Dissertationen zum Thema „Phytopathogenic microorganisms Biological control“

Rhizoctonia solani (AG-3), the causal agent of Rhizoctonia disease of potato, overwinters as sclerotia on potato tubers. To develop a biocontrol strategy based on the prevention of the sclerotial germination, an isolation of microorganisms colonizing sclerotia of infected potato tubers (cultivars Norland, Atlantic and Souris), was conducted. In vitro screening was used to select effective antagonistic fungi against Rhizoctonia solani. Fifty fungal isolates were selected in order to cover all identified genera and potato variety and examined for their ability to inhibit germination of sclerotia which were incubated with the test fungus for 14 days. Twenty-four (24) fungal isolates were retained based on their ability to reduce sclerotial viability by more than 50% as compared with 100% viability of untreated sclerotia. These 24 isolates were further examined for their ability to protect Table beet seedlings against the pathogen in greenhouse soils. Based on their ability to protect Table beet seedlings from Rhizoctonia infections and to increase the number of secondary roots and root length isolates, F2, F11, F132, F158, and F258 were screened and test their efficacy to increase beet seed germination in field soils. (Abstract shortened by UMI.)

Includes bibliographical references (leaves 102-114) The highly successful biological control of the citrophilous mealybug Pseudococcus calceolarie (Maskell) (CM) by the parasitic wasp Coccophagus gurneyi Compere in several countries led to the release of this parasitoid in the Riverland of South Australia as part of an integrated pest management program. However CM has not been successfully controlled in this region. The results of this study may help to explain the lack of effective biological control of CM in Riverland citrus.

The initial phase in the development of a biological control strategy is screening of biological control agents. Secondary to this phase is the establishment of accurate, effective application techniques. However, successful control requires a thorough understanding of all factors affecting the relationship between host plant, pathogen and other microbes. The purpose of this study was to screen and identify potential bacterial antagonists against Alternaria, a fungal citrus pathogen, attachment of the antagonists to bees, and bee dissemination of the antagonist to citrus flowers. A total of 568 bacterial epiphytes were screened on agar plates for antagonism against Alternaria. Only eight of these isolates, which were identified as Bacillus subtilis, B licheniformis, B. melcerons, B. polymyxa, B. thermoglycodasius, B. sphaericus, B. amiloliquefaciens, and B. coagulans, showed inhibitory effects on the growth of Alternaria. The most effective isolates were B. subtilis and B. licheniformis. Further screening was done with B. subtilis and B. subtilis commercial powder (Avogreen). These bacteria were sprayed on citrus flowers for colonisation studies. Mean populations of B. subtilis and the commercial powder recovered from the flowers were 104 and 103 cfu/stamen respectively. The organisms colonised the styler end and ovary of the flowers when observed under scanning electron microscope (SEM). Avogreen was placed in an inoculum dispenser, which was attached to the entrance of the hive. Honeybees emerging from the beehive acquired 104 cfu/bee. The powder attached to the thorax and thoracic appendages, as revealed by SEM. One active beehive was placed in an enclosure with fifteen flowering citrus nursery trees in pots for dissemination trials. Mean populations of commercial B. subtilis recovered from the flowers visited by bees were 104 cfu/stamen. Electron microscope studies revealed that the antagonist was colonising the styler end and ovary of the flowers. Field dissemination studies were unsuccessful due to low yields.
Dissertation (Magister Institutiones Agrariae)--University of Pretoria, 2006.
Plant Production and Soil Science
unrestricted

Dissertation (PhD)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The events that prepare the plant for, and follow plant-pathogenic interactions, are extremely complex and have been the topic of intensive investigation in recent years. These interactions involve a plethora of genes and proteins, and intricate regulation thereof; from the host and pathogen alike. Studying the contribution of single genes and their encoded proteins to the molecular dialogue between plant and pathogen has been a focus of plant molecular biologists. To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP) was recently cloned from Vitis vinifera. These proteins have the ability to inhibit fungal endopolygalacturonases (ePGs), enzymes which have been shown to be required for the full virulence of several fungi on their respective plant hosts. The activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive for the improvement of disease tolerance of crop species. The VvPGIP-encoding gene was subsequently transferred to Nicotiana tabacum for high-level expression of VvPGIP. These transgenic plants were found to be less susceptible to infection by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG inhibition by protein extracts from these lines correlated to the observed decrease in susceptibility to B. cinerea. This study expands on previous findings by corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse infection assays. Six transgenic tobacco lines and an untransformed wildtype (WT) were infected and the lesions measured daily from day three to seven, and again at day 15. The transgenic lines exhibited smaller lesions sizes from three to seven days post-inoculation, although these differences only became statistically significant following seven days of incubation. At this point, four of the six lines exhibited significantly smaller lesions than the WT, with reductions in disease susceptibility ranging between 46 and 69% as compared to the WT. Two of the lines exhibited disease susceptibility comparable to the WT. In these resistant plant lines, a correlation could be drawn between Vvpgip1 expression, PGIP activity and ePG inhibition. These lines were therefore considered to be PGIP-specific resistant lines, and provided ideal resources to further study the possible in planta roles of PGIP in plant defense. The current hypothesis regarding the role(s) of PGIP in plant defense is twofold. Firstly, PGIPs have the ability to specifically and effectively inhibit fungal ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively, unhindered action of these enzymes results in maceration of plant tissue and ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with the ability to activate plant defense responses. Thus, PGIPs limit tissue damage by inhibition of ePG; this inhibition results in activation of plant defense responses aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic plant lines overexpressing PGIP-encoding genes. However, none of these publications could expand on the current hypotheses regarding the possible in planta roles of PGIP in plant defense. In this study we used transgenic tobacco lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP. Transcriptomic and hormonal analyses were performed on these lines and a WT line, both before and following inoculation with Botrytis cinerea. Transcriptomic analysis was performed on uninfected as well as infected tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis with healthy, uninfected plant material, it became clear that genes involved in cell wall metabolism were differentially expressed between the transgenic lines and the WT. Under these conditions, it could be shown and confirmed that the gene encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was downregulated in the transgenic lines. Additionally, genes involved in the lignin biosynthetic pathway were affected in the individual transgenic lines. Biochemical evidence corroborated the indication of increased lignin deposition in their cell walls. Additionally, phytohormone profiling revealed an increased indole-acetic acid content in the transgenic lines. These results show that constitutive levels of PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may have a positive impact on the observed reduced susceptibilities of these plants. An additional role for PGIP in the contribution to plant defenses is therefore proposed. PGIP may directly influence defense responses in the plant leading to the strengthening of cell walls. This might occur by virtue of its structural features or its integration in the cell wall. These reinforced cell walls are thus “primed” before pathogen ingress and contribute to the decrease in disease susceptibility observed in lines accumulating high levels of PGIP. Transcriptional and hormonal analyses, at the localized response, were performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line. Several Botrytis responsive genes were found to be upregulated in both the WT and the transgenic lines. Although limited differential expression was observed between the two genotypes, the analyses identified a gene which was upregulated two-fold in the transgenic lines, as compared to WT. This was confirmed by quantitative Real-Time PCR. This gene is involved in the lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory effects on Botrytis spore germination. Phytohormone profiling revealed that the transgenic lines accumulated more of the defense-related hormone pool of jasmonates. These are formed via the 13-LOX pathway and have been shown to be important for the restriction of Botrytis growth at the site of infection. Collectively, the results from the infection analyses indicate that in these transgenic lines, both branches of the lipoxygenase pathway are differentially induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid could be observed, although this hormone did not accumulate to significantly higher levels. These results are the first report of differential induction of a defense-related pathway in pgip-overexpressing lines and substantiate the proposal that following ePG inhibition by PGIP, signaling which activates plant defense responses, takes place. Taken together, these results significantly contribute to our understanding of the in planta role of PGIP in plant defense responses.
AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en proteïene is by hierdie interaksies betrokke en aan komplekse reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en patogeen is ‘n sterk fokus van plant-molekulêre bioloë. Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's), ensieme wat benodig word vir die virulensie van verskeie fungi op hul gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel hierdie verskille slegs statisties beduidend geword het na sewe dae van inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT, van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie. Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies van PGIP in plantsiekteweerstandbiedendheid. Die huidige hipotese betreffende die funksie(s) van PGIP in plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie; maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel word met die oog op die vermindering van patogeenindringing. Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,. Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase (XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in die lignien-biosintetiese pad was ook in die individuele transgeniese lyne beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het. Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke van PGIP akkumuleer. Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer. Differensïele geenekspressie tussen die twee genotipes was taamlik beperk, maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR). Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en “colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante het gewys dat die transgeniese lyne verhoogde vlakke van die poel van jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer. Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons geïnduseer word. ‘n Verhoogde induksie van ‘n ander plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie. Hierdie resultate is die eerste wat onderskeidende induksie van ‘n siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer. Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind. Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die huidige kennis van die in planta-funksie van PGIP in plantsiekteweerstandsresponse.

Thesis (M. Tech) --Central University of Technology, Free State, 2008
Aphids (Hemiptera: Aphididae) are amongst the most destructive insects in agricultural crop production systems. This reputation stems from their complex life cycles which are mostly linked to a parthenogenetic mode of reproduction, allowing them to reach immense population sizes within a short period of time. They are also notorious as important and efficient vectors of several plant viral diseases. Their short fecund life cycles allow them to be pests on crops with a short growth period, e.g. lettuce (Lactuca sativa L.). It is common practice to provide this crop with some degree of protection from environmental extremes on the South African Highveld. Shadehouses are popular in this regard, but aphids are small enough to find their way into these structures, and their presence on lettuce is discouraged due to phytosanitary issues. In addition, the excessive use of insecticides is criticized due to the negative influence on human health, and because aphids can rapidly develop resistance. This necessitates the use of alternative control options in order to suppress aphid numbers. Biological control is popular in this regard and the use of predatory ladybirds (Coleoptera: Coccinellidae) is a popular choice. This study investigated the aphid and coccinellid species complex encountered under varying shadehouse conditions on cultivated head lettuce in the central Free State Province (South Africa). Their seasonality was also examined, along with variations in their population size throughout a one-year period. Finally, the impact of varying aphid populations on some physical characteristics of head lettuce was examined, and recommendations for aphid control (using naturally occurring coccinellid predators) were made. Two shadehouse structures were evaluated during this study. One was fully covered with shade netting and designed to exclude the pugnacious ant, Anoplolepis custodiens (Hymenoptera: Formicidae), while the other was partially covered with shade netting (on the roof area) allowing access to the ants. Six cycles of head lettuce were planted and sampled four times during each cycle. These were scheduled to monitor the seedling, vegetative and heading stage of lettuce. Four important aphid species were recorded on the lettuce, namely Acyrthosiphon lactucae, Nasonovia ribisnigri, Myzus persicae and Macrosiphum euphorbiae. Both structures harboured similar aphid and coccinellid species, but their population dynamics differed. A. lactucae dominated in the absence of A. custodiens in the fully covered structure (whole study), while N. ribisnigri dominated in the partially covered structure in the presence of these ants during the warmer months (December – January). M. euphorbiae replaced this species as the dominant species in the absence of A. custodiens (April – September). M. persicae occured during the winter (May – August) in the fully covered structure. Promising coccinellid predators were Hippodamia variegata and Scymnus sp. 1, and to a lesser extent, Exochomus flavipes and Cheilomenes lunata. However, the fully covered structure hampered the entrance of the larger adult coccinellid species, resulting in their lower occurrence. Aphid and coccinellid activity peaked during the summer months (October – January), and the fully covered structure attained the highest aphid infestation levels and coccinellid larval numbers during this time. On the other hand, aphid numbers were higher in the partially covered structure during the cooler months of the year (April – July) and this structure also harboured more adult coccinellids. In most cases, aphid infestation levels did not affect the amount of leaves formed. However, symptomatic damage in terms of head weight reduction did occur under severe infestation levels. Specific environmental conditions within a shadehouse structure concurrently contributed to this reduction, with less favourable conditions accelerating this condition. Results from this study have shown that even though the type of shadehouse structure does not influence the insect species complex found on lettuce, it does have an influence on detrimental and beneficial insect population dynamics. Aphid species infesting lettuce have been identified, along with coccinellid predators that could potentially be used in their control. Both types of structures had advantages and disadvantages, and therefore, decisions concerning shadehouses should not be focused on which type of structure to use, but rather which type of structure to use during different seasons of the year.

A total of six hundred and sixteen fungal and two hundred and thirty two bacterial isolates were obtained either from the sapwood of Pinus radiata or from other sources, including UV mutagenesis. All isolates were screened on Pinus radiata wood chips for their survival and colonisation attributes. Of these isolates, two hundred and eighty two failed to grow or caused permanent deep seated discolourations or decay and were eliminated from the study. The remaining five hundred and sixty six isolates were assessed for their antagonistic ability against sapstain. In a dual screen on Pinus radiata wood chips, one hundred and twelve fungal and four bacterial isolates inhibited the growth of the known sapstain fungus, Ophiostoma piceae. In a second biological control screen, on Pinus radiata wood blocks, isolates of Gliocladium viride, Gliocladium roseum, Trichoderma hamatum, Trichoderma harzianum, Trichoderma sp., Trichothecium roseum and an isolate of the Thelephoraceae proved inhibitory to the sapstain isolates Ophiostoma piceae and Sphaeropsis sapinea providing between 94 and 100% control. These isolates were considered for further examination in the field. The remaining isolates provided poor or inconsistent inhibition or were mould fungi and, therefore, not suitable for direct application. All fungal and bacterial isolates that had shown inhibitory ability in the initial biological control screen and the remaining non-staining bacteria were examined for their ability to produce non-volatile metabolites that were inhibitory to sapstain. The bacterial isolates were examined in a preliminary dual plate screen in which 91 isolates were identified as producing inhibitory compounds. The best of these bacterial isolates were screened, with the fungal isolates, in a non-volatile metabolite trial utilising filter sterilised culture filtrates. Isolates of Bacillus sp., Fusarium solani, Gliocladium roseum, Gliocladium virens, Trichoderma harzianum, Trichoderma sp., Trichoderma viride and Trichothecium roseum were found to be significantly inhibitory to the growth of Ophiostoma piceae at concentrations of 50% or less. However, the filtrates did not provide adequate sapstain control, when tested on Pinus radiata wood block, to prompt consideration for further examination in the field. Studies are currently examining several of these isolates for the production of biologically active compounds. The six most promising isolates, from the wood chip and wood block trials, were tested in the field for their ability to control sapstain on unseasoned Pinus radiata sapwood and/or peeled logs. These were Gliocladium viride (FK75), Trichoderma hamantum (FK561), Trichoderma harzianum (FK228), Trichoderma sp. (FK247), Trichothecium roseum (FK238) and an isolate of the Thelephoraceae (FK33). The fungi were prepared as mycelial/spore homogenates. For application to the timber, the homogenates were mixed with 0.2% Alcosorb gel, producing 108 cfu/ml suspensions, these suspensions were applied by dipping. Diluted homogenates, 108 cfu/ml, were applied as spray treatments to the logs. All of the biological control agent treatments reduced the level of sapstain on either the logs or timber with Trichoderma harzianum (FK228), Trichoderma sp. (FK247) and Trichothecium roseum (FK238) providing control equivalent to that of the fungicides NP-1 and Diffusol for portions of the trial. Trichoderma sp. (FK247) and Trichothecium roseum (FK238) gave sapstain control in excess of 90% for the first 30 days of the timber trial equalling the control provided by NP-1 and Diffusol. In another trial, Trichoderma harzianum (FK228) was more effective than NP-1, providing 60% sapstain control, after six months, on the internal tissue of Pinus radiata logs. The six isolates selected for the field trials were examined in additional studies. In a dual inoculation study, Trichoderma sp. (FK247) exhibited localised antibiotic ability causing the lysis of mycelium of sapstain fungi. There was no evidence of mycoparasitic action by any of the six isolates. Trichoderma harzianum (FK228), Trichoderma sp. (FK247) and Trichothecium roseum (FK238) were observed to degrade cellulose. However, neither these nor the other isolates caused a significant change in the mechanical properties of Pinus radiata timber when compared to untreated controls. Decreasing pH or the addition of nitrate were identified as having potential for the promotion of biological control agent growth. The potential of mixed biological control agent inoculations was also examined. While these results are preliminary, they are extremely encouraging and provide a basis from which future studies can develop.

Phomopsis convolvulus Ormeno, a foliar pathogen of field bindweed, is a good candidate to be developed as a bioherbicide. Large numbers of infective propagules were produced in shake-flask liquid fermentation with modified Richard's (V-8) medium and in solid-substrate fermentation with pearl barley grains. In complex liquid media, pycnidium-like structures were observed. Most conidia stored at $-$70$ sp circ$C remained viable and virulent for at least six months.
In controlled environment studies, a minimum of 18 hr of dew was required for severe disease development on inoculated plants. The addition of gelatin, Sorbo $ sp{ rm TM}$, or BOND$ sp{ rm TM}$ to the inoculum did not enhance the disease under various leaf wetness periods. A continuous dew period of 18 hr was superior to the cumulative effect of three interrupted 6 hr dew periods. Secondary inoculum was produced on diseased plants placed under moist conditions for 48 hr or more.
In greenhouse experiments, seedlings at the cotyledon and 3- to 5- leaf stage were severely diseased and killed when inoculated with 10$ sp9$ conidia/m$ sp2$. This inoculum density adversely affected the regenerative ability of 4 wk old seedlings and established plants, but few plants were killed. Inoculation of the healthy regrowth from plants previously inoculated with the fungus resulted in much less disease symptoms than expected.

Six pathogenic fungal species were isolated from naturally-infected Echinochloa species and evaluated as biological control agents for E. crus-galli, E. colona, and E. glabrescens in rice. Bipolaris sacchari, Curvularia geniculata, and Exserohilum monoceras were non-pathogenic to rice and caused high mortality of Echinochloa species. E. monoceras was selected for further study. Under regulated greenhouse conditions, an inoculum dose of 2.5 $ times$ 10$ sp7$ conidia/m$ sp2$ killed E. crus-galli and E. glabrescens seedlings while 5.0 $ times$ 10$ sp7$ conidia/m$ sp2$ caused 100% mortality of E. colona seedlings. The 1.5-leaf stage was the most susceptible growth stage for all three Echinochloa species. E. glabrescens was most susceptible to E. monoceras infection, E. crus-galli had an intermediate susceptibility, and E. colona was least susceptible. The optimum temperature for 100% mortality was between 20 and 30 C for all Echinochloa species, whereas the minimum dew period for 100% mortality was 16 h for E. colona, 12 h for E. crus-galli, and 8 h for E. glabrescens. Under screenhouse conditions and in the absence of an artificial dew period, over 90% of Echinochloa seedlings were killed when inoculum was sprayed in an oil emulsion or when applied as a dry powder to the water surface of a simulated paddy field. Maximum conidia production occurred on V-8 juice agar or centrifuged V-8 juice agar, at 28 C in the dark. No conidia were produced in liquid media. Of various agricultural products tested as solid substrates, the highest sporulation (1.81 $ times$ 10$ sp6$ conidia/g dry weight) occurred on corn leaves. Host range tests on 54 plant species in 43 genera and 19 families showed that Rottboellia cochinchinensis, was also highly susceptible to this fungus. Of the crops tested, only corn seedlings were lightly infected under optimum greenhouse conditions but no disease occurred on corn under field conditions. Bipolaris sacchari, Exserohilum monoceras, and E. oryzae

Amaranthus retroflexus L. (redroot pigweed) is a major weed of many crops in North America including corn, soybean, and potato. It can be readily controlled by chemical and cultural methods. However, some populations of A. retroflexus have developed resistance against the application of triazine herbicides. Biololical control could be an alternative method to control this weed species. In 1990, a Macrophoma sp. causing foliar lesions was isolated from redroot pigweed and the potential of this plant pathogenic fungus as a mycoherbicide was evaluated. Large numbers of infective propagules were produced in solid substrate fermentation with chickpeas. When inoculated with 10$ sp8$ or 10$ sp9$ conidia m$ sp{-2}$, plants at the cotyledon to 2-leaf stage showed the most severe damage. Disease developed over a wide range of dew period durations (6 hr to 24 hr) and temperature regimes (14 C to 26 C), and the most rapid and destructive disease development occurred following a 24-hr dew period at 18 C. In controlled environment studies, this Macrophoma sp. was pathogenic to the genus Amaranthus and the closely related genus Celosia.

Septoria ampelina causes a disease of grapes known as septoria leaf spot. This study was done to determined which of the fungicides currently used to control the various diseases of grapes, plus one experimental fungicide, is the most effective in controlling septoria leaf spot. Both in vitro and in vivo methods were used. In vivo studies examined the systemic and/or protectant activities of the fungicides. The systemic and protectant fungicides included Bayleton, Benlate, Elite (an experimental fungicide), Nova, Rovral and Rubigan. The protectant only fungicides included Captan, Dithane and Kocide. In vitro tests to determine the minimum inhibitory concentration (MIC) for each fungicide (e.g., the concentration of the fungicide that prevents the fungus from forming colonies on the PEA-fungicide medium), indicate that Benlate (MIC = 0.1 ppm) and Elite (MIC = 1.0 ppm) have the greatest potential'to control septoria leaf spot of grape. These are followed by Dithane, Nova and Rubigan (MIC = 2.0), which in turn are followed by Bayleton and Captan (MIC = 50.0 ppm). Kocide and Rovral did not inhibit fungal growth at concentrations up through 100 ppm. Although all the fungicides tested significantly reduced the incidence of septoria leaf spot in vivo, Benlate and Elite were the most effective fungicides (both in systemic and protectant application).
Department of Biology

Field and growth bench experiments were performed to assess the effect of a selective fungal pathogen of Abutilon theophrasti (velvetleaf) on various aspects of intra- and interspecific competition between this vigorous agricultural weed and soybean (Glycine max). In the absence of the foliar pathogen, Colletotrichum coccodes, A. theophrasti and soybean responded differently to the presence of conspecies or to individuals of the other species. In pure stand, the deleterious effects of intraspecific competition on reproductive output were substantially greater for A. theophrasti than for soybean, especially at lower monoculture densities. In mixtures, however, A. theophrasti reproductive performance was markedly higher than at equivalent monoculture densities, particularly at the lower mixture densities. Soybean reproduction at these lower mixture densities (10 to 20 plants m$ sp{-2}$) was severely curtailed compared with reproductive output at equivalent pure stand densities. A. theophrasti reproductive output was limited more by the presence of conspecies than by the presence of soybean, whereas the opposite trend was observed for soybean. In pure stand, application of C. coccodes had limited impact on either A. theophrasti or soybean yield. However, application of the fungal pathogen in A. theophrasti monocultures caused significant (30-44%) aboveground biomass reductions within five weeks of inoculation, in two of the three years in one field study. Eight weeks following C. coccodes inoculation, A. theophrasti biomass within inoculated monoculture plots did not differ significantly from biomass within uninoculated control plots, although height hierarchies were significantly more developed. In mixtures, C. coccodes applications caused reductions in A. theophrasti growth and reproduction when provided with an adequate dew period. Alternatively, soybean yield losses within inoculated mixture plots were generally lower than for uninoculated control plots, althoug

Colletotrichum coccodes (Wallr.) Hughes, a foliar pathogen of velvetleaf, is being developed as a bioherbicide. Formulation of living organisms for use as pest control products presents unique problems. This research has achieved the development of an adequate formulation of the pathogen by using kaolin clay or talcum powder (1:2.79 wt/wt) as the fillers to dry conidia. Formulated C. coccodes conidia stored at 4, 30C, or at room temperature in bags permeable to oxygen remained viable and able to infect velvetleaf plants at least six months in storage. Various reported germination stimulants increased germination of formulated conidia, although not significantly, whereas increasing concentrations of cutin resulted in subsequent decreases in germination and appressoria formation of fresh as well as formulated conidia. In controlled environment experiments, 14 day-old velvetleaf seedlings were severely diseased when stearic or oleic acids were added to conidia formulated in kaolin clay or talcum powder, respectively. Combinations of germination stimulants, cutinase and/or pectinase inducers did not significantly increase germination and appressoria formation of C. coccodes conidia. Germination of fresh and formulated conidia increased, although not significantly, with the addition of 1% sucrose.

Resistance responses of Abutilon theophrasti were investigated to determine defense mechanisms of the weed against Colletotrichum coccodes and to verify if some chemical suppression of the resistance mechanism could be exploited to enhance the virulence. Induced resistance in A. theophrasti has been confirmed in treatments with C. coccodes, benzothiadiazole, bentazon, and acifluorfen. Induction of peroxidase and phenylalanine ammonia lyase (PAL) activities in the leaves that did not contact with the inducing agents was observed after the localized stresses to the first leaf or the root of the plant with those agents. alpha-Amino-oxy acetic acid (AOA), 2-deoxy-D-glucose (DDG), mannose, oxalic acid, and analogues of oxalic acid and mannose were tested to enhance C. coccodes virulence. However, the compounds did not enhance C. coccodes virulence or affect A. theophrasti growth. Strong antifungal effects, poor inhibitory effects on plant defense mechanisms, or minor dependence of A. theophrasti on the defense mechanisms that the chemicals affected could be reasons. The efficacy of C. coccodes increased in the presence of 0.25 kg a.i. ha-1 bentazon more than when C. coccodes was applied alone, while the effect of glyphosate was minimal. Peroxidase activity was strongly induced by the treatment of C. coccodes and increased over time. PAL and activation of peroxidase was inhibited in the presence of bentazon, suggesting the synergy effect by bentazon is probably due to the suppression on the two defense-related enzymes. In conclusion, A. theophrasti exploits various biochemical and morphological types of defense mechanisms against C. coccodes infection. However, the activation of the defense responses can be suppressed or by-passed in an integrated weed management system.

Dissertation (PhD Agric)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Chrysanthemoides monilifera ssp. monilifera is a shrub indigenous to South Africa, which has become a serious weed of native vegetation in Australia. Endophyllum osteospermi is a microcyclic, autoecious, rust fungus that induces witches' brooms on C. monilifera ssp. monilifera. This rust is considered as a candidate biocontrol agent for use against C. monilifera ssp. monilifera in Australia. The vegetative growth and reproductive output of healthy branches on bushes with different levels of E. osteospermi infections were measured at three sites. The growth of healthy branches on infected bushes was 26- 81% less than that of healthy branches on uninfected bushes. The number of buds, flowering capitulae, fruiting capitulae, and cypselas on healthy branches of infected bushes was 35-75%, 45-90%, 15-99%, and 15-90% less, respectively, than those on uninfected bushes. At five sites, the infection levels and number of witches' brooms were determined every two months. The increase in number of witches' brooms per bush ranged between o and 282 within one year, with an average increase per bush of28 (SE ± 4.8) and 39 (SE ± 9.2) during two years. The average simple interest rate (rs) increase of infection levels for all bushes was 0.015 month-I (s.e. ± 0.0041, n = 72) and 0.0098 month" (s.e. ± 0.0073, n = 43) during two years. Aecidioid teliospores germinated between 10 and 20oe, with 15°e as optimum. Light, and particularly near-uv light, stimulated germination. A period of 6 to 8 hours of light was needed to obtain optimum germination levels. The temperature requirements for basidiospore development differed from that of aecidioid teliospore germination. Optimum was at 15°e, but a rapid decrease in basidiospore production occurred at higher temperatures, few developed at 19°e. Two nuclear divisions occurred within 12 hours of germination to produce a metabasidium with three or four nuclei. A third nuclear division occurred in the basidiospores between 24 and 48 hours. Plants inoculated under controlled conditions took 5 to 24 months before witches' brooms began to develop. A Geographic Information System (GIS) approach was used to model the potential distribution of E. osteospermi in South Africa, based on monthly average climate surfaces with parameters derived from the above experiments. The same model was applied to Australia to suggest a potential distribution of the rust if released in Australia. This potential distribution was similar to one generated using the climate matching computer programme CLIMEX©, but gave greater spatial accuracy. Both approaches indicate that E. osteospermi should establish in temperate Australia. Chrysanthemoides species, as well as other South African asteraceaus plants, were monitored for E. osteospermi between 1992 and 2003. Endophyllum osteospermi was recorded on C. monilifera ssp. monilifera, C. monilifera ssp. pisifera, C. monilifera ssp. rotundata, C. monilifera ssp. canescens, C. monilifera ssp. subcanescens, C. incana, an undescribed taxon of Chrysanthemoides, Osteospermum ciliatum, 0. polygaloides and 0. potbergense. Endophyllum dimorphothecae sp. nov. is described on Dimorphotheca cuneata. Aecidium elytropappi, which was recorded on Elytropappus rhinocerostis and Stoebe plumose, is transferred to Endophyllum as E. elytropappi comb. nov. Germination of aecidioid teliospores and penetration by basidiospores were observed on the surface of excised leaves of 32 plant species at 4 days after inoculation. Germinating aecidioid teliospores aborted on 14 plant species, whilst no penetration was attempted on a further 12. Penetration only occurred on 9. Therefore only these 9 plant species need to undergo traditional host specificity testing. Pending these results, E. osteospermi could be safely released in Australia for the biological control of C. monilifera ssp. monilifera.
AFRIKAANSE OPSOMMING: Chrysanthemoides monilifera ssp. monilifera 'n meerjarige wat inheems in Suid Afrika is, het 'n belangrike onkruid in Austalië geword. Endophyllum osteospermi 'n mikrosikliese, autoecious roesswam, induseer heksebesems op C. monilifera ssp. monilifera. Hierdie roesswam word as 'n potensiële biologiese beheeragent teen C. monilifera ssp. monilifera in Austalië beskou. Die vegetatiewe groei en voortplanting van gesonde takke op struike met verskillende vlakke van E. osteospermi infeksies is by drie lokaliteite gemeet. Groei van gesonde takke op geinfekteerde bosse was 26-81 % minder as die van gesonde takke op ongeïnfekteerde bosse. Die aantalokselknoppe, blommende capitulum, vrugdraende capitulum en pitvrugte op individuele gesonde takke van geïnfekteerde bosse was onderskeidelik 35-75%, 45-90%,15-99%, en 15-90% minder, as die op ongeïnfekteerde bosse. By vyf lokaliteite is die infeksievlakke en die aantal heksebesems elke twee maande vasgestel. Die toename in heksebesems van elke plant was tussen 0 en 282 binne eenjaar, met 'n gemmidel van 28 (SE ± 4.8) en 39 (SE ± 9.2) geduurende twee jaare. Die gemiddelde eenvoudige rentekoers (rs) toename in infeksievlakke van al die struike was 0.015 maand" (s.e. ± 0.0041, n = 72) en 0.0098 maand-1 (s.e. ± 0.0073, n = 43) gedurende twee jaare. Ontkieming van aecidioidteliospore het tussen 100e en 200e met 15°e as die optimum. Lig en veral naby-uv lig het ontkieming gestimuleer, terwylontkieming relatief swak was onder donker toestande. 'n Periode van 6 tot 8 uur lig was nodig vir optimale ontkiemingsvlakke. Die temperatuurvereistes vir basidiospoor ontwikkeling het verskil van die van aecidioid teliospoor ontkieming. Optimale was by 15°e, maar 'n vinnige afname in basidiospoorproduksie het by hoër temperature voorgekom, min het by 19°e voorgekom. Twee kernverdelings het binne 12 ure van die begin van ontkieming voorgekom om 'n metabasidium te produseer met drie of vier kerne. 'n Derde kern verdeling het in die basidiospore tussen 24 en 48 uur voorgekom. Plante wat onder beheeerde toestande geïnokuleer is het heksebesems 5 tot 24 maande na inokulasie ontwikkel. 'n Geografiese Inligtings Sisteem (GIS) benadering is gebruik om 'n model vir die potensiële verspreiding van E. osteospermi in Suid Afrika te ontwikkel, gebasseer op die maandelikse gemiddelde klimaatoppervlaktes met parameters wat vanaf bogenoemde eksperimente verkry is. Dieselfde model is in Austalië toegepas om 'n potensiële verspreiding van die roesswam voor te stel. Hierdie potensiële verspreiding was soortgelyk aan 'n program wat met die klimaats vergelykende rekenaarsprogram CLIMEX© ontwikel is, maar dit het groter ruimtelike akkuraatheid gemaak. Beide benaderings het aangedui dat E. osteospermi in Austalië behoort te vestig. Chrysanthemoides spesies, asook ander inheemse plante van die Asteraceae, is tussen 1992 en 2003 vir die voorkoms van E. osteospermi in Suid Afrika waargeneem. Endophyllum osteospermi is op C. monilifera ssp. monilifera, C. monilifera ssp. pisifera, C. monilifera ssp. rotundata, C. monilifera ssp. canescens, C. monilifera ssp. subcanescens, C. incana, 'n onbeskryfde taxon van Chrysanthemoides, Osteospermum ciliatum, a. polygaloides en a. potbergense waargeneem. Endophyllum dimorphothecae sp. nov. is beskryf op Dimorphotheca cuneata. Aecidium elytropappi, wat op Elytropappus rhinocerostis en Stoebe plumosa voorkom, is by Endophyllum ingesluit as E. elytropappi comb. nov. Ontkieming van aecidioidteliospore en penetrasie deur basidiospore op die oppervlak van verwyderde blare van 32 plant spesies 4 dae na inokulasie is waargeneem. Ontkiemende aecidioidteliospore het op 14 toets plant spesies ge-aborteer, terwyl geen penetrasie op 'n verdere 12 gepoog is nie. Penetrasie het slegs op 9 voorgekom. Derhalwe hoef slegs die 9 plant spesies tradisionele gasheer spesifisiteitstoetse te ondergaan. Afhangende van die resultate kan E. osteospermi dus met veiligheid in Austalië vir die biologiese beheer van C. monilifera ssp. monilifera vrygelaat word.

Thesis: Ph. D., Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution), 2015.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Zooplankton, such as copepods, are highly abundant environmental reservoirs of many bacterial pathogens. Although copepods are known to support diverse and productive bacterial communities, little is understood about whether copepods are affected by bacterial attachment and whether they can regulate these associations through mechanisms such as the innate immune response. This thesis investigates the potential role that copepod physiology may play in regulating Vibrio association and the community structure of its microbiome. To this end, the intrinsic ability of oceanic copepod hosts to transcriptionally respond to mild stressors was first investigated. Specifically, the transcriptional regulation of several heat shock proteins (Hsps), a highly conserved superfamily of molecular chaperones, in the copepod Calanusfinmarchicus was examined and demonstrated that Hsps are a conserved element of the copepod's transcriptional response to stressful conditions and diapause regulation. To then investigate whether copepod hosts respond to and regulate their microbiota, the transcriptomic response of an estuarine copepod Eurytemora affinis to two distinct Vibric species, a free-living strain (V. ordalii 12B09) and a zooplankton specialist (V. sp. F10 9ZB36), was examined with RNA-Seq. Our findings provide evidence that the copepod E. affinis does distinctly recognize and respond to colonizing vibrios via transcriptional regulation of innate immune response elements and transcripts involved in maintaining cuticle integrity. Our work also suggests that association with E. affinis can significantly impact the physiology of Vibrio colonists. Finally, the inter-individual variability of the C.finmarchicus microbiome was examined to identify how specifically and predictably bacterial communities assemble on copepods and whether host physiology influences the bacterial community structure. Our findings suggest that copepods have a predictable "core microbiome" that persists throughout the host's entrance into diapause, a dormancy period characterized by dramatic physiological changes in the host. However, diapausing and active populations harbor distinct flexible microbiomes which may be driven by factors such including the copepod's feeding history, body size, and bacterial interactions. This thesis work highlights the role of copepods as dynamic reservoirs of diverse bacterial communities and implicates copepod host physiology as an important contributor to the activity, abundance, and community structure of its microbiome.
by Amalia Aruda Almada.
Ph. D.

Many candidate mycoherbicides have shown promise in the laboratory or greenhouse, but most have been ineffective in the field. Factors limiting mycoherbicide efficiency include temperature and humidity. Results from this thesis indicate that solar radiation has both a damaging effect(reduction in germination)limiting efficacy and a photomorphogenic effect(appressorium induction)increasing efficacy. The study has also shown significant interaction between temperature and solar radiation on the survival of conidia of potential mycoherbistats. Therefore, solar radiation should be considered as third major component of the environment that should be considered when trying to produce mycoherbistats. With the findings presented in this thesis and further research on disease development under different conditions, in combination with the formulation of conidia in suitable UV protectants, a computer simulation modelling the conditions leading to epidemics caused by C.orbiculare, D.avenacea and R.alismatis could be constructed. It may be possible to manipulate fungal application time in order to expose conidia to doses of solar radiation that are not harmful to conidium germination and which stimulate appressorium formation. However, additional protection may be needed.
Doctor of Philosophy (PhD)

The fruit production is threatened by several bacterial diseases, such as fire blight of apple and pear, bacterial canker of kiwifruit, bacterial spot of stone fruits, and angular leaf spot of strawberry. The conventional pesticides that are available for the control of these diseases are mainly copper compounds and they have a limited efficacy and negative impact on environment. Therefore, there is a need to develop alternative and sustainable management tools. This Ph.D. Thesis contributes to the development of a novel microbial biopesticide based on lactic acid bacteria. Two Lactobacillus plantarum strains were selected due to their broad-spectrum activity. In order to improve the epiphytic survival of both strains in plants and to get more consistency in their biocontrol efficacy, a physiological adaptive strategy was defined to increase the water-stress tolerance. Also, a monitoring method was developed to evaluate the population dynamics of a L. plantarum strain.
La producció de fruita està afectada per diferents malalties bacterianes de quarantena com el foc bacterià de les pomeres i pereres, el xancre bacterià del kiwi, la taca bacteriana dels fruiters de pinyol i la taca angular de les fulles de maduixera. Els plaguicides disponibles pel seu control són principalment compostos cúprics els quals tenen una eficàcia limitada i un impacte negatiu en el medi ambient. Existeix la necessitat de desenvolupar eines de control alternatives i més sostenibles. Aquesta tesi contribueix en el desenvolupament d’un bioplaguicida microbià basat en bacteris de l’àcid làctic. Es van seleccionar dues soques de Lactobacillus plantarum amb activitat d’ampli espectre i es va definir una estratègia fisiològica d’adaptació per incrementar la tolerància a l’estrès per manca d’aigua i així millorar la supervivència epifítica a la planta. També es va desenvolupar un mètode de monitoratge per avaluar les dinàmiques poblacionals d’una soca de L. plantarum.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Among the species of known insects (approximately 900 thousands), at about 2% is considered eusocial, because they live in truly advanced societies. The leaf-cutting ants belong to eusocial insects group and reached the major level of the instinct through the cultivation of fungi. Nowadays, they seems to be the unique animal group that have developed an advanced agriculture, based on their symbiosis with fungi, that appeared at about 50 million years ago, long before human being had appeared and become cultivator. Cutting leaves that serves as substrate to fungi cultivation for feed, ensure a high economic value to leaf-cutting ants, especially when they compete with men. So, the aim of this work was to perform a study about the microorganisms communities associated to the fungi garden of the leaf-cutting ants, Atta sexdens, evaluating the possibility to control them by the antagonistic activity of endophyte microorganisms from Amazon plants. To enable these assays, 13 leaf-cutting ants colonies (at about 5 monts of age) containing all classes (queen, soldiers, males and workers) were collected and transferred to the Laboratório de Microrganismos LABGEMMA of the Universidade Federal do Amazonas UFAM. From these anthill, associated microorganisms were isolated, cultivated, identified (by molecular and classical methods) and preserved at appropriated conditions. The antagonisms assays were performed by the method of paired culture, using endophytic microorganisms as inhibitors of those anthill associated. In vivo assays were performed with lab assembled anthills, to evaluate the potential for biological control against them. The main isolated and identified anthills associated microorganisms were: Leucoagaricus gongylophorus; Bionectria ochroleuca; Aspergillus flavus; Trichoderma longibrachiatum; Fusarium solani, yeasts and gram positive and gram negative bacteria. Antagonisms assays against L. gongylophorus, T. longibrachiatum, A. flavus and one of the anthill yeasts were promissing as an alternative method to the anthills biological control.
Calcula-se que entre as espécies de insetos conhecidas (cerca de 900 mil), próximo de 2% são ditas eussociais, pois vivem em sociedades verdadeiramente avançadas. As formigas cortadeiras estão inseridas no grupo dos insetos eussociais e atingiram o que pode se chamar de apogeu do instinto por meio da agricultura de fungos. Até o momento, parece ser o único grupo de animais, além do homem, que desenvolveu uma agricultura avançada, que se baseia na simbiose mutualística com os fungos e surgiu há mais de 50 milhões de anos, ou seja, muito antes de o homem existir e se tornar agricultor. O fato de esses insetos cortarem folhas que servem de substrato para o cultivo do fungo do qual se alimentam as torna de grande importância econômica, sobretudo quando competem conosco. Dentro desse contexto, objetivou-se realizar um estudo sobre a microbiota associada ao jardim de fungos das formigas cortadeiras Atta sexdens, avaliando-se a possibilidade de controlá-los por meio da atividade antagonista de microrganismos endofíticos provenientes de plantas da Amazônia. Para viabilizar esses ensaios foram coletadas, em campo, treze colônias de formigas com aproximadamente cinco meses contendo todas as castas (rainha, soldados, machos e operárias) e encaminhado ao Laboratório de Microrganismos LABGEMMA da Universidade Federal do Amazonas UFAM. A partir desses formigueiros, os microrganismos associados foram isolados, cultivados, identificados (por métodos clássicos e moleculares), e preservados em meios e condições apropriadas. Os ensaios de antagonismo foram realizados pelo método de cultivos paralelos, in vitro , utilizando-se microrganismos endofíticos como agentes inibidores dos microrganismos associados aos formigueiros. Foram realizados ainda ensaios in vivo , utilizando-se formigueiros montados em laboratório, para avaliar o potencial dos endófitos no controle biológico dos formigueiros. Os principais microrganismos isolados e identificados como associados aos formigueiros foram: Leucoagaricus gongylophorus; Bionectria ochroleuca; Aspergillus flavus; Trichoderma longibrachiatum; Fusarium solani, leveduras e bactérias gram negativas e positivas. Os ensaios de antagonismo contra L. gongylophorus, T. longibrachiatum, A. flavus e contra uma das leveduras do formigueiro, foram promissores como métodos alternativos para o controle biológico dos formigueiros.

Thesis (Ph.D)--Stellenbosch University, 2000.
ENGLISH ABSTRACT: Copper oxychloride is a fungicide that is extensively used in vineyards in the Western Cape to treat and prevent fungal diseases. It is however not clear what the effects are on soil organisms, which play an important role in soil fertility, in South African soils. There is paucity of data linking results obtained in the laboratory to effects observed in the field, which will only become useful if a clear relation can be demonstrated. The aims of this study were to: ~ Determine the effects of copper oxychloride on field populations of earthworms and simultaneously monitor lysosomal membrane stability, measured as neutral red retention time (NRRT). ~ Validate experimental field studies by doing inventories of earthworm populations in long-term sprayed vineyards. ~ Determine the LC50 of copper oxychloride and simultaneously measuring NRRT, and linking them to the experimental field studies. ~ Conduct bioassays, burrowing activity- and soil-avoidance experiments to investigate their relations to earthworm population responses in the experimental field studies. Earthworms were sampled by hand-sorting in the field tests on treated and untreated field plots in the Western- (October 1998 - July 1999) and Northern Cape (April 1998 - October 1999). Soil samples and worms were analysed for copper contents and coelomocytes of live earthworms were used to conduct the neutral red retention assays. Acute toxicity tests were conducted over a period of 28 days during which the earthworms (Eisenia fetiday were exposed to different concentrations of copper oxychloride. Change in biomass and mortality were measured as endpoints, as well as NRRT. Bioassays, burrowing activity and soil-avoidance were conducted by exposing Aporrectodea caliginosa to grassland- and vineyard soil as well as grassland soil spiked with 60 J.1g.g-1copper in the form of copper oxychloride. Growth and mortality were recorded in the bioassays as well as copper concentrations In earthworm body tissues and substrates used over a period of28 days. Burrowing activity and soil-avoidance were determined by measuring the length of tunnels burrowed by A. caliginosa in soil profiles over a period of 4 days under different exposure regimes. Results from the field tests showed that spraying of copper oxychloride had a negative effect on earthworm populations at the prescribed application rates. NRR T in earthworms from the exposure plots was significantly (p<0.05) lower after just one spraying application. It was concluded that spraying copper oxychloride at prescribed application rates caused a decrease in field populations of earthworms and that NRRT was an early and reliable biomarker since it was indicative of later effects observed at the population level. Results obtained from the field inventory of earthworms in vineyards at Nietvoorbij, Robertson end Worcester confirmed data from the two field studies. The calculated LC50 of 882.78 I1g.g-1 for copper oxychloride and 519.40 I1g.g-1 for copper was ecologically relevant if a safety factor of 10 was applied. NRRT which manifested earlier than effects on biomass change in the acute toxicity tests, were significant when viewed against the background of responses of field populations of earthworms. From the bioassay experiments it was found that A. caliginosa exposed to copper oxychloride spiked soil had significantly (p<0.05) higher weight loss and mortality than those in grassland- and vineyard soil. This indicated that changes in biomass and mortality were indicative of population responses in the field and can be considered as ecologically relevant. Burrowing activity of A. caliginosa was significantly (p<0.05) lower in vineyard and copper oxychloride spiked soil than in grassland soil. Similarly in the soil avoidance experiments it was found that A. caliginosa avoided vineyard- and copper oxychloride contaminated soil. It is therefore concluded that burrowing activity and soil avoidance were ecologically relevant endpoints since they corresponded with population responses in the field. The study thus revealed that the long-term usage of copper oxychloride could have negative effects on earthworm populations. The spraying of copper oxychloride can have important implications on the sustainable use of agricultural soils since earthworms and other soil organisms play such an important role in soil fertility. The use of biomarkers and other ecotoxicological indicators can provide an early warning that soil organisms are under environmental stress.
AFRIKAANSE OPSOMMING: Die fungisied koperoksichloried word wyd gebruik in die Wes-Kaap om swamsiektes in wingerde te beheer en te voorkom. Dit is egter nie bekend wat die effek daarvan op Suid Afrikaanse grondbiota, wat 'n belangrike rol speel in grondvrugbaarheid, is nie. Daar is ook 'n tekort aan inligting wat die resultate van laboratoriumondersoeke in verband bring met veldstudies. Die doelstellings van die studie was om: ~ Die effek van koperoksichloried op erdwurmpopulasies in die veld te ondersoek en terselfdertyd membraanstabiliteit, as moontlike biomerker, gemeet as neutraal rooi retensietye (NRRT), te monitor. ~ Die geldigheid van eksperimentele veldstudies te toets deur ook grondanalises te doen in wingerde wat oor langtermyn met koperoksichloried bespuit is. ~ Die LC50 van koperoksichloried vir erdwurms te bepaal en terselfdertyd NRR T te meet asook om dié gegewens in verband te bring met die resultate van seisoenale veldstudies oor die uitwerking op erdwurmpopulasies. ~ Bio-evaluerings ("bioassays"), tonnelaktiwiteit- en vermydingseksperimente te onderneem en die verband tussen die toksiteitstoetse en populasieresponse, soos waargeneem in die veld, te ondersoek. Erdwurms is versamel deur handsortering tydens die veldtoetse in die Wes- (Oktober 1998 - Julie 1999) en Noord-Kaap (April 1998 - Oktober 1999) op kontrole en bespuite persele. Grondmonsters en erdwurms is spektrofotometries geanaliseer om koperinhoude te bepaal. Die selomosiete van lewende wurms is gebruik om NRR T te bepaal. Akute toksisiteitstoetse is uitgevoer oor 'n tydperk van 28 dae waartydens Eisenia fetida blootgestel is aan verskillende koperoksichloried konsentrasies. Veranderinge in biomassa en mortaliteit is bepaal asook NRRT. Bioevaluerings ("bioassays"), tonnelaktiwiteit- en vermydingseksperimente IS uitgevoer deur Aporrectodea caliginosa bloot te stel aan grasveld- en wingerdgrond asook grasveldgrond wat met koperoksichloried gekontamineer is. Groei en mortalitiet is bepaal in die "bioassays" asook koperkonsentrasies in die grond en erdwurm liggaamsweefsels oor 'n tydperk van 28 dae. Tonnelaktiwiteit en grondvermyding is bepaal deur die lengte van tonnels wat deur A. caliginosa gegrawe is te meet oor 'n tydperk van vier dae vir die verskillende blootgestelde groepe. Die resultate het aangedui dat koperoksichloriedbespuiting 'n negatiewe invloed het op erdwurmpopulasies teen die voorgeskrewe toedieningsprogram. NRRT in erdwurms van die blootstellingperseel, was beduidend (p<0.05) laer na 'n enkele bespuiting. Daar is verder bevind dat NRR T 'n betroubare en vroeë biomerker is, aangesien dit 'n aanduiding gegee het van latere effekte wat op populasievlak na vore getree het. Veldopnames in Nietvoorbij, Robertson en Worcester het die geldigheid van data verkry uit die veldstudies ondersteun. Die berekende LC50 van 882.78 ug.g" vir koperoksichloried en 519.40 ug.g" VIr koper was ekologies relevant indien 'n veiligheidsfaktor van 10 toegepas is. NRRT se ekologiese relevansie is bevestig deur dit te vergelyk met response wat in die veldtoetse waargeneem is. Deur bioevalueringseksperimente is bevind dat gewigsverlies en mortaliteit van A. caliginosa beduidend hoër was in koperoksichloried gekontamineerde grond as in die grasveld- (kontrole) en wingerdgronde. Veranderinge in biomassa en mortalitiet was aanduidend van populasieresponse soos waargeneem in die veldstudies en kan dus as ekologies relevante eindpunte beskou word. Tonnelaktiwiteit van A. caliginosa was beduidend (p<0.05) laer in wingerd- en koperoksichloried gekontamineerde grond as in grasveldgrond. Dieselfde is gevind in die grondvermydingstoetse waar A. caliginosa wingerd- en koperoksichloried gekontamineerde grond vermy het. Dit kan dus afgelei dat tonnelaktiwiteit en grondvermyding ook ekologies bruikbare eindpunte is aangesien dit verband hou met populasieresponse soos waargeneem in die veldstudies. Hierdie studie het getoon dat die herhaalde gebruik van koperoksichloried 'n nadelige invloed kan hê op erdwurmbevolkings. In die lig van die belangrike rol wat erdwurms en ander grondorganismes speel in grondvrugbaarheid kan die oormatige gebruik van hierdie fungisied ernstige implikasies inhou vir volhoubare benutting van landbougronde. Die gebruik van biomerkers en ander ekotoksikologiese eindpunte kan egter as vroeë waarskuwingsmetode dien dat die grondorganismes onder omgewingstres verkeer.

Research and application of microbial control of brown spot disease on the dragon fruit caused by fungi Neoscytalidium dimetiatum have important implications towards the safe and sustainable dragon fruit production. In this article, the research team has identified two strains of microorganisms capable of inhibiting fungi Neoscytalidium dimitiatum denoted A3, B7. Classification results determined that A3 belongs to Actinomyces group 3 with similarities of 100% (1500/1500 bp) with 16S rDNA segment of Streptomyces fradiae; B7 with similarities of 100% (1414/1414 bp) with 16S rDNA segment of bacteria Bacillus polyfermenticus and ensure biosafety when released into the environment
Nghiên cứu ứng dụng vi sinh vật kiểm soát bệnh đốm nâu trên cây thanh long do nấm Neoscytalidium dimetiatum gây ra có ý nghĩa quan trọng hướng tới ngành sản xuất thanh long an toàn và bền vững. Trong bài viết này nhóm nghiên cứu đã xác định được hai chủng vi sinh vật có khả năng ức chế nấm Neoscytalidium dimitiatum cao kí hiệu là A3, B7. Kết quả phân loại xác định chủng A3 thuộc nhóm xạ khuẩn 3 tương đồng 100% (1500/1500 bp) với đoạn 16S rDNA của Streptomyces fradiae; chủng B7 tương đồng 100% (1414/1414 bp) với đoạn 16S của vi khuẩn Bacillus polyfermenticus và đảm bảo an toàn sinh học khi phóng thích ra môi trường

A espécie Araucaria angustifolia, pertencente ao fragilizado Bioma Mata Atlântica, foi, durante décadas, uma das maiores fontes de madeira no Brasil. Essa espécie é de grande importância sendo fonte de alimento e matéria prima para a produção de móveis, polpa de celulose e vernizes. Devido à grande importância econômica e ambiental da araucária, estudos voltados à preservação e manejo da espécie se tornaram necessários. Este trabalho teve como finalidade isolar actinobactérias, presentes na rizosfera de araucária, antagônicos aos fungos Fusarium sp .e Armillaria sp. Estes patógenos são responsáveis por danos às raízes e sementes causando podridão e perda de mudas. Além disso, verificou-se também o efeito das actinobactérias sobre a germinação de esporos de Gigaspora rosea. Após a seleção dos melhores inibidores, identificação morfológica e seqüenciamento do gene 16S rRNA, verificou-se o efeito destes sobre o crescimento de Pinus taeda, na presença e na ausência do fungo ectomicorrízico (Suillus brevipes). Os isolados ainda foram avaliados quanto à produção de enzimas como quitinase, lipase, fosfatase, celulase, amilase e protease. Para o isolamento foram coletadas raízes de 15 árvores adultas presentes na mata nativa. Dez gramas de raízes frescas com resíduos de solo aderidos firmemente foram agitados por 30 minutos em solução salina 0,85 % padronizando como solo rizosférico aquele que soltou das raízes. Neste isolamento foram utilizadas duas metodologias: por plaqueamento e por separação física pela utilização de membrana de 0,45 micrômetros. Foram obtidos 33 possíveis actinobactérias. Os isolados foram testados quanto à capacidade antagônica pelo método de contato direto, inoculando-os a 30 mm de distância do fungo fitopatogênico Fusarium sp., em placas de petri contendo meio ISP2. Os halos de inibição foram medidos após 3, 5, 7 e 10 dias. De todos os isolados testados 6 mantiveram a inibição por 10 dias com manutenção de halos de inibição de até 4 mm contra um Fusarium sp. isolado de sementes de milho e 2 foram eficientes na inibição de Fusarium sp, patógeno de Pinus sp. A análise de inibição do fungo Armillaria sp, foi feito em meio líquido medindo o crescimento em mg/dia após 30 dias na presença de extratos de actinobactérias e também pela contagem de rizomorfas em placa de petri após 20 dias de incubação em contato direto com as actinobactérias. Verificou-se que de 28 isolados testados 24 foram capazes de inibir a produção de rizomorfas, destacando-se o isolado A43 que foi capaz de inibir ambos os fungos, Fusarium e Armillaria. Os esporos de Gigaspora rosea tiveram a taxa de germinação avaliada na presença de actinobactérias a partir da técnica de dupla camada. Todos os seis isolados testados foram capazes de estimular a germinação dos esporos desse fungo micorrizico. Nenhum dos seis isolados (A43, A43b, PNA, A64, A75 e A93) foi capaz de produzir fosfatases e lipases. Porém os isolados A93, A75, A64 e PNA produziram protease, quitinase e amilase. Em casa de vegetação, foi testado o efeito de seis isolados, na presença e não ausência de ectomicorriza (Suillus brevipes), sobre plântulas de Pinus taeda. Analisou-se o diâmetro, a altura, a massa seca da raiz e da parte aérea e também o fósforo da parte aérea. Destaca-se o isolado A43 que, quando na ausência de ectomicorriza, estimulou o desenvolvimento da planta em relação ao controletambém sem ectomicorriza, provocando aumento de 100 % para massa seca da parte aérea e da raiz. Os resultados encontrados neste trabalho poderão levar ao desenvolvimento de novas tecnologias, visando à identificação de novos metabólitos e novas técnicas de manejo, voltados ao controle de doenças de plantas, especialmente as espécies arbóreas.
The tree Araucaria angustifolia, belonging to the endangered Atlantic Forest biome, for many decades was the source of Brazilian wood. This species is also very important in providing food and feed, as well as raw material for joinery, cellulose pulp and varnish. Due to the economic and environmental importance of A. angustifolia, research projects involving the preservation and management of this species are becoming more urgent and necessary. The aim of this work was to isolate Araucaria rhizosphere actinobacteria with antagonic effects against the plant pathogens Fusarium sp. and Armillaria sp. These fungi cause root rot and seed damage, with the consequent loss of seedlings. Moreover, the effect of these actinobacteria on Gigaspora rosea spore germination was studied. After the selection of the best pathogen inhibitors, we also tested the effect of these microorganisms on Pinus taeda growth, in the presence or absence of the ectomycorrhizal fungus Suillus brevipes. The production of protease, chitinase, lipase, phosphatase, cellulase and amylase of these bacteria in culture media was also investigated. For the isolation of rhizosphere bacteria, we collected roots of 15 adult trees in a native forest. Ten grams of fresh roots with soil residues adhered to the surface were shaken in 0,85 % salt solution for 30 minutes. Two techniques were used, the dilution plate method and the coverage of the medium, utilizing a 0,45 µm membrane to separate these filamentous bacteria. About 33 actinobacteria were isolated. After isolation the actinobacteria were tested against the plant pathogenic fungi Fusarium spp., utilizing dual culture techniques and ISP2 medium. The inhibition halo was measured after 3, 5, 7 and 10 days. Six of our isolates maintained an inhibition zone measuring at least 4 mm against Fusarium sp. isolated from corn seed and 2 mm against the Fusarium, which causes root root of Pine trees. For the inhibition test of Armillaria, in liquid medium with the addition of culture extracts of actinobacteria, the growth in mg/day was measured after thirty days growth, and the number of rizomorphs produced in culture dishes after twenty days in dual culture with the actinobacteria was counted. Six bacteria proved to be antagonistic (A43, A43b, A64, PNA, A93 e A75), and only one had no effect. Possibly the elevation of the pH value played also a role in this situation. About 24 of 28 isolates inhibited the rizomorph production, especially the isolate A43 that showed a double antagonism against Fusarium and Armillaria. The dual layer test was used to investigate the reaction of the arbuscular mycorrhizal fungus Gigaspora rosea spore germination to the presence of actinobacteria. All the six actinobacteria stimulated the germination, but the germ tube did not grow straight forward as in the control. This result may indicate a negative effect against this arbuscular mycorrhizal fungus. None of the six isolates tested (A43, A43b, PNA, A64, A75 and A93) produced phosphatases and lipases, but A93, A75, A64 and PNA produced protease, amylase and chitinase. Isolates A43 and A43b did not produce any of the enzymes tested. This fact suggests that there is production of an antibiotic acting against the pathogenic fungi. Pinus taeda seedlings were grown under green-house conditions. After three months the stem diameter, shoot height, root and shoot dry weight and shoot phosphorus content were evaluated. Plants with ectomycorrhiza presented a significant growth promotion in comparison with the nonmycorrhizal ones. Among the actinobacteria in the absence of mycorrhiza only the isolate A43 produced a 100% growth enhancement in comparison with the control plant without ectomycorrhiza. The results presented in this dissertation could lead to the development of new technologies and new management techniques, with regard to the control of plant diseases, especially in tree species.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were v identified: S8-introgression (Triticum dicoccoides) = one RAPD and two SCARs; S13-translocation (Aegilops speltoides) = four RAPDs, three RGAPs and five SCARs; S15-translocation (Ae. peregrina) = one RAPD and two SCARs; S20-translocation (Ae. neglecta) = two RAPDs, two RGAPs and one SCAR. The markers are already being employed in current projects aiming to map and shorten these translocations. Some of the markers can be combined in multiplex reactions for more effective mass screening. No repeatable markers could be identified for the four remaining translocations (S12 from Ae. sharonensis; S14 from Ae. kotschyi; Smac from Ae. biuncialis and Lr19-149-299 from Thinopyrum ponticum).

A cultura do guaranazeiro, (Paullinia cupana var. sorbilis) espécie nativa da região amazônica, é de grande importância para o Brasil, tanto do ponto de vista econômico quanto social. Atualmente, a comercialização do guaraná em rama (sementes torradas) é a maneira mais comumente utilizada na produção de xaropes e extratos de guaraná utilizados principalmente pelas indústrias de bebidas. O Brasil é o único produtor de guaraná no mundo, com destaque para o Estado do Amazonas e a Bahia. Entretanto, a produção de guaraná na região amazônica, centro de origem da planta, vem sendo cada vez mais afetada por condições fitossanitárias desfavoráveis, como a presença da antracnose, doença causada pelo fungo Colletotrichum spp.., que não se constitui um problema na Bahia. A finalidade da presente pesquisa foi de comparar microbiota endofítica de sementes dos dois estados na detecção de possíveis causas que poderiam explicar as diferenças de sanidade, além de procurar uma alternativa sustentável para a utilização de microrganismos endofíticos, que vivem no interior dos tecidos vegetais, e tem sido descritos como agentes de controle biológico de doenças através de vários mecanismos, dentre eles a produção de enzimas líticas. Assim, o presente estudo teve por objetivo isolar e identificar bactérias e fungos endofíticos associados à sementes de guaranazeiros provenientes do Amazonas e da Bahia. Esses microrganismos foram também avaliados quanto ao potencial biotecnológico na produção de enzimas e controle da antracnose. Para o isolamento, as sementes passaram por um processo de desinfecção. A análise da produção enzimática foi detectada por meios de culturas específicos e para o estudo da atividade antagonística contra o Colletotrichum sp. (L1) foram realizados ensaios in vitro por meio de cultura pareada. A identificação dos isolados foi realizada por sequenciamento parcial do gene 16S rDNA para isolados bacterianos, e da região ITS do rDNA para isolados fúngicos. Não foram encontradas diferenças significativas no número de isolados bacterianos em sementes das duas procedências. Fungos em pequeno número (6) foram isolados apenas de sementes da Bahia. Desses isolados foram selecionadas 102 bactérias, sendo 48 provenientes do Amazonas e 54 da Bahia. Nos testes de antagonismo houve redução de 32% a 64% do crescimento do fungo patogênico Colletotrichum por bactérias endofíticas e 8% a 52% pelos fungos endofíticos. Com relação à atividade enzimática, 13 bactérias apresentaram atividade amilolítica, 5 celulolítica, 11 pectinolítica, e 24 proteolítica. Em relação aos fungos endofíticos, todos apresentaram pelo menos uma atividade das enzimas testadas, com exceção de atividade proteolítica. Bactérias dos gêneros Bacillus, Paenibacillus, Ochrobacterium, Rhizobium e Microbacterium foram isolados como endofíticos de sementes de guaraná. Em relação a isolados fúngicos foram encontrados representantes dos gêneros Lasiodiplodia e Fusarium. Embora não tenham sido encontradas diferenças substanciais com relação aos endófitos isolados, para explicar a incidência de antracnose no Amazonas em relação à Bahia, os resultados demonstram o potencial biotecnológico de microrganismos endofíticos de sementes de guaraná na produção de enzimas e para o controle biológico da antracnose
Guarana (Paullinia cupana var. sorbilis) a plant from the Amazon region is of great importance mainly from the Amazonas state both from economic and social points of view. Currently roast seed extracts have been used for the production of soft drinks and medicinal purposes. Brazil is the only guarana producer in the world, being the states of Amazonas and Bahia the main producers in the country. However, guarana production in Amazonas state has been increasingly affected by unfavorable conditions due the plant disease anthracnose caused by fungi from the genus Colletotrichum, which is not a problem in Bahia state. The aim of the present research was to compare the endophytic microbiota from seeds derived from the two states, to detect possible causes which could explain plant sanity differences in both regions and also to use endophytic microorganisms as biological controllers, a sustainable alternative to chemical products to control the disease. Endophytic bacteria and fungi from guarana seeds obtained from Bahia and Amazonas regions were isolated and studied as antagonists against Colletotrichum sp. strain L1 and also for enzymes production. The isolates identification was performed by partial sequencing the 16S rDNA region for bacterial isolates and rDNA ITS region for fungal isolates. Fungal morphological characterization was also done. We detect few differences between the number of bacteria isolated from Bahia and Amazonas seeds. Although only reduced number of seeds were analyzed, only six fungal isolates were obtained, all from Bahia. A total of 102 bacteria isolates, 48 from Amazonas and 54 from Bahia were selected. Antagonistic tests and enzymatic activities showed that from bacteria tested, 13, 5, 11 and 24 isolates were able to produce amylolytic, cellulolytic, pectinolytic and proteolytic activities respectively. All the six fungal isolates produced the tested enzymes with exception of proteases. From the positive tests, Bacillus, Paenibacillus, Ochrobacterium, Rhizobium and Microbacterium were the genera isolated from guarana seeds and the genera Lasiodiplodia and Fusarium were found among the endophytic fungi. Although no clear distinctions among isolated endophytes were found to explain anthracnose differences in Amazonas and Bahia, the results demonstrated the biotechnological potential of selected endophytic microbiota from guarana seeds for enzyme production and in vitro control of Colletotricum, the causal agent of guarana anthracnose

A citricultura é uma atividade rural extremamente importante no contexto sócio-econômico brasileiro. A laranja representa 49% de toda a produção brasileira de frutas. Entretanto, anualmente milhares de toneladas de frutos são perdidas devido à ação de fitopatógenos. A Mancha Preta dos Citros (MPC) tem sido responsável por grandes prejuízos em várias regiões produtoras de citros no mundo, sendo designada como barreira fitossanitária, principalmente no mercado Europeu. Para o consumo in natura, a aparência dos frutos é fator essencial, onde a MPC compromete a comercialização de frutas frescas. O controle químico de patógenos de plantas é geralmente o mais utilizado para minimizar os danos causados pela MPC, apesar do uso de tais produtos implicar em altos custos, tanto para os agricultores com para o meio ambiente, causando contaminação do solo e da água e aumentando a pressão de seleção sobre a população do patógeno. Dessa forma, o controle biológico torna-se uma alternativa atrativa, como uma estratégia que permite um menor impacto ambiental aliado à proteção da planta contra o patógeno. No entanto, para sua aplicação, são necessários estudos relacionados à utilização de técnicas de biocontrole, como por exemplo, a utilização de microrganismos produtores de enzimas hidrolíticas. Tais enzimas, como as quitinases, endoglicanases e β- glicosidases têm a capacidade de digerir a parede celular de fungos e bactérias. Neste trabalho, 24 isolados de G. citricarpa foram avaliados quanto à sensibilidade aos fungicidas usados em campo para controle da doença: piraclostrobina e carbendazim, nas dosagens de 0,5; 1 e 2 mg i.a./mL, a fim de verificar o efeito da pressão de seleção causada pelo uso contínuo destes compostos. Dois dos isolados apresentaram resistência ao carbendazim em todas as dosagens testadas, indicando que o uso desse agroquímico pode selecionar mutantes resistentes, resultando na não eficiência deste produto no controle da MPC. Uma alternativa para minimizar este tipo de efeito seria a utilização deste composto em combinação com outros princípios ativos. As atividades celulolíticas e quitinolíticas de 96 isolados de fungos filogeneticamente distintos foram avaliadas para a seleção de potenciais agentes de controle biológico. Os quatro isolados que apresentaram maior atividade de cada uma destas enzimas, além de duas linhagens de Trichoderma, foram testados como potencial de biocontrole de G. citricarpa em um experimento com folhas laranja ‘Valência’, comparando a ação destes com a de fungicidas comerciais. Apesar de o melhor controle do patógeno ter sido observado em folhas tratadas com piraclostrobina, dois isolados fúngicos mostraram eficiência similar ao fungicida, inibindo o desenvolvimento de G. citricarpa, o que sugere a possibilidade de futura utilização de métodos de controle biológico para a Mancha Preta dos Citros.
Citriculture is an extreme important rural activity in social and economical national context in Brazil. Orange is 49% of total brazilian fruit production. However, thousands of tons are lost due to the action of phytopathogens annually. The Black Spot of Citrus (BSC) is responsible for great lost in various citrus producers regions all around the world, being already designed as a phytosanitary barrier, mainly in European market. For in natura fruit consuming, the fruit esthetic is a limiting factor, where BSC compromises the market of fresh affected fruits. Chemical control of plant pathogens is the most commonly way used to minimize damages in citriculture by BSC, although the application of such products implies in high costs, not only for farmers but also for environment, causing soil and water contamination and increasing the selection pressure on pathogen population. On this way, the biocontrol became an attractive way, as a strategy that permits a minor environmental impact besides the plant protection against phytopathogens. For this application, are necessary researches based on utilization of biocontrol techniques, as for example, the using of microorganism producers of hydrolytic enzymes. Such enzymes, like quitinases, endoglicanases and β-glicosydases are able to digest the fungal and bacterial cell wall. In this work, 24 strains of G. citricarpa were evaluated about the sensibility to fungicides used in field for BSC control: piraclostrobin and carbendazin, in dosages of 0,5, 1,0 and 2,0 mg a.i./mL, aiming to verify the effect of selection pressure caused by continuous use of this compounds. Two of these strains presented resistance to carbendazim in all evaluated dosages, showing that the use of this agrochemical may select resistant individuals, resulting a non-efficiency of this compound for BSC control. An alternative to minimize this kind of effect must be the application of this compound in combination to others active principles. The celullolitic and chitinollitic activities of 96 fungi strains widely spread phylogenetically were evaluated for selection of potential biological control agents. Four strains that presented major activity of each enzyme, besides two Trichoderma lineages, were tested as potential biological control of G. citricarpa in an experiment with ‘Valência’ orange leaves, comparing the action of these biocontrolers with commercial fungicides. Although a better pathogen control was achieved in leaves treated with piraclostrobin, two fungi strains revealed to have the similar efficiency to fungicide, inhibiting the development of G. citricarpa, suggesting the possible future utilization of biocontrol methods to Black Spot of Citrus.

Devido à percepção dos consumidores sobre o impacto da utilização de pesticidas sobre o ambiente e saúde humana, além da aquisição de resistência por parte dos fitopatógenos, a sociedade tem exercido pressões que levaram ao estabelecimento de políticas governamentais que restringem a utilização de fungicidas levando agricultores e pesquisadores a considerar a aplicação de técnicas de controle biológico de fitopatógenos fúngicos. Guignardia citricarpa é o agente causal da pinta preta dos citros, que é de grande importância econômica, pois interfere na produção e causa depreciação estética dos frutos, acarretando em prejuízos principalmente na comercialização de frutos in natura para o mercado externo. Neste contexto, o objetivo deste trabalho foi a avaliação in vitro do potencial de linhagens de Saccharomyces cerevisiae, utilizadas em processos fermentativos, como agentes de biocontrole contra G. citricarpa. Através de ensaio em placa, foi evidenciado que entre as linhagens de S. cerevisiae testadas (BG-1, CR-1, CAT-1, KD-1, K-1 e PE-2), a CR-1 demonstrou a maior atividade antagônica contra o fitopatógeno, causando 73% de inibição do crescimento micelial. Também foi demonstrado que as linhagens são capazes de produzir voláteis de ação fungistática inibindo em até 83% o desenvolvimento do patógeno. O filtrado de cultura autoclavado e não autoclavado, bem como as células inativadas termicamente, obtidas a partir do crescimento da linhagem CR-1 por 24 h em meio YEPD, não causaram redução do crescimento vegetativo do fungo. A produção de enzimas hidrolíticas extracelulares (quitinases, β-1,3-glucanases e proteases) pela levedura não foi detectada em meio YEPD contendo glicose ou preparação de parede celular de G. citricarpa, nos tempos de cultivo avaliados. Baseado nas informações obtidas, foi possível constatar que as linhagens de S. cerevisiae, em especial a linhagem CR-1, são potenciais antagonistas para o controle de G. citricarpa. O possível mecanismo utilizado para inibição pela levedura é a produção de voláteis, no entanto, outros mecanismos não podem ser descartados. Desta forma, o presente trabalho mostra o potencial de S. cerevisiae no controle de G. citricarpa em frutos de laranja na pós-colheita.
Due to the consumers perception about the impact caused by pesticides utilization over the environment and human health, besides the acquisition of resistance for part of the phytopathogens, the society has exercised pressures that had led to the establishment of governmental politics that restrict the use of fungicides leading agriculturists and researchers to consider the application of techniques of biological control of plant pathogenic fungi. Guignardia citricarpa is the causal agent of citrus black spot that has a great economic importance, therefore interfering in production and causing aesthetic depreciation of the fruits that can interfere with commercialization of fresh-fruit in the external market. In this context, the aim of this work was to evaluate in vitro the potential of Saccharomyces cerevisiae strains, used in fermentative process, as biocontrol agents against G. citricarpa. Through plate assay it was evidenced that among the tested strains of S. cerevisiae (BG-1, CR-1, CAT-1, KD-1, K-1 and PE-2), the strain CR-1 was the one that demonstrated the greatest antagonic activity against the phytopathogen, causing 73% of micelial growth inhibition. It was also demonstrated that the strains were able to produce volatile compounds with fungistatic action inhibiting up to 83% the development of the pathogen. The autoclaved and not autoclaved culture filtrate, as well as the termical inactivated cell obtained from the growth of strain CR-1 in YEPD medium for 24 h, did not cause reduction in the fungal vegetative growth. The production of extracellular hydrolytic enzymes (chitinases, β-1,3-glucanases and proteases) by the yeast was not detected in YEPD medium with glucose or cell wall preparation of G. citricarpa at the evaluated times. Based upon the obtained information it was possible to evidence that the strains of S. cerevisiae, specially the strain CR-1, are potentials antagonists for the control of G. citricarpa. The possible mechanism used for inhibition by yeast is the volatile production, however other mechanisms cannot be discarded. Thus, the present work shows the potential of S. cerevisiae to control G. citricarpa in orange fruits in postharvest.

Este trabalho teve como principais objetivos avaliar os efeitos dos agentes bióticos (Saccharomyces cerevisiae, Bacillus thuringiensis, Lentinula edodes e Agaricus blazei), e abióticos (UV-C, irradiação gama, acibenzolar-S-metil, quitosana, ácidos acético e salicílico) na proteção de mamões contra C. gloeosporioides, bem como estudar os mecanismos bioquímicos de resistência ativados no tecido vegetal, em resposta ao tratamento com os agentes de maior eficiência, além de investigar os efeitos destes sobre o desenvolvimento in vitro do fungo. Para tanto, mamões cv. Golden foram inoculados com C. gloeosporioides através de injeção subcuticular de 15 µL da suspensão de esporos e, após 10 h, tratados com os diferentes agentes bióticos e abióticos. Para avaliar a possibilidade de indução de resistência pelos agentes, mamões foram também inoculados após 24, 48 e 72 h dos tratamentos. Os frutos foram armazenados a 25 ºC / 80 %UR por 7 dias e, avaliados diariamente quanto a incidência e severidade da podridão. Ao final do período de armazenamento, efetuou-se a avaliação dos parâmetros físico-químicos (cor de casca e de polpa, firmeza, sólidos solúveis, pH e acidez total). Quando de interesse, as atividades de peroxidase, β-1,3-glucanase e quitinase foram também investigadas. In vitro, avaliou-se o crescimento micelial, a germinação de conídios e a esporulação do fungo em resposta aos diferentes tratamentos. Os resultados mostraram que a irradiação gama (0,75 e 1 kGy) reduziu a incidência e a severidade da antracnose. Não houve efeito da UV-C no controle da podridão e todas as doses testadas causaram danos na casca dos frutos. O acibenzolar-S-metil reduziu em mais de 50 % a incidência e a severidade da podridão, além de induzir a maior atividade das enzimas peroxidase, quitinase e β-1,3-glucanase, e não alterar as características físico-químicas dos frutos. O ácido acético, a 2,5 µL L-1, reduziu a severidade e a incidência das lesões nos frutos. A quitosana (1, 2 e 4 %) reduziu significativamente a severidade da antracnose, e a 4 % foi também eficiente em reduzir a incidência da podridão. Concentrações acima de 0,25 % suprimiram a esporulação de C. gloeosporioides nas lesões. No entanto, os frutos tratados com 2 e 4 % de quitosana não amadureceram normalmente, permanecendo com a coloração da casca verde até o final do período de armazenamento. S. cerevisiae (20 mg mL-1) e B. thuringiensis (7,5 mg mL-1), aplicadas 24 h antes da inoculação do patógeno, reduziram a incidência da antracnose nos frutos, mas não o acúmulo de proteínas relacionadas à patogênese. Os cogumelos (A. blazei e L. edodes) e o ácido salicílico não foram eficientes em reduzir a incidência e a severidade da antracnose. In vitro, a irradiação gama, a UV-C, os ácidos acético e salicílico, a quitosana, S. cerevisiae e L. edodes inibiram o crescimento micelial do fungo. A germinação de conídios foi reduzida pelas irradiações gama e UV-C, pelos ácidos acético e salicílico e pela quitosana. Esses resultados demonstram a possibilidade dos agentes estudados serem utilizados no manejo da antracnose, bem como na redução da utilização ou da dosagem de fungicidas empregados no controle da doença.
This work had as main objectives evaluate the effect of biotic and abiotic agents (Saccharomyces cerevisiae, Bacillus thuringiensis, Lentinula edodes and Agaricus blazei), and abiotic (UV-C, gamma irradiation, acibenzolar-S-methyl, chitosan, acetic and salicylic acids) on the protection of papaya fruits against C. gloeosporioides, and study the biochemical mechanisms of resistance activated in the tissues in response to the treatment with the agents exhibiting better efficiency. The effects of the agents on the in vitro development of the fungus were also investigated. For this, papaya fruits cv. Golden were inoculated with C. gloeosporioides through subcuticular injection of 15 µL of the spore suspension and after 10 h treated with the different biotic and abiotic agents. To evaluate the possibility of resistance induction by the different agents, fruits were also inoculated 24, 48 and 72 h after treatments. The fruits were stored at 25 ºC / 80 %RH for 7 days and evaluated daily for the incidence and severity of the anthracnose. At the end of the storage period, the evaluation of the physical-chemical parameters (skin and flesh color, firmness, total soluble solids, pH and tritatable acidity) was carried out. The peroxidase, β- 1,3-glucanase and chitinase activities were also investigated when need. In vitro, mycelial growth, conidium germination and sporulation of the fungus in response to the different treatments were also evaluated. The results showed that the gamma irradiation (0.75 and 1 kGy) reduced the anthracnose incidence and severity. The UV-C did not have effect on the control of the rot and all the doses caused damages in the skin of the fruits. The acibenzolar-S-methyl reduced in more than 50 % anthracnose incidence and severity, and induced the highest activity of peroxidase, chitinase and β-1,3-glucanase, and did not modify the physical-chemical characteristics of the fruits. The acetic acid at 2.5 µL L-1 reduced rot severity and incidence. The chitosan (1, 2 and 4 %) significantly reduced the rot severity, and at 4 % was also efficient in reducing anthracnose incidence. Chitosan concentrations above 0.25 % suppressed the sporulation of C. gloeosporioides in the lesions. However, the fruits treated with chitosan at 2 and 4 % did not ripen normally, remaining with green skin until the end of the storage period. S. cerevisiae (20 mg mL-1) and B. thuringiensis (7.5 mg mL-1), applied 24 h before the pathogen inoculation, reduced anthracnose incidence, but did not change the activities of pathogenesis related proteins. The mushrooms (A. blazei and L. edodes) and the salicylic acid were not efficient in reducing the incidence and the severity of anthracnose. In vitro, gamma irradiation, UV-C, acetic and salicylic acids, chitosan, S. cerevisiae and L. edodes inhibited the mycelial growth. The conidium germination was reduced by gamma and UV-C irradiation, acetic and salicylic acids and chitosan. These results show that these agents can be utilized for anthracnose management, and on the reduction in the use or dosage of fungicides utilized on the anthracnose control.

Colletotrichum spp. su značajni prouzrokovači bolesti biljaka u svetu i u našoj zemlji, u polju i u skladištu. Na plodovima jabuke prisutne su dve vrste ovog roda, C. acutatum i C. gloeosporioides. Pomenute vrste uzrokuju ekonomski značajne gubitke posle berbe plodova jabuke, tokom skladištenja, transporta i plasmana na tržište. Nedoumice sa kojima se naučna javnost suočava kada je u pitanju ovaj rod jeste precizno utvrđivanje sistematske pozicije i definisanje vrsta i nižih kategorija. Klasične fitopatološke metode ne omogućavaju preciznuividentifikaciju do nivoa vrste. Otuda su molekularni pristupi sve zastupljeniji u identifikaciji Colletotrichum spp. U suzbijanju skladišnih patogena pretežno se primenjuju hemijski fungicidi. Zbog nepovoljnih toksikoloških svojstava i pojave rezistentnosti, primena hemijskih fungicida se sve više redukuje, a njihova upotreba posle berbe plodova zabranjena je u većini zemalja. Stoga, za održivi razvoj poljoprivredne proizvodnje neophodna su istraživanja usmerena ka otkrivanju mikroorganizama i prirodno sintetisanih materija koje imaju potencijala za primenu u biološkoj zaštiti.Cilj ovog rada je da se utvrdi zastupljenost Colletotrichum spp. na uskladištenim plodovima jabuke u R. Srbiji, kao i pouzdanost klasičnih i molekularnih metoda za njihovu identifikaciju do nivoa vrste i nižih kategorija. Utvrđivanje filogenetske pozicije i genetičke udaljenosti izolata je takođe svrstano u ciljeve istraživanja. Nadalje, cilj je i da se izdvoje mikroorganizami i etarska ulja koja ispoljavaju antifungalno delovanje na Colletotrichum spp. Zatim, da se za mikroorganizam sa najizraženijim antifungalnim delovanjem definišu uslovi kultivacije (sastav hranljive podloge i trajanje) koji maksimizuju njegovu antifungalnu aktivnost i utvrdi način njegovog delovanja.Utvrđeno je da su Colletotrichum spp. redovno prisutni na uskladištenim plodovima jabuke u Vojvodini i delovima zapadne, centralne i jugoistočne Srbije i da je njihova zastupljenost u odnosu na druge fitopatogene gljive 7,8-10%. Zbog osetljivosti C. gloeosporioides na niske temperature, C. acutatum postaje sve dominantnija vrsta ovog roda na uskladištenim plodovima jabuke. Razlikovanje C. acutatum i C. gloeosporioides je moguće na osnovu fenotipskih karakteristika kolonija (u slučaju hromogenih izolata C. acutatum), dužine konidija, rasta kolonija na 5 i 35°C i brzine rasta kolonija na različitim podlogama. Oblik i širina konidija i optimalna temperatura rasta su nedovoljno pouzdani kriterijumi za identifikaciju do nivoa vrste. PCR metodom uz primenu prajmera specifičnih za vrstu uspešno se identifikuju C. acutatum i C. gloeosporioides. Primenom univerzalnih prajmera ITS1 i ITS4 amplifikuju se rDNK-ITS sekvence ovih izolata. Analizom sekvenci izrađuju se filogenetska stabla visoke stabilnosti i jasno razdvajaju C. acutatum i C. gloeosporioides, a u okviru C. acutatum se odvajaju nehromogeni i hromogeni izolati. S. hygroscopicus, S. aureus, B. cereus, P. aeruginosa i B. subtilis sojevi N146, ST 1/III, Č13 i QST 713 ispoljavaju antifungalnu aktivnost na Colletotrichum spp. in vitro i in vivo. S. hygroscopicus obezbeđuje zaštitu plodova na nivou sa hemijskim fungicidima (trifloksistrobin, boskalid+piraklostrobin, pirimetanil+flukvinkonazol, kaptan). Kultivacijom S. hygroscopicus u podlozi sa 15,07 g/l glicerola, 5,28 g/l ekstrakta kvasca i 0,81 g/l fosfata, uvtrajanju 3-4 dana, postiže se maksimalno antifungalno delovanje na Colletotrichum spp. Antifungalno delovanje S. hygroscopicus zasnovano je na produkciji ekstracelularnih, termostabilnih metabolita. Dvomesečno skladištenje trtiranih plodova na 2±0,5°C ne slabi antifungalno delovanje S. hygroscopicus. Etarska ulja origana i timijana ispoljavaju snažno inhibitorno delovanje na Colletotrichum spp.
Colletotrichum spp. are significant plant pathogens worldwide in field, as well as on stored fruits. Two species of this genera, C. acutatum and C. gloeosporioides, can occur on apple fruits. The species cause significant economic losses on apple fruits after harvest, during storage, transport and marketing. Scientific community faces confusion in defining precise systematic position of this genera, as well as in identification to the species level and lower categories. Conventionalviiiphytopathological methods do not provide precise identification to the species level. Thus, molecular approaches are taking the lead in Colletotrichum spp. identification. Post-harvest pathogens are mainly managed by chemical fungicides. Due to adverse toxicological properties and resistance occurrence, use of chemical fungicides is being reduced, and their application after harvest is prohibited in most countries. Therefore, studies regarding detection of microorganisms and naturally synthetized substances with a potential for application in biological control are necessary for sustainable development of agriculture.The aim of this study was to determine frequency of Colletotrichum spp. on stored apple fruits in the Republic of Serbia as well as reliability of conventional and molecular methods in their identification to the species level and lower categories. Determination of phylogenetic position and molecular distance of the isolates were also set as investigation goals. The aim was also to determine microorganisms and essential oils with antifungal activity against Colletotrichum spp. Defining of cultivation conditions (nutrient medium composition and duration) for the microorganism with the most pronounced antifungal activity which maximize its activity and defining its mode of action were also set as goals.It was found that Colletotrichum spp. are commonly present on stored apple fruits in Vojvodina Province and in western, central and southeastern parts of Serbia with a share of 7.8-10% among other phytopathogenic fungi. Due to susceptibility of C. gloeosporioides to low temperatures, C. acutatum is becoming dominant species of this genus on stored apple fruits. Discrimination between C. acutatum and C. gloeosporioides is possible on the basis of phenotypic characteristics of colony (in the case of chromogenus isolates of C. acutatum), conidium length, colony growth at 5 and 35°C and colony growth rate on different media. Conidium shape and width and optimal growth temperature are insufficient criteria for identification to the species level. PCR method using species-specific primers is reliable for identification of C. acutatum and C. gloeosporioides. rDNK-ITS sequences of the isolates can be successfully amplified with universal primers, ITS1 and ITS4. Analyses of the sequences alow construction of highly stable phylogenetic trees with distinctively separated C. acutatum and C. gloeosporioides clades, and also additional clades of non-chromogenum and chromogenum C. acutatum isolates. S. hygroscopicus, S. aureus, B. cereus, P. aeruginosa and B. subtilis strains N146, ST 1/III, Č13 and QSTix713 exhibit antifungal activity against Colletotrichum spp. in vitro and in vivo. S. hygroscopicus ensures fruit protection at the same level as chemical fungicides (trifloxistrobin, boscalid+pyraclostrobine, pyrimethanil+flukvinconazol, captan). Cultivation of S. hygroscopicus in the medium with 15.07 g/l glycerol, 5.28 g/l yeast extract and 0.81 g/l phosphates, for 3-4 days, ensures maximal antifungal activity against Colletotrichum spp. Antifungal activity of S. hygroscopicus is based on production of extracelular, thermostable metabolites. Two-month storage of treated apple fruits at 2±0.5°C does not reduce antifungal activity of S. hygroscopicus. Oregano and thyme essential oils exhibit strong inhibitory effects on Colletotrichum

O estudo de fungos endofíticos tem sido intensificado nos últimos anos e abrange principalmente a descrição de novas espécies, o potencial de utilização no controle biológico de fitopatógenos e a produção de metabólitos secundários com atividades biológicas diversas. No entanto, a interação e a possível função destes organismos em plantas de clima tropical são pobremente estudadas e pouco compreendidas. A cana-de-açúcar é uma das principais culturas do Brasil e nos últimos anos está recebendo especial atenção devido à produção de etanol para uso como biocombustível. Um grande avanço no conhecimento das comunidades microbianas endofíticas desta planta tem sido alcançado, abrindo a possibilidade de estudo sobre as funções destes organismos na interação endofítica, bem como de seu potencial biotecnológico. Uma espécie que coloniza consistentemente os tecidos de cana-de-açúcar é o fungo Epicoccum nigrum. Este fungo é conhecido por sua atividade de biocontrole de fitopatógenos e por apresentar metabolismo secundário altamente desenvolvido e diverso. Nesse contexto, este trabalho teve por objetivos: 1) estudar a diversidade genética de isolados endofíticos de Epicoccum de cana-de-açúcar e de outros hospedeiros; 2) desenvolver um sistema de transferência de genes repórteres, visando à análise da interação in vitro e in vivo com cana-deaçúcar; 3) avaliar a atividade antimicrobiana in vitro de isolados endofíticos de Epicoccum contra diferentes fitopatógenos e patógenos humanos; 4) obter mutantes insercionais com atividade antimicrobiana ausente, visando à identificação de genes envolvidos no metabolismo secundário. O trabalho foi iniciado com o isolamento de Epicoccum de cana-de-açúcar. A análise por meio de abordagem polifásica resultou na separação dos isolados em dois taxa distintos (Grupo 1, E. nigrum; Grupo 2, Epicoccum sp.), demonstrando que a classificação de E. nigrum como uma única espécie variável deve ser reavaliada. A seguir, transformantes resistentes a higromicina B e expressando GFP foram obtidos por meio de transferência gênica mediada por A. tumefaciens. Ensaios de colonização de cana-de-açúcar in vitro e in vivo com a linhagem selvagem e transformada demonstraram a natureza não patogênica das linhagens e que E. nigrum é capaz de colonizar endofiticamente e persistir nas folhas desta planta. A análise da atividade antimicrobiana revelou extensa variação fisiológica, a qual foi correlacionada com a variabilidade genética dos isolados. Uma biblioteca de agrotransformantes de E. nigrum e Epicoccum sp. foi caracterizada quanto à perda da atividade antimicrobiana. A análise das seqüências flanqueadoras do T-DNA obtidas por TAIL-PCR a partir dos mutantes revelou que a inserção ocorreu em diferentes locais do genoma, muitos com elevada similaridade com proteínas hipotéticas de fungos, algumas sem função conhecida, e outras contendo domínios conservados envolvidos em diferentes funções celulares, como regulação da expressão gênica, transporte, obtenção de energia e outras funções enzimáticas. A análise do extrato orgânico por meio de bioautografia revelou a perda da atividade antimicrobiana de alguns mutantes insercionais, em comparação ao extrato da linhagem selvagem. O grande número de mutantes obtidos e caracterizados constitui uma ferramenta importante para o estudo molecular do metabolismo secundário deste fungo endofítico, auxiliando o entendimento da biossíntese de diversos compostos com estruturas complexas produzidos por esta espécie.
The study of endophytic fungi has increased in last few years and includes mainly the description of new species, biological control and production of compounds with biological activity. However, due the fact that few studies have been done, the role of these microorganisms inside the host plant from tropical areas is poorly understood. Sugarcane is one of the most important crop in Brazil, mainly due the biofuel production. Therefore, studies have been done to better understanding the role of endophytic microbial diversity inside the sugarcane plants, allowing the possibility to use this interaction in sugarcane production and also find endophytic isolates with biotechnological potential. Previous studies have shown that an important sugarcane endophytic fungus is Epicoccum nigrum, which species has been associated to biological control of many phytopathogens and also production of different secondary metabolites. In this way, the aims of the present study were to 1) study the genetic diversity of sugarcane endophytic Epicoccum; 2) develop a genetic transformation system for Epicoccum spp., allowing the in vitro and in vivo study of the interaction with sugarcane; 3) evaluate the in vitro antimicrobial activity of Epicoccum; 4) obtain mutants defective to antimicrobial activity and identification of genes associated to this activity. The polyphasic approach indicated that the evaluated Epicoccum population present two different genotypes (Group 1: E. nigrum and Group 2: Epicoccum sp.), suggesting that the classification of E. nigrum in only one species should be revised. Mutants resistant to hygromicin B and expressing GFP gene were obtained by transformation with Agrobacterium tumefaciens. In vitro and in vivo sugarcane colonization showed that the Epicoccum isolates and mutants were able to settling endophytically inside sugarcane without induce disease symptoms, and live up to leaves. The results shown that the antimicrobial activity was related to genetic variability, and this activity was better characterized by analysis of a obtained mutant library of E. nigrum and Epicoccum sp. The TAIL-PCR analysis revealed that the T-DNA introduced into the mutants was inserted in different regions of the fungi genome, truncating different genes those coding fungi hypothetical proteins, conserved domain associated to cellular function, such as genetic regulation, energy obtaining and others enzymatic activity. The analysis by bioautography indicated that some mutants lose the antimicrobial activity, allowing the correlation between the truncated genes and antimicrobial compound production. The evaluation of this mutant library showed that different genes are associated to antimicrobial compound production and may be an important tools to study the secondary metabolism in this endophytic fungus, allowing the understanding of biochemical pathway associated to biosynthesis of complex molecules produced by this fungus.

Microrganismos endofíticos são definidos como organismos que habitam em pelo menos durante um período de seu ciclo vital, o interior de um vegetal, sem causar aparentemente nenhum dano a este. Na busca de novos organismos e novos metabólitos secundários, um estudo foi conduzido visando avaliar a diversidade química de bactérias endofíticas por meio do isolamento e identificação de metabólitos secundários produzido por bactérias endofíticas de etnovariedades de mandioca, mantidas por tribos indígenas da Amazônia brasileira. Sessenta e sete bactérias endofíticas de mandioca foram selecionadas e submetidas à uma seleção através de testes de antagonismo in vitro. A bactéria endofítica, Bacillus pumilus foi a que apresentou forte ação inibitória contra os fitopatógenos Rhizoctonia solani, Pythium e Sclerotium rolfsii. Essa bactéria foi identificada através do sequenciamento de um fragmento do gene de 16S rRNA e por meio da análise de ácidos graxos (FAME). A obtenção dos metabólitos da bactéria selecionada foi realizada através da extração dos meios de cultura com os solventes hexano, acetato de etila e diclorometano. Após a concentração dos extratos, seus constituintes químicos foram realizados através de cromatografia em camada delgada, cromatografia em coluna, cromatografia gasosa acoplada ao espectrômetro de massas CG-EM) e cromatografia líquida acoplada ao espectrômetro de massas (CL-EM). Os extratos obtidos por meio de diclorometano e acetato de etila continham diferentes componentes químicos que mostraram atividade inibitória contra os três fitopatógenos testados. O método cromatográfico CL-EM permitiu a identificação de uma substância antifúngica produzida pela bactéria endofítica, Bacillus pumilus, conhecida como pumilacidina.
Endophytic microorganisms are defined as organisms that inhabit the interior of a vegetable at least during a period of the vital cycle, without causing any apparent damage. In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the chemical diversity of endophytic bacteria through the isolation and identification of secondary metabolites produced by endophytic bacteria of cassava cultivated by Brazilian Amazon Indian tribes. Sixty seven endophytic bacteria isolated from cassava were screened using in vitro antagonisms tests. An endophytic bacterium, the Bacillus pumilus, which showed a strong inhibitory activity against Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii was selected. This bacterium was identified by sequencing of 16S rRNA genes and by FAME. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy. The bacterial metabolites were extracted from the culture media using the solvents hexan, ethyl acetate and dichloromethane. After concentrate the extracts their chemical constituents were analyzed using thin layer chromatography (TLC), chromatography column, gas chromatography coupled with mass spectrometry (GC/MS) and liquid chromatography coupled with mass spectrometry (LC/MS). The extracts obtained with dichloromethane and ethyl acetate contained different chemical compounds that showed inhibitory activity against the three plant-pathogenic fungi tested. The LC/MS method allowed the identification of an antifungal compound produced by the B. pumilus, which is known as pumilacidin.

Fomes lignosus est un champignon parasite des racines de l'hevea (hevea brasiliensis). Dans le cadre de la lutte biologique contre ce pathogène, trois champignons antagonistes ont été étudiés : lentinus squarrosulus, cerrena meyenii et gloeophyllum striatum. Les caractères culturaux et les conditions optimales de développement des quatre champignons ont été étudiés

A pinta preta dos citros tem como agente causal o fungo G. citricarpa. A doença causa depreciação estética dos frutos e praticamente inviabiliza a exportação de laranja in natura principalmente para a União Européia, por ser considerada uma doença quarentenária. Frutos colhidos contendo infecções quiescentes, embora sem sintomas, podem desenvolvê-los durante o processo de exportação. A utilização de fungicidas é o principal método de controle utilizado em pré e pós-colheita. No entanto, o custo do controle químico é significativamente alto e já foi relatado o surgimento de isolados resistentes. A levedura S. cerevisiae é capaz de inibir o desenvolvimento in vitro de G. citricarpa e o antagonismo se deve a produção de compostos orgânicos voláteis de ação fungistática. Em virtude da procura por novos métodos de controle menos danosos à saúde humana e meio ambiente, dos prejuízos causados pela doença e do potencial dos compostos voláteis no controle da doença na pós-colheita, o trabalho visou identificar os compostos voláteis produzidos pela levedura e elucidar os mecanismos envolvidos na inibição do fitopatógeno. Esse conhecimento é fundamental para o desenvolvimento de técnicas inovativas de controle. A produção de compostos voláteis antimicrobianos por S. cerevisiae foi dependente do substrato utilizado para o seu cultivo, sendo a glicose, sacarose e maltose fontes de carbono favoráveis. Foi verificado através da análise em SPME-GC-MS que a levedura produziu em meio BDA, principalmente compostos pertencentes ao grupo dos álcoois, constituindo 95% da composição total, além de ésteres em menor proporção. A mistura artificial de voláteis mimetizou os efeitos dos voláteis produzidos pela levedura, sendo os valores de MIC50 e MIC100 de 0,48 µL mL-1 e 2,84 µL mL-1, respectivamente. A exposição do fitopatógeno aos compostos voláteis reduziu a síntese de proteínas e inibiu a atividade de enzimas associadas à morfogênese (quitinase, b-1,3-glucanase, lacase e tirosinase). Os compostos voláteis desencadearam o processo de estresse oxidativo no fungo, fato observado através da elevação da atividade de enzimas (superóxido dismutase e catalase) associadas a mecanismos de detoxificação de espécies ativas de oxigênio. A análise das proteínas diferencialmente expressas de G. citricarpa, através de eletroforese bidimensional, indicou que os compostos voláteis alteraram a expressão de 40 proteínas, sendo 29 inibidas e 11 superexpressas. A mistura artificial de voláteis também foi ativa contra fungos de diversos grupos taxonômicos, no entanto, não apresentou atividade sobre as bactérias testadas. Os compostos 3-metil-1-butanol e 2- metil-1-butanol foram os mais ativos quando utilizados isoladamente contra Colletotrichum gloesporioides, Penicillium digitatum, Sclerotinia sclerotiorum e Fusarium oxysporum. Sementes de feijão fumigadas com a mistura artificial de voláteis exibiram redução na incidência de S. sclerotiorum. A mistura também exibiu efeito nematicida sobre Meloidogyne javanica. Portanto, os resultados demonstram que os voláteis identificados em S. cerevisiae afetam a síntese de proteínas e a atividade de enzimas associadas ao crescimento vegetativo de G. citricarpa. Finalmente, fumigação utilizando compostos voláteis apresenta potencial no controle de G. citricarpa em pós-colheita, bem como de outros organismos de importância agronômica. O trabalho também contribuiu com novos elementos sobre o papel dos voláteis na interação entre microrganismos na natureza.
The citrus black spot has as causal agent the fungus G. citricarpa. The disease causes aesthetic depreciation of fruits and practically makes them unviable for exportation mainly to European Union where the disease is considered a quarantine pest. Harvested fruits can have quiescent infections although without symptoms and can develop them during the exportation process. The use of fungicides is the main chemical control method used in pre and post-harvest, however the cost is significantly high and it was already described the development of fungicide resistant strains. The yeast S. cerevisiae is able to inhibit the in vitro G. citricarpa growth, and the antagonism is due to production of volatile organic compounds (VOCs) of fungistatic effect. Because of the search for new control methods less harmful for human health and environment, the losses caused by the disease and the potential of the VOCs in the control of the pathogen in the post-harvest, the aim of this work was to identify the VOCs produced by the yeast and to elucidate the mechanisms involved in the inhibition of the fungus. This knowledge is essential for the development of innovative control techniques. The results showed that the production of antimicrobial VOCs by S. cerevisiae was dependent on the substrate used, being glucose, sucrose and maltose favorable carbon sources. It was verified through analysis in SPME-GC-MS that the yeast, grown on PDA medium, produced mainly compounds belonging to the group of alcohols (95% of the total composition), besides esters in smaller proportion. An artificial volatile mixture reproduced the effects of the VOCs produced by the yeast, being the values of MIC50 and MIC100 of 0.48 µL mL-1 and 2.84 µL mL-1, respectively. The phytopathogen exposition to the VOCs reduced protein synthesis and inhibited the activity of morphogenesis associated enzymes like chitinase, b-1,3-glucanase, laccase and tyrosinase. The VOCs initiated the oxidative stress process, which was observed through the increase in the activity of superoxide dismutase and catalase, enzymes associated to the detoxification of active oxygen species. The analysis of the differentially expressed proteins of G. citricarpa, through two-dimensional gel electrophoresis, indicated that the VOCs modified the expression of 40 proteins, being 29 down regulated and 11 up regulated. The artificial mixture of VOCs was also active against fungi of several taxonomic groups, however, there were no activity against bacteria. The compounds 3-methyl-1-butanol and 2-methyl-1-butanol were the most active when tested alone against Colletotrichum gloesporioides, Penicillium digitatum, Sclerotinia sclerotiorum and Fusarium oxysporum. Bean seeds fumigated with the artificial mixture of VOCs showed reduction in S. sclerotiorum incidence. In addition, the VOCs exhibited nematicidal effect against Meloidogyne javanica. The results demonstrate that the VOCs produced by S. cerevisiae affect protein synthesis and the activity of enzymes associated to the vegetative growth in G. citricarpa. Thus, the fumigation using VOCs presents potential in the control of citrus black spot disease in post-harvest as well as of other organisms of agronomic importance. The present workalso contributed with new information regarding the role of the VOCs in the interactions among microorganisms in nature.

There is a growing recognition globally that many agrochemicals are hazardous to humans, animals and the environment. Therefore, there is a need to substitute these chemical products with biological and physical treatments, and to change agronomic practices in order to control pests and diseases in agriculture. The primary objective of this thesis was to develop and test in laboratory, field and commercial packhouses trials as alternative control measures against green mould of citrus (caused by Penicillium digitatum Pers: Fr. Sacc) and Penicillium molds of litchi (caused by several Penicillium). A South African isolate of P. digitatum, isolated from an infected orange fruit, was found to be resistant to imazalil (the standard postharvest fungicide used in South Africa). Sixty yeast and 92 Bacillus strains were screened for their antagonistic activity against this isolate of P. digitatum. None of the yeasts or Bacillus isolates produced a curative action against P. digitatum on oranges. However, yeast Isolate B13 provided excellent preventative control of P. digitatum, superior to all the Bacillus isolates, when it was applied to citrus fruit prior to artificial inoculation with P. digitatum. Electron microscopy showed that yeast Isolate B13 inhibited conidial germination of P. digitatum. For the control of P. digitatum pre-harvest, trees were sprayed with a yeast, Isolate B13, a few months or a few days before harvest. However, this treatment alone proved to be ineffective in providing preventative control of green mould on Valencia oranges. For the control of P. digitatum preharvest, trees were treated with potassium silicate for a full season. Regular potassium silicate treatments resulted in a significant preventative control of P. digitatum infection on both navel and Valencia oranges. Treatment of Eureka lemons with potassium silicate as a postharvest treatment for the control of P. digitatum resulted in reduced disease lesion diameters when applied preventatively or curatively. Electron microscopy showed that potassium silicate inhibited germination of P. digitatum conidia and growth of its mycelium. Hot-water dip treatment at 50-58°C for 60-180 seconds (in increments of 15 seconds), significantly reduced infection development in inoculated wounds of Valencia oranges compared with control fruit treated with tap water, without causing any rind damage. The integration of the yeast, a hot water dip and potassium silicate pre-and postharvest applications provided control of P. digitatum control in multiple packhouse trials. The control achieved by the yeast Isolate B13 or hot-water, and potassium silicate in the packhouse alone was superior or equivalent to that provided by imazalil. A similar study was also carried out to determine possible control measures for Penicillium sp. on litchis. In this study, a total of 23 yeast and 13 Bacillus isolates were obtained from litchi fruit surfaces. Ten yeast and 10 Bacillus isolates that had shown good efficacy against P. digitatum of citrus were added to these for screening against Penicillium sp. of litchis. None of the yeasts or Bacillus isolates produced a curative action against Penicillium sp. infection on litchis. However, several yeast isolates (YL4, YL10, YLH and B13) resulted in reduced severity of the pathogen, when applied preventatively, compared with an untreated control. The yeast isolates were superior to all the Bacillus isolates, when applied to litchis prior to artificial inoculation by Penicillium infection on litchis. Potassium silicate as a postharvest treatment for the control of the pathogen caused reduced lesion diameters when applied preventatively or curatively to naturally infected litchis. The results presented in this thesis highlight the use of biological, physical and agronomic practices singly or in combination as an alternative control strategy against citrus postharvest green mould. This thesis also provides an insight into expanding these strategies, partly or fully, for the control of other postharvest Penicillium infections using litchi as an example.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Sphaeropsis sapinea and Diaporthe ambigua are important pathogens of forest and orchard tree species, respectively. Some isolates of S. sapinea are co-infected with two dsRNA viruses, SsRVl and SsRV2. Isolates of D. perjuncta (formerly thought to be D. ambigua) are infected with a positive-stranded RNA virus known as DaRV. While S. sapinea is. infected with a heterogeneous mixture of dsRNA elements of different sizes, D. perjuncta is infected with a single virus. This presents excellent opportunity for biocontrol of Diaporthe. The aim of this study was to assess these three viruses for possible application as biological control agents of S. sapinea and D. ambigua. This was' done by transfecting these with in vitro-produced RNA from the cloned viral genomes and assessing the pathogenicity of the transfected isolates on apples and apple trees. Attempts to transfect S. sapinea spheroplasts with SsRVl and SsRV2 failed. Co¬transfection of S. sapinea spheroplasts with both viruses also failed. Three isolates of D. ambigua and a single isolate of a Phomopsis sp. were successfully transfected with DaRV. Attempts to transfect the same fungi with a mutant of DaRV, bearing six codons for histidine immediately downsteam of an AUG thought to be a start codon for the translation of ORFl, failed. DaRV was originally thought to be isolated from D. ambigua. The fungal isolates transfected with DaRV were thought to be D. ambigua. The transfectants did not resemble the naturally-infected isolate. The ITS regions from the ribosomal DNA operon of these isolates were amplified using ITS 1 and ITS4 primer pair. The blast search revealed that the ITS sequence of the naturally-infected isolates are identical to D. perjuncta. One virus-free isolate was identified as a Phomopsis sp. while three other virus-free isolates were identified as D. ambigua. A PCR-based RFLP was developed to differentiate the naturally-infected D. perjuncta isolates from the virus¬free Phomopsis sp. and D. ambigua isolates. In the growth and pathogenicity studies, a DaRV-transfected, wild-type and negative control isolate of one Phomopsis and three D. ambigua isolates, were used. The DaR V -transfected Phomopsis sp. had a higher growth rate than the wild-type isolate. This DaRV-transfected Phomopsis sp. was more virulent on apples than the wild-type isolate. The wild-type isolate was slightly more virulent than the DaR V -transfected Phomopsis sp. on apple trees. There were no significant differences in growth rates between the DaRV-transfected and wild-type isolates of D. ambigua CMW5587 and D. ambigua CMW5287. There were no significant differences in virulence on apples between the DaRV-transfected and wild-type isolates of these fungi. The DaRV-transfected D. ambigua CMW5287 was more virulent than the wild-type isolate on apple trees. The DaRV-transfected D. ambigua CMW5587 had the same virulence as the wild-type isolate on both apples and apple trees. The DaRV-transfected D. ambigua CMW5288 had a slower growth rate than the wild-type isolate. There were no significant differences in virulence on apples between these isolates. The wild-type isolate of this isolate was significantly more virulent on apple trees than the DaRV-infected isolate. Although transfection was successfully done, the effects of DaRV on the Phomopsis sp. and D. ambigua isolates are not conclusive. In order to obtain conclusive results, virus-free isolates of D. perjuncta must be transfected. During the course of this study, there were no available virus-free isolates of this fungus.
Dissertation (PhD)--University of Pretoria, 2006.
Genetics
Unrestricted

Phytophthora vignae, causal agent of stem and root rot of cowpea (Vigna unguiculata), was reported for the first time in Sri Lanka. The pathogen was found in cowpea field soils from 3 of 5 geographic regions sampled. Only one site however, had plants exhibiting disease symptoms. Of the eight cowpea varieties grown in Sri Lanka, four were shown to be relatively resistant; all other legumes inoculated were completely resistant. Two morphologic and physiologic races of P. vignae were identified among the 24 isolates recovered, based on differential pathogenicity on cowpea varieties. Bacteria isolated from field soils, and other known bacterial biocontrol agents, inhibited P. vignae in culture, but only three Sri Lankan isolates considerably suppressed the disease in greenhouse tests. Volatile substances produced by most bacteria inhibited mycelial growth and sporangial production by P. vignae. The increased pH of the exposed medium suggested the involvement of ammonia. Volatile inhibitors were produced by these bacteria in soil, but only with added substrate; Strain DF-3101 also reduced oospore germination in soil. Cowpea plants inoculated with the VA mycorrhizal (VAM) fungus Glomus intraradices in P. vignae-infested soil were larger than non-mycorrhizal plants, but only at low levels of the pathogen. VAM colonization was reduced at high levels of the pathogen, and root infection by the pathogen was reduced by VAM. The fungicides metalaxyl, fosetyl-Al, Banrot, and Manzate-200DF reduced in vitro mycelial growth, but at different concentrations. Sporangia formation and germination, and oogonia formation by P. vignae, was reduced significantly by metalaxyl and fosetyl-Al. In greenhouse tests, metalaxyl, even at low concentrations, reduced disease; Fosetyl-Al was effective at high concentrations; Manzate-200DF was effective as a soil drench but not as a foliar spray; Banrot effectively reduced disease at 50 mg a.i./L. Exposure of a bacterial biocontrol agent to these fungicides in vitro did not affect its capacity to subsequently produce volatile inhibitors, but exposure to 10 ug/ml of metalaxyl and 50 ug/ml of Manzate-200DF reduced its capacity to subsequently inhibit mycelial growth of P. vignae.
Graduation date: 1991

The following body of research provides a detailed overview of the interactive effects of biocontrol agents and environmental factors and how these influence both the host plant and pathogen populations within hydroponic systems. Pythium and other zoosporic fungi are pathogens well suited to the aquatic environment of hydroponics. Motile zoospores facilitate rapid dispersal through fertigation water, resulting in Pythium becoming a yield reducing factor in most hydroponic systems and on most crops. With increasing trends away from pesticide use, biocontrol is becoming an ever more popular option. Unfortunately, much of our knowledge of biocontrol agents and their formulation can not be directly transferred to the widely differing environments of hydroponic systems. Paulitz (1997) was of the opinion that if biocontrol was to be successful anywhere, it would be in hydroponics. This is primarily due to the increased ability, in hydroponics, to control the growing environment and to differentiate between the requirements of the pathogen versus those of the host plant and biocontrol agent. Key environmental factors were identified as soil moisture, root zone temperature, form of nitrogen and pH. A review of the literature collated background information on the effects of biocontrol agents and environmental manipulation on plant growth and disease severity in hydroponic systems. A commercial formulation of Trichoderma (Eco-T(R1)) was used as the biocontrol agent in all trials. Dose responses in Pythium control and plant growth stimulation in lettuce were first determined using a horizontal trough system (closed system). In such systems optimum application rates were found to be lower than in field application (1.25x10[to the power of 5] spores/ml). This is probably because Trichoderma conidia are not lost from the system, but re-circulate until being transported into the root zone of a host plant. No significant growth stimulation was observed, although at high doses (5x10[to the power of 5] and 2.5x10[to the power of 5] spores/ml) a significant reduction in yield was recorded. Possible reasons for this growth inhibition are suggested and a new theory is proposed and investigated later in the thesis. In an open system of cucumber production (drip irrigated bag culture) no statistically significant results were initially obtained, however, general trends still showed the occurrence of positive biocontrol activity. The initial lack of significant results was mostly due to a poor knowledge of the horticulture of the crop and a lack of understanding of the epidemiology behind Trichoderma biocontrol activity. These pitfalls are highlighted and, in a repeat trial, were overcome. As a result it could be concluded that application rates in such systems are similar to those used in field applications. Management of soil moisture within artificial growing media can aid in the control of Pythium induced reductions in yield. A vertical hydroponic system was used to determine the interactive effects of soil moisture and Trichoderma. This system was used because it allowed for separate irrigation regimes at all 36 stations, controlled by a programmable logic controller (PLC). With lettuce plants receiving optimum irrigation levels, no significant reduction in yield was observed when inoculated with Pythium. However, after Pythium inoculation, stresses related to over- or under-watering caused significant yield losses. In both cases, Trichoderma overcame these negative effects and achieved significant levels of disease control, especially under higher soil moisture levels. Growth stimulation responses were also seen to increase with increasing soil moisture. Similar results were obtained from strawberry trials. These results show that Pythium control is best achieved through the integration of Trichoderma at optimum soil moisture. However, where soil moisture is above or below optimum, Trichoderma serves to minimize the negative effects of Pythium, providing a buffering capacity against the effects of poor soil moisture management. Pythium, root zone temperature and form of nitrogen interact significantly. In greenhouse trials using horizontal mini troughs with facilities for heating or cooling recirculating water, nitrate fertilizer treatments resulted in statistically significant results. Lettuce growth was highest at 12°C, although no significant differences in yield were observed between 12-24°C. Pythium was effective in causing disease over the same temperature range. Pythium inoculation did not result in yield reduction at 6 and 30°C. Trichoderma showed a slight competitive advantage under cooler temperatures (i.e., 12 degrees C), although significant biocontrol occurred over the 12-24 degrees C range. Ammonium fertilizer trials did not generate statistically significant data. This is possibly due to complex interactions between root temperature, ammonium uptake, and competitive exclusion of nitrification bacteria by Trichoderma. These interactions are difficult to replicate over time and are probably influenced by air temperature and available light which are difficult to keep constant over time in the system used. However, the data did lead to the first clues regarding the effects of Trichoderma on nitrogen cycling as plants grown with a high level of ammonium at high temperatures were seen to suffer more from ammonium toxicity when high levels of Trichoderma were added. In further trials, conducted in the recirculating horizontal mini trough system, it was determined that Trichoderma applications resulted in an increase in the percentage ammonium nitrogen in both the re-circulating solution and the growing medium. This was a dose-related response, with the percentage ammonium nitrogen increasing with increasing levels of Trichoderma application. At the same time an increase in ammonium in the root tissue was observed, corresponding with a decrease in leaf nitrate levels and an increase in levels of Cu, Na, Fe and P in leaf tissue. In independent pot trials, populations of nitrifying bacteria in the rhizosphere were also seen to decrease with increasing Trichoderma application rates. This led to the conclusion that the increase in ammonium concentration was as a result of decreased nitrification activity due to the competitive exclusion of nitrifying bacteria by Trichoderma. The possibility that Trichoderma functions as a mycorrhizal fungus and so increases the availability of ammonium for plant uptake is not discarded and it is thought that both mechanisms probably contribute. Water pH provides the most powerful tool for enhancing biocontrol of Pythium by Trichoderma. Trichoderma shows a preference for more acidic pHs while Pythium prefers pHs between 6.0 and 7.0. In vitro tests showed that Trichoderma achieved greater control of Pythium at pH 5.0, while achieving no control at pH 8.0. In greenhouse trials with the recirculating horizontal mini trough system, yield losses resulting from Pythium inoculation were greatest at pH 6.0 and 7.0, with no significant reduction in yield at pH 4.0. Biocontrol activity showed an inverse response with greatest biocontrol at pH 5.0.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2003.

Tese de Doutoramento em Ciências (Especialidade em Biologia)
The olive leaf spot (OLS) and the olive knot (OK) diseases are key constraints to olive production, due to their high incidence and related losses. However, none of the available control measures are effective against both diseases. This work aims to characterize the phyllosphere fungal communities, which reside in and on leaf/twig tissues of olive tree, and to understand their role in conferring host protection against these two diseases. Fungal communities of cultivars displaying differences on disease susceptibility were assess by culture-dependent approach and compared either among asymptomatic and symptomatic plant tissues or among different levels of disease incidence. The isolation of fungal communities was performed in autumn and spring. The relationship between foliar composition on fungi, secondary metabolites and host susceptibility was also evaluated. Phyllosphere fungal community revealed to be rich and abundant, comprising species belonging mainly to Ascomycota phyla and Cladosporiaceae family. Endophytic and epiphytic communities were distinct and affected primarily by season. In addition, climatic factors and the presence of disease were important in shaping epiphytes, whereas plant organ and genotype (at cultivar level) were the major drivers of endophytes. The interplay between the pathogen, the plant and its indigenous microbiota, also seemed to be critical for the establishment of fungal communities in the olive phyllosphere. The level of disease incidence was linked to host cultivar and to fungal and metabolite (phenolic and volatile compounds) composition of their leaves. Thus, it is possible that cultivar susceptibility might be in part related with the composition of fungal and metabolites. Some key fungal taxa and metabolites were identified to play an important role in conferring cultivar susceptibility/tolerance to OLS disease. Similarly, several fungal taxa were found to be specific to either asymptomatic or symptomatic plant tissues, suggesting their competitive or cooperative activity with the pathogen. Further investigations are still required to identify the functional role of these fungi and metabolites in conferring host plant protection to OLS and OK diseases.
O olho-de-pavão e a tuberculose são importantes ameaças à produção olivícola, devido à sua incidência e perdas relacionadas. Não existe nenhum método de luta que se tenha mostrado eficaz contra estas duas doenças. Este trabalho tem como objetivo caracterizar a comunidade fúngica da filosfera da oliveira, que reside interna e externamente nas suas folhas/ramos, de forma a compreender o seu papel na proteção da planta contra estas duas doenças. A comunidade fúngica foi avaliada em cultivares que apresentam diferenças de suscetibilidade às doenças, recorrendo a métodos culturais, e comparada entre material assintomático e sintomático ou entre diferentes níveis de incidência de doença. O isolamento de fungos foi realizado durante o outono e a primavera. Foi ainda avaliada a relação entre a composição foliar de fungos e de metabolitos secundários, e a suscetibilidade da planta às referidas doenças. A comunidade fúngica da filosfera mostrou ser rica e abundante, incluindo espécies pertencentes maioritariamente ao filo Ascomycota e à família Cladosporiaceae. A composição da comunidade endofítica foi distinta da epifítica, e mostrou ser fortemente influenciada pela estação do ano. Vários fatores climáticos e a presença de doença foram ainda cruciais na estruturação dos epifíticos, enquanto o órgão e o genótipo da planta (cultivar) influenciaram também a composição de endófitos. A interação entre o patogénico, a planta e a sua flora microbiana nativa, também revelou ser crítica para o estabelecimento das comunidades fúngicas na filosfera da oliveira. O nível de incidência de doença mostrou estar relacionado com a cultivar, e com a composição de fungos e metabolitos (fenóis e voláteis) das suas folhas. Este resultado sugere que a suscetibilidade da cultivar possa estar relacionada com a sua composição em fungos e metabolitos, tendo, alguns deles, mostrado ter um papel importante na suscetibilidade/ tolerância da cultivar ao olho-depavão. Algumas espécies fúngicas mostraram também estar fortemente associados quer a material sintomático ou assintomático, sugerindo que possam estabelecer relações de competição ou cooperação com o patogénico. Estudos adicionais são ainda necessárias para identificar a função destes fungos e metabolitos na proteção da oliveira contra o olho-de-pavão e a tuberculose da oliveira.
This research was partially supported by FEDER funds through COMPETE (Programa Operacional Factores de Competitividade) and by national funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the project EXCL/AGR-PRO/0591/2012. This work was supported by FCT under the project UID/MULTI/04046/2013. T. Gomes thanks FCT, POPH-QREN and FSE for PhD SFRH/BD/98127/2013 grant; and also the COST Action FA1405 for a short-term scientific mission (STSM) grant.

This M. Sc. thesis is based on using of mycoparasitic fungi Trichoderma virens, Clonostachys rosea f. catenulata in biological control against phytopathogenic fungi Sclerotinia sclerotiorum, Rhizoctonia solani, Botrytis cinerea and Fusarium solani. The efficacy of mycoparasitic fungi against pathogens was evaluated in dual cultural tests. The strains of T. virens and C. rosea f. catenulata isolated from soils in the Czech Republic were tested in the experiment. Reference strain was GL 21 fungus T. virens reisolated from commercially available bio-preparation SoilGard and strain C. rosea f. catenulata reisolated from Prestop Mix. All the strains were tested for biological and production properties. All strains are able to colonize the substrate and to suppress the growth and development of pathogens. Strain GL 21 of T. virens was used for seed coating of variety Scirocco in combination with products Guar gum and Carboxymethyl cellulose, which served as a carrier for stick on conidia. After 3 days, the effect of fungus T. virens was evaluated on energy of germination, development of roots of grain. The grain health was determined after 7 days. The fungus T. virens has a positive effect on the grains germination and grain health. During the vegetation the influence of seed coating by T. virens was observed on growth and development of spring wheat. The parameters such as number of plants per m2, tiller numbers, plants health, stand height, number of grains in the spike and thousand grain weight (TGW) were evaluated. During the vegetation the fungus T. virens has positive effect on the plant height.