Masters Degrees (Plant Pathology)

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Maize root rot in South Africa.
(1995) Hugo, Elbé.; Rijkenberg, Fredericus Hermanus Johannes.
Numerous fungal species are known to infect maize roots and eventually cause rot. The spectrum of fungi differs over localities as well as their relative frequencies. Fungi isolated from discoloured root tissue and root tissue without visible discolouration were classified as root pathogens and root colonizers, based on their isolation frequency from the respective samples. Exserohilum pedicellatum, Macrophomina phaseolina and Fusarium oxysporum were classified as the major root pathogens and Phoma spp., Curvularia spp. and F. chlamydosporum as root colonizers. Fungi classified as root pathogens tended to occur early in the growing season in juvenile tissue as opposed to root colonizers which occurred later. Treatments in an existing long-term water stress trial included no stress (irrigation), normal rainfall, stress until flowering, stress after flowering until mid grain-fill, stress after flowering and total stress. Root pathogens were isolated at higher frequencies in the no stress and normal rainfall treatments than in the total stress treatment. A positive linear relationship between the water stress index and the isolation frequency of M. phaseolina was obtained. Negative, non-linear relationships were, however, recorded for E. pedicellatum, F. oxysporum and F. moniliforme. The effect of tillage practices on disease severity was carried out in field trials at two localities. Tillage practices applied included rip, plough, chisel plough and no-till treatments. Significant differences between isolation frequencies and tillage treatments were recorded for F. oxysporum at Bloekomspruit and Trichoderma spp., Alternaria spp. and M. phaseolina at Mmabatho. Differences in isolation frequency of fungi involved in maize root rot, were determined in a crop rotation system where maize was rotated with soybeans, sunflowers and groundnuts. Crop rotation had a significant effect on the isolation frequency of Thielavia ferricola, E. pedicellatum, F. moniliforme and F. graminearum. The effect of crop rotation, however, seems to be complex since fungi were affected differently in the various treatments.
The epidemiology and control of Capsicum viruses in Natal.
(1995) Budnik, Krzysztof.; Da Graca, John Vincent.; Laing, Mark Delmege.
Virus diseases pose a serious threat to commercial pepper (Capsicum annuum L.) production in Natal. Following a survey of the principal capsicum-producing areas, potato virus Y (PVY) was found to be the predominant virus infecting peppers, often reaching 100% incidence. Currently, TSWV incidence and CMV levels are insignificant with respect to pepper crops in Natal. Thus, the diverse ecological and epidemiological factors which determine PVY infection of peppers were investigated. The potential host range of PVY was established in a glasshouse study. Seed from solanaceous weeds commonly occurring in vegetable-producing areas of Natal was collected. Seedlings were mechanically inoculated with a pepper strain of PVY and assayed for infection using double-antibody sandwich ELISA. Nicandra physaloides L., Solanum elaegnifolium Cav., S. nigrum L., S. velosum L. and S. aculeastrum L. were found to be susceptible to PVY infection. In addition, a field survey of over 100 samples of commonly occurring weed species growing in or adjacent to capsicum crops in the Pietermaritzburg and South Coast regions of Natal was carried out. Several weed species were found to be naturally infected with PVY, including Acanthospermum hispidum DC., Bidens pilosa L., N physaloides and S. nigrum. The spread of PVY into a pepper crop on the Natal South Coast was monitored during 1993. Virus spread was rapid, with PVY first detected in pepper seedling one week after planting, suggesting a nearby source of the virus. A survey of the wild vegetation prior to planting of the crop, revealed that populations of N physaloides may be the primary sources of PVY infecting the crop. Large virus-infected S. nigrum populations appeared later in the season, suggesting its role in maintaining high levels of PVY during periods when no pepper cultivation takes place. In addition to identifying possible virus reservoirs, several virus control measures were investigated, demonstrating ways of avoiding or minimising infection. The effects of insecticides, oil sprays (Virol), insect repellents (AzatinTM), yellow polyethylene traps and plastic mulches on virus incidence within peppers were evaluated in field experiments. Results of weekly sprays of the insecticide mercaptothion at 5%, increased virus incidence in peppers by 15% when compared to the untreated control. Similarly, the effects of insecticide applications on pepper yields and quality were negative. Results of applications of Virol at 1 % and Azatin™ at 1.5% did not differ from those of the unsprayed control. Mulching was most effective by reducing virus incidence in treated plots by 50% and resulted in a yield increase of 62% and a 40% increase in fruit quality. The use of yellow sticky traps reduced virus incidence by 35%, with a yield increase of 25% and a 24% improvement in fruit quality, when compared to the untreated control. Both mulching and the use of yellow sticky traps reduced the number of aphids trapped within the plots. In order to assist the development of capsicum cultivars resistant to PVY infections, a screening method was developed to determine susceptibility levels of a breeding population. Two rating procedures were investigated based on disease severity of the whole plant and on the fruit (chilli pods). The technique was effective in detecting small incremental increases in susceptibility within a breeding population, provided that an adequate positive selection pressure is applied. Using this technique breeders may be able to define a large breeding population to those parents exhibiting a genetic base most suitable for resistance development and eliminate those which exhibit low frequencies of resistance genes. Based on the results obtained, an integrated virus management strategy is suggested, including the elimination of virus sources and the use of cultural practices which facilitate a reduction in virus spread.
Cultivation of exotic and local mushroom species for commercial production.
(1995) Adey, Samantha.; Laing, Mark Delmege.
The project was undertaken primarily to determine the potential of growing Stropharia rugoso-annulata and Volvariella volvacea at a commercial level under South African conditions. Termitomyces umkowaani, an indigenous mushroom, was also investigated. Mushroom culturing, spawn production and optimizing fruiting substrates were determined. The feasibility of commercial and subsistence production in KwaZulu-Natal was investigated. Of the seven strains of S. rugoso-annulata purchased from CBS (Baarn, the Netherlands) that were tested, Strain 289.85 was the most vigorous. The best agar medium for culturing of S. rugoso-annulata was potato dextrose agar (PDA) followed by malt extract agar (MEA) and vegetable juice agar (V8). Maximum growth of the mushroom mycelium occurred from Day 7 to Day 14, as expected. The best spawn substrate tested was sorghum, and the best fruiting substrates tested were banana leaves and maize stover. As S. rugoso-annulata is a white-rot fungus capable of degrading lignin, this result was expected. S. rugoso-annulata grows best on substrates with a low C:N ratio. Strain 1665 of V. volvacea was the best of the three strains obtained from CBS. The best culture medium for V. volvariella was PDA amended with 1 % straw. V8 agar alone supported poor growth. In trials for suitable spawn and fruiting substrates for V. volvacea, banana leaves supported the best mycelial growth. Mycelium grown on V8 amended with 1% maize stover showed the greatest expansion on the substrates tested. A strain of T. umkowaani was isolated from a fresh basidiocarp collected in Pietermaritzburg, KwaZulu-Natal. Growth of T. umkowaani was most rapid on a Basal Medium but mycelial vigour was inferior to that of mycelium grown on Basal Medium amended with rabbit faeces, especially at the level of 2.5%. High levels of contamination occurred when more rabbit faeces was added to the Basal Medium. Microbial contamination was a major limiting factor to the productivity of this project. The primary sources of culture contamination were due to mites, inadequate sterile technique and a high level of Trichoderma spores in the Department due to an ongoing Trichoderma biocontrol project. Placing the bags of cultures on trays with the edges smeared with petroleum jelly effectively kept the mites away from the cultures. The contamination of a stock culture with Penicillium was solved with the use of Benomyl-amended agar media. Contamination problems in spawn production were the result of the inoculation process (especially when conducted by one person), the initial leaking of cotton wool caps, polypropylene bag seams and micropores and cracks in the walls of polypropylene bags. Treating the cotton wool in the caps with Busan 30A prevented the entry of contaminant bacteria and fungi through the caps. The problem of leaking seams of the bags was solved by using a longer heating period on the bag-sealer machine. SEM studies confirmed the presence of micropores and stress fractures in the walls of the bags used in this project. This problem can be solved by using high quality polypropylene with a reduced content of plasticiser. A systems analysis of exotic mushroom production, the process of mushroom production, steps in the process, sales and constraints in the South African context, and possible solutions are discussed. Spawn production by small growers is not economically feasible due to the capital required. Access to capital is a constraint of production in controlled environments but should not limit outdoor production. However, outdoor production is constrained by climatic requirements of the fungi. Based on optimum temperatures, GIS maps of KwaZulu-Natal were generated to display the potential areas for outdoor production. Overlap of suitable regions for production of S. rugoso-annulata and V. volvacea is limited. Outdoor production of V. volvacea will be limited to Northern coastal regions for only four months of the year. A computerized decision support system was developed to answer questions of feasibility of production to the mushroom growers, based on the requirements of the mushrooms. A current lack of marketing of speciality mushrooms is considered to be a major constraint to sales and therefore potential production, particularly for subsistence farmers.
Control of insect-transmitted viruses in cucurbit crops in KwaZulu- Natal.
(1998) Cradock, Kenwyn Roan.; Da Graca, John Vincent.; Laing, Mark Delmege.
The production of cucurbits (Cucurbitaceae) in KwaZulu-Natal faces the constant threat of viral diseases. These can be so severe as to severely limit or prevent production in the latter part of the growing season (December-April). The important viruses in this regard are zucchini yellow mosaic potyvirus (ZYMV), watermelon mosaic 2 potyvirus (WMV2), watermelon mosaic potyvirus - Morocco strain (WMV-M), papaya ringspot potyvirus - type W (PRSV-W), cucumber mosaic cucumovirus (CMV), and squash mosaic comovirus (SqMV). The potyviruses and CMV are vectored by aphids (Homoptera: Aphididae) and SqMV is vectored by cucumber beetles (Coleoptera: Coccinelidae). PRSV and SqMV were found to be absent from the region, while CMV was found not to be a serious threat to cucurbit production. ZYMV, WMV2 (now confirmed to occur in South Africa) and WMV-M are the major viral pathogens of cucurbits in KwaZulu-Natal. The distribution of these viruses and methods for their control were investigated. Investigations of aphid morphology using the scanning electron microscope were undertaken to determine if taxonomic studies could be conducted using this form of microscopy. The best form of specimen preparation was the cryo-fixation technique, which resulted in less collapse of the body wall and general damage to the specimen when compared to the critical point drying technique. Due to the lack of mobility of the specimen while viewing, this form of microscopy is rejected as a means of identifying aphids to the species level. ZYMV was found to occur in a number of weed species (Galinsoga parviflora, Malva parviflora, Amaranthus sp., Solarium spp.), which could serve as reservoirs of virus. WMV-M and CMV were also found in some weed species. All tests for the potyviruses and SqMV were done using the double-antibody sandwich (DAS) ELISA technique. CMV was tested for using indirect ELISA tests. A third of the plants tested were found to be infected with more than one virus which could have implications for disease severity. Disease severity was found to increase at about midway through the growing season (December- January). This was concurrent with a massive increase in the general aphid population in the experimental area. As no aphids were seen on the cucurbits in the fields, these vectors are believed to be transient inhabitants of the crop at first testing and then rejecting the plants as a food source. All control measures applied in the trial were aimed at reducing the numbers of aphids in the plots. Aphids were trapped using yellow sticky traps. Cucurbita pepo (zucchini) was used in the trials due to its bush growth habit and good virus symptom expression. The success of the treatments was determined by monitoring the numbers of aphids present in the plots, and the use of a rating scale which assessed the severity of virus disease in the plots. The two best treatments were the white reflective mulch and the straw mulch. In the cultivar trial which assessed ten different cultivars for their virus resistance/tolerance. The best three cultivars were 'SQ 229', 'Puma', and 'SQ 228'. 'SQ 229' and 'SQ 228' were withdrawn from the market by the seed company for unknown reasons. From the results obtained from these investigations, a disease management programme can be suggested. All cucurbit crops should be grown over a white reflective mulch, drip irrigation should be used to reduce agitation of the plants which could unnecessarily disturb feeding aphids, and a resistant or tolerant cultivar should be used in the latter half of the growing season. The effectiveness of any treatment can be assessed by comparing the number of aphids caught with the number caught in the control plots.
Factors affecting germination and growth of sugarcane transplants.
(1998) Goodall, Julia Louise.; Laing, Mark Delmege.; Bailey, Roger.
Transplants are produced and sold in South Africa for the planting of seedcane supply plots (nurseries), commercial fields, and for gap filling. The most important factor constraining the use of transplants is the low germination of single-budded setts (SBS) planted in polystyrene trays. The main aims of this project were to develop practical methods for optimising germination and to control pathogens without adversely affecting germination. Seedcane quality, cane age, storage and treatments using heat, chemicals and fungicides affected germination and growth. Germination of SBS from old seedcane was significantly higher when taken from the top than from the middle and bottom of the stalk. Storage of seedcane for three and eight days after harvest adversely affected germination and growth. Topping of stalks three days before harvest increased germination potential, but results were variable, depending on cane age, cane quality and further treatments. Treatment of SBS at both 50°C for 120 minutes and 52°C for 30 minutes controlled Clavibacter xyli subsp. xyli (C. x. xyli) (the causal organism of ratoon stunting disease) more effectively than treatment of whole setts. After treatment of SBS at 52°C for 30 minutes, germination was greater than that after treatment at 50°C for 120 minutes, and C. x. xyli was eliminated from stalks of six out of seven varieties. Treatment of SBS at 52°C for 10 minutes significantly improved both germination and plant growth. Treatment of SBS for 10 minutes after addition of ethephon to the hot water significantly increased germination compared with the untreated control, but not compared with treatment with hot water alone. After treatment of SBS with fungicides, germination was highest after treatment with Eria® (Novartis), a chemical with two active ingredients, namely carbendazim and difenoconazole. Compared with no treatment and the short hot water treatment, treatment with Eria® in hot water (52°C) significantly improved germination and plant growth in both unsterilised and sterilised medium. Treatment of SBS and drenching of trays with a solution of propamocarb-HCl and benomyl had no effect on germination or growth, indicating the limited role of systemic infections and soilborne pathogens in germination failure. However, germination and growth were significantly increased when the same SBS were also treated with Eria®, suggesting that germination was predominantly increased by the plant growth regulator activities of its active ingredients. When used separately, both difenoconazole and carbendazim significantly increased germination, and difenoconazole significantly increased plant growth. The conclusion drawn from these results is that germination failure of SBS in trays is mainly due to the inappropriate hormonal balance for germination within .the SBS, rather than systemic infections or infection by soilborne pathogens. Therefore, germination and growth can be optimised by using mature, good quality seedcane, and by treatment of SBS with chemicals that adjust the hormonal balance in the bud region to one appropriate for germination.
The epidemiology and control of crucifer chocolate spot.
(1998) Machaba, Kulani Patrick.; Laing, Mark Delmege.; Wallis, Frederick Michael.
In the 1991/92 season, a leaf spotting disease of crucifer seedlings appeared at a number of nurseries in Kwazulu-Natal (KZN) Province. Towards the end of 1996, the disease had also been detected in Western and Northern Provinces, and even in Zambia. The disease appeared in nurseries during warm humid conditions. It initially started as small, water-soaked spots on leaves, which later expanded and coalesced, forming halo regions surrounded by a dark brown to black ring. Due to the typical dark brown spots, the disease was named crucifer chocolate spot (CCS). From the studies conducted by Qhobela & Laing (1994), the causal agent of CCS was found to be a Xanthomonas campestris pathovar that was in symbiotic association with a unique bacteriophage. Koch's postulates were conducted. The causal agent of CCS was inoculated on cabbage seedlings (6-8 weeks old), and the seedlings subjected to post-inoculation periods of approximately 48 hours at 28-30°C, and relative humidity (RH) of ≥70%. The best method of inoculation was achieved when the leaves were pricked with pins prior to application of the inoculum (≥10(7)cfu/ml). The latent period of CCS varied from 2-6 days, depending on RH, temperature, nutritional status of the host and other factors within the crop pathosystem. Artificial inoculation studies showed the host range of CCS X. campestris to be similar to X. campestris pv. armoraciae and X. campestris pv. campestris. All the pathogens were able to infect cabbage, cauliflower, broccoli and lettuce plants during artificial inoculation. A hypersensitive response was elicited on pepper, tomato, and tobacco plants. Electron microscopic studies showed epiphytic colonization by CCS X. campestris, and also the colonization of xylem, phloem and parenchyma tissues. Although xylem tissues were also colonized, the V-shaped lesions and vein blackening characteristics typical of X. campestris pv. campestris were not observed. Profuse multiplication of the pathogen resulted in rupturing of cell organelles and cell membranes. Different crucifer cultivars from two seed companies were found to have varying levels of susceptibility to CCS disease during artificial inoculation, with levels of leaf area infected ranging from 0% to 34%. Disease severity was also affected by seasonal variations. None of the seed-lots tested in this research were found to be infected by CCS X. campestris, although some were infected by other xanthomonads. Chemical seed treatment (soaking in bactericide solutions at 30°C for two hours) with biocides; e.g., Kocide 101, copper oxychloride, and copper ammonium carbonate was more effective than hot water treatment (50°C for 30 min). All seed treatments lowered pathogen levels, however, none resulted in complete control of seedborne inoculum. Percentage germination of seed was low for chemical treatment compared to the hot water treatment. Disease levels were reduced with increased rates of Ocean 3.1.3 (38) fertilizer applied, with significant differences in the percentage leaf area infected at obtained at application levels of 400 ppm. Increased levels of N, P, K, Zn and Cu in leaf tissues were associated with low levels of disease severity. Copper bactericides; e.g., Kocide 101, Copper Count N, Copper Ammonium Carbonate B and Copper oxychloride were more effective than quaternary ammonium compounds in reducing disease levels (≤30%). The combination of copper bactericides and mancozeb improved the performance of the copper compounds. Application of copper bactericides up to four times the recommended dosage did not result in increased efficacy. CCS was found in 80% of KZN nurseries surveyed between December 1995 and December 1996. During this period, it was not detected from Mpumalanga and Northern Provinces. Factors that contributed to CCS disease outbreaks include favourable climatic conditions (≥ 40% RH, ≥24°C), wounding of seedlings by pests, and nutrient stress.
Development of concentration and detection techniques for Cryptosporidium and Giardia and the significance of these protists in KwaZulu-Natal.
(1999) Jarmey-Swan, Claire.; Howgrave-Graham, Alan R.; Bailey, Ian.
Cryptosporidium and Giardia are waterborne parasitic protozoons that have been associated with some diarrhoeal illness in most parts of the world. They are considered of major importance for drinking water safety as waterborne outbreaks still occur regularly in both developed and developing countries, sometimes with fatal consequences. To determine their incidence in the KwaZulu-Natal population, an epidemiological review of pathology laboratory data was compiled. Both protists were found to be endemic although their occurrence did not appear to correlate with climatic factors such as rainfall, season or year, possibly indicating that other factors such as personal hygiene, potable water supply, sanitation and education probably have a more significant impact here rather than waterborne transmission. The results, however, may not be representative of the entire population as detection techniques are not standardised and data were only collected from laboratories willing to supply in the Durban and Pietermaritzburg areas which represent under 10% of laboratories in KwaZulu-Natal that test for Cryptosporidium and Giardia. These laboratories, however, perform most of the testing from throughout KwaZulu-Natal and are situated in the metropolitan areas. In addition, poor recording of patient details made conciling of data difficult. Evaluation of a calcium carbonate flocculation, membrane filtration and membrane dissolution technique for concentrating Cryptosporidium oocysts and Giardia cysts from water is necessary to accurately quantify (oo)cysts in water. Methods currently used result in varying recoveries and the best method had to be identified and enhanced for this study. Greater (oo)cyst losses occurred as turbidity increased irrespective of the method used. The calcium carbonate flocculation method proved to have the best recovery for all water types and is recommended for use in the regular routine monitoring of smaller volumes of water. Pre-filtration prior to flocculation had the potential to make microscopy easier although losses of (oo)cysts still occurred. Sucrose flotation following flocculation reduced recovery whilst pH adjustment to 6 ± 0.5 following flocculation improved recovery and is recommended with turbid water samples. A cheap and simple detection method using the slide immunoenzymatic assay (SIA) which is based on the principles of enzyme linked immunosorbent assay (ELISA), was adapted to detect Cryptosporidium and Giardia in potable and turbid water concentrates. The results were reproducible and sensitivity was improved using a spectrophotometer. A multi-phase SIA system, using reagents in liquid and dry ready-to-use forms, was investigated and the potential exists for its further development. Unfortunately this method does not indicate the viability status of (oo)cysts therefore a novel method to do so was developed using fluorescein diacetate (FDA) and tetramethyl red (TMR) labelled anti- Giardia monoclonal antibodies. This combination stained viable cysts green internally with a red wall while non-viable cysts stained red only. While the use of FDA overestimates viability, any error would err in favour of safety, and could be complemented with fluorescein isothiocyanate / propidium iodide for confirmation of viability status. As Cryptosporidium and Giardia were found to be present in the KwaZulu-Natal population, monitoring of water bodies, water supplies, wastewaters and sludges, using the enhanced flocculation procedure and immunofluorescence assay, was undertaken to determine their source. Oocysts and cysts were detected in dam, river and raw waters, treated effluent and sludge samples. No oocysts or cysts were detected in the treated water samples although this may be due to the inability of the method used to detect low numbers of (oo)cysts. This research confirmed the occurrence of Cryptosporidium and Giardia in the KwaZulu- Natal population and water matrices. The optimum concentration method for use with water samples was established and further enhanced for use with turbid waters while a simpler and cheaper means of detecting (oo)cysts and a novel viability-detection stain for cysts were developed.
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.
Utilization of Bacillus spp. as plant probiotics.
(2000) Yobo, Kwasi Sackey.; Laing, Mark Delmege.; Wallis, Frederick Michael.
Numerous microorganisms produce beneficial effects on plant developmentwhen applied to crop seeds or incorporated into soil. Research efforts worldwide over the past two decades have renewed commercial interest in plant growth promoting rhizobacteria (PGPR).With successes being recorded in PGPR research, it is expected that within the next few years, more commercial PGPR products will be available on the market. In particular, commercial PGPR could be advantageous to plant nurseries if they enabled earlier sale of plants, more rapid turnover of seedlings and further crop production cycles. Trials were carried out to evaluate the growth stimulation and biological control abilities of Biostart™, a Bacillus-based plant probiotic comprising seven Bacillus spp. and Biostart® 2000 (a combination of three of the Bacillus spp.). The first priority was to determine the survival pattern of six Biostart™ Bacillus spp., namely B. chitinosporus, B. uniflagellatus, B. laterosporus, B. pumilus, B. subtilis and B. licheniformis in potting soil in the presence or absence of a crop plant, i.e., cucumber, with or without NutriStart-AC. Bacterial numbers in pots in the absence of cucumber seedlings, with or without NutriStart-AC, declined slowly but steadily. Population sizes in pots without NutriStart-AC decreased steadily from Day 1 to Day 14 for all six Bacillus spp. and thereafter remained constant between 6.19 and 6.15 log cfu g -1 of wet soil for all six Bacillus spp. up to termination of the experiment on Day 35. A similar effect was observed in pots supplemented with one gram of NutriStart-AC. In the presence of cucumber seedlings, population sizes in pots without NutriStart-AC supplement declined faster until Day 14 than those in the NutriStart-AC supplemented pots. Populations remained stable after Day 14 for all six Bacillus spp. in the NutriStart-AC unsupplemented pots, while there was a variation in population sizes among Bacillus spp. in pots supplemented with NutriStart-AC. Growth stimulation trials in tunnels were carried out using four crops, i.e., lettuce, tomato, sorghum and beans. Seed treatment and seed treatment plus drenching with or without NutriStart-AC were evaluated. All Bacillus spp. used stimulated plant growth. Growth stimulation was more pronounced with a 4% NutriStart-AC supplement. Growth stimulation was best in lettuce, with Biostart® 2000. There was an increase of 466% compared to the dry biomass of the water control lettuce seedlings. The lowest responses were recorded in sorghum and beans. Three tomato cultivars, i.e., Roma, Floradade and Rodade and a pepper cultivar Thai were evaluated for growth stimulation by applying Biostart™ as seed treatment and seedling drench. The highest growth stimulation, 96%, was obtained using B. licheniformis on Roma as a seedling drench. Growth response was better in Roma and Floradade cultivars than in the Rodade cultivar. Pepper plants drenched with Biostart™ Bacillus spp., and supplemented weekly with a 4% NutriStart-AC suspension, showed increased fruit yield. Using B. subtilis, a 533% increase in fruit yield was recorded when seedlings were supplemented weekly with a 4% NutriStart-AC suspension. Similar results were recorded using an unidentified Bacillus strain CM-33 (433%) and B. licheniformis (333%). In a nematode control trial, no galls were found on the roots of treated and untreated control seedlings inoculated with Meloidogyne spp. Early inoculation of seedlings might have failed because there were no roots for the nematodes to attack at the time of inoculation. In a biological control trial, Biostart™ Bacillus spp. were applied by seed treatment and seedling drench to control Rhizoctonia causing damping-off of marigold, cabbage and eucalyptus seedlings. Biostart™ was ineffective under the conditions of this trial. Most seedlings died seven days after pathogen inoculation and by Day 21 about 90% of the seedlings were dead. The results presented in this thesis have some practical applications to seedling growers in South Africa, especially in growth promotion. Applying Biostart™ probiotic Bacillus spp. may increase the turnover of seedlings in nurseries. More trials are needed if the growth promotion and biological control potentials of Biostart™ probiotic Bacillus spp. are to be fully exploited.
Aspects of management of poplar rust in South Africa.
(2001) Hawke, Georgina Frances.; Laing, Mark Delmege.
An assessment of infection on poplar clones grown at the Lion Match Company Redclyff Nursery in Seven Oaks, KwaZulu-Natal was made to ascertain the nature of infection of the three common species of Melampsora infecting poplars in South Africa. These three species are M. larici-populina, M. medusae and the hybrid, M. medusa-populina. Their contrasting wall echinulations were used to differentiate these species using a scanning electron microscope. A visual rating scale measuring percentage leaf area infected (LAI) was used to determine disease severity. Rust development was slow in new material obtained from New Zealand, indicating rust resistance. This new material was not infected with M. larici-populina. Plant material from Europe showed severe susceptibility to M. larici-populina. Melampsora medusae-populina was the most prevalent species found at Lion Match Company plantations, Seven Oaks, KwaZulu-Natal, South Africa in the survey conducted from January to April, 1998. A once-off survey of rust infected poplar leaves from ten different locations (over 1 500 km apart) was conducted to ascertain the effect of geographical and meteorological conditions on the presence and severity of rust on poplars. The most popularly grown clones in South Africa are Clone 65/29, Clone 65/31, Clone 1488, Clone 129 and the old clone Populus deltoides var. missouriensis. Clone 65/31 had the greatest severity of disease (10.4%) closely followed by Clone 1488 (9.5%). Clone 129 had the least amount of disease (1.8%). The most common rust species occurring in South Africa was M. larici-populina. The hybrid, M. medusae-populina, was the least prevalent, although race studies found this species to be the most virulent. Four trials were conducted to determine the potential of fungicides to control rust infections on poplars. Sixteen fungicides were tested. Naturally infected poplar trees of the clone 65/31, grown in pots, were used as test material. The first trial had 16 fungicide treatments and an untreated control. Four treatments were significantly more effective than others: Alto (cyproconazole) (at 0.3 ml/L) with and without the adjuvant Armoblen 600 (at 0.75 ml/L), Anvil (hexaconazole) (at 0.2 ml/L) and Early Impact (flutriafol + carbendazim) (at 0.6 ml/L), respectively. Oxycarboxin appeared to have enhanced disease progression. Two experimental strobilurins, Stroby WG and Quadris FL (kresoxim-methyl, BASF and azoxystrobin, Zeneca) (at 0.12 ml/L, and O.4 ml/L, respectively) and a new class of fungicide, Astor WG40 (experimental, Novartis) (at 2g/L) controlled rust poorly. Four treatments were used in the second trial: Quadris as a foliar spray (O.4 ml/L), Impact applied on superphosphate granules «1 ml + 5g)/tree), and two controls; superphosphate alone (5g/tree) and untreated. The key finding of the second trial was that Impact gave complete control as a granular application over a 56 day period. Superphosphate alone enhanced rust development slightly. A third trial was conducted which corroborated results obtained in the first two trials: Alto plus Armoblen 600 was the best treatment, Early Impact the next best, then Alto, Anvil, superphosphate coated with Impact, Impact alone, the untreated control, Duett and lastly, superphosphate alone. The superphosphate treatment again slightly increased the disease percentages. A fourth trial was conducted with different rates of Alto (0.1, 0.2, 0.3, 0.5, 0.7 ml/L, and an untreated control), applied with the standard rate of Armoblen 600. All rates of Alto gave control of the disease, the highest rate being the most effective. A 3 x 3 x 3 factorial design was used to determine the effect of nitrogen (N), phosphorus (P) and potassium (K) on the growth of poplar trees and development of rust infection. Nitrogen (limestone ammonium nitrate (LAN) at 28% N) was applied at 0, 15.5 and 31 kg/ha, K (KCI at 50% K) was applied at 0, 16.7 and 33.3 kg/ha and P (single superphosphate at 10% P) at 0, 5.3 and 10.6 kg/ha. Over one year the single best tree grew 4.1 m, having received a treatment of 31 kg N/ha, 10.6 kg P/ha and 16.7 kg K/ha. This same treatment gave the best mean growth of 3.1 m. The poorest treatment was 15.5 kg N/ha, 5.3 kg P/ha and 33.3 kg K/ha, with a mean of 1.7m in growth. The treatment of 15.5 kg N/ha, O kg P/ha and 33.3 kg K/ha resulted in the lowest disease level with a mean of 23.5% leaf area infected (LAI). Treatment with 31 kg N/ha, O kg P/ha and 33.3 kg K/ha resulted in the highest disease level with 39.2% LAI. The results suggested that higher N applications increased disease susceptibility, although this trend was not significant.
Studies on the application of biocontrol agents for the control of seedling diseases.
(2002) Omarjee, Jeh-han.; Laing, Mark Delmege.
The controlled environment of greenhouses, the high value of crops, and the limited number of registered fungicides offer a unique niche for the biological control of plant diseases. During the past ten years, over 80 biocontrol products have been marketed worldwide. A large percentage of these that have been developed in greenhouses could predominate over chemical pesticides in the same way that biological control of greenhouse insects predominated in the United Kingdom. A review of the literature was undertaken to obtain information on biocontrol agents with specific reference to Trichoderma and Gliocladium spp. Literature on the application, types of formulations, limitations in formulation, registration and commercialization of these biocontrol agents were obtained. Trichoderma harzianum Strain KMD has been used successfully as a biological control agent against several soil-borne plant pathogens. Biological control agents should possess several desirable characteristics, including, ease of preparation and application, stability during transport and storage, abundant production of viable propagules and good shelf-life. A strain of Trichoderma harzianum KMD with potential biocontrol activity was used to determine the effect of culture conditions on spore shelf-life. The influence of four growing media were investigated on the spore ultrastructure and shelf-life, using a basal salts medium with C:N ratios of 3 and 14, and pH's of 4.0 and 7.0. Mycelial development and sporulation were positively affected by acidic conditions (pH 4.0). The effect of these culture parameters on viability and shelf-life were evaluated by counting colony forming units (c.f.u) before and after seven days of storage at 75% relative humidity (rH): The effect of carbon concentration on spore viability after seven days of storage was also determined by increasing concentrations of glucose while a constant C:N ratio of 3 or 14 at pH 4.0 was maintained at a 75% rH. Increasing carbon concentration and C:N ratios increased spore production times. Spore viability was greatest when harvested from a medium with a C:N of 14 at pH 4.0 even when storage time was increased to 45 days and rH was reduced to 12%. Ultrastructural studies showed that spores had two cell wall layers, with the outer being more electron-dense than the inner layer. This layer is the spore's first defense against adverse conditions. Spores obtained from this medium were larger, germinated better and had a longer shelf-life than spores from C:N 3 medium, possibly because the two cell wall layers acted as a thicker barrier against adverse conditions. Increasing carbon concentration, while maintaining a constant C:N ratio of 3 or 14 at pH 4.0 slowed down spore production. Viability of spores were similar when introduced on media with variable carbon concentrations but fixed C:N ratios. The ultrastructural differences and shelf-life studies, confirmed empirical results from liquid fermentation studies, that the pH and C:N ratio of the medium upon which spores of T harzianum KMD strain KMD were produced have critical effects on physical and chemical structure of the spores and viability. This, in turn, affects critical parameters for biocontrol agents spore germination and shelf-life. Ultrastructural studies of mycoparasitism of T harzianum KMD on a soil-borne pathogen, Rhizoctonia solani were investigated. The modes of antagonistic action by Trichoderma in biological control have not been fully elucidated. However several mechanisms have been described, such as mycoparasitism, antibiotics, production of inhibitors, which have been identified and shown to suppress soil-borne pathogens. Mycoparasitic activities of T harzianum KMD against R. solani were studied using in vitro bioassays and Scanning electron microscopy (SEM). The fungal growth in dual cultures revealed that T harzianum KMD made hyphal contact with the pathogen within four days of inoculation, leading to an inhibition of pathogen growth. SEM observations showed that T harzianum KMD bound firmly to R. solani hyphae by coiling around the hyphae. Penetration of the pathogens hyphae occurred by the formation of hooks, haustoria and appressoria-like structures by T harzianum KMD, followed by cell disruption. The pathogen's hyphae disintegrated and collapsed upon contact with T harzianum KMD. It is hypothesized that the outcome of the interaction of antagonist and pathogen was most likely determined by initial hyphal contact that triggered a series of events in pathogen destruction. An experimental trial was undertaken to evaluate various formulations if T harzianum KMD and Gliocladium virens Strain MM1 for growth stimulation and biocontrol of R. solani and Pythium sp. on a variety of crops under greenhouse conditions using three application techniques at various dosages. Preparations of isolates of biocontrol agents T harzianum KMD, G. virens MM1 and Bacillus subtilis Strain AW57 were evaluated for their efficacy in enhancing growth and preventing damping-off caused by Pythium sp. and R. solani on a variety of crops namely cabbage, cucumber, Namaqualand daisy and Eucalyptus. Percentage survival and plot weights were measured after 3-4 weeks of growth. The preparations that were used included chlamydospores of biocontrol fungi in milled oats, powders containing conidia in an experimental compound, an oil base, and a commercial product. Formulations of bacteria were prepared with and without Nutristart. The evaluation of three delivery methods were used namely, a seed coating using an adhesive, Pelgel®, capping (a preparation is capped on the surface and incorporated into planting media) and as a drench (preparation drenched on seed at planting). Various dosage levels 0.25, 0.5, 1, 5 and 10g/1 of each formulation was mixed with water and drenched on seed at planting. Growth promotion of seedlings varied for the different formulations of different biocontrol organism. Overall, plot weight was significantly increased on all crops tested. Plant growth of seedlings was consistently increased by all conidial formulations of T. harzianum KMD and G. virens MMI. The best application technique that effectively delivered the biocontrol agents to the target was seed treatment followed by drenching and capping. Most formulations significantly increased plot weight on all seedlings ranging from 2000-5000% when compared to controls and percentage survival was comparable to the controls. In most instances it was recorded that all biocontrol organisms effectively enhanced growth of seedlings equally well irrespective of other main effects. Most formulations of the different biocontrol organisms significantly reduced damping-off caused by Pythium sp. on eucalyptus and Namaqualand daisy. Formulations of T. harzianum KMD prepared with chlamydospores in milled oats and prepared with conidia effectively reduced damping-off on eucalyptus and Namaqualand daisy by 8-31% when compared to the controls. It was observed that biocontrol organisms T. harzianum KMD and G. virens MMI effectively reduced damping-off better than B. subtilis AW57. To effectively reduce damping-off caused by Pythium sp. seed treatment was the best application technique to deliver the biocontrol agent to the target. Biocontrol of damping-off caused by R. solani was achieved on all crops by all formulations of T. harzianum KMD, G. virens MMl and B. subtilis AW57. Disease was reduced by 1000 fold with the application of biocontrol organisms when compared to disease controls. Conidial formulations performed better in reducing disease than formulations prepared with chlamydospores applied as a drench or a seed treatment. In most instances the best dosage to apply formulations were doses that ranged from l-5g/1 for both growth stimulation and biocontrol of soil-borne pathogens. Severe stunting of seedlings occurred at high dosages of 109/I. The compatibility of the biocontrol agent T harzianum KMD with selected fungicides were determined on a variety of crops under greenhouse conditions. A commercial formulation of T harzianum KMD was used for this investigation. An in vitro assay was used to determine the sensitivity of T harzianum KMD to a range of rates of two fungicides, Benlate® and Previcur®. Trichoderma harzianum KMD was found least sensitive to both fungicides after 15 days of incubation at 25°C. The compatible mutants resulted in a lack of sporulation even when induced with UV light. Greenhouse trials were then carried out on cabbage, cucumber, Namaqualand daisy, eucalyptus and tomato. It was confirmed that T harzianum KMD achieved better growth and biocontrol activity against R. solani and Pythium sp. when applied without fungicides to infested and non-infested composted pine bark. Trichoderma harzianum KMD was only compatible to fungicides when applied as a seed treatment prior to planting. As a disease integrated management programme, seed treatment application of T harzianum KMD may be compatible with fungicides for control of damping-off of seedling diseases caused by R. solani and Pythium sp. The effect of environmental stress (oxidative injury, cold and drought) on the growth enhancement of a variety of greenhouse crops by a commercial formulation of T harzianum KMD was evaluated. In an absence of a disease colonization by T harzianum KMD on maize and cucumber roots in rhizotron studies increased root area by 3104 mm(2) and 1787, 48 mm(2) respectively. Oxidative stress was carried out by applying 0.05% NaOCl, to cabbage, cucumber and tomato seeds. This stress did not reduce vigor of seedlings and hence the effect of subsequent treatment with T harzianum KMD on stressed seeds was not determined. Treatments of imbibed but unemerged seeds of cucumber, tomato and white grain maize in cold temperatures (5-100C night/day) for varying periods reduced subsequent growth. Seeds treated with cold stress and T harzianum KMD did not display any growth enhancement. On cabbage, cucumber, tomato and white grain maize seeds sown in various media, which induced various levels of drought and water logging conditions, were not enhanced when seeds were coated with T harzianum KMD. Overall, T. harzianum KMD did not enhance growth under stressed conditions of oxidative injury, cold and drought. The results presented in this thesis shows that T harzianum KMD has potential against soilborne pathogens namely Pythium sp. and R. solani under greenhouse conditions. Applying conidial formulations of T harzianum KMD using seed treatment and applying it at the correct dosage may increase the turnover of seedling production in nurseries. Trichoderma harzianum KMD can replace toxic fungicides and. fumigants under greenhouse conditions. More trials and research are needed on a wider variety of crops and diseases if growth promotion and biological control of T harzianum KMD are to be fully exploited.
Evaluation of growth media parameters for the cultivation of selected biological control agents.
(2002) Phillips, Shivaani.; Laing, Mark Delmege.; Hunter, Charles Haig.
Trichoderma harzianum kmd, Gliocladium virens MMI and Colletotrichum gloeosporioides C6 are potential biological control agents. Trichoderma harzianum kmd and and G. virens MMI have been shown to have excellent growth stimulation and disease suppressive characteristics by the Biocontrol for Africa team of researchers at the University of Natal, Pietermaritzburg. Colletotrichum gloeosporioides C6 has been shown to have effective control of the invasive weed, Hakea sericea (Shrad.). The aim of this dissertation was to establish a method which was most effective for the mass production of the biological control agents (BCAs). Various parameters and the impact of carbon-to-nitrogen and total organic carbon (TOC) on the growth of the BCAs were investigated. Fingerprinting and detection of mutations between strains of Colletotrichum gloeosporioides C6 of different ages were attempted using AFLPs for patenting purposes. Pine shavings and molasses were used in the semi-solid fermentation of T harzianum kmd, G. virens MMI and C. gloeosporioides C6. A 70% (v/v) ethanol soak was the most effective pretreatment in the removal of resin off the pine shavings as well as eradication of contamination. Parameters tested were pH, C:N ratios and TOCs. The optimal pH range for T harzianum kmd and C. gloeosporioides C6 was between pH 6.5 and 7. The optimal pH for G. virens MMI was pH6. Various C:N ratios and TOCs produced highly significant differences in spore yield and mycelial biomass (P
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.
Seed-borne pathogens and the bean weevil (Acanthoscelides obtectus) in bean (Phaseolus vulgaris) seed and their effect on seed germination and vigour.
(2003) Siyoum, Nazareth A.; Laing, Mark Delmege.; Modi, Albert Thembinkosi.
Two semi-selective media, MT and MSP, were compared for their ability to recover cells of the bacteria Xanthomonas axonopodis pv. phaseoli (Xap) and Pseudomonas syringae pv. phaseolicola (Psp), the causal organisms of bean common and halo blights respectively. Isolates Sx70 and Sp75 (for Xap and Psp respectively) were plated on the media by dilution plating. Greater number of Xap colonies on MT than on MSP was obtained, but the number of Psp colonies was not significantly different on the two media. Four media, XCP1, MT, MT(new), and TSM were also compared for the number of contaminants and Xap colonies grown on them, using data obtained from ISTA/ISHI Comparative test for Xap (2002), which was conducted at the Agricultural Research Council (ARC) in Roodeplaat, north of Pretoria. In most cases, the number of colonies was similar in XCPI, MT, MT(new), but bigger in TSM. The number of contaminants was smaller in MT than in MT(new). Bean plants (Phaseolus vulgaris), variety PAN 146, were grown and inoculated with the bacteria Xap and Psp, and two virus isolates of bean common mosaic virus (BCMV). Uninoculated controls were also grown. Germination and vigour tests were conducted on the seeds harvested from these plants. Germination was significantly lower for seeds harvested from Psp inoculated plants, while seed vigour was reduced both for seeds harvested from Psp and Xap inoculated plants. Neither germination nor vigour was affected for seeds from BCMV inoculated plants. Late and early matured seeds from the BCMV inoculated plants were compared for the virus transmission and emergence. There was greater virus transmission and reduced emergence in the late matured seeds than in the early matured ones. Seeds harvested from Psp and BCMV inoculated plants and uninoculated controls were unexpectedly found to be infested with the bean weevil (Acanthoscelides obtectus (Say)). The BCMV infected batch of seeds appeared to repel weevil attack, whereas 25% of the uninfected batch of seeds were attacked.
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.
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.
Biocontrol of three fusarial diseases.
(2004) Kidane, Eyob Gebrezgiabher.; Laing, Mark Delmege.
Over the past one hundred years, research has repeatedly demonstrated that phylogenetically diverse microorganisms can act as natural antagonists of various plant pathogens. Interest in biological control research continues reflecting the desire of multiple constituencies to develop sustainable methods for controlling plant diseases. The review of the literature comprises information on the epidemiology, economic importance and the different control options available against Fusarial diseases of cabbage, maize and pine, and the safety of microorganisms intended for use as biocontrol agents, their management and strategy of control. Trichoderma and Bacillus isolates used as biocontrol agents were obtained mainly from the rhizosphere of cabbage, maize and pine with a view that they would be adapted to those habitats where they would eventually to be used as innundative biocontrol agents. Preliminary selection was made based on in vitro antagonism of those isolates towards Fusarium oxysporum f. sp. conglutinans (Wollenweb.) W.C. Snyder & H.N. Hans. Ultrastructural studies of mycoparsitism of Trichoderma Isolates ET23, ET13 and Trichoderma harzianum Eco-T® which caused significant reduction in disease incidence and severity on later study under greenhouse conditions, were investigated on the vascular pathogen, F. oxysporum f. sp. conglutinans. Although the mode of action of the three isolates wall not fully elucidated, certain mechanisms such as mycoparasitism and antibiosis or production of antimicrobial substances, which cause cell wall degradation and lysis, have been identified. Twenty Trichoderma and 18 Bacillus isolates which showed antagonism towards F. oxysporum f. sp. conglutinans were tested against the same pathogen on cabbage under greenhouse conditions. Trichoderma isolates were delivered to the soil in two different ways, i.e., seed treatment and drenching, while Bacillus isolates were only drenched as spore suspensions. More than two-third of the biocontrol isolates caused significant reductions in disease incidence and severity of the vascular wilt disease. Application of Trichoderma isolates by drenching resulted in better control of the disease than when applied as a seed treatment. Of the 38 Bacillus and Trichoderma isolates tested against the cabbage yellows fungus, three Trichoderma and four Bacillus isolates were selected for further testing against Fusarium sp. and Rhizoctonia solani Kuhn on maize and Fusarium circinatum on Pinus patula seedlings. Since none of the Fusarium isolates obtained from diseased kernels and cobs of maize were pathogenic to the two maize cultivars, yellow maize and PAN 6479, provided by Pannar® seed company, biocontrol experiments on Fusarium diseases of maize could not be conducted. Only Trichoderma Isolate ET23 and T. harzianum Eco-T® were found to significantly control Rhizoctonia preemergency damping-off on maize while none of the Bacillus isolates caused significant increase in seedling emergence. In the test against F. circinatum on pine, in most cases, significant reduction in seedling mortality was observed in the first 4 to 8wk, however, after 12wk they were no longer effective. Improvement in the survival of pine seedlings were observed when T. harzianum Eco-T® was applied prior to the introduction of F. circinatum. There was almost a direct relationship between the inoculation time and percentage of survival of seedlings. Prior inoculation gives the biocontrol agent time to colonize the potential infection courts for the pathogen in the root area and to be established in the rhizosphere of the pine seedlings. It has been reported that the inconsistent and poor performance of biocontrol agents in the field can be improved with the use of mixtures of biocontrol agents to mimic the naturally suppressive soils which comprise numerous saprophytic microorganisms. However, these organisms have co-evolved for many years that they are adapted to live together in the same soil ecosystem. Therefore, when combinations of biocontrol organisms are used, the compatibility between these isolates is important. Compatibility tests between and among Bacillus and Trichoderma isolates were carried out in vitro. The tests revealed that the Bacillus and Trichoderma isolates are not all compatible. Trichoderma Isolate ET13 showed antagonism towards Isolates ET23 and T. harzianum Eco-T®; Bacillus Isolates B81 and BF011 were slightly antagonistic to Isolates EXR and JR01, and Isolate JR01 was slightly antagonistic to Isolate EXR. Comparisons of single versus mixtures of Bacillus or Trichoderma isolates showed that mixtures of Bacillus or Trichoderma isolates did not result in significantly greater reduction in disease incidence and severity of cabbage yellows.
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.
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.

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