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Thesis (M. Sc. Agriculture (Plant Protection)) -- University of Limpopo, 2021 / Mimusops zeyheri tree groves made up of seventeen trees collected from communities in Southern Africa were used in this study. The trees had high morphological variations in terms of growth rate, fruit (size and taste) and leaf (shapes and sizes) and their identification was made by communal people from where the trees were collected using their morphological characteristics. Generally, this evergreen tree is drought‒tolerant, salt‒tolerant and highly resistant to root-knot (Meloidogyne spp.) nematodes, along with various other pests. This could probably be attributed to high concentration of latex in aboveground organs. Some typical fungal rust symptoms have been observed believed to be the cause of high leaf abscission in certain groves. Currently, there is no report of leaf rust disease on M. zeyheri plants and the mechanism of resistance to other pests is not documented. The objective of this study was to (i) identify the pathogen associated with M. zeyheri leaf rust symptoms using morphological technique and to (ii) determine levels and types of potential defence chemicals and endophytes in M. zeyheri. Samples of M. zeyheri leaves showing rust like symptoms were collected from University of Limpopo, South Africa (23°53”10’S, 29°44”15’E) during summer in September 2018. Light compound microscope and electron microscope were used in the identification of the leaf rust spores. The species identity of the seventeen M. zeyheri trees that form a grove collection at University of Limpopo was confirmed using internal transcribed spacer (ITS) of ribosomal nuclear DNA. DNA extraction and sequencing was done with the help of Inqaba Biotechnologies. Obtained DNA sequences were aligned using CLUSTALX (2.0), with the phylogenetic tree constructed through the neighbour-joining method (NJM) in MEGA v. 5.1 programme. Evolutionary distances were computed using the Juke– Cantor method. Phytochemicals in leaves were identified and quantified using Liquid chromatography–mass spectrometry (LC–MS) at ARC-VOP. The pustules on the collected leaves contained reddish brown spores. The uredospores were oval and ellipsoidal under a light microscope. The size of spores ranged between 35-37 × 24-26 µm. The cell walls showed bilaminate structures with the outer layer hyaline. The warts were rod shaped with one subequatorial germ pore. The most common identified endophytic fungi observed in all M. zeyheri leaves were Teratosphaeria species, Zeloasperium species, Pezizomycotina. In addition, endophytes such as Cladosporium species, Aspergillus species, Phyllosticta species and Epicoccum species were also identified to be associated with some M. zeyheri trees. There were significant differences on the level of tannins, flavonoids, proteins and phenolics among the M. zeyheri trees. The highest level of tannins was 7.2151 mg/g and the lowest being 2.7232 mg/g. The highest level of flavonoids was 1.1537 mg/g the lowest being 0.0123 mg/g. The level of phenolics among the trees ranged from 2.4749 mg/g to 1.5788 mg/g. Protein content ranged from 5.3100% to 2.7967% among the trees. Very high levels of tannins, flavonoids, phenolics among the trees when compared with others studies indicate the potential role of these metabolites in previously reported resistance of M. zeyheri to a number of pests. The morphological characteristics of the identified leaf rust pathogen causing rust symptoms on M. zeyheri in South Africa is more similar to Maravalia species previously isolated from M. caffra. And as such, this finding is paramount, as it is the first report of association between the pathogen and the plant. Teratosphaeria species has been associated with stem cancer in Eucalyptus trees, its presence in M. zeyheri species in asymptomatic plants is a major find since the tree is common in Limpopo and Mpumalanga, Provinces well known for forestry production making M. zeyheri a potential host for pathogens of commercial forestry production. Other endophytes observed such as Aspergillus species, Phyllosticta species and Cladosporium species have been shown to protect plants against some pests and pathogenic organism. Further studies to determine the direct role of identified phytochemicals and endophytes in the resistance of M. zeyheri to pests needs to be conducted

No / Until now little attention has been paid to the development of military capabilities designed to target food crops with biological warfare agents. This book represents the first substantive study of state-run activities in this field. It shows that all biological warfare programs have included a component concerned with the development of anti-crop agents and munitions. Current concern over the proliferation of biological weapons is placed in the context of the initiative to strengthen the Biological and Toxin Weapons Convention. The author concludes that the risks posed by this form of warfare can be minimized by the implementation of regimes concerning the peaceful use and control of plant pathogens that pose a risk to human health and the environment.

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

Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs. / AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en inokulumbronne van die Focep-populasie nie. Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium fistulosum) berig. Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre metodes ontwikkel kan word vir die identifisering van die mees virulente en wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie aspekte te bestudeer. Om die eerste doel van die studie te bereik is die genetiese diversiteit en evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F. oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424) en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426). VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en 0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde (IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese materiaal tussen isolate gedui. Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area (NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS (RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer, maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het. Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F. oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word, aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad het. Die derde doel van die studie was om vas te stel of saad en saailinge inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was. Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die meestal hoogs virulente isolate uit volwasse bolle.

Fungos endofíticos estão presentes em plantas de diversos ambientes e produzem compostos com amplas propriedades químicas e aplicações, tanto na área médico-farmacológica quanto na agronômica. Entretanto, ainda há muito a ser investigado sobre seu potencial biotecnológico, principalmente em locais pouco explorados. As ilhas apresentam um ambiente particular e altamente vulnerável, tornando-as locais promissores na busca de organismos pouco comuns ou ainda endêmicos. Sendo assim, neste trabalho foi realizado o isolamento e estudo químico e biológico de fungos endofíticos isolados de 2 espécies plantas do gênero Anthurium da Ilha de Alcatrazes-SP. Para isto, fragmentos foliares das plantas A. loefgrenii (HRCB 46467) e A. alctrazense (HRCB 46465 - endêmica da ilha) foram inoculados em 10 meios de cultivo com diferentes composições, resultando no isolamento de 106 fungos endofíticos. Por meio de análises químicas por MALDI-TOF-MS e ensaio biológico contra fitopatógenos, foram selecionados 3 fungos para estudo. Estes foram identificados por técnicas morfológicas e moleculares como sendo Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) e Aureobasidium melanogenum (P7AF2F3). No estudo dos metabólitos secundários de P. citrinum foi isolado o composto citrinina, o qual apresentou atividade inibitória do crescimento micelial dos fitopatógenos Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0,48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) e Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Outras frações obtidas do meio metabólico de P. citrinum (fração F3a3 e citrinina) apresentaram atividade inibitória (100% de inibição) à formas promastigotas de Leishmania infantum. No estudo dos metabólitos secundários de P. simplicissimum foi obtida a fração F2b, ativa contra L. infantum (100% de inibição), da qual foram isolados os compostos andrastina A e penicisoquinolina, sendo o primeiro relato de sua produção por esta espécie, além de outros 5 compostos ainda não identificados. No estudo dos metabólitos secundários de A. melanogenum foi isolado o composto metil-orselinato, relatado pela primeira vez para este fungo. Do mesmo fungo foi obtida a fração F1d2l ativa contra L. infantum (100% inibição), da qual foram isolados 2 compostos ainda não identificados. Este é o primeiro relato de fungos isolados de antúrios da ilha de Alcatrazes, bem como do estudo de seus metabólitos secundários. Este trabalho apresenta contribuição no conhecimento sobre fungos endofíticos e seu potencial metabólico, com aplicações nas áreas agronômica e médico-farmacológica. / Endophytic fungi are present in plants in various environments and produce compounds with wide chemical properties and applications, both in the medical and in agronomic field. However, much remains to be investigated about their biotechnological potential, especially in unexplored places. Islands have a particular and highly vulnerable environment, making them promising sites in search of unusual or endemic organisms. Thus, this work represents the isolation and chemical and biological study of endophytic fungi isolated from 2 species of plants of the genus Anthurium of Alcatrazes island-SP. For this, leaf fragments from plants A. loefgrenii (HRCB 46467) and A. alcatrazense (HRCB 46465 - endemic plant from the Island) were inoculated onto 10 culture media with different composition, resulting in the isolation of 106 endophytic fungi. Three strains were selected to be studied through chemical analysis by MALDI-TOF-MS and bioassay against phytopathogens. These were identified by morphological and molecular techniques as Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) and Aureobasidium melanogenum (P7AF2F3). In the study of secondary metabolites of P. citrinum it was isolated the compound citrinin, which showed inibitory activity against the plant pathogens Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0.48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) and Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Other fraction obtained from the metabolic extract of P. citrinum (F3a3 fraction and citrinin), showed inibitory activity (100% inibition) to Leishmania infantum promastigotes. In the study of secondary metabolites of P. simplicissimum it was obtained F2b fraction, active against L. infantum (100% inhibition), which were isolated andrastin A and penicisoquinoline compounds, the first report of its production for this species, as well 5 unidentified compounds. In the study of secondary metabolites from A. melanogenum was isolated the methyl-orsellinate compound, first reported for this fungus. From the same strain was obtained F1d2l fraction, active against L. infantum (100% inhibition), from which were isolated 2 unidentified compounds. This is the first report of fungi isolated from Alcatrazes Island anthuriums and the study of their secondary metabolites. This study presents contribution to knowledge of endophytic fungi and their metabolic potential with applications in the medical and agronomic fields.

Fungos endofíticos estão presentes em plantas de diversos ambientes e produzem compostos com amplas propriedades químicas e aplicações, tanto na área médico-farmacológica quanto na agronômica. Entretanto, ainda há muito a ser investigado sobre seu potencial biotecnológico, principalmente em locais pouco explorados. As ilhas apresentam um ambiente particular e altamente vulnerável, tornando-as locais promissores na busca de organismos pouco comuns ou ainda endêmicos. Sendo assim, neste trabalho foi realizado o isolamento e estudo químico e biológico de fungos endofíticos isolados de 2 espécies plantas do gênero Anthurium da Ilha de Alcatrazes-SP. Para isto, fragmentos foliares das plantas A. loefgrenii (HRCB 46467) e A. alctrazense (HRCB 46465 - endêmica da ilha) foram inoculados em 10 meios de cultivo com diferentes composições, resultando no isolamento de 106 fungos endofíticos. Por meio de análises químicas por MALDI-TOF-MS e ensaio biológico contra fitopatógenos, foram selecionados 3 fungos para estudo. Estes foram identificados por técnicas morfológicas e moleculares como sendo Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) e Aureobasidium melanogenum (P7AF2F3). No estudo dos metabólitos secundários de P. citrinum foi isolado o composto citrinina, o qual apresentou atividade inibitória do crescimento micelial dos fitopatógenos Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0,48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) e Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Outras frações obtidas do meio metabólico de P. citrinum (fração F3a3 e citrinina) apresentaram atividade inibitória (100% de inibição) à formas promastigotas de Leishmania infantum. No estudo dos metabólitos secundários de P. simplicissimum foi obtida a fração F2b, ativa contra L. infantum (100% de inibição), da qual foram isolados os compostos andrastina A e penicisoquinolina, sendo o primeiro relato de sua produção por esta espécie, além de outros 5 compostos ainda não identificados. No estudo dos metabólitos secundários de A. melanogenum foi isolado o composto metil-orselinato, relatado pela primeira vez para este fungo. Do mesmo fungo foi obtida a fração F1d2l ativa contra L. infantum (100% inibição), da qual foram isolados 2 compostos ainda não identificados. Este é o primeiro relato de fungos isolados de antúrios da ilha de Alcatrazes, bem como do estudo de seus metabólitos secundários. Este trabalho apresenta contribuição no conhecimento sobre fungos endofíticos e seu potencial metabólico, com aplicações nas áreas agronômica e médico-farmacológica. / Endophytic fungi are present in plants in various environments and produce compounds with wide chemical properties and applications, both in the medical and in agronomic field. However, much remains to be investigated about their biotechnological potential, especially in unexplored places. Islands have a particular and highly vulnerable environment, making them promising sites in search of unusual or endemic organisms. Thus, this work represents the isolation and chemical and biological study of endophytic fungi isolated from 2 species of plants of the genus Anthurium of Alcatrazes island-SP. For this, leaf fragments from plants A. loefgrenii (HRCB 46467) and A. alcatrazense (HRCB 46465 - endemic plant from the Island) were inoculated onto 10 culture media with different composition, resulting in the isolation of 106 endophytic fungi. Three strains were selected to be studied through chemical analysis by MALDI-TOF-MS and bioassay against phytopathogens. These were identified by morphological and molecular techniques as Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) and Aureobasidium melanogenum (P7AF2F3). In the study of secondary metabolites of P. citrinum it was isolated the compound citrinin, which showed inibitory activity against the plant pathogens Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0.48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) and Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Other fraction obtained from the metabolic extract of P. citrinum (F3a3 fraction and citrinin), showed inibitory activity (100% inibition) to Leishmania infantum promastigotes. In the study of secondary metabolites of P. simplicissimum it was obtained F2b fraction, active against L. infantum (100% inhibition), which were isolated andrastin A and penicisoquinoline compounds, the first report of its production for this species, as well 5 unidentified compounds. In the study of secondary metabolites from A. melanogenum was isolated the methyl-orsellinate compound, first reported for this fungus. From the same strain was obtained F1d2l fraction, active against L. infantum (100% inhibition), from which were isolated 2 unidentified compounds. This is the first report of fungi isolated from Alcatrazes Island anthuriums and the study of their secondary metabolites. This study presents contribution to knowledge of endophytic fungi and their metabolic potential with applications in the medical and agronomic fields.

The increasing concerns about chemical pesticides that are environmentally hazardous and the continuous development of resistance by palhogens to chemical pesticides have led to this study. Many studies have shown that some Gram-negative bacteria, such as Pseudomonas flouresens, control plant diseases and promote plant growth. In this study Gram positive bacteria, Bacillus sp., were chosen because of their ability to produce endospores. Endospores can be used in stable, dry formulations. The advantage of using endospores is their ability to survive harsh conditions such as droughts and high temperatures, which give a long shelf life to the biological control agent. Bacillus isolates were recovered from the rhizosphere of 12 different crops, and were subsequently screened in vitro for their antimicrobial activity. Of 130 isolates, 87 exhibited antimicrobial activity against the test organisms: Rhizoctonia solani, Pythium sp., Phytophthora cinnamoni, Fusarium sp., and single representatives of Gram negative and Gram positive bacteria, namely, Erwinia carotovora and Staphylococcus aureus respectively. The Bacillus isolates B77, B81 and B69 inhibited all the test organisms investigated, which suggests that they produced broad spectrum antimicrobial compounds or more than one antimicrobial compound. Of the isolates that showed antimicrobial activity, 78 of them did not inhibit Trichoderma harzianum K D, which is a registered biological control agent; indicating their potential for combined application. Selected Bacillus isolates were tested for the biological control of R. solani under greenhouse conditions in wheat, cabbage, tomato, maize, and cucumber seedlings. Bacillus isolates were applied as seed treatments, and the inoculated seeds were planted in R. solani infested speedling trays. Shoot dry weight measurement of seedlings indicated that 12 out of 19 Bacillus isolates showed significantly different shoot dry weight in wheat whereas all the isolates tested in tomato and cucumber gave significantly different shoot dry weight. No significantly different shoot dry weight was obtained for maize or cabbage. Seed emergence findings indicated that none of the Bacillus isolates gave significantly different emergence percentage on wheat, cabbage, tomato, and maize but all of them showed significantly different emergence percentage on cucumber. The results indicate that both the pathogen and the biological control agents exhibited varying levels of specificity on each crop tested. The biological control potential of the best Bacillus isolates was tested on bean and maize crops in the field. Green bean and maize seeds were coated with the selected Bacillus isolates and then sown under field conditions. For each isolate, four replicate treatment plots were established, with and without a R. solani inoculum. Percentage emergence, plant survival levels to harvesting and yield of maize cobs and green beans pods were measured. For all parameters measured the positive and negative controls were not significantly different thereby rendering the results for the entire field study inconclusive. However, Bacillus isolates B77, BII, R5 and R7 improved green bean pod yield and Bacillus Isolate B8I increased maize yield, indicating their potentials as plant growth promoting rhizobacteria (PGPR). / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Various Trichoderma and Bacillus spp. have been documented as being antagonistic to a wide range of soilborne plant pathogens, as well as being plant growth stimulants. Successes in biological control and plant growth promotion research has led to the development of various Trichoderma and Bacillus products, which are available commercially. This study was conducted to evaluate the effect of six Trichoderma spp. and three Bacillus spp. and their respective combinations, for the biological control of Rhizoctonia solani damping-off of cucumber and plant growth promotion of dry bean (Phaseolus vulgaris L.). In vivo biological control and growth promotion studies were carried out under greenhouse and shadehouse conditions with the use of seed treatment as the method of application. In vitro and in vivo screening was undertaken to select the best Trichoderma isolates from 20 Trichoderma isolated from composted soil. For in vitro screening, dual culture bioassays were undertaken and assessed for antagonisms/antibiosis using the Bell test ratings and a proposed Invasive Ability rating based on a scale of 1-4 for possible mycoparasitic/hyperparasitic activity. The isolates were further screened in vivo under greenhouse conditions for antagonistic activity against R. solani damping-off of cucumber (Cucumis sativus L.) cv. Ashley seedlings. The data generated from the in vivo greenhouse screening with cucumber plants were analysed and grouped according to performance of isolates using Ward‟s Cluster Analysis based on a four cluster solution to select the best isolates in vivo. Isolates exhibiting marked mycoparasitism of R. solani (during ultrastructural studies) viz, T. atroviride SY3A and T. harzianum SYN, were found to be the best biological control agents in vivo with 62.50 and 60.06% control of R. solani damping-off of cucumber respectively. The in vitro mode of action of the commercial Trichoderma product, Eco-T®, and Bacillus B69 and B81 suggested the production of antimicrobial substances active against R. solani. In vitro interaction studies on V8 tomato juice medium showed that the Trichoderma and Bacillus isolates did not antagonise each other, indicating the possibility of using the two organisms together for biological control and plant growth promotion studies. Greenhouse studies indicated that combined inoculation of T. atroviride SYN6 and Bacillus B69 gave the greatest plant growth promotion (43.0% over the uninoculated control) of bean seedlings in terms of seedling dry biomass. This was confirmed during in vivo rhizotron studies. However, results obtained from two successive bean yield trials in the greenhouse did not correlate with the seedling trials. Moreover, no increase in protein or fat content of bean seed for selected treatments was observed. In the biological control trials with cucumber seedlings, none of the Trichoderma and Bacillus combinations was better than single inoculations of Eco-T®, T. atroviride SY3A and T. harzianum SYN. Under nutrient limiting conditions, dry bean plants treated with single and dual inoculations of Trichoderma and Bacillus isolates exhibited a greater photosynthetic efficiency that the unfertilized control plants. Bacillus B77, under nutrient limiting conditions, caused 126.0% increase in dry biomass of bean seedlings after a 35-day period. Nitrogen concentrations significantly increased in leaves of plants treated with Trichoderma-Bacillus isolates. However, no significant differences in potassium and calcium concentrations were found. Integrated control (i.e. combining chemical and biological treatments) of R. solani damping-off of cucumber seedlings proved successful. In vitro bioassays with three Rizolex® concentrations, viz., 0.01g.l-1, 0.1g.l-1 and 0.25g.l-1 indicated that the selected Trichoderma isolates were partly sensitive to these concentrations whereas the Bacillus isolates were not at all affected. In a greenhouse trial, up to 86% control was achieved by integrating 0.1g.l-1 Rizolex® with T. harzianum SYN, which was comparable to the full strength Rizolex® (1g.l-1) application. Irrespective of either a single or dual inoculations of Trichoderma and/or Bacillus isolates used, improved percentage seedling survival as achieved with the integrated system, indicating a synergistic effect. The results presented in this thesis further reinforce the concept of biological control by Trichoderma and Bacillus spp. as an alternative disease control strategy. Furthermore, this thesis forms a basis for Trichoderma-Bacillus interaction studies and proposes that the two organisms could be used together to enhance biological control and plant growth promotion. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

With an increasing realization that many agrochemicals are hazardous to animals and humans, came the desire to replace these chemical agents with biological approaches that are more friendly to the environment and human health. Microorganisms play an important role in plant disease control, as naturally occurring antagonists. Microorganisms may also have beneficial effects on plant development when applied to plant roots. Research efforts worldwide have recorded successes in biological control and growth stimulation on many crops, particularly when using members of the genera Bacillus and Trichoderma. Their use on citrus rootstock could be advantageous to nurserymen and growers in reducing the incidence of seedling mortality and increasing production. To achieve these objectives, laboratory and tunnel experiments were conducted to develop effective biocontrol agents for citrus seedlings and cuttings. Nineteen 0 ut 0 f 23 Trichoderma isolates tested in vitro against Phytophthora p arasitica sp showed antagonistic activity by hyperparasitism and four out of eight Bacillus isolates resulted in antagonism by forming inhibition zones. The positive in vitro activity of Trichoderma and Bacillus isolates on Phytophthora provided motivation step for further trials in the greenhouse to evaluate their biological control activity on citrus seedlings and cuttings. A greenhouse trial was carried out to evaluate the biological control potential of 23 Trichoderma isolates (drenched at 5 x 105 spores / rnI) and two Bacillus isolates (drenched at 1 X 106 or 1 X 108 colony forming units (CFU) / rnI) to suppress Phytophthora parasitica sp. of rough lemon (Citrus jambhirini Lush.) seedlings. Five isolates ofTrichoderma (AA12, AA5, Trichoderma harzianum (AA16), SY3F and Eco-T~ were highly effective in suppressing Phytophthora root rot, with AA12 providing the best control. The Bacillus isolates also suppressed the pathogen but were not as effective as the Trichoderma isolates. This trial was used to test for growth stimulation activity by some of the biocontrol agents. To verify these results, a further trial was carried out to evaluate growth stimulation capabilities in the absence of any pathogen. Trichoderma Isolates AA13 and AA17 caused no 111 change in seedling growth, while other Trichoderma and Bacillus isolates had an inhibitory effect on the seedling growth. This trial indicated that the biocontrol activity was affected by inoculum densities, and as a result in vitro sporulation capacity was evaluated. TrichodermaIsolate AA16 was the largest spore producer, followed by Eco-T®. Spore production was lowest from Trichoderma isolates AA4 and AA12. Growth stimulation responses of Trichoderma Isolates AA4, AA16, Eco-TID and SYN6 were further studied at four different doses (1 X 103, 1 X 104, 5 X 105 or 1 X 106 spores / ml) on rough lemon and trifoliate orange seedlings. Trifoliate oranges responded positively to 1 X 104 and 5 X 105 spores / ml of Eco-TID, but rough lemon responded negatively to all dosages of the Trichoderma isolates applied. This indicates that the inoculum density responses may be host specific. Higher population density of 1 X 106 spores / ml of all tested Trichoderma isolates had a stunting effect on seedling growth of both species. Based on t he positive results 0 f individual applications of some Trichoderma and Bacillus isolates, of the biological control agents on rough lemon seedlings against Phytophthora parasitica in an earlier greenhouse trial, their combined effect in the control of the pathogen was performed. Before carrying out a greenhouse trial, activities of the isolates to be combined were evaluated in vitro. This trial showed that Trichoderma Isolates AA16 and Eco-T®were compatible. Trichoderma isolates AA16 and Eco-T®were also found to be compatible with Bacillus Isolates B77, B81 and PHP. As a result, further in vivo trials were conducted. The tunnel trials were carried out as two separate experiments: In the first experiment, a combination of two Trichoderma Isolates A A 16 and Eco-T®was conducted assayed at 5 X 105 or 1 X 106 spores / ml, on rough lemon seedling, and cuttings and trifoliate orange and sour orange seedlings. A combination of Trichoderma isolate AA16 and Eco-T®at 5 X 105 spore / ml increased significantly the new flush biomass of rough lemon cuttings compared to AA16 alone, but was not different from Eco-TID alone. The combination of AA16 and Eco-T® achieved no change of biomass of rough lemon and trifoliate orange seedlings. The combination of AA16 and Eco-TID did not increase the root biomass of sour orange compared to AA16 or Eco-r® alone. The combination of AA16 and Eco-r® at higher doses (1 x 106 spores / ml) showed significantly better suppression of Phytophthora root rot of rough lemon cuttings but did not show disease suppression in all seedling species verities tested. In a second experiment, individual and combined effects of Trichoderma isolates (drenched at 5 X 105 spores / ml) with Bacillus isolate (drenched at 1 X 106 colony forming units (CFU) / ml) for suppression of Phytophthora root rot on rough lemon and trifoliate orange seedlings was performed. The combination of Trichoderma Isolate AA16 and Bacillus Isolate B81 increased root biomass on rough lemon seedlings compared to the combination of Trichoderma AAI6 or Bacillus PHP but was not significantly different to Trichoderma AA16 alone. Bacillus PHP combined with Trichoderma AA16 or singly had no effect on rough lemon seedlings. Combining Trichoderma Eco--r® and with Bacillus B8I or PHP did not increase biomass of rough lemon seedlings compared to Trichoderma Isolate Eco--r® alone. There was no statistically significant differences in the effects of the combinations of the Trichoderma and Bacillus isolates compared to their individual applications on the biomass of trifoliate oranges. This study established the antagonistic potential of several South African isolates of Trichoderma and Bacillus as a viable alternative to agrochemicals for controlling Phytophthora parasitica. The growth stimulation capabilities of Trichoderma isolates in terms of seedling development was also demonstrated. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.