No description available.

No description available.

The production of azadirachtin and other related limonoids were investigated using plant cell culture systems of Neem (Azadirachta indica). The production of antifeedant compounds in callus lines was monitored by no-choice bioassays with Schistocerca gregaria with instar nymphs. Growth of two callus lines produced from leaves of a Ghanaian neem tree were monitored and the growth patterns displayed the typical lag, stationary and exponential phases. Growth and product formation, as measured by antifeedant bioassays with S.gregaria, showed non-growth associated product formation. Azadirachtin was isolated from callus derived from a Ghanaian neem tree by Prof. E.D. Morgan, University Keele using the standard procedure of solvent partitioning and column chromatography. Biological activity of the partitioning fractions was measured with antifeedant tests. Azadirachtin was identified by chromatography on three independent systems (Supercritical Fluid Chromatography (SFC), High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography). The yield of azadirachtin was 0.0007% based on dry weight of callus. Neem leaf explants collected from wild neem trees could be successfully surface sterilised by treating with 0.1% HgCl2 for 5 min followed by 10% Bleech (Care Products, Sri Lanka) or NaOCl (Sigma, UK) for 10 min. New callus lines were initiated from leaves collected from wild neem trees by incubating on Maintenance Medium (Kearney et al., 1994) at 25°C in the dark. Fifteen callus lines thus initiated were extracted with ethanol under reflux, quantified and screened for the production of azadirachtin, nimbin and salannin by reversed phase HPLC. There was no correlation between azadirachtin, nimbin and salannin yields of callus lines and those of seeds collected from same neem trees. The genetic variation of callus lines was examined by iso-enzyme electrophoresis using three enzymes, malate dehydrogenase, alcohol dehydrogenase and diaphorase and no variation was observed among the callus lines examined.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is an economically important pathogen on canola (Brassica nanups L.). Due to the increasing public concerns of fungicide usage and the limitations of cultural methods, biological control is emerging as a promising alternative to control S. sclerotiorum in an environmentally-friendly way. Several bacterial strains, mainly Bacillus spp., were isolated from canola and wheat plants. Eight Gram-positive bacterial strains were inhibitory against mycelial growth of S. sclerotiorum in intro. Three Bacillus spp., one Staphylococcus spp. and one Pseudomonas spp. (control) were tested in whole plant assays, and all of them achieved significant (P<0.05) disease suppression. An experiment in the greenhouse to investigate the optimal time of application showed that the highest disease suppression was achieved when B. amyloliquefaciens BS6 was inoculated at the same time as the pathogen. The field experiment (Trial two) in 2003 showed a significant (P<0.05) reduction of disease incidence and severity in bacteria-pre-treated plots, compared to control plots, even though the survival of bacterial populations of Pseudomonas chlororaphis PA-23 and B. amyloliquefaciens BS6 were low on the petals’ surface. The bacterial strains were effective against S. sclerotiorum under laboratory, greenhouse, and field conditions, and these results also suggest that antibiosis and plant induced resistance might be involved in the disease suppression. To identify antibiotic-related genes in these potential biocontrol bacterial agents, polymerase chain reaction (PCR) with specific primers was used. The sequencing of PCR products and BLAST search in the gene bank showed that P. chlororaphis PA-23 contains biosynthetic genes for phenazine-1-carboxylic acid, pyrrolnitrin and probably 2,4-diacetylphloroglucinol, and that B. thuringiensis/cereus BS8, B. cereus L and B. mycoides S contain zwittermicin A self-resistant gene. The significance of the presence of these genes is discussed. Induced resistance mediated by S. sclerotiorum and B. amyloliquefaciens BS6 was evaluated in a greenhouse study followed by analysis of the phenolic compounds from canola leaf extracts by high performance liquid chromatography (HPLC). Pre-treatment with bacteria significantly (P<0.05) reduced the disease symptoms on canola plants. The HPLC results indicated that the disease suppression from the bacterial pre-treatment was related to the induction of secondary metabolites in canola leaves. The HPLC results also showed that the inoculum of S. sclerotiorum was also associated with the induction of these compounds in canola leaves. The knowledge accumulated during this study has shown that the bacterial biocontrol agents tested have a great potential in controlling sclerotinia stem rot of canola. The understanding of the biocontrol mechanisms involved in the disease suppression would help optimize their biocontrol efficiency. Therefore, further studies on the role of bacterial antibiotics in disease suppression, and better understanding of plant induced resistance mediated by pathogen and bacterial agents are needed. / October 2004

Fusarium head blight (FHB) is an important disease of wheat and other small grain cereals. The principal pathogen associated with FHB in Manitoba is Gibberella zeae (Schwein.) Petch (anamorph = Fusarium graminearum Schwabe). Currently there no biological control agents registered for the control of FHB. The overall objects of the project were to identify isolates that may be used in the biological control of FHB and to investigate the interaction between the identified biocontrol agents and G. zeae.In this study, 150 bacteria and 29 fungi were isolated from soil, wheat heads and crop debris from southern Manitoba. An additional 10 isolates of Trichoderma harzianum were obtained from the Canadian Collection of Fungal Cultures, CCFC (Ottawa, Ontario). The T. harzianum isolate, T-22 (RootShield) was included as a positive control. All were screened for inhibition of Gibberella zeae using confrontation plate assays in vitro and seed, wheat head, and straw assays in planta. Only 6% of bacterial and 45% of fungal isolates tested in the confrontation plate assays. Of the 6% of the bacterial isolates none significantly reduced FHB disease on wheat heads or reduced perithecial production on wheat straw, and had a negative effect on seed germination, therefore were not further evaluated. Chrysosporium sp. and Penicillium spp. and Trichoderma harzianum, were the fungal species that inhibited the growth of G. zeae by more than 50%. Of which Trichoderma isolates were the most effective and were able to over-grow G. zeae. Fourteen of the 18 isolates tested, including six Trichoderma and two Chrysosporium isolates, significantly reduced perithecial production on wheat straw by 52-89% compared to the control. From this study, Trichoderma harzianum was identified as most potentially effective candidate for the biocontrol of Gibberella zeae. Spore suspensions and cell-free filtrates of Trichoderma harzianum isolates were evaluated for their effectiveness in reducing perithecial and ascospore production of Gibberella zeae on wheat straw. Five T. harzianum isolates, including T-22 (RootShieldTM), reduced perithecial formation by 70% or more. Perithecial reduction was highest (96-99%) when T. harzianum spore suspension or cell-free filtrate was applied to straw 24 hours prior to inoculation with G. zeae. Control was less effective when T. harzianum was applied at the same time (co-inoculated) or 24 hours after G. zeae. Field trials showed significant reduction of perithecia on residues treated with T. harzianum prior to placement on the soil surface. Identification of those compounds in the cell-free filtrate most likely to affect biocontrol was accomplished through the use of cluster analysis, ordination and regression methods. It was found that isolates that produce similar levels of biocontrol had similar chemical composition. Ultrastructural changes were observed primarily in the exterior cells of the outer cell wall. Cytoplasmic degradation, invagination of the plasma cell membrane and thin cell walls were observed in the treated samples. Immature perithecia were overgrown by T. harzianum 15 days after co-inoculation. Few perithecia were overgrown at later stages. The perithecia affected by T. harzianum collapsed 21 days after inoculation (dai), compared to the perithecia in the untreated samples which collapsed 28 dai.

No description available.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is an economically important pathogen on canola (Brassica nanups L.). Due to the increasing public concerns of fungicide usage and the limitations of cultural methods, biological control is emerging as a promising alternative to control S. sclerotiorum in an environmentally-friendly way. Several bacterial strains, mainly Bacillus spp., were isolated from canola and wheat plants. Eight Gram-positive bacterial strains were inhibitory against mycelial growth of S. sclerotiorum in intro. Three Bacillus spp., one Staphylococcus spp. and one Pseudomonas spp. (control) were tested in whole plant assays, and all of them achieved significant (P<0.05) disease suppression. An experiment in the greenhouse to investigate the optimal time of application showed that the highest disease suppression was achieved when B. amyloliquefaciens BS6 was inoculated at the same time as the pathogen. The field experiment (Trial two) in 2003 showed a significant (P<0.05) reduction of disease incidence and severity in bacteria-pre-treated plots, compared to control plots, even though the survival of bacterial populations of Pseudomonas chlororaphis PA-23 and B. amyloliquefaciens BS6 were low on the petals’ surface. The bacterial strains were effective against S. sclerotiorum under laboratory, greenhouse, and field conditions, and these results also suggest that antibiosis and plant induced resistance might be involved in the disease suppression. To identify antibiotic-related genes in these potential biocontrol bacterial agents, polymerase chain reaction (PCR) with specific primers was used. The sequencing of PCR products and BLAST search in the gene bank showed that P. chlororaphis PA-23 contains biosynthetic genes for phenazine-1-carboxylic acid, pyrrolnitrin and probably 2,4-diacetylphloroglucinol, and that B. thuringiensis/cereus BS8, B. cereus L and B. mycoides S contain zwittermicin A self-resistant gene. The significance of the presence of these genes is discussed. Induced resistance mediated by S. sclerotiorum and B. amyloliquefaciens BS6 was evaluated in a greenhouse study followed by analysis of the phenolic compounds from canola leaf extracts by high performance liquid chromatography (HPLC). Pre-treatment with bacteria significantly (P<0.05) reduced the disease symptoms on canola plants. The HPLC results indicated that the disease suppression from the bacterial pre-treatment was related to the induction of secondary metabolites in canola leaves. The HPLC results also showed that the inoculum of S. sclerotiorum was also associated with the induction of these compounds in canola leaves. The knowledge accumulated during this study has shown that the bacterial biocontrol agents tested have a great potential in controlling sclerotinia stem rot of canola. The understanding of the biocontrol mechanisms involved in the disease suppression would help optimize their biocontrol efficiency. Therefore, further studies on the role of bacterial antibiotics in disease suppression, and better understanding of plant induced resistance mediated by pathogen and bacterial agents are needed.

Fusarium head blight (FHB) is an important disease of wheat and other small grain cereals. The principal pathogen associated with FHB in Manitoba is Gibberella zeae (Schwein.) Petch (anamorph = Fusarium graminearum Schwabe). Currently there no biological control agents registered for the control of FHB. The overall objects of the project were to identify isolates that may be used in the biological control of FHB and to investigate the interaction between the identified biocontrol agents and G. zeae.In this study, 150 bacteria and 29 fungi were isolated from soil, wheat heads and crop debris from southern Manitoba. An additional 10 isolates of Trichoderma harzianum were obtained from the Canadian Collection of Fungal Cultures, CCFC (Ottawa, Ontario). The T. harzianum isolate, T-22 (RootShield) was included as a positive control. All were screened for inhibition of Gibberella zeae using confrontation plate assays in vitro and seed, wheat head, and straw assays in planta. Only 6% of bacterial and 45% of fungal isolates tested in the confrontation plate assays. Of the 6% of the bacterial isolates none significantly reduced FHB disease on wheat heads or reduced perithecial production on wheat straw, and had a negative effect on seed germination, therefore were not further evaluated. Chrysosporium sp. and Penicillium spp. and Trichoderma harzianum, were the fungal species that inhibited the growth of G. zeae by more than 50%. Of which Trichoderma isolates were the most effective and were able to over-grow G. zeae. Fourteen of the 18 isolates tested, including six Trichoderma and two Chrysosporium isolates, significantly reduced perithecial production on wheat straw by 52-89% compared to the control. From this study, Trichoderma harzianum was identified as most potentially effective candidate for the biocontrol of Gibberella zeae. Spore suspensions and cell-free filtrates of Trichoderma harzianum isolates were evaluated for their effectiveness in reducing perithecial and ascospore production of Gibberella zeae on wheat straw. Five T. harzianum isolates, including T-22 (RootShieldTM), reduced perithecial formation by 70% or more. Perithecial reduction was highest (96-99%) when T. harzianum spore suspension or cell-free filtrate was applied to straw 24 hours prior to inoculation with G. zeae. Control was less effective when T. harzianum was applied at the same time (co-inoculated) or 24 hours after G. zeae. Field trials showed significant reduction of perithecia on residues treated with T. harzianum prior to placement on the soil surface. Identification of those compounds in the cell-free filtrate most likely to affect biocontrol was accomplished through the use of cluster analysis, ordination and regression methods. It was found that isolates that produce similar levels of biocontrol had similar chemical composition. Ultrastructural changes were observed primarily in the exterior cells of the outer cell wall. Cytoplasmic degradation, invagination of the plasma cell membrane and thin cell walls were observed in the treated samples. Immature perithecia were overgrown by T. harzianum 15 days after co-inoculation. Few perithecia were overgrown at later stages. The perithecia affected by T. harzianum collapsed 21 days after inoculation (dai), compared to the perithecia in the untreated samples which collapsed 28 dai.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is an economically important pathogen on canola (Brassica nanups L.). Due to the increasing public concerns of fungicide usage and the limitations of cultural methods, biological control is emerging as a promising alternative to control S. sclerotiorum in an environmentally-friendly way. Several bacterial strains, mainly Bacillus spp., were isolated from canola and wheat plants. Eight Gram-positive bacterial strains were inhibitory against mycelial growth of S. sclerotiorum in intro. Three Bacillus spp., one Staphylococcus spp. and one Pseudomonas spp. (control) were tested in whole plant assays, and all of them achieved significant (P<0.05) disease suppression. An experiment in the greenhouse to investigate the optimal time of application showed that the highest disease suppression was achieved when B. amyloliquefaciens BS6 was inoculated at the same time as the pathogen. The field experiment (Trial two) in 2003 showed a significant (P<0.05) reduction of disease incidence and severity in bacteria-pre-treated plots, compared to control plots, even though the survival of bacterial populations of Pseudomonas chlororaphis PA-23 and B. amyloliquefaciens BS6 were low on the petals’ surface. The bacterial strains were effective against S. sclerotiorum under laboratory, greenhouse, and field conditions, and these results also suggest that antibiosis and plant induced resistance might be involved in the disease suppression. To identify antibiotic-related genes in these potential biocontrol bacterial agents, polymerase chain reaction (PCR) with specific primers was used. The sequencing of PCR products and BLAST search in the gene bank showed that P. chlororaphis PA-23 contains biosynthetic genes for phenazine-1-carboxylic acid, pyrrolnitrin and probably 2,4-diacetylphloroglucinol, and that B. thuringiensis/cereus BS8, B. cereus L and B. mycoides S contain zwittermicin A self-resistant gene. The significance of the presence of these genes is discussed. Induced resistance mediated by S. sclerotiorum and B. amyloliquefaciens BS6 was evaluated in a greenhouse study followed by analysis of the phenolic compounds from canola leaf extracts by high performance liquid chromatography (HPLC). Pre-treatment with bacteria significantly (P<0.05) reduced the disease symptoms on canola plants. The HPLC results indicated that the disease suppression from the bacterial pre-treatment was related to the induction of secondary metabolites in canola leaves. The HPLC results also showed that the inoculum of S. sclerotiorum was also associated with the induction of these compounds in canola leaves. The knowledge accumulated during this study has shown that the bacterial biocontrol agents tested have a great potential in controlling sclerotinia stem rot of canola. The understanding of the biocontrol mechanisms involved in the disease suppression would help optimize their biocontrol efficiency. Therefore, further studies on the role of bacterial antibiotics in disease suppression, and better understanding of plant induced resistance mediated by pathogen and bacterial agents are needed.

Phytophthora cinnamomi Rands is a common and devastating pathogen of cultivated proteas worldwide. Webb (1997) described a Sudden Death plant disease of proteas in Western Australia (WA) protea plantations. Proteas that suffer the syndrome display symptoms such as stunted growth, wilting, chlorosis and often death. In the current study, a number of protea plantations in the southwest of WA were visited to quantify the extent that P. cinnamomi was attributing to deaths of cultivated proteas. The survey indicated that P. cinnamomi is the major cause of Sudden Death in proteas. A range of other fungi (Fusarium, Botryosphaeria, Pestalotiopsis, Alternaria) and pests (nematodes, mealy bug, scale insects) were also identified to be contributing to protea death and decline in WA plantations. In many cases the factors contributing to protea disease appeared complex, with a range of physical factors or nutritional imbalances commonly associated with these pathogens and pests. As P. cinnamomi was the major cause of death of cultivated proteas the remainder of the experiments described in this dissertation investigated its control in horticultural plantings. Biofumigation has the potential to become an important technique in an overall integrated management approach to P. cinnamomi. In this thesis, biofumigation refers to the suppression of pathogens and pests by the incorporation of Brassica plants into the soil. Two biofumigants (Brassica juncea (L.) Czern., B. napus L.) were screened for their effect on the in vitro growth of five common Phytophthora species (P. cinnamomi, P. cactorum (Lebert & Colin) Schroeter., P. citricola Sawada, P. cryptogea Pethyb. & Laff. and P. megasperma Drechsler). Growth was determined by the measuring dry weight and radial growth of vegetative hyphae. B. juncea was found to be superior in its suppressive effect compared to B. napus. There was also significant variation in the sensitivity of the Phytophthora species to the suppressive effects of the biofumigants. P. cinnamomi was the most sensitive of the five species investigated. Where the rates of the biofumigant were sufficient to suppress growth of Phytophthora, the suppressive effect was mostly fungicidal. To determine how B. juncea and B. napus affect the infective ability and survival of P. cinnamomi, their effects on sporangia and chlamydospores production in soil was investigated in vitro. P. cinnamomi colonised Miracloth discs were added to soil amended with the two Brassica species, before being removed every two days over an eight day period for the determination of sporangia production, chlamydospore production and infective ability. Only the soils amended with B. juncea significantly reduced sporangia production in P. cinnamomi. Both Brassica species increased the percentage of aborted or immature sporangia and reduced the infective ability of the pathogen. Neither Brassica species had any effect on zoospore release or chlamydospore production in P. cinnamomi. Soil cores and soil leachate were collected from biofumigant-amended field soils to determine the inoculum potential and infective ability of the pathogen under glasshouse conditions. Amending the soil with both Brassica species had an immediate suppressive effect on the inoculum potential and infective ability of the P. cinnamomi. However, after this initial suppression there was a gradual increase in the recovery of the pathogen over the monitoring period of four weeks. To determine if the suppression would result in decreased disease incidence in a susceptible host, Lupinus angustifolius L. seeds were planted in the biofumigant amended soil. B. juncea amended soils reduced the disease incidence of P. cinnamomi by 25%. B. napus had no effect on disease incidence in L. angustifolius. Although the current study had demonstrated that biofumigants could suppress the growth, sporulation and infection of P. cinnamomi, it was unclear if this would equate to a reduction in disease incidence when applied in the field. A field trial was conducted on a protea plantation in the southwest of Western Australia that compared biofumigation with B. juncea to chemical fumigation (metham sodium) and soil solarisation. The three soil treatments were used in an integrated management approach to control P. cinnamomi that included the use of a hardwood compost, mulch and water sterilisation. All treatments were monitored during their application to ensure the treatments were conducted successfully. The three soil treatments significantly reduced the recovery of the pathogen and the infective ability of the pathogen to a soil depth of 20 cm. Metham sodium was the most suppressive soil treatment and soil solarisation was the least suppressive treatment. Only the metham sodium treatment resulted in a significant reduction in the incidence of root rot in Leucadendron salignum P.J. Bergius x laureolum (Lam.) Fourc (c.v. Safari Sunset) over the monitoring period of three years. Another field trial was conducted on the same protea plantation to compare the effectiveness of B. juncea and B. napus, without the use of other control strategies, to reduce the incidence of P. cinnamomi infection of Leucadendron Safari Sunset. The concentration of isothiocyanates was monitored for seven days after the incorporation of the biofumigants. Although both Brassica species reduced the recovery and infective ability of the pathogen, neither biofumigant reduced the incidence of root rot in Leucadendron Safari Sunset. In conclusion, P. cinnamomi is the most common and devastating pathogen in WA protea plantations. The current study demonstrated that P. cinnamomi is sensitive to the suppressive nature of biofumigants. Biofumigants can suppress the in vitro growth, sporulation, infective ability of P. cinnamomi and reduce the incidence of the disease caused by the pathogen in the glasshouse. Of the two Brassica species investigated, B. juncea was superior in its ability to control P. cinnamomi compared to B. napus. When applied in the field, biofumigation using B. juncea was found to be more suppressive that soil solarisation, but not as effective as metham sodium.