Masters Degrees (Plant Pathology)

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Characterization of potato virus Y (PVY) isolates infecting solanaceous vegetables in KwaZulu-Natal (KZN), Republic of South Africa (RSA)
(2009) Ibaba, Jacques Davy.; Gubba, Augustine.
Potato virus Y (PVY) is an economically important virus worldwide. In South Africa, PVY has been shown to be a major limiting factor in the production of important solanaceous crops, including potato (Solanum tuberosum L.), pepper (Capsicum annuum L.), tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana spp). The variability that PVY displays, wherever the virus occurs, merits the study of the isolates occurring in KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). This characterization will provide a clear understanding of strains/isolates from local vegetables and how they relate to the other PVY strains already identified, as well as information that can be used to manage the diseases they cause. Hence, the aim of this project was to study the biological and genetic properties of PVY isolates infecting potato, tomato and pepper in KZN. Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and reverse transcription polymerase chain reaction (RT-PCR) using primers specific to all PVY strains were used to detect the virus in plant material showing PVY-like symptoms collected from various locations in KZN. A total of 39 isolates (18 isolates infecting tomato, 12 infecting potato and 9 infecting pepper) were further differentiated into strains by means of ELISA using strain specific antibodies and RT-PCR using primers specific to the different strains of PVY identified around the world. All PVY isolates infecting tomato and pepper tested positive for the ordinary PVYO strain with both ELISA and RT-PCR. PVY isolates infecting potato were more diverse and comprised the PVYN, PVYNTN and PVYNWilga strains, with mixed infections noted in some cases. The biological properties were studied by mechanically inoculating Chenopodium quinoa, Nicotiana tabacum cv Xanthi, N. tabacum cv Samsun, N. glutinosa, and N. rustica with leaf extracts from plants infected with the different PVY strains detected in this study. All inoculated C. quinoa plants did not show symptoms. All tobacco plants showing symptoms were tested for the presence of PVY by means of ELISA using monoclonal antibodies targeting all strains and electron microscopy using the leaf dip technique. Not all the inoculated tobacco tested positive with ELISA. The symptoms observed were therefore divided into PVY-related and PVY non- related. PVY-related symptoms included vein clearing, mosaic chlorosis, stunting, and vein necrosis. PVY non-related symptoms included wrinkles and leaf distortions. Potyvirus-like particles of about 700 nm were observed under the transmission electron microscope (TEM) from plants showing PVY-related symptoms while rod shaped viral particles of sizes varying between 70 and 400 nm were observed from plants showing non-PVY related symptoms. A portion of the virus genome (1067 bp) covering part of the coat protein gene and the 3’ non-translated region (NTR) of three PVYO isolates infecting tomato, one PVYO isolate infecting pepper and one PVYNWilga isolate infecting potato were amplified, cloned and sequenced. The 5’ NTR, P1, HC-Pro and part of P3 regions (2559 bp) of a PVYN isolate infecting potato were also amplified, cloned and sequenced. Sequence data was compared with selected PVY sequences from different geographical locations around the world. These were available on the NCBI website and subsequently used for phylogenic analyses. The sequenced genomic regions of the PVYN isolate were found to be 99% similar to the New Zealand PVYN isolate (GenBank accession number: AM268435), the Swiss PVYN isolate CH605 (X97895) and the American PVYN isolate Mont (AY884983). Moreover, the deduced amino acid sequence comparison of the genomic regions of the PVYN isolate revealed the presence of five distinct amino acids residues. The three amino acid residues (D205, K400, and E419), which determine the vein necrosis phenotype in tobacco, were also identified. The coat protein and 3’ NTR sequences of all KZN PVYO isolates infecting pepper and tomato were closely similar to each other than to KZN PVYNWilga isolate infecting potato. The phylogenic analysis clustered the KZN PVYN isolate with the European sublineage N, PVYNWilga isolate infecting potato with the American PVYO isolate Oz (EF026074) in the O lineage and all PVYO isolates infecting tomato and pepper in a new sublineage within the O lineage. Taken together, these results point to the presence of PVY in solanaceous vegetables cultivated in KZN and they lay the foundation for the formulation of effective control measure against PVY diseases in KZN.
Shoot apex culture of Acacia mearnsii (De wild)
(2007) Thompson, Iain Mungo.; Laing, Mark Delmege.
Research into the micropropagation of black wattle in South Africa is important for two reasons. Firstly micropropagation technology allows breeders to select and propagate mature tissue, which in turn allows them to better capture selected traits. Secondly, tissue culture may control the highly invasive nature of black wattle. If triploid black wattle can be developed, foresters will then have to rely on clonal propagation to supply material for their growing operations. This research was part of the Institute for Commercial Forestry’s Acacia mearnsii vegetative propagation programme. The main focus of this research was to overcome various problems associated with direct organogenesis of ex vitro material. The shoot apex region was used as the explant in all studies because this region is thought to harbour relatively few internal microbial contaminants and is of sufficient size to withstand stresses associated with micropropagation. The initial research was focussed on the screening of sterilants, searching for a viable alternative to mercuric chloride. Surface sterilisation is integral to any micropropagation technique. This process should do the least amount of plant damage, whilst reducing microbial contamination to an acceptable level. Explants were cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 BA and monitored for signs of contamination and shooting. Household bleach proved an excellent alternative to mercuric chloride because it did significantly less damage to the explants than mercuric chloride and is handled easily. There was no significant effect of sterilant exposure time on explant decontamination levels, whilst the shortest exposure time resulted in significantly higher levels of shoot development than the other two times tested. The results of this initial research was developed into a protocol and utilised in subsequent investigations. Due to a considerable variation in the success of the developed surface sterilisation protocol according to different times of the year, a further investigation into the effects of season and mother plant material on shoot apex culture of Acacia mearnsii was undertaken. The success of any tissue culture technique depends on a large array of ex vitro and in vitro variables. The objective of this research was to determine the ii effect of two ex vitro variables, season and mother plant, on shoot apex culture of Acacia mearnsii. Explants from individual mother plants were cultured on MS medium supplemented with 2.0 mg L-1 BA during four separate seasons and monitored for signs of contamination and shooting. Spring was found to be the best harvesting season because spring explants showed significantly higher decontaminated explant levels and shooting levels than explants harvested in the other three seasons. The effect of mother plant selection on the performance of Acacia mearnsii explants during shoot apex culture was also found to be significant, especially with regard to shooting levels. Finally factors influencing shoot elongation of A. mearnsii during shoot apex culture were investigated. In the past, induction of shoot elongation during micropropagation of A. mearnsii was attained through the addition of plant growth regulators and other supplements to the basal culture medium. However, some micropropagation methods in other species have utilised red light as a means of promoting shoot elongation. The objective of this study was to test the effects of an alternative basal medium, red light and differing concentrations of chemical additions to the culture medium on shoot elongation of Acacia mearnsii during shoot apex culture. Four independent experiments were undertaken comparing: shoot elongation on Woody Plant Medium (WPM) to the MS basal medium control; shoot elongation under a red cellophane box compared to control culture light conditions; shoot elongation on media supplemented with various concentrations of GA3 to the un-supplemented control and shoot elongation on media supplemented with combinations of BA and IBA compared to a control. Although no significant effects were observed, many trends were noted. The results indicated that there was no advantage to using WPM instead of MS medium when attempting to elongate shoots, rejuvenated through shoot apex culture of A. mearnsii, whilst the effect of GA3 showed a negative trend. The effects of red light and some BA and IBA combinations showed positive trends on the elongation of initiated shoots. This research successfully addressed some of the problems associated with micropropagation of A. mearnsii. Shoot apex culture shows promise and further research into this technique should be considered. A viable surface sterilant alternative to mercuric chloride was successfully identified. This alternative is not only iii safer to use but shows a large reduction in phytotoxic effects. The effects of season and mother plant on shoot apex culture was successfully investigated, resulting in a better understanding of mother plant influences on tissue culture as well as the identification of an optimum season for explant selection. Finally two possible shoot elongation promoters were identified for further research and a more affordable alternative to red light sources and screens was identified.
Studies on brown rust (Puccinia melanocephala) of sugarcane in South Africa.
(2009) Ramouthar, Prabashnie Vengetsamy.
The first serious outbreak of brown rust of sugarcane caused by Puccinia melanocephala Syd. & P. Syd. was reported in India in 1907. It was first reported in South Africa (SA) in 1941 on the variety Co301 and is now present in almost all the sugarcane growing areas of the world. In SA, it is now described as an important disease of sugarcane, causing yield losses of up to 26% in susceptible varieties. Within the SA sugar industry, rust is controlled through the use of resistant varieties as it is the most economical method of control. However, most of the newer varieties that are being released have an intermediate resistance rating for rust. An integrated management approach for the control of rust is therefore being investigated. Aspects investigated in this study included environmental conditions required for development of the disease i.e. epidemiology, the use of silicon (Si) as a cultural control method against brown rust and identification of gene sequences expressed in response to brown rust infection. For the epidemiology study, inoculated plants were incubated in a dew chamber at different temperatures and leaf wetness periods. The choice of leaf wetness duration and temperature was based on urediniospore germination studies. The optimum temperature for urediniospore germination and disease development at > 98% relative humidity was found to be between 20 and 25°C with nine hours of leaf wetness. Silicon has been shown to reduce the incidence of diseases and pests in a number of crops. The ability of sugarcane to accumulate Si and the location of Si deposition was established using two uptake and deposition trials. Different concentrations of Si were applied to the plant and accumulation in the roots, stalks, old leaves and young leaves was determined using inductively coupled plasma optical emission spectrometry, with accumulation found to be roots > old leaves > stalks > young leaves. Silicon deposition in the leaves was determined using energy dispersive X-ray mapping on freeze dried specimens and significant differences were found between the upper epidermis, lower epidermis and mesophyll with the most Si being deposited in the lower epidermis. For disease severity, plants were naturally infected with rust and rated weekly. A significant decrease in disease severity and area under disease progress curve was noted when the Si concentration increased, indicating that Si has potential in reducing rust incidence. Currently, the most reliable and economical method of managing brown rust is with the use of resistant varieties. Identification of resistance within breeding lines is therefore important. For this part of the study, suppression subtractive hybridization was used as a tool to identify differentially expressed genes between a susceptible and resistant variety and a susceptible and intermediate variety, in response to brown rust infection. Two efficient subtracted cDNA libraries were generated and differentially expressed sequences were identified within each library. The results of this study show potential for the development of molecular markers which could be used for the early identification of brown rust resistance during the breeding process. This study forms a firm basis on which an integrated management strategy, for the management of brown rust in the SA sugar industry, could be designed. The cDNA sequences identified could be further investigated and used to develop molecular markers to select for rust resistant varieties, the epidemiology results together with further field data could be used to develop a disease prediction model and Si has potential in the field to reduce brown rust severity.
Studies on Sclerotinia sclerotiorum (Sclerotinia stem rot) on soybeans.
(2007) Visser, Dael Desiree.; Caldwell, Patricia May.
Soybeans, Glycine max, are an economically and strategically important crop in South Africa (SA). In order to meet local demands, large imports of soybeans are required, e.g., in the 2005/2006 soybean production period, 842 107 tonnes of oilcake were imported. Due to an increase in soybean production throughout the world, diseases that affect this crop have also increased in incidence and severity. Sclerotinia sclerotiorum, the causal organism of sclerotinia stem rot (SSR), is an important yield limiting disease of soybeans, as well as numerous other crops. The pathogen was first reported in SA in 1979. However, it was only in 2002 that this fungus was considered a major pathogen of soybeans in SA. The research reported in this thesis was conducted to investigate the epidemiology of S. sclerotiorum and examine numerous potential control methods for this pathogen, i.e., resistant cultivars, biocontrol, chemical control and seed treatments. A S. sclerotiorum isolate was obtained from sunflowers in Delmas, Mpumulanga, SA, in the form of sclerotia. This isolate was cultured and sent for identification and deposition in the Plant Protection Research Institute collection. This isolate, in the form of mycelia, was used for the duration of the study. For epidemiology studies, the effect of temperature, leaf wetness duration (LWD) and relative humidity (RH) were examined for their effect on rate of pathogen development. Twenty four combinations of temperature (19°C, 22°C, 25°C and 28°C), LWD (24, 48 and 72 hr) and RH (85 and 95%) were investigated. No interaction between temperature, LWD and RH was found. Temperature alone was the only factor that affected disease development. At 22°C, the rate of pathogen development (0.45 per unit per day) was significantly higher than all other temperatures, indicating that this temperature is optimum for disease development. Thirteen different soybean cultivars, i.e., LS6626RR, LS6710RR, LS666RR, LS555RR, LS6514RR, LS678RR, Prima 2000, Pan 626, AG5601RR, AG5409RR, 95B33, 95B53 and 96B01B, commercially grown in SA were investigated for their reaction to S. sclerotiorum. Prima 2000, 96B01B, 95B33 and AG5409RR were considered to be the least susceptible as they showed a significantly low rate of pathogen development (0.28, 0.28, 0.24, 0.23 per unit per day, respectively) and produced a significantly low number of sclerotia (3.03, 3.42, 3.21, 2.38, respectively). LS6626R and LS666RR may be considered most susceptible because of their significantly high rate of pathogen development (0.45 and 0.42 per unit per day, respectively) and high sclerotia production (8.16 and 7.50, respectively). Regression analysis showed a positive correlation coefficient (R2=0.71) between rate of growth of the pathogen and number of sclerotia produced, indicating that a higher rate is associated with a higher number of sclerotia. In vitro dual culture bioassays were performed to identify the biocontrol mechanisms of the biocontrol agents, EcoT® (a seed treatment) and Eco77® (a foliar treatment), against hyphae and sclerotia of S. sclerotiorum. Ultrastructural studies revealed that mycoparasitism is the probable mode of action as initial signs of hyphae of EcoT® and Eco77® coiling around hyphae of S. sclerotiorum were observed. Surface colonization of sclerotia by hyphae of EcoT® and Eco77® was also observed. In vitro antagonism of EcoT® against S. sclerotiorum on soybean seed was performed to determine pre-emergence and post-emergence disease. There was no significant difference in percentage germination between seeds treated with EcoT® and plated with the pathogen, untreated seeds and no S. sclerotiorum, and the control (i.e. no EcoT® and no pathogen). However, percentage non infected seedlings from seeds not treated with EcoT® was significantly lower, suggesting that EcoT® may be successfully used as a seed treatment for the control of SSR. In vivo trials were performed to investigate the effect of silicon (Si) alone, and in combination with Eco77®, on the effect of the rate of disease development. Plants treated with Eco77® had a significantly lower rate of disease development (0.19 per unit per day for plants treated with Eco77® and S. sclerotiorum and 0.20 per unit per day for plants treated with Eco77®, S. sclerotiorum and Si), compared to plants not treated with Eco77® (0.29 per unit per day for plants treated with S. sclerotiorum and 0.30 per unit per day for plants treated with S. sclerotiorum and Si), regardless of the application of Si. Similarly, plants treated with Eco77® had a significantly lower number of sclerotia (0.46 for plants treated with Eco77® and S. sclerotiorum and 0.91 for plants treated with Eco77®, S. sclerotiorum and Si), compared to plants not treated with Eco77® (3.31 for plants treated with S. sclerotiorum and 3.64 for plants treated with S. sclerotiorum and Si). The significantly lower rate of disease development coupled with a significant reduction in sclerotia showed that Eco77®, and not Si, was responsible for reducing the severity of SSR. A strong positive correlation between rate of disease development and the number of sclerotia produced (R2=0.79) was observed. For the investigation of various fungicides for the control of S. sclerotiorum, in vitro trials to determine the potential of three different fungicides at different rates, i.e., BAS 516 04F (133 g a.i. ha-1), BAS 516 04F (266 g a.i. ha-1), BAS 512 06F (380 g a.i. ha-1) and Sumisclex (760 g a.i. ha-1) were initially conducted. The control (non-amended PDA) had a significantly higher area under mycelial growth curve (243.0) than all fungicides tested. BAS 516 04F (at both concentrations) and BAS 512 06F completely inhibited the mycelial growth of S. sclerotiorum. Sumisclex inhibited the fungus by 89.07%. For in vivo trials, preventative treatments, i.e., BAS 516 04F (133 g a.i. ha-1), BAS 516 04F (266 g a.i. ha-1), BAS 512 06F (380 g a.i. ha-1), curative treatment, i.e. Sumisclex (760 g a.i. ha-1) and a combination preventative/curative treatment, i.e., BAS 512 06F (380 g a.i. ha-1)/Sumisclex (570 g a.i. ha-1) were investigated. No significant difference in disease severity index (DSI) was found between fungicide treatments and the inoculated control. BAS 512 06F and BAS 512 06F/Sumisclex had significantly lower grain yields (6.09 g and 5.96 g, respectively) compared to all other treatments. There was a positive correlation coefficient (R2=0.76), between DSI and grain yield, indicating that a high DSI is correlated with low grain yield. Trials to evaluate the effect of commercially available and currently unregistered seed treatments for the control of S. sclerotiorum on soybean seeds in vivo and in vitro were performed. Seed germination tests were performed to determine if seed treatments had any negative effects on seed germination in vitro. All seed treatments tested, i.e., BAS 516 03F (8, 16 and 32 ml a.i. 100 kg-1 seed), BAS 512 00F (7.5, 15 and 32 ml a.i. 100 kg-1 seed), Celest XL (100, 125, 200 and 250 ml a.i. 100 kg-1 seed), Sumisclex (5 and 10 ml a.i. 100 kg-1 seed), Benomyl (150 g a.i. 100 kg-1 seed), Captan (240 ml a.i. 100 kg-1 seed), Thiulin (180 g a.i. 100 kg-1 seed) and Anchor Red (300 ml a.i. 100 kg-1 seed), showed no negative effect on seed germination. For in vivo trials, BAS 516 03F (16 and 32 ml a.i. 100 kg-1 seed), BAS 512 00F (7.5, 15 and 32 ml a.i. 100 kg-1 seed), Celest XL (100, 125, 200 and 250 ml a.i. 100 kg-1 seed), Sumisclex (5 and 10 ml a.i. 100 kg-1 seed), Benomyl and Anchor Red had significantly similar percent germination and percent seedling survival as the untreated/uninoculated control. These seed treatments should be recommended for the control of S. sclerotiorum, as they protected seed during germination and subsequent seedling development. BAS 516 03F (8 ml a.i. 100 kg-1 seed) should not be recommended for the control of SSR, as it gave the lowest percent germination and percent seedling survival. The results presented in this thesis have helped to identify optimal environmental conditions for the development of S. sclerotiorum, which is important for the development of forecasting models for disease control. The least and most susceptible cultivars of those tested have been identified. Biocontrol using Eco77® as a foliar application showed great potential. The effect of Si needs to be further investigated, including the testing of more frequent applications and higher concentrations. The fungicides tested in this research did not show any potential for the control of SSR. However, the spray programme tested is for the control of soybean rust (Phakopsora pachyrhizi), and was investigated for its potential for the control of SSR. The spray programme, fungicide application and rating scale needs to be modified, to determine the true potential of these fungicides for the control of SSR. Numerous seed treatments have shown potential for the control of seed infection by SSR. Due to difficulties in producing ascospores, which are the primary source of inoculum for this pathogen in the field, all studies in this research were conducted with mycelia and not ascospores. The production, collection and storage of ascospores needs to be thoroughly investigated, and research conducted with ascospores.
Biological control of Phytophthora root rot of citrus seedlings and cuttings.
(2005) Abraham, Abraha Okbasillasie.; Laing, Mark Delmege.; Bower, John Patrick.
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.
Development of fungal biological control of four agriculturally important pests, Sitophilus oryzae, Trialeurodes vaporariorum, Planococcus ficus and Eldana saccharina, in South Africa.
(2005) Chambers, Craig Brian.; Laing, Mark Delmege.
The use of entomopathogenic fungi to control agriculturally important pests, both in greenhouses and in the field, has been demonstrated by various authors for a number of years. This has been brought about by the development of resistance in certain pest species to chemical applications and a growing public awareness of the safety implications of residual insecticides. Several entomopathogenic fungi were tested against four insect pests found in the Republic of South Africa (RSA), the greenhouse whitefly, Trialeurodes vaporariorum, the rice weevil, Sitophilus oryzae, the grapevine mealybug, Planococcusficus and the sugarcane stem borer, Eldana saccharina. Further concentration, temperature and humidity studies were conducted with selected isolates on the rice weevil, S. oryzae. Sitophilus oryzae is considered one ofthe most important pests of stored grain. Several fungal isolates were tested against the rice weevil, four of which, B1, PPRI 6690, PPRI 6864 and PPRI 7067, were selected for further testing based on the mortality results over a 21 d period. Varying conidial concentrations were applied and at high doses of 1x10 -6 conidia ml -1 with mortality rates of to 84% achieved. LT 50 values ranged from 6 - 68d. Increased spore concentration resulted in an increase in overall mortality. Temperature and humidity was found to affect the infection potential of the four isolates tested. Four temperatures ranging from 15 - 30°C were tested. The highest mortality rates were obtained at 25°C where mortality ranged from 46 - 65% in 14d. Mortality rates decreased with decreasing temperature, and no mortality was recorded at 30°C. Temperature was found to significantly alter the LT 50 values, increasing the LT 50 with decreasing temperatures. Decreasing the humidity resulted in an increased LT 50 and a reduction in the overall mortality rates. The mortality of S. oryzae ranged according to the RH and isolate. Isolates Bland PPRI 6690 resulted in the highest mortalities of 80 and 83% at 92.5% RH, with LT 50's of 6.3d and 6.4d, respectively. Several entomopathogenic fungi were tested against T vaporariorum, P. ficus and E. saccharina, three key pests of South African crops. Nine fungal isolates were tested against the greenhouse whitefly, T vaporariorum, with mortalities ranging from 26.7 - 74.7% over 14d. Beauveria bassiana Isolates Bl and PPRl 6690 produced the highest mortality rates and were recommended for further pathogenicity testing against T. vaporariorum. Planococcus ficus is a common pest ofvineyards in the Western Cape Province, South Africa. Nine entomopathogenic fungi were screened against P.ficus, only two of which produced mortality. Eldana saccharina is a stalk borer, which infests sugarcane in large areas of Southern Africa. Five isolates were tested against second and third instar larvae, three of which, B1, PPRl 6864 and PPRl 6690 resulted in mortalities. Mean percentage mortality was low for all three isolates. From the study it was evident that two of the isolates tested, Bland PPRI 6690 (B. bassiana), showed potential against three of the four pests, and two isolates of Lecanicillium lecanii caused mortality in P. ficus. Further research and understanding of the effect of environmental conditions, spore concentration and epizootic potential would result in the further development of these isolates as future biological control agents.
Chemical control of soybean rust (Phakopsora pachyrhizi) on soybeans.
(2005) Du Preez, Eve Diane.; Caldwell, Patricia May.; Laing, Mark Delmege.
Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. is an aggressive wind dispersed fungal disease which has spread around the world at an alarming rate in the last decade. The disease was first reported in South Africa (SA) in 2001. It has become well established in the province of KwaZulu-Natal. Reports are occasionally made from eastern Mpumalanga, late in the growing season, in years with good rainfall. Yield losses of 10 - 80% have been reported due to SBR infection. Literature was reviewed to better understand the pathogen in an attempt to find suitable disease management strategies. Many strategies involve delaying, rather than preventing, SBR infection. Of the two strategies to prevent infection, the use of fungicides was the only option for disease control in SA, as no resistant cultivars are available. Field trials were conducted to determine which fungicides are effective in controlling SBR. Further research was conducted to determine the timing, frequency and rate of fungicide applications for optimal control of SBR. Trials were evaluated for disease severity, seed yield and the effect of fungicides on seed quality. Fungicides from the triazole class of the sterol biosynthesis inhibiting group of fungicides were found to be the most effective in controlling SBR. A fungicide from the strobilurin group was found to be less effective than the triazoles at the suggested rate, but was found to be as effective when evaluated at higher dosage rates. Triazoles premixed with fungicides from the benzimidazole and strobilurin groups were also effective in controlling SBR. Timing of application was found to be critical for strobilurin fungicides, but not for triazole fungicides, which have a curative ability, unlike strobilurins. Strobilurin fungicides applied preventatively, before the appearance of disease symptoms were as effective as triazole fungicides applied after disease symptoms, but before infection levels had reached 10%. Across both wet and dry seasons two fungicide applications applied at 21d intervals at the R2 growth stage resulted in effective disease control. In wet seasons, a third fungicide application resulted in yields that were higher, albeit not statistically significant, than two fungicide applications. Assessments of individual fungicides for optimal dosage rate found that registered rates were already optimal for some fungicides, but for others it appeared as if alterations were necessary to the rate suggested for registration. This study was one of the first to extensively evaluate the efficacy of the new triazole and strobilurin fungicides on SBR control. The results have been shared globally, but particularly with newly affected countries in South and North America. Although this research has been groundbreaking, there are still many aspects of fungicide control which need to be studied in order to further optimise chemical control of SBR.
Testing for microbiologically active compounds extracted from members of the family laminaceae and other indigenous plants.
(2005) Gurlal, Prenitha.; Laing, Mark Delmege.; Drewes, Siegfried Ernst.
The Labiatae is a large family that occurs worldwide and have species that are adapted to almost all habitats and altitudes. Plectranthus is in this family. Plectranthus species are beautiful South African shrubs. The genus Plectranthus belongs to subfamily Nepetoideae of tribe Ocimeae. The test microorganisms were chosen carefully as each one belonged to a different taxonomic group of fungi and bacteria. Biologically active mono- and sesquiterpenoids are frequently found in many species of Plectranthus but there are little published data that directly link the presence of specific compounds in a species with the traditional uses of that species. Various Plectranthus spp. were collected and dried, followed by chemical extraction using various solvents. Dichloromethane extracts of P. fruticosus and P. ecklonii were screened for antibacterial and antifungal activities using the agar well and trench diffusion methods. It was found that both methods produced inconsistent results. The trench method required a bigger volume of plant extract to be filled into the well, hence, better biological activity was observed with that method. The well method required a smaller volume therefore poor activity was noted with this assay. The size of inhibition zones are dosage dependent. Overall, both plant extracts exhibited antibacterial but no antifungal properties. The pure compound (1), 11-Hydroxy-2-(4-hydroxybenzoyl)-5,7,9(11),13-abietatetraen-12-one, isolated from P. ecklonii was found to be the same as compound (10) which was isolated from P. lucidus. P. hadiensis was extracted using dichloromethane and hexane. The dichloromethane extract proved to contain much higher biological activity than the hexane extract. Three pure compounds, identified as diterpenes, were isolated from the crude dichloromethane extract of P. hadiensis. 6,7-Dihydroxyroyleanone-6,7,12-trihydroxy-8,12-abietadiene-ll,14-dione (2) and 7(alpha)-formoxy-6(beta)-hydroxyroyleanone (3) exhibited good antibacterial and antifungal activity but not against all the test organisms. The remaining pure compound, 7(alpha)-acetoxy-6(beta)hydroxyroyleanone (4), exerted good antifungal activity. This was measured by the inhibition zone which measured up to 14mm when compound 4 was tested against S. sclerotiorum. When testing the hexane extract against the Bacillus formulations, the pellets that were suspended once in Ringer's solution produced bigger inhibition zones than the pellets that were suspended twice. This could be due to bacterial cells washing out of the suspension. The dichloromethane extract of P. praetermissus proved to be very active against X campestris, producing an inhibition zone of 8 - 20mm. Two pure compounds were isolated from the crude extract and identified as diterpenes. Compound 5, 20(10--> 5)-abeo1( 10),6,8,11,13-abietapentaene-11,12,16-triol, and compound 6, 11,12,15-trihydroxy-20( 10-->5)-abeo-abieta-1-(10),6,8,11,13-pentaene are both known compounds which have previously been isolated from Salvia apiana. P. cilatus was extracted with chloroform and tested against various microorganisms for antifungal and antibacterial activities. It showed poor biological activity overall, except against S. sclerotiorum. The crude dichloromethane extract of P. zuluensis exhibited good antibacterial activity, which was limited to the Gram negative test organism. The extract produced an inhibition zone of 10-12mm when tested against X campestris. Pure compound 7, 2-hydroxy-4,6dimethoxyacetophenone, exerted excellent inhibition against B. subtilis and S. sclerotiorum. Neither compound 8, 1,2,4-trimethoxy-5-(2-propenyl)-benzene, nor compound 7, inhibited Candida spp., F. oxysporum and R. solani. Two diterpenes were isolated from the aerial plant parts of P. lucidus with dichloromethane and their structures elucidated by spectroscopic means. The pure compound 9, 11-hydroxy19-( 3-methyl-2-butenoyl)-5,7,9(11), 13-abietatetraen-12-one, showed moderate antifungal activity whereas compound 10, 11-hydroxy-2-(4-hroxybenzoyl)-5,7,9(11),13-abietatetraen12- one, showed high antifungal activity against R. solani, S. sclerotiorum and F. oxysporum. The crude and the pure compounds (formerly isolated from P. parviflorus) showed inhibition against X campestris. The dichloromethane extracts of P. purpuratus subsp. purpuratus and P. purpuratus subsp. tongaensis exhibit similar levels of biological activity when tested against the same test organisms. Poor antibacterial activity was noted with both extracts. However, excellent antifungal activity was depicted when both plant extracts were tested against F. oxysporum, R. solani and S. sclerotiorum. However, the highest biological activity was noted by R. solani which was totally inhibited by both dichloromethane extracts. The pure compound (11) isolated from P. purpuratus subsp. purpuratus was found to have the same chemical structure as compound (9) previously isolated from P. lucidus. The bioautography assay was used to detect and activity-guide the fractionation of antimicrobial compounds from all the Plectranthus spp. tested. The TLC fingerprint showed a zone of clearing around the lower bands of P. fruticosus and P. ecklonii when the plate was sprayed with a suspension of B. subtilis. This result is consistent with the agar well diffusion method. Clear zones were also noted on some bands of the extracts of P. zuluensis, P. ciliatus, P. hadiensis and P. praetermussis. Clear zones indicate inhibition of growth. Other plant extracts tested for biological activity were from the family Lamiaceae, however, not of the genus Plectranthus. Persicaria senegalensis, Pycnostachys reticulata and Ficus sur possessed moderate biological activity overall. It is interesting to note that P. senegalensis and F. sur exert high biological activity against Candida spp. This could be useful as herbal remedies for yeast infections.
Studies on Phakopsora pachyrhizi, the causal organism of soybean rust.
(2006) Nunkumar, Archana.; Caldwell, Patricia May.; Pretorius, Zacharias Andries.
Phakopsora pachyrhizi H. Syd and P. Syd, the causal organism of soybean rust (SBR) was first reported in Japan in 1902. In 1934 the pathogen was found in several other Asian countries and as far south as Australia. In India, SBR was first reported on soybeans in 1951. There have been several early reports of SBR in equatorial Africa but the first confirmed report of P. pachyrhizi on the African continent was in 1996 from Kenya, Rwanda and Uganda. Since then, the pathogen has spread south with reports from Zambia and Zimbabwe in 1998 and in Mozambique in 2000. In February 2001, P. pachyrhizi was first detected on soybeans near Vryheid, in Northern KwaZulu-Natal, South Africa (SA). As the season progressed, the disease was observed in other parts of the province, and epidemic levels were found in the Cedara, Greytown, Howick and Karkloof production regions. Soybean rust subsequently spread to Amsterdam and Ermelo in the Highveld region of SA. The disease reappeared in SA in March 2002. It is now established that the pathogen is a threat to soybean production in the country with yield losses in the region of 10-80%. A literature review on SBR investigating the taxonomy of the pathogen, its morphology, symptoms, host range, infection process, epidemiology, control options and the economic importance of P. pachyrhizi was complied to provide the necessary background information to conduct research under local conditions and to assist in interpretation of results of experiments. Epidemiological trials were conducted at the University of KwaZulu-Natal under controlled environmental conditions in a dew chamber and conviron. Development of P. pachyrhizi on the susceptible cultivar (LS5995) was quantified in combinations of seven temperatures (15,19,21,24,26,28 and 30°C) and five leaf wetness durations (LWD) (6,9,12,14 and 16hrs) at three relative humidities (RH) (75%, 85% and 95%). Studies indicate that optimum temperature for uredospore infection is 21-24°C with a LWD greater than 12hrs and RH 85-95%. The number of pustules as well as lesion size on the abaxial and adaxial leaf surface increased with increasing LWD at all the RH values tested. Infection did not occur on plants incubated at 15°C and 30°C at 85% or 95%RH whereas at 75%RH infection did not occur on plants incubated at 15°C, 19°C and 30°C regardless of LWD. Number of pustules per lesion produced at 75%, 85% and 95%RH was highest at 24°C and showed a gradual increase with increasing LWD. Lesion size on both leaf surfaces increased after 12hrs LWD at 24°C at 75% and 85%RH whereas at 95%RH lesion size increased after 14hrs LWD at 24°C. Exposure of uredospores to ultraviolet light which is equivalent to ultraviolet C (sunlight) which is < 280nm, shows a decrease in germination (7%). Under continuous darkness, the germination percentage was found to range from 58% after 48 hrs. Germination was found to peak at 16hrs in darkness with a gradual decrease as time increased whereas germination under ultraviolet light was highest after 6hrs with a gradual decrease with increased exposure to light. Germ tube lengths were found to be shorter when exposed to ultraviolet light (107µm) compared to controls kept in the dark (181µm). Results obtained clearly show a negative effect of ultraviolet light on the germination and germ tube length of uredospores. A 0.1 ml suspension of uredospores on 1.25% water agar Petri dishes was exposed to cycles of 14h ultraviolet light and 10h darkness for 48h. Results indicate an increase in germination percentage of uredospores when exposed to 10h of darkness following a 14h period under ultraviolet light. Controlled environmental studies were conducted to determine alternative hosts of P. pachyrhizi in SA. The control used in this experiment was Prima 2000, a susceptible cultivar to soybean rust. Seven legume plants [Cajanus cajan (L.) Huth, Glycine max (L.) Merr, Lablab purpureus (L.) Sweet, Lupinus angustifolius (L.) Finnish, Phaseolus vulgaris (L.), Pueraria lobata (M&S) Wild and Vigna unguiculata (L.) Walp] and three dry bean lines (Bonus; OPS-RS2 and PAN 159) showed typical SBR symptoms when rated after 21 days post inoculation with uredospores for percentage disease severity. Disease severity was significantly different within the alternative hosts, but G. max, P. vulgaris and P. lobata were not significantly different from Prima 2000 (control). A uredospore suspension of 2.5 x 10(5) uredospores ml(-1) from plants that showed typical SBR symptoms was made and inoculated on to Prima 2000, a susceptible soybean cultivar. Uredospores from pustules on G. max, L. purpureus, L. angustifolius, P. vulgaris, P. lobata, V. unguiculata, Bonus and PAN 159 produced viable uredospores on PRIMA 2000. These plants are considered alternative hosts of P. pachyrhizi. Effect of leaf age on susceptibility of soybean to SBR was tested under controlled environmental conditions. Mean number of lesions as well as lesion size were greater on younger leaves than on older leaves of plants at the same physiological age. Plants at the early vegetative and reproductive stages had a significantly lower number of lesions as well as a smaller lesion size. Plants at the V6 and R1 growth stages were significantly more susceptible to P. pachyrhizi than plants at other developmental stages. Trichoderma harzianum Rifai, Eco-77® a commercial biological control product, was evaluated for its efficacy as a biological control agent of P. pachyrhizi. Trichoderma harzianum sprayed at the standard concentration on infected soybean plants was significantly more effective in controlling P. pachyrhizi than plants sprayed at 1/2X and 2x the standard concentration. This was noted in both Trial 1 and 2. Data indicate that spraying the filtrate two days after inoculation produces less disease.
The metabolic fate of sucrose in intact sugarcane internodal tissue.
(2000) McDonald, Zac.; Botha, Frikkie Coenraad.; Huckett, Barbara Isobel.
The study was aimed at determining the metabolic fate of sucrose in intact sugarcane internodal tissue. Three aspects of the fate of sucrose in storage tissue of whole plants formed the main focus of the work. These were the rate of sucrose accumulation in the developing culm, the characterisation of partitioning of carbon into different cellular organic fractions in the developing culm and the occurrence of sucrose turnover in both immature and mature stem tissues. Specific attention was paid to confirming the occurrence of sucrose turnover in both immature and mature internodal tissue. This sucrose turnover has been described previously in both tissue slices and cell suspension cultures. However, certain results from previous work at the whole plant level have indicated that sucrose turnover does not occur in mature internodal tissue. Radiolabeled carbon dioxide (14CO2) was fed to leaf 6 of sugarcane culms of a high sucrose storing variety (Saccharum spp. hybrid cv. Nco376). All plants were of similar age (12 months) and were grown under similar conditions. The movement and metabolic fate of radiolabeled sucrose was determined at four time points, (6 hours, 24 hours, 7 days and 6 weeks) during a 6 week period. The metabolic fate of sucrose was determined in internodes number 3, number 6 and number 9. Internode 3 was found to have a relatively high hexose sugar content of 42 mg glc&fruc fw g-1 and a low sucrose content of 14 mg suc fw g-1. In contrast the sucrose content of internode 9 was much higher at 157 mg suc fw g-1 and the hexose sugar content much lower at 4.3 mg glc&fruc fw g-1. Based on previous work, the sugar content of internode 3 and internode 9 are characteristic of immature and mature tissues respectively. Internode 6 occupies an intermediary position between internode 3 and 6 with its sucrose content higher than its hexose sugar content, but with the hexose sugar content still being notable at 15 mg glc&fruc fw g-1. Although the metabolic fate of sucrose within sink tissue was the focal point of the study, the experimental design also allowed for certain aspects of sucrose production in the source to be investigated. The average photosynthetic rate for leaf 6 in full sunlight was estimated at 48 mg CO2 dm-2 s -1. During photosynthesis, only 30% of the fixed carbon was partitioned into the storage carbohydrate pool while the remaining 70% was partitioned into sucrose for immediate export from the leaf. This high rate of carbon fixation combined with a high rate of carbon export is characteristic of C4 plants such as sugarcane. On entering the culm, translocation of radiolabeled sucrose was predominantly basipetal with relatively little acropetal translocation. The majority of the radiolabeled carbon was found to be stored in mature internodes. No significant loss of radiolabeled carbon was observed in mature and elongating internodes over the study period. A 22% loss of total radiolabeled carbon was observed in immature internodes over the same period. This can probably be attributed to the higher rates of cellular respiration known to occur in immature tissues. There appear to be three phases of sucrose accumulation in the developing culm. Initially, the accumulation rate in rapidly growing tissue, as internode 3 develops into internode 6, is relatively low. This is followed by a rapid increase in the rate of sucrose accumulation during internode elongation, as internode 6 becomes internode 9. Finally, a decrease in the rate of sucrose accumulation is observed during late maturation, as internode 9 becomes internode 12. Determination of the sucrose content in internodes 3, 6 and 9 revealed that there is a notable increase in sucrose content during internode maturation. It is proposed that the higher sucrose content of mature tissue is not merely a consequence of the longer growth period of mature tissue, but is due to the increased rate of sucrose accumulation observed during internode elongation. Short-term (24 hours) analysis of carbon partitioning revealed that intemodal maturation was associated with a redirection of carbon from non-sucrose cellulal organic fractions to sucrose storage. In immature internodes only 20% of the total radiolabeled carbon was present in the sucrose pool 24 hours after feeding. In elongating internodes the figure increased to 54% while in mature internodes as much as 77% of the total radiolabeled carbon was retained in the sucrose pool. Concomitant with the increased carbon partitioning into stored sucrose down the developing culm is a decrease in carbon partitioning into the hexose sugar pool. In immature tissue, 42 % of the total radiolabel is present in the hexose sugar pool, while in mature tissue the percentage drops to 11%. This decrease is probably indicative of decreased levels of carbon cycling between the sucrose and hexose sugar pool as a result of internode maturation. Internode maturation was also found to be associated with a decrease in the amount of carbon in the water insoluble matter pool and the amino acid/ organic acid/ sugar phosphate pool. Thus, internode maturation is associated with a redirection of carbon from total respiration to sucrose storage. Long-term (6 weeks) analysis of carbon partitioning confirmed that sucrose storage in mature tissue is greater than that in immature tissue. From the 6 hour time point to the 6 week time point, an 87% reduction in the stored radiolabeled sucrose content was observed in immature internodes. During the same period only a 25% reduction in the stored radiolabeled sucrose was observed in mature internodes. Radiolabel loss from the radiolabeled sucrose pool in both mature and immature internodes was accounted for by relative radiolabel gains in other cellular organic fractions. At all time points during the study, and in all three tissues studied, radiolabel was found in the sucrose pool, the hexose sugars pool, the ionic pool and the water insoluble matter pool. The occurrence of radiolabel in the non-sucrose tissue constituents suggests that sucrose turnover is occurring in both immature, and mature internodal tissue.
Development of free-living diazotrophic (FLD) inoculants and their effects on crop growth.
(2008) Kifle, Medhin Hadish.; Laing, Mark Delmege.
In this study several free-living diazotrophs (FLD) were isolated and screened for their nitrogen fixing ability on a range of crops grown in greenhouse, hydroponics and field trials. Rhizosphere isolates of free-living diazotrophs (FLD) may be effective biofertilizer inoculants, and may improve plant health where crops are grown with little or no fertilizer, as is the case in the Developing World. FLD isolates from rhizospheric soils in KwaZulu-Natal were assessed by growing them on N-free media, which is a key isolation method. They were then evaluated for their nitrogenase activity by quantifying ethylene production from acetylene by gas chromatography (GC). The free living isolates that produced greater quantities of ethylene were detected by an acetylene reduction assay (ARA). These were further assessed for colony formation on N-free media with different carbon sources, and at a range of temperatures (20, 25 and 300C) and pH values (6.0, 7.0 and 8.0). Isolates G3 and L1 were identified using DNA sequencing by Inqaba Biotechnical Industries (Pty) Ltd as Burkholderia ambifaria Coenye et al, and Bacillus cereus Frankland, respectively. These isolates grew significantly better on an ethanol medium, at temperatures of 20, 25 and 300C and pHs of 6.0, 7.0 and 8.0. Isolates B3 (Burkholderia sp.) and D6 (Bacillus cereus Frankland) also grew well on an ethanol medium, but only at 200C and at a pH of 6.0 and 7.0, respectively, while Isolate E9 (Burkholderia cepacia Frankland) grew well on an ethanol medium only at 300C, and pH 6.0 and 7.0. Temperature and pH strongly influence FLD growth on N-free media using different carbon sources. Further trials were conducted to screen the best isolates under greenhouse condition, using both seed treatments and drenching application techniques onto several crops. The drenching application resulted in an increase in the growth and N-total of all the evaluated crops, relative to an unfertilized control. Growth and N-total of maize and sorghum increased with seed treatments, but did not increase the growth of lettuce and zucchini. Drenching of FLD isolates at 106cfu ml-1, applied on weekly basis, resulted in an increase in the growth of lettuce. Increased doses and frequency of application of the FLD bacteria resulted in a decrease in lettuce growth. This led to the conclusion that application of FLD bacteria at high doses and short intervals may create a situation where the applied FLD bacteria and the resident rhizosphere microbes compete for root exudates. High doses at low frequencies and low doses at high frequencies may be more effective on lettuce. Inoculation of Isolate L1 (B. cereus) at 106cfu ml-1 or in combination with Eco-T® (Trichoderma harzianum Rifai), significantly increased growth of lettuce. This result may have been due to nitrogen fixation, or to secretion of growth promoting substances by both the FLD and T. harzianum, and to biocontrol effects of Eco-T®. Application of Isolate L1 (B. cereus) at 106cfu ml-1 with or without Eco-T® was an effective tool for enhancing plant growth and nitrogen fixation. An FLD, Isolate L1 (B. cereus), was applied to lettuce plants together with a complete hydroponics fertilizer at 25% strength (Ocean Agriculture 3:1:3 (38) Complete), with the N level at 25mg l-1. These plants grew significantly better than the control plants grown on 25% of normal NPK fertilization, or with an inoculation of L1 alone. This indicates that it may be possible to integrate FLD applications with the application of low levels of commercial fertilizers, which is what resource poor farmers can afford.
Isolation and molecular characterisation of tomato spotted wilt virus (TSWV) isolates occuring in South Africa.
(2006) Sivparsad, Benice.; Gubba, Augustine.
Tomato spotted wilt virus (TSWV), a Tospovirus, is one of the ten most economically destructive plant viruses worldwide, causing losses exceeding one billion U.S. dollars annually on several crops. In South Africa (SA), TSWV has become an important virus in many economically important crops. The main objective of this research project was to isolate, identify and characterise TSWV isolates occurring in SA. A review of current literature assembled background information on TSWV molecular biology, epidemiology, transmission, detection and control. A TSWV isolate infecting pepper (Capsicum sp.) occurring in KZN was isolated and partially characterised. The virus was positively identified as TSWV using the enzyme-linked immunosorbent assay (ELISA) and the presence of typical necrotic TSWV symptoms on Nicotinia rustica L. Symptomatic leaves were harvested and the virus was partially purified using standard procedures. Under the transmission electron microscope (TEM), typical quasi-spherical and dumbbell-shaped particles of 80-100nm in diameter were observed in negatively stained preparations of both crude and purified virus samples. In negatively stained ultra-thin virus infected leaf sections, an abundance of mature viral particles (100nm) housed in the cisternae of the endoplasmic reticulum (ER) were observed among typical viroplasm inclusions (30nm) and hollow tubules (200-300nm). A viral protein migrating as a 29kDa band, which corresponds to the TSWV nucleocapsid (N) protein, was observed after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Total plant RNA, isolated from N. rustica displaying typical symptoms was subjected to reverse-transcription polymerase chain reaction (RT-PCR) using .primers specific to the nucleocapsid (N) gene. An expected 760bp product was amplified. The results obtained in this study confirm the presence of TSWV in infected pepper plants from KZN. The genetic diversity of TSWV isolates occurring in SA was examined. The nucleocapsid (N) gene sequences of six SA TSWV isolates originating from Gauteng, KwaZulu-Natal, North West, Limpopo and Mpumulanga provinces were determined and used in a phylogenetic tree comparison with TSWV isolates occurring in different geographical locations in the world. Nucleotide sequence comparisons of the N gene revealed high levels of similarity between the SA isolates and TSWV isolates from Asia and Europe. SA isolates showed a high degree of sequence similarity (99-100%) which was reflected in their distinct clustering pattern. The resistance of tomato (Lycopersicon escuJentum Mill.) plants with natural and transgenic resistance against mechanical inoculation with TSWV isolates occurring in SA was evaluated. The Stevens cultivar which has natural resistance conferred by the Sw-5 gene and the transgenic 13-1 line, which expresses the nucleocapsid (N) protein gene of the TSWV-BL isolate, was used as test cultivars. Plants were assessed for TSWV resistance using a disease severity rating scale and measurements of virion accumulation levels (A405nm). There were no significant differences among the reactions produced by the six TSWV isolates on the test plants. Although both plants were susceptible to the SA TSWV isolates by exhibiting similarly high viral accumulation levels, the transgenic tomato line showed milder disease severity compared to the natural resistant cultivar. Results suggest that transgenic resistance is a more effective approach in the control of TSWV in SA. The information generated in this study will be useful in formulating effective control measures using genetic engineering approaches for this economically important virus. Such approaches will be used as a tool to make strategic decisions in an integrated control programme for ISWV.
Evaluation of the potential use of antagonistic microbes on grass species, turf and pasture, for disease control and growth stimulation.
(2003) Cunningham, Debra M.; Laing, Mark Delmege.; Caldwell, Patricia May.
Public tendency, of late, is to reduce liberal use of harmful synthesized chemicals for promoting plant health. Today, biological control is becoming a commonly cited disease control option. Biological control agents (BCAs) not only control disease , but also promote plant growth. Application of biological control is based largely on knowledge of control mechanisms employed by antagonists, as well as the means of application that will ensure that an antagonistic population is established. Knowing the advantages is not the only factor that should be considered before application commences as, the disadvantages must be clearly outlined and explored further before a constructive decision as on implementation of biological control. A literature review was undertaken to provide the necessary technical information about biological control, its potential uses, methods of application, mechanisms of action employed, advantages and disadvantages associated with biological control application, public perceptions and the potential future of biological control. Diseases encountered within the KwaZulu-Natal Midlands on pasture and turf grasses were determined by a once-off survey conducted over 1999/2000. The aim of the survey was to determine broadly the management practices of farmers and groundsmen in KwaZulu-Natal and the potential impact of these on the occurrence of weeds, insects and diseases. The survey also addressed the level of existing knowledge about biological control and willingness to apply such measures. In the pasture survey, farmers were questioned about: soil type, grass species common used, irrigation , fertilization and liming, grazing programs and weed, insect and disease occurrences and control measures implemented. The same aspects were addressed in a survey to a representative sample of groundsmen (turfgrass production) , including also: topdressing, greens base used, drainage systems, mowing practices and decompaction principles. The survey showed correlation between pest incidence and management practices implemented. In terms of pest control, both farmers and groundsmen indicated a stronger preference to the use of herbicides , insecticides and fungicides. Use of fungicides for disease control by farmers is considered an often unfeasible expense, rather more emphasis was placed on implementing cultural control methods. At present farmers do not apply biological control strategies, but they did indicate much interest in the topic. Alternatives to current, or lack of current, disease management strategies are important considerations, with two new diseases identified in the KwaZulu-Natal Midlands just within the period of this thesis. Biological control strategies are implemented by 8% of the groundsmen surveyed, with emphasis being placed on augmenting the already present natural predators rather than the introduction of microbial antagonists. Although often mis-diagnosed by farmers Helminthosporium leaf spot is a common disease in the KwaZulu-Natal Midlands on Pennisetum clandestinum (kikuyu), This disease reduces pasture quality and detracts from the aesthetic appearance and wearability of turfgrasses. Helminthosporium leaf spot is incited by a complex of causal agents , Bipolaris was confirmed as the casual agent of Helminthosporium leaf spot on kikuyu at Cedara. Disease control by two BCAs, Bacillus (B. subtilis Ehrenberg & Cohn.) and Trichoderma (T. harzianum Rifai), as commercial formulations was tested against the fungicide, PUNCH EXTRA®. In vitro, Trichoderma was shown to be aggressive in controlling Bipolaris sp. In vivo, disease control achieved with Trichoderma kd was comparative with PUNCH XTRA® but not statistically different (P>=0.05). Trichoderma and Bacillus provided better disease control in comparison to an untreated control. Improved growth of Lolium sp. was determined in vitro, with Trichoderma kd and Bacillus B69 treatments. The microbe-based treatments accounted for growth stimulation, with significant (P<=O.05) growth differences noted. A microbial activator, MICROBOOST®was added to the treatments to improve microbial efficiency. Improved plant growth with MICROBOOST® applications was shown. Improved growth associated with microbial treatments, Trichoderma harzianum kd; Bacillus subtilis B69 and Gliocladium virens Miller, Gibens, Foster and con Arx. ,was also determined in vivo at Cedara, on L.perenne L., Festuca rubra L. and Agrostis stolonifera L. Establishment of a suppressive soil with antagonistic microbes resulted in significant (P<=O.05) effects on final grass coverage (except G. virens), as well increased root and shoot lengths (P<=O.05). Increased germination rates, as expressed in vitro, were not shown in vivo. Microbial activity with the application of MICROBOOST® showed little effect on germination but increased root and shoot lengths significantly (P<=O.05). Increased weed growth associated with the treatments (except G. virens) was considered a drawback of the microbial-treatments. Microbial treatments were also applied to pasture grasses. An in vitro grazing trial was established at Cedara, using L. multiflorum L. to evaluate the microbe-based treatments Trichoderma kd, Bacillus B69 and G. virens for improved pasture establishment and for increased grazing preference by Dohne Merino sheep. Trichoderma kd was associated with increased dry and wet biomass , but lower dry matter yields in comparison to the control. Only G. virens accounted for a higher dry matter percentage than the control. However, differences between the control and the microbial treatments was very small and not significant (P>=0.05). Of the three grazing observations made, sheep showed no grazing preference to plots with or without microbial treatments In general, the body of this research has shown that microbial treatments have the potential for increased disease control and growth stimulation of grasses. However, lack of significant differences between microbial treatments and controls has raised the question as to effect of external factors on microbial activity and survival, especially in vivo. This raises the question as to the validity of the use of microbial treatments where growth conditions cannot be controlled , remembering that the cost of establishment must be covered by the economic returns from utilization.
In vitro and in vivo screening of Bacillus spp. for biological control of Rhizoctonia solani.
(2003) Kubheka, Bongani Petros.; Hunter, Charles Haig.; Laing, Mark Delmege.
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).
Studies on the biocontrol of seedling diseases caused by Rhizoctonia solani and Pythium sp. on sorghum and tef.
(2003) Tesfagiorgis, Habtom Butsuamlak.; Laing, Mark Delmege.; Caldwell, Patricia May.
Rhizoctonia solani and Pythium spp. are aggressive soil-borne fungal pathogens responsible for seed rot and seedling damping-off of many crops. With increased environmental and public concern over the use of chemicals, biological control of these diseases has been attracting more attention. However, success with this strategy depends on the development of effective antagonists, which requires repeated in vitro and in vivo tests. Bacillus spp. were isolated from a soil sample obtained from a field where sorghum and tef had been grown for at least two years. Potential Bacillus isolates were screened for their ability to inhibit in vitro growth of R. solani and Pythium sp. Among 80 isolates tested, endospore forming Bacillus spp. H44 and H51 gave highest antifungal activity against the two test-pathogens in three consecutive tests. Results demonstrated that both H44 and H51 have potential as biocontrol agents against diseases caused by these two pathogenic fungi. The interaction between three isolates of Trichoderma (T. harzianum Eco-T, Trichoderma spp. SY3 and SY4) and Pythium sp. were investigated using in vitro bioassays together with environmental scanning electron microscopy (ESEM). Visual observation on the dual culture tests revealed that hyphal growth of Pythium was inhibited by these antagonists soon after contact between the two organisms within 3-4 days of incubation. The ESEM investigations showed that all three isolates of Trichoderma grew toward the pathogen, attached firmly, coiled around and penetrated the hyphae of the pathogen, leading to the collapse and disintegration of the host's cell wall. Degradation of the host cell wall was postulated as being due to the production of lytic enzymes. Based on these observations, antibiosis (only by Eco-T) and mycoparasitism (by all three isolates) were the mechanisms of action by which in vitro growth of Pythium sp. was suppressed by these Trichoderma isolates. The reduction of seedling diseases caused by R. solani and a pythium sp. were evaluated by applying the antagonists as seed coating and drenching antagonistic Bacillus spp. (B81, H44 and H51) and Trichoderma (T. harzianum Eco-T and Trichoderma spp. SY3 and SY4). On both crops, R. solani and Pythium sp. affected stand and growth of seedlings severely. With the exceptions of H51, applications all of isoltes to seeds reduced damping-off caused by R. solani in both crops. Application of Eco-T, H44 and SY3 to sorghum controlled R. solani and Pythium sp. effectively by yielding similar results to that of Previcur®. On tef, biological treatments with Eco-T and SY4 reduced seedling damping-off caused by R. solani and Pythium sp., respectively, by providing seedling results similar to the standard fungicides, Benlate® and Previcur®. Most other treatments gave substantial control of the two pathogens on tef. Overall, Bacillus sp. H44 and T harzianum Eco-T were the best biocontrol agents from their respective groups in reducing damping-off by the two pathogens. In all instances, effects of application method on performance of biocontrol agents and adhesive on emergence and growth of seedlings were not significant. A field trial was conducted at Ukulinga Research Farm at the University of Natal, Pietermaritzburg, South Africa, to determine efficacy of biological and chemical treatments on growth promotion and reduction of damping-off incited by R. solani and Pythium sp., and to evaluate the effects of a seed coating material, carboxymethyl cellulose (CMC), on seedling emergence and disease incidence. Seeds of sorghum and tef were treated with suspensions of antagonistic Bacillus H44 or T harzianum Eco-T, or sprayed with fungicides, Benlate® or Previcur®. Application of Benlate® and Previcur® during planting significantly increased the final stand and growth of sorghum seedlings. Seed treatments with both H44 and Eco-T substantially controlled damping-off caused by Pythium, resulting in greater dry weights of seedlings than the standard fungicide. However, they had negative effects when they were tested for their growth stimulation and control of R. solani. The CMC had no significant effect on germination and disease levels. These results showed that these antagonists can be used as biocontrol agents against Pythium sp. However, repeated trials and better understanding of the interactions among the antagonists, the pathogens, the crop and their environment are needed to enhance control efficiency and growth promotion of these antagonists. Some of these biocontrol agents used in this study have the potential to diseases caused by R. solani and Pythium sp. However, a thorough understanding of the host, pathogen, the antagonist and the environment and the interactions among each other is needed for successful disease control using these antagonists.
The use of potato and maize disease prediction models using automatic weather stations to time fungicide applications in KwaZulu-Natal.
(2003) Van Rij, Neil Craig.; Caldwell, Patricia May.; Savage, Michael John.; Quinn, Nevil Wyndham.; Laing, Mark Delmege.
Maize grey leaf spot (GLS), caused by Cercospora zeae-maydis, and potato late blight (LB), caused by Phytophthora infestans, are foliar diseases of maize and potato, two of the most widely grown crops in KwaZulu-Natal (KZN), after sugarcane and timber. Commercial maize in KZN accounts for just on 4.3% of the national maize crop. This is worth R563 million using an average of the yellow and white maize price for the 2001/02 season (at R1 332.87 ton(-1)). In 2003 KZN produced about 5% of the national potato crop (summer crop: 7531 300 10kg pockets from 2243 hectares). This equates to a gross value of R89.4 million based on an average price of R1 188 ton(-1) in 2001. Successful commercial production of maize and potatoes depends upon control of these diseases by translaminar fungicides with highly specific modes of action. This study extends an existing model available for timing of fungicide sprays for GLS and tests and compares two LB models for two calendar-based spray programmes. The study also evaluated the use of an early blight model which is caused by Alternaria solani, and over the single season of evaluation showed potential for use in KZN. For the GLS model it was found that a number of refinements are needed, e.g., the amount of infected maize stubble at planting and not the total amount of maize residue at planting. Based on two years' data, it was found that for the LB models there are no significant differences in levels of control between using a predicted fungicide programme and a calendar-based programme. The importance of knowing initial infection sites, and hence initial inoculum, was demonstrated. This led to the creation of a KZN LB incidence map, now being used to more accurately time the start of a preventative spray programme and to time the inclusion of systemic fungicides in the preventative spray programme. This study has contributed to the further development and expansion of the Automatic Weather Station Network (AWSN) at Cedara, which now comprises 15 automatic weather stations in KZN. The AWSN is currently used to aid farmers and advisers in decision-making regarding fungicide spray timing for GLS and LB.
Evaluation of integrated control of postharvest grey mould and blue mould of pome fruit using yeast, potassium silicate and hot water treatments.
(2012) Mbili, Nokwazi Carol.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
The public concern over synthetic pesticides in foods and the environment has created an interest to find effective and safe non-fungicide means of controlling postharvest pathogens. The overall objective of this thesis was to evaluate the effect of potassium silicate, yeast antagonists and hot water dip treatment to control postharvest grey mould and blue mould of pome fruits, caused by Botrytis cinerea and Penicillium expansum, respectively. Botrytis cinerea and Penicillium expansum were isolated from infected strawberry and pear fruits, respectively. These isolates were found to be non-resistant to YieldPlus® (Anchor yeast, Cape Town, South Africa), a biofungicide containing a yeast Cryptococcus albidus. A total of 100 epiphytic yeast isolates were obtained from the fruit surface of “Golden Delicious” apples and “Packham’s Triumph” pears, and screened against B. cinerea and P. expansum. Fifteen yeast isolates reduced grey mould incidence by > 50%, when applied four hours before inoculation with B. cinerea. Similarly, seven yeast isolates reduced blue mould incidence by > 50%, when applied four hours before inoculation with P. expansum. YieldPlus® and yeast Isolate YP25 provided the best control of B. cinerea, while Isolate YP60 and YieldPlus® provided the best control of P. expansum on “Golden Delicious” apples. A mixture of YP25 and YP60 provided complete control of both B. cinerea and P. expansum, when applied to “Golden Delicious” apples before inoculation with either B. cinerea or P. expansum. Electron microscopy studies showed that yeast Isolates YP25 and YP60 inhibited the mycelial growth of B. cinerea and P. expansum, respectively. Preventative and curative application of potassium silicate resulted in reduced incidence of B. cinerea or P. expansum of “Golden Delicious” apples. Electron microscopy studies indicated that potassium silicate inhibited the growth of B. cinerea and P. expansum. Furthermore, treatment of “Golden Delicious” apples with either potassium chloride or potassium hydroxide resulted in reduced incidence of both B. cinerea and P. expansum. In vivo tests showed that the disease incidence of P. expansum and B. cinerea on “Golden Delicious” apples was reduced by hot water dip treatments at 58-60°C for 60 to 120 seconds, compared with the control fruit treated with sterile distilled water, without causing skin damage. The use of potassium silicate, yeasts (Isolates YP25 and YP60), YieldPlus® and the antagonists mixture (YP25+YP60) in combination, resulted in the control of B. cinerea and P. expansum of “Golden Delicious” apples compared with Imazalil® treated fruit.
Biological control of gastrointestinal nematodes of small ruminants, using Bacillus thuringiensis (Berliner) and Clonostachys rosea (Schroers).
(2011) Baloyi, Mahlatse Annabella.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
Gastrointestinal nematode parasites cause great losses in the production of small ruminants through reduced productivity and the cost of preventive and curative treatments. Because of the threat of anthelmintic resistance, biological control of sheep nematodes has been identified as an alternative to anthelmintic drugs. Bacillus thuringiensis (Bt) (Berliner) and Clonostachys rosea (Schroers) have been widely studied as biocontrol agents. B. thuringiensis has been used for the biocontrol of insects and C. rosea has been successfully used as biocontrol agent of Botrytis cinera (De Bary) in plants. B. thuringiensis and C. rosea strains were isolated from soil collected from the Livestock Section at Ukulinga Research Farm, University of KwaZulu Natal, Pietermaritzburg. Twenty-five strains of Bt and 10 strains of C. rosea were successfully isolated. The Bt colonies were identified by their circular, white, flat and undulate character, and the gram-positive and rod-shaped endospores. C. rosea was identified by white colonies on Potato-dextose agar and the characteristic conidiophores, which were branched and showed phialides at the tips. In vitro screening of the isolates was undertaken to select the best isolates. The isolates that caused significantly greater mortality were Bt isolate B2, B10 and B12 and C. rosea isolates P1, P3 and P8. These isolates caused substantial nematode mortality in both faeces and water bioassay. Nematode counts were reduced by 28.5% to 62% and 44% to 69.9% in faecal bioassay for Bt and C. rosea, respectively. In the water bioassay, nematode counts were reduced by 62% to 85% for Bt and by 62.7% to 89.3% for C. rosea. The best inoculum level at which the best isolates were most effective, and the optimum frequency of application were determined. The trial was conducted using bioassays with faeces and water. Inoculum levels of 10(6), 10(8), 10(10), 10(12) spores ml-1 for Bt and 10(6), 10(8) and 10(10) conidia ml-1 for C. rosea was used in the faecal bioassay. The inoculum levels tested in water bioassay were 10(6), 10(8), 10(10) and 10(12) spores ml-1 for Bt and 10(9), 10(10), 10(11), 10(12) conidia ml-1 for C. rosea. In the faecal bioassay, B2 was the most effective Bt isolate at an inoculum level of 10(10) spores ml-1. Isolate P3 was the best C. rosea isolate at 10(8) conidia ml-1. In the water bioassay, Isolate P3 caused a mortality of 85% at inoculum levels of 10(9), 10(10) and 10(11) conidia ml-1. The performance of biological control agents in the field is sometimes inconsistent. Combining different biocontrol agents may be a method of improving their reliability and performance. However, the combination of most of the isolates was antagonistic, with efficacy less than that of either individual biocontrol agent. In particular, Isolate P3 was more effective when used alone than when combined with any other isolates. Therefore, the combination of biocontrol agents does not always result in synergistic interaction. There were some additive interactions between two bacterial isolates, and with one bacterial and fungal combination. The effect of feeding the best of the biocontrol agents, or diatomaceous earth (DE), was evaluated in sheep. Two doses of Bt (1g and 2g kg-1BW) and C. rosea (1g kg-1BW) reduced the numbers of L3 nematode larvae in sheep faeces. The DE product (at 15% of feed) also reduced L3 numbers but it was less effective than either the Bt or the C. rosea products. Nematode counts were reduced by 74.6%, 75.1%, 84.6%, 68.5% and 27.5% for Bt 1g kg-1BW, Bt 2g kg-1BW, C. rosea (1g kg-1 BW), DE and control, respectively.
Marker-assisted selection for maize streak virus resistance and concomitant conventional selection for Downy Mildew resistance in a maize population.
(2013) Mafu, Nothando Fowiza.; Laing, Mark Delmege.; Derera, John.; Naidoo, Roobavathie.
Maize streak virus (MSV) disease, transmitted by leafhoppers (Cicadulina mbila, Naude), and maize downy mildew (DM) disease caused by Peronosclerospora sorghi (Weston and Uppal) Shaw, are major contributing factors to low maize yields in Africa. These two diseases threaten maize production in Mozambique, thus the importance of breeding Mozambican maize varieties that carry resistance to these diseases. Marker-assisted selection (MAS) was employed to pyramid MSV and DM disease resistant genes into a single genetic background through simultaneous selection. Firstly, it was essential to determine the genetic diversity of MSV disease resistance in 25 elite maize inbred lines to aid in the selection of suitable lines for the introgression of the msv1 gene; and subsequently, to introduce the msv1 resistance gene cluster from two inbred lines, CM505 and CML509, which were identified as the ideal parental lines for the introgression of MSV disease resistance into a locally adapted Mozambican inbred line LP23 that had DM background resistance. Pyramiding the resistance genes by the use of simple sequence repeat (SSR) molecular markers to track the MSV gene cluster was investigated in 118 F3 progeny derived from crosses of CML505 x LP23 and CML509 x LP23. High resolution melt (HRM) analysis using the markers umc2228 and bnlg1811 detected 29 MSV resistant lines. At the International Maize and Wheat Improvement Centre (CIMMYT) in Zimbabwe, MSV disease expression of the 118 F3 progeny lines was assessed under artificial inoculation conditions with viruliferous leafhoppers and the effect of the MSV disease on plant height was measured. Thirty-seven family lines exhibited MSV and DM (DM incidence ≤50) disease resistance. Individual plants from a total of 41 progeny lines, that exhibited MSV disease severity ratings of 2.5 or less in both locations within each of the F3 family lines, were selected based on the presence of the msv1 gene based on SSR data, or field DM disease resistance, and were then advanced to the F4 generation to be fixed for use to improve maize hybrids in Mozambique for MSV resistance. Simultaneous trials were run at Chokwe Research Station in Mozambique for MSV and DM disease assessment, under natural and artificial disease infestation, respectively. Thus the MSV and DM genes were effectively pyramided. Lines with both MSV and DM resistance were advanced to the F4 generation and will be fixed for use to improve maize hybrids in Mozambique for MSV and DM resistance, which will have positive implications on food security in Mozambique. This research discusses the results of combined selection with both artificial inoculation and the three selected SSR markers. It was concluded that a conventional maize breeder can successfully use molecular markers to improve selection intensity and maximise genetic gain.
Genomics of quantitative resistance to brown rust (Puccinia melanocephala) in a sugarcane breeding population.
(2012) Mhora, Terence Tariro.; Danson, Jedidah W.; Rutherford, Richard Stuart.
The Sugarcane Industry contributes approximately 400 000 jobs and ZAR 8 billion annually to South Africa’s economy. Due to climate change and the subsequent threat posed by disease, these figures have been on the decline. Brown rust, a contributor to this decline is caused by the basidiomycete Puccinia melanocephala Syd. and P. Syd., which previously resulted in 50% yield losses in susceptible varieties. This highlighted the need for improved screening and breeding techniques which will result in the replacement of susceptible varieties. The objectives of this study were to: a) Adopt and optimise a glasshouse whorl inoculation screening technique applicable for mass screening of large populations. b) Develop a rapid and cost effective rust resistance screening technique using detached leaves. c)Utilise two flanking marker sets (R12H16 and 9O20-F4-PCR primers) for the rust resistance Bru1 gene in a diagnostic polymerase chain reaction (PCR) to identify rust resistant genotypes lacking Bru1 and possessing either quantitative resistance or an alternative major qualitative resistance gene. d) Correlate rust phenotypic data to AFLP marker data for the Linkage Disequilibrium (LD2) mapping population. e) Utilise suppression subtractive hybridization (SSH) profiling on rust challenged genotypes to discover differentially expressed genes between susceptible and resistant (susceptible Bru1 negatives taken away from resistant Bru1 negatives); and resistant genotypes (resistant Bru1 positives taken away from resistant Bru1 negatives). 4 Results from the glasshouse whorl inoculation trials showed the technique could be reliably used to screen large populations, as two independently conducted pot trials showed highly correlated rust ratings. A visually assessed detached leaf assay (DLA) was developed using selected genotypes. Chlorophyll fluorescence and SPAD readings were used in the DLA to determine the leaf photochemical efficiency (PIABS) with relation to chlorophyll content, resulting in reduced assessment time of at least two days. PCR diagnostics revealed 31% of LD2 did not possess either flanking marker, 8% had one or the other marker, and 61% had both markers. The overall rust phenotypic ratings (rating scale of 0-10) and Bru1 status of the genotypes was used to group the population, with the Bru1 negative genotypes containing all three rating categories (resistant 0-3.5; intermediate 3.51-6.5; susceptible 6.51-10); while the Bru1 positive genotypes were all resistant. The phenotypic data was correlated to AFLP data using the Pearson product-moment correlation coefficient and stepwise multiple linear regression employed to build marker based models to use for predicting non-Bru1 mediated resistance. SSH analysis was then subsequently conducted on genotypes selected on the basis of Bru1 status and AFLP correlation data. Two subtraction cDNA libraries were constructed and the cDNA inserted into electro-competent Escherichia coli cells. PCR on transformed cells revealed cDNA inserts ranging from 200- 1300bp. BLAST analysis of the cDNA sequences indicated the presence of high proportions of disease and drought stress related sequences in the libraries. Analysis of the sequences in both libraries showed that the resistant Bru1 negative genotypes contained oxidative stress related sequences which were however absent in the Bru1 positive resistant genotypes. The library comparing the Bru1 negative resistant genotypes against the Bru1 negative intermediate and susceptible genotypes showed a higher proportion of differentially expressed sequences coding for putative disease resistance proteins, highlighting their presence in the resistant genotypes. Both subtraction libraries also contained high proportions of a leucine rich repeat protein coding cDNA which contained a conserved domain homologous to that of a disease resistance protein conferring resistance to Pseudomonas syringae in Arabidopsis thaliana. The outcomes of this study will subsequently enable an improved understanding of sugarcane-rust resistance mechanisms and improved breeding and screening techniques for sugarcane by identifying SSH and AFLP markers linked to rust resistance QTLs or alternative R genes.

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