Read the abstract in the section 00front of this document. / Thesis (PhD (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is an important pest of citrus in South Africa and challenging to manage due to its inconspicuous nature. An effective method currently being employed for the area-wide suppression of the FCM is the Sterile Insect Technique (SIT) and the effective dispersal of sterile moths is very important for success with SIT. This study was conducted in the Addo area of the Sundays River Valley (Eastern Cape) where the programme is commercially used. In this study, sterile male moths were released in different orchards on a citrus farm, and in nearby veld at different times of the year, and their dispersal was monitored through the use of pheromone traps. Various climatic factors were monitored. This provided insight into the local dispersal of sterile male FCM adults in response to abiotic cues (particularly climatic factors). The movement of the FCM in four citrus cultivars, namely lemons, navel and Valencia oranges and mandarins and in the nearby veld (open field), was determined at six different stages of the year. Results clearly indicated that sterile FCM movement is concentrated within citrus orchards, as very few moths were trapped beyond 30 m from the release point, particularly in navel and Valencia orchards. Of the climatic factors measured, minimum and maximum temperatures had the most significant influence on FCM dispersal, and based on the results, various recommendations are made for the releases of sterile FCM in an area-wide SIT management programmes on citrus. A better understanding of the dispersal capabilities of the FCM in an agricultural system, under different conditions and at different times of the year, is invaluable not only in improving release strategies in an SIT programme but in planning future control strategies against the FCM.

The false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a serious pest of citrus and other crops in Sub-Saharan Africa. The introduction of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) Cryptogran and Cryptex (biopesticides) has proven to be very effective in the control of FCM. However, markedly lower susceptibility of some codling moth (CM), Cydia pomonella (L.) populations to Cydia pomonella granulovirus (CpGV-M), another granulovirus product used in the control of CM’s in Europe have been reported. Genetic differences between FCM populations in South Africa have also been established. It is therefore possible that differences in the susceptibility of these geographically distinct FCM populations to CrleGV-SA might also exist. To investigate this phenomenon, a benchmark for pathogenecity was established. In continuation of previous work with Cryptogran against the 1st and 5th instar FCM larvae, dose-response relationships were established for all five larval instars of FCM. In surface dose-response bioassays, the LC50 values for the 2nd, 3rd and 4th instars were calculated to be 4.516 x 104, 1.662 x 105 and 2.205 x 106 occlusion bodies (OBs)/ml, respectively. The LC90 values for the 2nd, 3rd and 4th instars were calculated to be 4.287 x 106, 9.992 x 106 and 1.661 x 108 OBs/ml, respectively. Susceptibility to CrleGV-SA was found to decline with larval stage and increase with time of exposure. The protocol was used in guiding bioassays with field collected FCM larvae. Laboratory assays conducted with Cryptogran (at 1.661 x 108 OBs/ml) against field collected FCM larvae from Addo, Kirkwood, Citrusdal and Clanwilliam as well as a standard laboratory colony, showed a significant difference in pathogenecity in only one case. This significant difference was observed between 5th instars from the Addo colony and 5th instars from the other populations. Four geographically distinct FCM colonies from Addo, Citrusdal, Marble Hall and Nelspruit were also established. Since Cryptogran and Cryptex are always targeted against 1st instar FCM larvae in the field, further comparative laboratory assays were conducted with the Addo colony and an old laboratory colony. Cryptogran was significantly more pathogenic than Cryptex against both the Addo and the old colony. However, a high level of heterogeneity was observed in responses within each population.

Thesis (M.Sc. Agriculture (Horticulture)) -- University of Limpopo, 2019 / Cucurbitacin-containing phytonematicides and a variety of unidentified soil microbes in suppressive soils (Biomuti) had been consistent in suppression of population densities of root-knot (Meloidogyne spp.) nematodes on various crops. However, information on suppressive effects of cucurbitacin-containing phytonematicides and Biomuti on citrus growth and suppression of the citrus nematode (Tylenchulus semipenetrans) had not been documented. The objective of this study therefore, was to determine the interactive effects of Nemarioc-AL and Nemafric-BL phytonematicides and Biomuti on growth and nutrient elements in leaf tissues of Poncirus trifoliata rootstock seedlings under greenhouse and field conditions. Uniform six-month-old citrus rootstock seedlings [Du Roi Nursery (Portion 21, Junction Farm, Letsitele)] were transplanted in 4 L plastic bags filled with growing mixture comprising steam-pasteurised (300°C for 1 h) loam and compost (cattle manure, chicken manure, sawdust, grass, woodchips and effective microorganisms) at 4:1 (v/v) ratio and placed on greenhouse benches. A 2 × 2 × 2 factorial experiment with the first, second and third factors being Nemarioc-AL phytonematicide (A) and Nemafric-BL phytonematicide (B) and Biomuti (M), were arranged in randomized complete block design, with 10 blocks. The treatment combinations were A0B0M0, A1B0M0, A0B1M0, A0B0M1, A1B1M0, A1B0M1, A0B1M1 and A1B1M1, with 1 and 0 signifying with and without the indicated factor. Treatments were applied at 3% dilution for each product as substitute to irrigation at a 17-day application interval. Under greenhouse conditions, seedlings were irrigated every other day with 300 ml chlorine-free tap water. Under field conditions, the study was executed using similar procedures to those in the greenhouse trial, except that the citrus seedlings were transplanted directly into the soil of a prepared field and seedlings were irrigated using drip irrigation for 2 h every xxi other day. At 64 days after transplanting, plant growth variables were measured and foliar nutrient elements were quantified using the Inductively Coupled Plasma Optical Emission Spectrometry (ICPE-9000). Data were subjected to analysis of variance using SAS software. Significant second and first order interactions were further expressed using the three-way and two-way tables, respectively. At 64 days after the treatments, under greenhouse conditions Nemarioc-AL × Nemafric-BL × Biomuti interaction was not significant (P ≤ 0.05) on plant variables of seedling rootstocks in both experiments. In contrast, the Nemarioc-AL × Biomuti interaction was highly significant (P ≤ 0.01) on stem diameter, contributing 52% in TTV of the variable in Experiment 1 (Table 3.1), whereas in Experiment 2 the interaction was highly significant on dry shoot mass, contributing 33% in TTV of the variable (Table 3.2). Relative to untreated control, the two-way matrix showed that the Nemarioc-AL × Biomuti interaction, Nemarioc-AL phytonematicide and Biomuti each increased stem diameter by 1%, 12% and 5%, respectively (Table 3.3). Relative to untreated control, the two-way matrix table showed that Nemarioc-AL × Biomuti interaction increased dry shoot mass by 10%, whereas Nemarioc-AL phytonematicide and Biomuti each increased dry shoot mass by 23% and 17%, respectively (Table 3.4). Nemarioc-AL × Nemafric-BL × Biomuti interaction was not significant (P ≤ 0.05) for all plant growth variables in both experiments. However, Nemarioc-AL × Nemafric-BL interaction was significant for leaf number and stem diameter contributing 45% and 29% in TTV of the respective variables in Experiment 2 (Table 4.1). Relative to untreated control, two way matrix table showed that the Nemarioc-AL × Nemafric-BL interaction and Nemafric-BL phytonematicides each increased stem diameter by 8% and 11% respectively, whereas Nemarioc-AL phytonematicides reduced stem diameter by 2% (Table 4.2). Also using two-way matrix table showed that Nemarioc-AL and Nemafric xxii BL phytonematicides each increased leaf number by 1% and 7% respectively, whereas the Nemarioc-AL × Nemafric-BL interaction increased leaf number by 6% (Table 4.2). Nemafric-BL × Biomuti interaction was significant for stem diameter contributing 29% in TTV of the respective variable in Experiment 2 (Table 4.1). Using two-way matrix table showed that Nemafric-BL × Biomuti interaction and Nemafric-BL phytonematicide each increased stem diameter by 7%, whereas Biomuti alone reduced stem diameter by 6% (Table 4.3). Under greenhouse conditions, the second order Nemarioc-AL × Nemafric-BL × Biomuti interaction was highly significant for foliar Mg, contributing 5% in TTV of the variable in Experiment 1 (Table 3.4). Relative to untreated control, the three-way matrix table showed that the three factors, Nemafric BL phytonematicide and Biomuti each reduced Mg by 33%, 35% and 53%, respectively, whereas Nemarioc-AL phytonematicide increased Mg by 12% (Table 3.5). Nemarioc-AL × Biomuti interaction was highly significant for foliar Mg, contributing 9% in TTV of the variable in Experiment 1 (Table 3.4). Relative to untreated control, the two-way matrix table showed that the Nemarioc-AL × Biomuti interaction and Nemafric-BL phytonematicide reduced Mg by 42% and 12%, respectively, whereas Nemarioc-AL phytonematicide alone increased Mg by 14% (Table 3.6). Nemarioc-AL × Biomuti interaction was highly significant for foliar Ca and Mg, contributing 59 and 4% in TTV of the respective variables in Experiment 1 (Table 3.4). Also using two-way matrix table showed that Nemarioc-AL phytonematicide and Biomuti separately reduced Ca by 12% and 22% respectively, whereas the Nemarioc-AL × Biomuti interaction increased Ca by 1% (Table 3.7). Relative to untreated control, the Nemarioc-AL × Biomuti interaction, Nemarioc-AL phytonematicide and Biomuti reduced foliar Mg by 26%, 21% and 33%, respectively (Table 3.7). Nemafric-BL × Biomuti interaction was highly significant for foliar Mg and P, contributing 50 and 21% xxiii in Experiment 1, whereas in Experiment 2 the interaction was significant for foliar Ca and Mg, contributing 41% and 38% in TTV of the respective variables (Table 3.4). Relative to untreated control, the two-way matrix table showed that Nemafric-BL phytonematicide and Biomuti individually reduced Mg by 60% and 51%, respectively, whereas the Nemafric-BL × Biomuti interaction reduced Mg by 38% (Table 3.8). Also, in the two-way matrix table the Nemafric-BL × Biomuti interaction and Nemafric-BL phytonematicide each reduced Mg by 13% and 2%, respectively, whereas Biomuti alone increased P by 17% (Table 3.8). Relative to untreated control, Nemafric-BL phytonematicide and Biomuti reduced Ca by 29% and 18%, respectively, whereas Nemafric-BL × Biomuti interaction reduced Ca by 14% (Table 3.9). Using two-way matrix table showed that Nemafric-BL phytonematicide and Biomuti separately reduced Mg by 21%, whereas the Nemafric-BL × Biomuti interaction reduced Mg by 16% (Table 3.9). Interaction of Nemarioc-AL × Nemafric-BL × Biomuti had no significant effect on K, Na and Zn in both experiments. Under field conditions, the second order Nemarioc-AL × Nemafric-BL × Biomuti interaction was not significant for all the nutrient elements in Experiment 1. Nemarioc-AL × Biomuti was significant for Ca, K and highly significant for Mg and P, contributing 31, 8, 23 and 19% in TTV of the respective variables in Experiment 1 (Table 4.4). Relative to untreated control, two-way matrix table showed that Nemarioc-AL phytonematicide and Biomuti each increased Ca by 15% and 26% repectiviely, whereas the Nemarioc-AL × Biomuti increased Ca by 17% (Table 4.5). Interaction of Nemarioc-AL × Biomuti, Nemarioc-AL phytonematicide and Biomuti each reduced Mg by 48%, 70% and 37% (Table 4.5). Also using two-way matrix table showed that Nemarioc-AL phytonematicide and Biomuti each increased P by 4% and 5% respectively, whereas the Nemarioc-AL × Biomuti interaction increased P by 50% (Table 4.5). Realative to untreated control, xxiv Biomuti and Nemarioc-AL phytonematicide each reduced K by 10% and 5% respectively, whereas the Nemarioc-AL × Nemafric-BL interaction reduced K by 38% (Table 4.7). Nemafric-BL × Biomuti interaction was highly significant for Mg and Zn, contributing 11% and 29% in TTV of the respective variables in Experiment 1 (Table 4.4). Relative to untreated control, two-way matrix table showed that Nemarioc-AL phytonematicide and Biomuti separately increased Mg by 1% and 19% respectiviely, whereas the Nemafric-BL × Biomuti interaction reduced Mg by 43% (Table 4.6). Nemafric-BL × Biomuti interaction, Nemafric-BL phytonematicide and Biomuti each reduced Zn by 35%, 31% and 64% (Table 4.6). Using three-way matrix table showed that the Nemarioc-AL × Nemafric-BL × Biomuti, Nemarioc-AL × Nemafric-BL, Nemarioc-AL × Biomuti and Nemafric-BL × Biomuti interactions each increased Ca by 44%, 18%,10% and 24% (Table 4.8). Further the matrix showed that Nemarioc-AL, Nemafric-BL phytonematicides and Biomuti each increased Ca by 25%, 31% and 23% (Table 4.8). Under both greenhouse and field conditions, although second and first order interactions were not consistent of various variables, results demonstrated that the three products interacted significantly for various products. In conclusion, the study suggested that these innovative products could be used in combination with Biomuti to stimulate plant growth but had antagonistic effects on accumulation of nutrient elements in P. trifoliata rootstock seedlings, suggesting that the products should be applied separately. / Agricultural Research Council-Universities Collaboration Centre and the National Research Foundation (NRF)

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, 2003. / Plant Production and Soil Science / unrestricted

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Fruit and Leaf Spot Disease (FLSD) of citrus, caused by Phaeoramularia angolensis, is found only in 18 countries in Africa, the Comores Islands in the Indian Ocean and Yemen in the Arabian peninsula. The major citrus export countries in Africa are Morocco, South Africa, Swaziland, and Zimbabwe. Zimbabwe is the only country affected by FLSD. FLSD is a disease of major phytosanitary and economic importance and its devastating effect on citrus is highlighted by the fact that the damage is cosmetic, which renders the fruit unmarketable. Total crop losses are not uncommon in Kenya. The aims of the present study, therefore, was was to determine the occurrence of P. angolensis in Zimbabwe and neighbouring Mozambique, to compare these isolates with the Cercospora Fresen. isolates from Swaziland and South Africa, to determine the epidemiology of the pathogen and to implement an effective control strategy to prevent the spread of FLSD. Leaf samples with citrus canker-like lesions collected in the early 1990’s in Zimbabwe were found to be infected by the fungus, Phaeoramularia angolensis. Surveys were undertaken to determine the spread and intensity of FLSD in Zimbabwe and Mozambique. In Zimbabwe, P. angolensis was limited to an area above the 19° south latitude, predominantly the moist areas and not the low-lying drier parts of the country. In Mozambique, no P. angolensis symptoms were found. Observations during the survey indicated that no proper management systems were implemented by Zimbabwean growers. A cercosporoid fungus causing a new Fruit and Leaf Spot Disease on Citrus in South Africa was identified. From morphological and rDNA sequence data (ITS 1, 5.8S and ITS 2), it was concluded that the new disease was caused by Cercospora penzigii, belonging to the Cercospora apii species complex. The genera Pseudophaeoramularia and Phaeoramularia are regarded as synonyms of Pseudocercospora, and subsequently a new combination was proposed in Pseudocercospora as P. angolensis. Cercospora gigantea was shown to not represent a species of Cercospora, while Mycosphaerella citri was found to be morphologically variable, suggesting that it could represent more than one taxon. A control strategy for the control of FLSD was evaluated in the study. The data showed that P. angolensis in Zimbabwe can be managed successfully by the removal of all old and neglected orchards, and on timely fungicide applications. Trifloxystrobin + mancozeb + mineral spray oil (20 g + 200 g + 500 ml/100 l water) applied in November, January and March was the most effective treatment. Three applications of benomyl + mancozeb + mineral spray oil (25 g + 200 g + 500 ml/100 l water) applied during the same period, was the second most effective treatment, and two applications (November and January) of trifloxystrobin + mineral spray oil (20g + 500 ml/100 l water) and difenoconazole (40 g) per 100 l/water applied twice in November and January, the third most effective treatment. The spore trap and weather data showed that P. angolensis needs high moisture and temperatures in excess of 25°C for disease development. It is concluded that P. angolensis in Zimbabwe can be managed successfully by implementing a holistic approach, which should be supported by the authorities, organised agriculture and all technical personnel involved in citrus production. / AFRIKAANSE OPSOMMING: Blaar- en vrugvleksiekte (BVVS) op sitrus, veroorsaak deur Phaeoramularia angolensis, kom in 18 lande in Afrika voor asook die Comores Eilande in die Indiese Oseaan en Yemen op die Arabiese skiereiland. Marokko, Suid Afrika, Swaziland en Zimbabwe is die belangrikste uitvoerders van sitrus in Afrika. Van dié lande het slegs Zimbabwe blaar en vrugvleksiekte op sitrus. Hierdie siekte is van fitosanitêre en ekonomiese waarde en die nadelige effek van die siekte, wat slegs kosmetiese van aard is, is venietigend aangesien vrugte onbemarkbaar is. Totale opbrengsverliese is nie ongewoon in lande soos Kenya nie. Die doelwitte van die studie was dus om die voorkoms van P. angolensis in Zimbabwe te bepaal, om die Cercospora Fresen. isolate vanaf Swaziland en Suid-Afrika met mekaar te vergelyk, om die epidemiologie van die siekte vas te stel en om ‘n effektiewe beheermaatreël teen die siekte te ondersoek. Blaarmonsters met kankeragtige letsels wat in die vroeë 1990’s in Zimbabwe gevind is, het getoon dat die blare geinfekteer is met die swam, Phaeoramularia angolensis. Ondersoeke is geloots om die verspreiding en intensiteit van BVVS in Zimbabwe en Mosambiek te bepaal. In Zimbabwe was gevind dat P. angolensis beperk was tot gebiede bo die 19° Suid breedtegraad, wat die hoër vogtiger gebiede insluit eerder as die droeër, laagliggende gebiede. Geen P. angolensis simptome kon in Mosambiek gevind word nie. Tydens die opnames was dit duidelik dat geen geskikte beheerstrategieë toegepas word deur Zimbabwe se produsente nie. ‘n Nuwe cercosporoid swam, wat blaar en vrugvleksiekte op sitrus is in Suid Afrika veroorsaak is geidentifiseer. Morfologiese en rDNA volgorde (ITS 1, 5.8S en ITS 2) data het getoon dat die siekte veroorsaak word deur Cercospora penzigii wat tot die Cercospora apii spesie kompleks behoort. Die genus Pseudophaeoramularia kan as sinoniem van Pseudocercospora beskou word en ‘n nuwe kombinasie word voorgestel in Pseudocercospora as P. angolensis. Cercospora gigantea het getoon dat dit nie ‘n spesie van Cercospora kon verteenwoordig nie terwyl Mycosphaerella citri varieërend voorkom en meer as een takson kan verteenwoordig. ‘n Beheerstrategie vir die beheer van BVVS is ondersoek. Die data wys dat P. angolensis in Zimbabwe doeltreffend beheer kan word deur die uitroeiing van ou en verwaarloosde bome, en deur goed beplande fungisied bespuiting. Trifloxystrobin + mancozeb + minerale spuitolie (20 g + 200 g + 500 ml/100 l water), wat in November, Januarie en Maart toegedien is, was die mees effektiefste behandeling. Drie bespuitings van benomyl + mancozeb + minerale spuitolie (25 g + 200 g + 500 ml/100 l water) wat oor dieselfde tydperk toegedien is, was die naas beste behandeling. Trifloxystrobin (20 g) + minerale spuitolie (500 ml) per 100 l/water en difenoconazole (40 g) per 100 l/water, beide as twee bespuitings toegedien in November en Januarie, het die derde beste resultaat opgelewer. Die spoorlokval en klimatologiese data het getoon dat P. angolensis vogtige toestande en temperature hoër as 25°C benodig vir siekteontwikkeling. Die afleiding uit die studie is dat P. angolensis suksesvol beheer kan word indien ‘n holistiese benadering gevolg word en alle rolspelers naamlik die owerheid, georganiseerde landbou en tegniese personeel die proses ondersteun.

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.

Thesis (MScAgric (Horticulture))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Creasing, also known as albedo breakdown, is a preharvest disorder that affects the albedo of citrus fruit causing creases on the surface of the fruit. It is a recurrent problem in Navel and Valencia oranges and can cause individual orchard losses which often exceed 50%. Although the contributing factors are known, the physiological basis of creasing development is unresolved and the current control measures do not prevent creasing satisfactory. Hence, better control measures and further understanding of the physiology of creasing development is required. The objective of this two-year study was to determine if the position of fruit in a tree, light and carbohydrate manipulation techniques, and albedo mineral nutrients influence creasing development. Furthermore, the most effective application timing of gibberellic acid (GA3) with the least negative effect on fruit rind colour development and the effectiveness of cytokinins, other products and different root biostimulants to reduce creasing incidence were evaluated. The position of fruit in the tree and light influenced the development of creasing and the distribution of mineral nutrients in the albedo. Creasing incidence was higher on the south side than on the north side of the tree and fruit from the inside sub-sectors had a greater creasing incidence compared to fruit from the outside sub-sectors. The shady part of outside fruit was more creased compared to the sunny part of the fruit and covering fruit with brown paper bags increased creasing severity. The light manipulation techniques used on the leaves and fruit increased the nitrogen (N), phosphorus (P), potassium (K) and manganese (Mn) concentrations in the albedo and differences in the albedo mineral nutrients amongst the sub-sectors evaluated were observed, but creasing severity or creasing incidence was not significantly correlated with the albedo mineral concentrations at harvest. Albedo mineral concentrations earlier in the season may play a role in creasing development, as creasing severity was significantly correlated with copper (Cu), K, and Mn concentrations in the albedo during stage II of fruit development. Creasing incidence and albedo mineral concentrations were not affected by any of the carbohydrate manipulation techniques used in this study. The incidence and severity of creasing was significantly reduced, with a minor negative effect on fruit rind colour development, by the application of GA3, from mid November to mid January. Localised fruit application of CPPU [N-(2-chloro-4-pyridyl)-N-phenylurea], MaxCel (6- Benzyladenine) and CPPU in combination with calcium after physiological fruit drop reduced the incidence and severity of creasing, although creasing incidence was not significantly different from the control. The application of Messenger®, AVG (aminoethoxyvinylglycine) and different root biostimulants did not reduce creasing incidence. The results showed that cytokinins could reduce creasing incidence and justify further studies on application and uptake efficiency. The use of different root biostimulants are not recommended, but it is suggested that treatment effects may be more pronounced over a longer period. / AFRIKAANSE OPSOMMING: Kraakskil is ‘n vooroes abnormalitiet wat die albedo van sitrusvrugte affekteer, deur krake op die oppervlak van vrugte te veroorsaak. Dit is ‘n algemene probleem in Navel en Valencia lemoene en kan boordverliese van tot 50% of soms hoër veroorsaak. Alhoewel die bydraende faktore bekend is, is die fisiologiese basis van kraakskil ontwikkeling onopgelos en die beskikbare beheermaatreëls is nie bevredigend nie. Dus, beter beheermaatreëls en ‘n beter begrip van die fisiologie van kraakskil ontwikkeling is nodig. Die doel van die twee-jaar studie was om te bepaal of die posisie van vrugte in ‘n boom, lig en koolhidraat manipulasie tegnieke en minerale elemente in die albedo, kraakskil ontwikkeling beïnvloed. Die mees effektiewe toedieningstyd van gibberelliensuur (GA3) sonder ‘n negatiewe effek op vrugkleur is bepaal en die effektiwiteit van sitokiniene, ander produkte en verskillende wortel biostimulante om kraakskil voorkoms te verminder, is geëvalueer. Die posisie van vrugte in ‘n boom en lig het kraakskil ontwikkeling en die verspreiding van minerale element in die albedo beïnvloed. Kraakskil voorkoms was hoër aan die suidekant van die boom as aan die noordekant en vrugte in die binnekant van die boom het ‘n groter kraakskil voorkoms as vrugte in die buitekant van die boom gehad. Die skadukant van buitevrugte het meer kraakskil gehad as die sonkant en die toemaak van vrugte met ‘n bruin papiersak het die graad van kraaksil verhoog. Die lig manipulasie tegnieke wat op die blare en vrugte gebruik is, het die stikstof (N), fosfaat (P), kalium (K) en mangaan (Mn) konsentasies in die albedo verhoog en verskille in die albedo minerale elemente tussen sub-sektore is waargeneem, maar betekenisvolle korrelasies is nie tussen die graad en voorkoms van kraakskil en die albedo minerale element konsentrasies by oestyd waargeneem nie. Albedo minerale element konsentrasies vroeër in die seisoen mag ‘n rol speel by kraakskil ontwikkeling, omdat die graad van kraakskil betekenisvol gekorreleer was met albedo koper (Cu), K, en Mn konsentrasies tydens fase II van vrugontwikkeling. Kraakskil voorkoms en albedo minerale element konsentrasies is nie deur enige van die koolhidraat manipulasie tegnieke geaffekteer nie. Die voorkoms en graad van kraakskil is betekenisvol verlaag, met ‘n geringe negatiewe effek op vrugkleur, deur die toediening van GA3 vanaf mid November tot mid Januarie. Gelokaliseerde vrugtoedienings van CPPU [N-(2-chloro-4-piridiel)-N-phenielureum], MaxCel (6- Bensieladenien) en CPPU saam met kalsium na fisiologiese vrugval het die voorkoms en graad van kraakskil verlaag, alhoewel kraakskil voorkoms nie betekenisvol van die kontrole verskil het nie. Die toediening van Messenger®, AVG (amino etoksievinielglisien) en veskillende wortel biostimulante het nie kraakskil voorkoms verlaag nie. Die resultate het getoon dat sitokiniene kraakskil voorkoms kan verlaag en verdere studies op die toediening en opname effektiwiteit word aanbeveel. Die gebruik van verskillende wortel biostimulante word nie aanbeveel nie, maar die effek behoort meer sigbaar te wees na ‘n langer periode van behandeling.

Orientador: Jesus Aparecido Ferro / Coorientador: José Belasque Júnior / Banca: Lúcia Maria Carareto Alves / Banca: Franklin Behlau / Resumo: As bactérias pertencentes ao gênero Xanthomonas constituem um dos grupos de fitopatógenos mais importantes na natureza, com capacidade de infectar aproximadamente 120 tipos diferentes de plantas monocotiledôneas e 270 dicotiledôneas. A bactéria Xanthomonas citri subsp. citri (Xcc) é o agente causal do cancro cítrico, uma das principais doenças dos citros. Restrita à Flórida, a mancha bacteriana dos citros, causada por Xanthomonas alfalfae subsp. citrumelonis (Xacm), afeta principalmente o citrumelo 'Swingle'. A bactéria Xanthomonas fuscans subsp. aurantifolii (Xfa) é o agente causal das cancroses B e C, encontradas apenas na América do Sul. As técnicas mais utilizadas na diferenciação e caracterização dos patógenos bacterianos, hibridização DNA-DNA (DDH) e sequenciamento da região do DNA que codifica o RNA ribossomal 16S (rRNA 16S), são onerosas, trabalhosas, lentas e de resultados restritos, não permitindo a comparação dos resultados obtidos entre diferentes estudos laboratoriais. Por isso, a técnica de análise de sequências multilocus (MLSA), considerada rápida, barata e confiável, tem sido recomendada para a caracterização e delimitação de espécies de patógenos bacterianos, tais como as do gênero Xanthomonas, permitindo o estabelecimento das relações filogenéticas por meio de genes housekeeping. Portanto, o objetivo do trabalho foi caracterizar os isolados de Xanthomonas (Xcc-A, Xfa-B, Xfa-C e Xacm) por meio da técnica MLSA. Foram utilizados 27 isolados de Xanthomonas patogênicas a citros, sendo quatro de X. citri subsp. citri (Xcc), três de X. fuscans subsp. aurantifolli B (Xfa-B), dezenove de X. fuscans subsp. aurantifolli C (Xfa-C) e um de X. alfalfae subsp. citrumelonis (Xacm). Os genes atpD, dnaK e fusA foram utilizados como genes housekeeping e para a reação da PCR... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bacteria belonging to the genus Xanthomonas are one of the main groups of phytopathogens in nature, capable to infecting about 120 different monocots and 270 dicots plants. Xanthomonas citri subsp. citri (Xcc) is the causal agent of bacterial citrus canker, one of the major diseases of citrus. Restricted to Florida, citrus bacterial spot, caused by Xanthomonas alfalfae subsp. citrumelonis (Xacm), affects mainly citrumelo 'Swingle'. Xanthomonas fuscans subsp. aurantifolii (Xfa) is the causal agent of canker B and C, found only in South America. The most widely used techniques for the differentiation and characterization of bacterial pathogens, DNA-DNA hybridization (DDH) and analysis of DNA sequences coding for small subunit ribosomal RNAs (16S rRNA), are costly, cumbersome, slow and give limited results, do not allowing the interlaboratorial comparisons of the results. Therefore, the technique of multilocus sequence analysis (MLSA), which is considered fast, cheap and reliable, has been recommended for characterization and delineation of species of bacterial pathogens, such as the genus Xanthomonas, allowing the establishment of phylogenetic relationships between them through housekeeping genes. Therefore, the objective of this study was to characterize strains of Xanthomonas (Xcc-A, Xfa-B, Xfa-C and Xacm) using the MLSA technique. We used 27 strains of Xanthomonas pathogenic to citrus, being four Xcc, three Xfa-B, 19 Xfa- C and one Xacm. The PCR reaction was performed using the specific primers to amplify the atpD, dnaK and fusA genes. PCR products were purified, sequenced and the sequences were analyzed using the software CodenCode Aligner version 3.7.1. Phylogenetic analysis was performed using Maximum Likelihood based on the model of Tamura-Nei and using the software MEGA 5. Analyses ... (Complete abstract click electronic access below) / Mestre

Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is one of the major citrus pests of economic importance for South Africa’s citrus industry. It is endemic to Africa, and therefore a phytosanitary pest with zero tolerance by most export markets. The cryptic nature of T. leucotreta makes visual inspection an inefficient method for detecting neonate larvae in fruit in the packhouse. Therefore, a more accurate method for sorting infested fruit at the packhouse, particularly for newly infested fruit could ensure market access. A recent study showed that fruit infested by T. leucotreta emit a chemical profile different from that of a healthy fruit. Several studies provide evidence that parasitoids locate their hosts feeding on fruit by exploiting the novel chemical profiles produced due to host herbivory. The aim of this study was to evaluate the potential of using the naturally occurring behaviour of a larval parasitoid Agathis bishopi (Nixon) (Hymenoptera: Braconidae) for detection of T. leucotreta infested fruit, by determining which compound in infested fruit is attractive to parasitoids. Ytube olfactometer and flight-tunnel bioassays with healthy and T. leucotreta infested fruit showed a significantly stronger response of A. bishopi female parasitoids to infested fruit. Among the volatile compounds associated with T. leucotreta infested fruit, D-limonene elicited the strongest attraction to A. bishopi female parasitoids. Attraction of mated A. bishopi female parasitoids to T. leucotreta infested fruit and D-limonene significantly increased after oviposition experience. Behavioural responses of A. bishopi female parasitoids that were associated with T. leucotreta infested fruit were investigated to determine which behaviours are distinct and interpretable. Probing and oviposition behaviours were the most noticeable and were only elicited on infested fruit when parasitoids contacted T. leucotreta frass, indicating that chemical compounds in frass are short-range cues used for final host location. Since production of D-limonene by fruit is elevated due to herbivory by different pests including mechanical injury on fruit, response of A. bishopi female parasitoids to compounds in frass offers a more specific and potentially useful mechanism for development of a detection system for T. leucotreta infested fruit. Chemical analysis of T. leucotreta frass and conditioning A. bishopi parasitoids to respond behaviourally to compounds in frass is proposed.