field cropping constraints for domestication of an endangered, wild medicinal plant, Siphonochilus aethiopicus, (Schweif.) B.L. Burt. Extensive literature review and careful observations of plant growth behavior during two years of crop trials overturned several long-held but erroneous claims that have consistently appeared in the scholarly literature, and revealed previously undocumented plant growth characteristics. S. aethiopicus (Schweif.) B.L. Burt. is a rhizomatous corm, not a rhizome. Field growth observations demonstrated clearly that the false stem and leaves grow continuously from emergence in September to senescence in April-May; the corm retains its tuberous roots during winter senescence, and is genetically preprogrammed to shoot in September. Flowers may emerge throughout the growing season (not only initially prior to shoot emergence), typical leaf count is 11-15, not 6-8 as previously reported, numbers that remain constant even when the plant height increases by 20-30% under shade, and leaf distichy is independent of the sun’s course and is unaffected by mother corm orientation. S. aethiopicus proved to be unusually resistant to common field diseases and pests, and resilient to severe hail. The responses of S. aethiopicus were tested in a series of field trials to the effects of levels of compost, field spacing, size of planting material, addition of biocontrol agents, different degrees of shading, and factorials of the macronutrients Nitrogen, Phosphorous and Potassium. Spacing-Composted chicken litter combinations were tested in 2005-2006 in factorial combination with Spacing at 15 cm-4.5 kg ha-1, 20 cm-7.5 kg ha-1, 30 cm-10 kg ha-1, and 40 cm-15.5 kg ha-1, and these treatments were randomized with 4 Corm planting sizes (height by base diameter in mm): Small (S, 12.38 mm x 12.6 mm), Medium Small (MS, 29.65 mm x 27.93 mm), Medium Large (ML, 38.48 mm x 37.78 mm) and Large (L, 52.37 mm x 44.10 mm). 2005-2006 ANOVA tests showed significant differences between Spacing-Compost and Corm Size for the total harvest biomass measure, with 30 cm and 40 cm spaces better than 15 cm spacing, and Corm Size MS, ML and L all better than S, and ML better than MS. Total Corms harvested per block and ii Survival Percentage were similarly significant for Corm Size, but not Spacing. Corms smaller than the Small criteria were raised separately, under optimal conditions in a nursery. In a separate 2005-2006 Compost-only trial ANOVA tests did not find significant differences between compost levels. In 2006-2007 we tested Spacing separately at 5, 10, 15, 20, 30 and 40 cm between planted corms in each plot. We tested Compost levels separately, with 0, 5, 10 and 15 kg ha-1 compost per plot. In 2006-2007 only the ML and L sizes were used in an even mix. There were no significant differences between treatments due to high experimental error, but measurement across all production parameters showed a clear trend towards best performance at spacing between 20 and 40 cm. Overall the results from the Spacing, Compost-level and Corm Size trials suggest that 30 cm is perhaps the optimal field spacing, higher compost levels tend to give better results, and the ML and L corm sizes perform better in open-sun field trials. These parameters are recommended for further field studies and production. The effects of two commercial strains of Trichoderma spp were tested at recommended doses applied to S. aethiopicus. T. harzianum Strain B77 was used as a drench at planting in comparison with a Control and a fungicide in 2005-2006. There were no significant differences between treatments for Harvested Biomass or Survival Percentage. B77 did perform significantly better than the Fungicide in the Total Corm measurement, but neither treatment was significantly different from the Control. In sum, there was a weak trend towards a greater number of output corms as a result of the application of the biocontrol agent. In both 2005-2006 and 2006-2007 we tested T. harzianum Strain kd applied as a drench at planting, with a second drench at 4 weeks. In 2006-2007 there were no significant differences between treatments, but the trend was towards better performance as a result of the drench at planting only. In 2005-2006 open field trials had shown that S. aethiopicus is susceptible to sunburn and Erwinia soft rot when grown in the full sun. Therefore, we tested the effect of various shadecloth densities and colours on production performance in 2006-2007. Treatments were Control (full sun), 40% White (TiO2) (23% shade), 40% Grey (28-30% shade), Light Black (40%), Medium Black (50%), Dark Black (80%), and Red (40%). There were no significant differences between treatments, but the trends indicated that the Control (full sun) and Dark Black (80% shade) performed the worst. Colour of shade did not appear to be important, and plants under all the shadecloths with 40-50% shade grew best. In a factorial trial different levels of Nitrogen, Phosphorous, and Potassium (NPK)were tested, over two seasons. Four levels of each input were used: N at 0 (Control), 40 kg ha-1 (N1), 80 kg ha-1 (N1), and 120 kg ha-1 (N3). P levels were 0 (Control) 60 kg ha-1 (P1) ,120 kg ha-1 (P2) and 200 kg ha-1 (P3). K levels were 0 (Control), 100 kg ha-1 (K1), 200 kg ha-1 (K2), and 400 kg ha-1 (K3). In 2005-2006 there were no significant differences between treatments. In 2006-2007 data there were significant results for Nitrogen only within each repetition. However, significance disappeared when combining across repetitions. We then ran a Bootstrap re-sampling analysis of both 2005-2006 and 2006-2007 data (data were analyzed separately because of different plot sizes and corm numbers in the two years), looking at the optimal level of each macronutrient tested against all combinations of the other two. Though significant results were obtained for each individual level of each macronutrient against the others in combination, the difference between the confidence intervals was not significant. However, there was a clear trend: the optimum N levels were between 40 and 80 kg ha-1; optimum P level was 0 (the Control) and optimum K levels were between 100 and 200 kg ha-1. Tests of handling during harvest, storage, and planting yielded additional useful information for small scale commercial farmers. The optimal harvest time is May, when the false stem and leaves are senescing and yellow, but still upright and visible. Harvest is facilitated by moistening the soil to minimize breaking off of tuberous roots, with simple field washing to remove compacted soil highly recommended. Harvested corms and tuberous roots should be stored under air-restricted, cool conditions because the tuberous roots contain high moisture and will shrivel quickly when left exposed to air, and excessively dried corms will eventually die. Senesced mother corms should be discarded at harvest. Corms are genetically preprogrammed to shoot, so should be planted in September in soft soil, with 1-2 cm of soil coverage. The studies provide a framework for developing the basic agronomy for the domestication and commercial crop production of an endangered medicinal plant species. / Thesis (M.Sc.Agric.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.
Edmonds, Gareth John.
30 October 2014
Five potential resistance-inducing chemicals were applied to two sugarcane varieties (N12 and N27) in a pot trial with the aim of inducing resistance to nematodes in naturally-infested soil. BION® (acibenzolar-S-methyl), methyl jasmonate, cis-jasmone and 2,6-dichloroisonicotinic acid (INA) were applied as a foliar spray and suSCon® maxi (imidacloprid) applied to the soil. All chemicals were tested at two rates and plants were sprayed one week prior to being harvested at 7, 9 and 11 weeks of age. Meloidogyne and Pratylenchus infestation of sett and shoot roots was determined at each harvest. The activity of four pathogenesis-related proteins was examined at 7, 9 and 11 weeks using separate assays, these enzymes where chitinase, β-1,3-glucanase, peroxidase and polyphenol oxidase. Methyl jasmonate treatment produced significant increases in β-1,3-glucanase, chitinase and peroxidase activity. All other elicitor treatments showed little difference in enzyme activity from the Control. The effect of each treatment on plant growth was examined by recording the dried root and shoot biomass of each plant. No significant differences were seen (p<0.05; Holm-Sidak test). However, root and shoot dried biomass was highest in the N12 variety treated by suSCon® maxi. The infection of sugarcane with Ustilago scitaminea (sugarcane smut) is commonly identified visually by the presence of a smut whip. Identification of sugarcane smut infection can be determined prior to whip development by staining tissue sections with lactophenol cotton blue and examining plant tissues microscopically. This allows for a rapid determination of smut infection which can aid breeding programs. Smut infection is achieved in vitro by soaking sugarcane setts in smut spores collected from infected whips. Four methods of inoculation were examined. The method that most consistently caused infection involved allowing setts to germinate for 24 hours, before puncturing a bud with a toothpick, followed by submerging the sett in 1x10⁸ smut spores per mℓ. An elicitor of systemic acquired resistance called BION®, and an insecticide with resistance-inducing properties called Gaucho® (imidacloprid) were used as a sett soak treatments to induce resistance to sugarcane smut. The effect of each treatment at three concentrations on plant germination and growth was examined in the NCo376 variety. Smut spore germination on agar was examined in the presence of both treatments at three concentrations. Sugarcane setts were treated with a concentration that did not significantly reduce the germination of smut spores or sugarcane setts. Plants were infected with smut post treatment and allowed to grow for approximately one month until plants were between 8 and 10 cm in height. Smut infection was assessed by cutting longitudinal sections through the base of the shoot and staining each section with cotton blue lactophenol. Treatment with BION® and Gaucho® did not reduce smut infection. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.
Development of an enzyme-linked immunosorbent assay (ELISA) for field detection and discrimination of Fusarium circinatum from Fusarium oxysporum and Diplodia pinea in pine seedlings.Mkhize, Phumzile. 18 September 2014 (has links)
Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus elliottii, Pinus patula and Pinus radiata. Infections in South Africa (SA) are largely on seedlings, and result in fatal seedling wilt. Accurate and quick detection systems suitable for field use are needed to monitor the spread of the disease and optimize fungicide applications. Detection of F. circinatum is currently based on visual observations of typical symptoms. However, symptoms are not unique to the pathogen and can be caused by other biotic and abiotic stress factors. Nucleic acid-based identification techniques using PCR are available for different fungal species. These are sensitive and accurate, but they are expensive and require skilled biotechnologists to conduct the assays. In this study an enzyme-linked immunosorbent assay (ELISA) was developed to identify F. circinatum in infected seedlings. This optimized ELISA is able to discriminate between F. circinatum and two other fungi that frequently affect pine. This method has advantages over other assays because of its ease of operation and sample preparation, sensitivity and the ability to run multiple tests simultaneously. Mycelium-soluble antigens from Diplodia pinea (=Sphaeropsis sapinea), F. circinatum and F. oxysporum were prepared in nutrient broth. Analysis of these antigens on SDS-PAGE indicated the presence of common antigens between the different fungal pathogens. Some antigens were expressed more by some isolates than by others. Separate groups of chickens were immunised with mycelium-soluble antigens from D. pinea, F. circinatum and F. oxysporum and exo-antigen from F. circinatum prepared in nutrient broth. A 34 kDa protein purified from SDS-PAGE specific for D. pinea was also used for immunisation. Five sets of antibodies were obtained including anti-D. pinea, anti-F. circinatum, anti-F. oxysporum, anti-F. circinatumexo and anti-D. pinea 34 kDa antibodies, respectively. Reactivity of these antibodies was evaluated against antigens prepared in nutrient broth using western blotting and ELISA. Western blot analysis indicated that immuno-dominant antigens for F. circinatum were larger than 34 kDa and their reactivity was not the same between different isolates. Each of the antibodies prepared using mycelium-soluble antigens showed increased reactivity when detecting its own specific pathogen, but cross-reactivity was observed. Anti-D.pineaantibodies showed minimal cross-reactivity with antigens from F. circinatum and F. oxysporum. Anti-F. circinatum antibodies cross-reacted with antigens from F. oxysporum but showed little cross-reactivity with D. pinea antigens. Anti-F. oxysporum antibodies showed more cross-reactivity towards antigens from F. circinatum than those from D. pinea. No reactivity was observed when anti-F. circinatum-exo antigen and anti-D. pinea 34 kDa antibodies were used in immuno-blotting analysis. Evaluation of antibody reactivity using indirect ELISA showed patterns similar to those observed on western blotting, where anti-D. pinea, anti-F. circinatum and anti-F. oxysporum antibodies showed the same cross-reactivity relationships. Anti-F. circinatum and anti-F. oxysporumantibodies showed a significant difference when reacting with antigens isolated from other pathogens including D. pinea, F. circinatum, F. oxysporum, F. solani, F. graminearum and F. culmorum (P = 0.001). No significant difference was observed when the antigens were detected with anti-D. pinea antibodies. Reactivity of anti-F. circinatum-exo and anti-D. pinea34 kDa antibodies was mostly similar to that of non-immune antibodies and showed no significant difference between detection of different antigens. Pine seedlings were artificially infected with the three fungal pathogens using a spore concentration of 1 – 1 x 106conidiaml-1.Infection was monitored using scanning electron microscopy. Results showed increased levels of mycelium growth on the stem and roots of the F. circinatum and F. oxysporum infected seedlings and on the leaves and stem in the case of D. pinea infected seedlings. These plant parts were used in ELISA tests for the detection of antigens. Isolation of antigens from the plant materials involved crushing plant parts in buffer and centrifugation of the suspension. The supernatant obtained was directly used in the assay. ELISA tests prepared in this study were sensitive enough to detect infection caused by 1 conidium ml-1at two weeks post inoculation. A positive reaction for detection of F. circinatum and F. oxysporum was indicated by an ELISA reading above an optical density at 405 nm. The plant material used in ELISA tests were further analysed using PCR. Results indicated that there was no cross-infection between seedlings and served as a confirmation of the disease-causing pathogen. This indicated that cross-reactivity observed was due to other factors such as common epitopes on the major antigens. Use of an ELISA dip-stick or ELISA using these antibodies should provide an easy, fast field test to identify infections of pine, discriminating between F. circinatum, F. oxysporum and D. pinea. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.
Kifle, Medhin Hadish.
22 September 2014
Inoculation with diazotrophic bacteria is well documented as a means to enhance growth and increase yields of various crops, especially when used as an alternative or a supplement to the use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. Nitrogen is the most limiting nutrient for increasing crop productivity, and the use of chemical sources of N fertilizers is expensive, and may contribute to environmental pollution. Therefore, there is a need to identify diazotrophic inoculants as an alternative or supplement to N-fertilizers for sustainable agriculture. The search for effective diazotrophic bacterial strains for formulation as biofertilizers has been going on for over 40 years and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 195 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within the KwaZulu-Natal Province, Republic of South Africa. Ninety five of the isolates were selected for further screening because they were able to grow on N-free media using different carbon sources. Isolates that were very slow to grow on N-free media were discarded. Of these, 95 isolates were screened in vitro for growth promotion traits tests including tests for ammonia production and acetylene reduction. The best 20 isolates that were also able to reduce acetylene into ethylene were selected for growth-promotion trials on maize under greenhouse conditions. Of the 20 isolates, ten isolates enhanced (P = 0.001) growth of maize above the Un-inoculated Control. Molecular tests were conducted to identify the ten most promising isolates selected in the in vitro study. In the greenhouse study, these diazotrophic isolates were screened for their ability to enhance various growth parameters of maize (Zea mays L.), following various inoculation techniques (drenching, seed treatment, foliar spray and combination of these). Inoculations with the five best diazotrophic isolates by various methods of application increased dry weight and leaf chlorophyll content (P < 0.001, P = 0.001), respectively, compared to the Untreated Control. Although, all methods of application of diazotrophic inoculants used in this study resulted in increased dry weight and leaf chlorophyll content, combined methods of application (seed treatment + drenching) and sole application (seed treatment) were significantly more (P < 0.05) efficient. The best five most promising isolates were identified for growth promotion of maize under greenhouse conditions. They were also assessed for their effects on germination of wheat in vitro and were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of wheat (Triticum aestivum L.). These five isolates were also investigated for their potential to enhance growth and yields of maize and wheat crops in field trials, when combined with a low dose of nitrogenous fertilizer. These isolates were further studied for their contribution for enhancing plant growth through nitrogen fixation by predicting N content in leaves using a chlorophyll content meter (CCM-200) and correlated to extractable chlorophyll level at R2 = 0.96. In this study, relative to the Un-inoculated Control, the best five isolates enhanced growth of maize and wheat when combined with a 33% N-fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry weight of maize and wheat; and enhanced root and shoot development of these crops in both greenhouse and field conditions. The best contributions of diazotrophic bacteria was achieved by Isolate LB5 + 0% NPK (41%), V9 + 65% NPK (28.9%), Isolate L1 + 50% NPK (25%), Isolate L1 + 25%NPK (22%) and LB5 + 75% NPK (15%) undergreenhouse conditions. At 30 or 60 DAP, isolates with 33%N-fertilizer caused relatively higher dry weight than the 100%NPK. Inoculation of Isolate StB5 without 33N% fertilizer cuased significant (P<0.005) increases in stover dry weight. In field studies, inoculation of diazotrophic bacteria alone or with 33%N-fertilizer resulted in relatively greater increases of dry weight, stover dry weight, number of spikes and yield at different growth stages higher than the Un-inoculated or Unfertilized Control. However, the increases were not statistically significant. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising yields. The isolates obtained in this study can effectively 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. / Ph.D. University of KwaZulu-Natal, Pietermaritzburg 2013.
Du Preez, Eve Diane.
Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. is an aggressive wind dispersed fungal disease which has spread around the world at an alarming rate in the last decade. The disease was first reported in South Africa (SA) in 2001. It has become well established in the province of KwaZulu-Natal. Reports are occasionally made from eastern Mpumalanga, late in the growing season, in years with good rainfall. Yield losses of 10 - 80% have been reported due to SBR infection. Literature was reviewed to better understand the pathogen in an attempt to find suitable disease management strategies. Many strategies involve delaying, rather than preventing, SBR infection. Of the two strategies to prevent infection, the use of fungicides was the only option for disease control in SA, as no resistant cultivars are available. Field trials were conducted to determine which fungicides are effective in controlling SBR. Further research was conducted to determine the timing, frequency and rate of fungicide applications for optimal control of SBR. Trials were evaluated for disease severity, seed yield and the effect of fungicides on seed quality. Fungicides from the triazole class of the sterol biosynthesis inhibiting group of fungicides were found to be the most effective in controlling SBR. A fungicide from the strobilurin group was found to be less effective than the triazoles at the suggested rate, but was found to be as effective when evaluated at higher dosage rates. Triazoles premixed with fungicides from the benzimidazole and strobilurin groups were also effective in controlling SBR. Timing of application was found to be critical for strobilurin fungicides, but not for triazole fungicides, which have a curative ability, unlike strobilurins. Strobilurin fungicides applied preventatively, before the appearance of disease symptoms were as effective as triazole fungicides applied after disease symptoms, but before infection levels had reached 10%. Across both wet and dry seasons two fungicide applications applied at 21d intervals at the R2 growth stage resulted in effective disease control. In wet seasons, a third fungicide application resulted in yields that were higher, albeit not statistically significant, than two fungicide applications. Assessments of individual fungicides for optimal dosage rate found that registered rates were already optimal for some fungicides, but for others it appeared as if alterations were necessary to the rate suggested for registration. This study was one of the first to extensively evaluate the efficacy of the new triazole and strobilurin fungicides on SBR control. The results have been shared globally, but particularly with newly affected countries in South and North America. Although this research has been groundbreaking, there are still many aspects of fungicide control which need to be studied in order to further optimise chemical control of SBR. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005
Phakopsora pachyrhizi H. Syd and P. Syd, the causal organism of soybean rust (SBR) was first reported in Japan in 1902. In 1934 the pathogen was found in several other Asian countries and as far south as Australia. In India, SBR was first reported on soybeans in 1951. There have been several early reports of SBR in equatorial Africa but the first confirmed report of P. pachyrhizi on the African continent was in 1996 from Kenya, Rwanda and Uganda. Since then, the pathogen has spread south with reports from Zambia and Zimbabwe in 1998 and in Mozambique in 2000. In February 2001, P. pachyrhizi was first detected on soybeans near Vryheid, in Northern KwaZulu-Natal, South Africa (SA). As the season progressed, the disease was observed in other parts of the province, and epidemic levels were found in the Cedara, Greytown, Howick and Karkloof production regions. Soybean rust subsequently spread to Amsterdam and Ermelo in the Highveld region of SA. The disease reappeared in SA in March 2002. It is now established that the pathogen is a threat to soybean production in the country with yield losses in the region of 10-80%. A literature review on SBR investigating the taxonomy of the pathogen, its morphology, symptoms, host range, infection process, epidemiology, control options and the economic importance of P. pachyrhizi was complied to provide the necessary background information to conduct research under local conditions and to assist in interpretation of results of experiments. Epidemiological trials were conducted at the University of KwaZulu-Natal under controlled environmental conditions in a dew chamber and conviron. Development of P. pachyrhizi on the susceptible cultivar (LS5995) was quantified in combinations of seven temperatures (15,19,21,24,26,28 and 30°C) and five leaf wetness durations (LWD) (6,9,12,14 and 16hrs) at three relative humidities (RH) (75%, 85% and 95%). Studies indicate that optimum temperature for uredospore infection is 21-24°C with a LWD greater than 12hrs and RH 85-95%. The number of pustules as well as lesion size on the abaxial and adaxial leaf surface increased with increasing LWD at all the RH values tested. Infection did not occur on plants incubated at 15°C and 30°C at 85% or 95%RH whereas at 75%RH infection did not occur on plants incubated at 15°C, 19°C and 30°C regardless of LWD. Number of pustules per lesion produced at 75%, 85% and 95%RH was highest at 24°C and showed a gradual increase with increasing LWD. Lesion size on both leaf surfaces increased after 12hrs LWD at 24°C at 75% and 85%RH whereas at 95%RH lesion size increased after 14hrs LWD at 24°C. Exposure of uredospores to ultraviolet light which is equivalent to ultraviolet C (sunlight) which is < 280nm, shows a decrease in germination (7%). Under continuous darkness, the germination percentage was found to range from 58% after 48 hrs. Germination was found to peak at 16hrs in darkness with a gradual decrease as time increased whereas germination under ultraviolet light was highest after 6hrs with a gradual decrease with increased exposure to light. Germ tube lengths were found to be shorter when exposed to ultraviolet light (107µm) compared to controls kept in the dark (181µm). Results obtained clearly show a negative effect of ultraviolet light on the germination and germ tube length of uredospores. A 0.1 ml suspension of uredospores on 1.25% water agar Petri dishes was exposed to cycles of 14h ultraviolet light and 10h darkness for 48h. Results indicate an increase in germination percentage of uredospores when exposed to 10h of darkness following a 14h period under ultraviolet light. Controlled environmental studies were conducted to determine alternative hosts of P. pachyrhizi in SA. The control used in this experiment was Prima 2000, a susceptible cultivar to soybean rust. Seven legume plants [Cajanus cajan (L.) Huth, Glycine max (L.) Merr, Lablab purpureus (L.) Sweet, Lupinus angustifolius (L.) Finnish, Phaseolus vulgaris (L.), Pueraria lobata (M&S) Wild and Vigna unguiculata (L.) Walp] and three dry bean lines (Bonus; OPS-RS2 and PAN 159) showed typical SBR symptoms when rated after 21 days post inoculation with uredospores for percentage disease severity. Disease severity was significantly different within the alternative hosts, but G. max, P. vulgaris and P. lobata were not significantly different from Prima 2000 (control). A uredospore suspension of 2.5 x 10(5) uredospores ml(-1) from plants that showed typical SBR symptoms was made and inoculated on to Prima 2000, a susceptible soybean cultivar. Uredospores from pustules on G. max, L. purpureus, L. angustifolius, P. vulgaris, P. lobata, V. unguiculata, Bonus and PAN 159 produced viable uredospores on PRIMA 2000. These plants are considered alternative hosts of P. pachyrhizi. Effect of leaf age on susceptibility of soybean to SBR was tested under controlled environmental conditions. Mean number of lesions as well as lesion size were greater on younger leaves than on older leaves of plants at the same physiological age. Plants at the early vegetative and reproductive stages had a significantly lower number of lesions as well as a smaller lesion size. Plants at the V6 and R1 growth stages were significantly more susceptible to P. pachyrhizi than plants at other developmental stages. Trichoderma harzianum Rifai, Eco-77® a commercial biological control product, was evaluated for its efficacy as a biological control agent of P. pachyrhizi. Trichoderma harzianum sprayed at the standard concentration on infected soybean plants was significantly more effective in controlling P. pachyrhizi than plants sprayed at 1/2X and 2x the standard concentration. This was noted in both Trial 1 and 2. Data indicate that spraying the filtrate two days after inoculation produces less disease. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.
Studies of integrated control of selected root diseases of sunflowers using Trichoderma harzianum (ECO-T®) and siliconElungi, Konis. January 2009 (has links)
The soil-borne fungi Rhizoctonia solani Kuhn and Sclerotinia sclerotiorum De Bary are ubiquitous plant pathogens with a wide host range. They are among the most widespread and destructive diseases of many crops, including sunflowers. Although in many cases, the use of chemicals appears to be the most economical and efficient means of controlling plant pathogens, their environmental concerns and the development of tolerance in pathogen populations have led to drastic reduction in their usage and increased the need to find alternative means of disease control. The potential benefits of applying Trichoderma harzianum Rifai and silicon (Si) nutrition to plants have been extensively reviewed. In this study, the ability of T. harzianum (Eco-T®), soluble silicon, and their combination was evaluated on sunflower (Helianthus annuus L.), for their potential to suppress pathogenic strains of R. solani and S. sclerotiorum. The ability of this crop to take up and accumulate Si in different plant parts was also investigated. In vitro assessment of fungal responses to Si in PDA showed that both R. solani and S. sclerotiorum were inhibited in the presence of Si. More inhibition was observed as the Si concentration increased with a relative increase in pH. Maximum growth inhibition was observed at 3000 mg ;-1 – 6000 mg ;-1 of PDA. No difference in inhibition between the two pathogens was observed, thus confirming the fungitoxic/suppressive ability of high Si concentrations to fungal growth. In addition, in vivo trials showed that the Si concentration of 200 mg ;-1 applied weekly significantly increased the dry weight of plants inoculated with R. solani and S. sclerotiorum and was therefore considered the optimum concentration. Assessments on in vitro antifungal activities of Eco-T® on R. solani and S. sclerotiorum, showed that Eco-T® significantly inhibited mycelial growth, in both dual culture methods and volatile and non-volatile compounds produced by Eco-T®. In addition, the combination of Eco-T® and Si was most effective in suppressing damping-off and increasing plant dry weight of sunflower seedlings in the greenhouse. The combination of Si and Eco-T® significantly increased percentage germination, number of leaves and head dry weight of the sunflower cultivars tested. Silicon alone increased growth but was unable to control R. solani and S. sclerotiorum effectively. Rhizotron studies showed that S. sclerotiorum infected the host through the roots and the stem, whereas R. solani only infected the host through the roots. A study on Si uptake and distribution showed that sunflower accumulates Si in various plant tissues. Analysis of plant tissues revealed that more Si was accumulated in leaves > stems > roots, with the Si levels in leaves being significantly higher than in stems and roots. In conclusion, Si alone could be used to increase growth but was unable to control R. solani and S. sclerotiorum. However, Si together with Eco-T® provides an environmentally friendly alternative for the control of R. solani and S. sclerotiorum, and enhanced plant growth and yield. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.
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