A detailed study of the leaf blotch disease, caused by Mycosphaerella nubilosa (Cke.) Hansf. on Eucalyptus regnans F. Muell (Mountain Ash) and E. delegatensis R.T. Baker (Syn. E. gigantea Hook. F., Alpine Ash) was made. The taxonomy of the fungus was studied(illustrations provided) and a previously unidentified imperfect state (Colletogloeum nubilosum) was described along with the spermagonial state (Asteromella). The histology and mode of infection process by ascospores and conidia was examined using Scanning Electron Microscopy, and wax and epoxy resin sections. The development of the fungus within the host was studied up to the stage of mature pseudothecia. The fungus was isolated from diseased tissue and Koch's postulates were carried out to prove pathogenicity. Detailed development of the leaf blotch and twig canker symptoms was followed in the field and in glasshouse conditions. The effect of substrate and environmental conditions on growth and sporulation of the fungus was studied. Controlled environment chambers and glasshouses were used. Several day/night temperature combinations (12/6, 18/12, 24/18 and 30/24C) as well as several light levels (50, 75, 150, 200, 300 and 650 μE) were investigated. The optimum temperature for infection was 24/18C and least infection occurred at 12/6C. The optimum light level for infection was 300 to 650 μE while the slowest infection was obtained at 50 and 75 μE. Under constant temperature conditions infection was most severe at 18C for both E. delegatensis and E. regnans. Wetness periods under sprinklers of one to seven days were compared with similar incubation periods in polythene bags. One-day incubation produced very little infection compared with 2, 4 or 7 days. Incubation in polythene bags gave better results than sprinklers. Experiments showed that leaf and stem infection was not affected by nutrient levels. Leaf expansion rates for both the eucalypt species were followed for twelve weeks to determine the optimum leaf size for infection. Fungicide trials in the field and in-vitro suggested that several chemotherapeutants may be effective in controlling the disease. Diseased leaves from field trials were assessed for ascospore discharge to determine fungal viability. From this study it was shown that diseased (inoculated)"plants lost significantly more leaves than controls. If conditions were optimum for infection, tips of inoculated plants were killed, leading to stem distortion, branching and reduced growth. In general, the fungus causes severe damage on susceptible Eucalyptus spp. in sapling stages.

Botrytis cinerea and Sclerotinia sclerotiorum are the two most serious pathogens on kiwifruit in New Zealand. Because of the pesticide regulations in some of the countries to which New Zealand exports fruit, total protection from Botrytis stem end rot with current dicarboximide fungicides is not possible. The aim of this thesis was to investigate biological control measures for Botrytis stem end rot and Sclerotinia diseases of kiwifruit. More than 1000 microorganisms, isolated from the leaves and flowers of kiwifruit during spring and autumn, and selected from BCAs reported to be effective against B. cinerea and./or S. sclerotiorum, were tested in vitro for their antagonistic ability against B. cinerea and S. sclerotiorum. Successful antagonists were those that, in dual culture on agar plates, produced a zone of inhibition, an area of browning of the pathogens, or grew rapidly over the pathogens and inhibited their growth. The fifty most promising isolates from the initial screen were tested on fruit for their ability to reduce Botrytis and Sclerotinia fruit rots. Mature kiwifruit were artificallv wounded and dual inoculated with a spore suspension of one of the fifty test organisms and either a conidial suspension of B. cinerea or a mycelial suspension of S. sclerotiorum. Following 8-12 weeks incubation in a cool store, fruit were assessed for Botrytis or Sclerotinia induced rot. Isolates of Bacillus spp., Epicoccum purpurascens, Pseudomonas sp. and Trichoderma. spp. reduced, Botrytis fruit rot from 92% (inoculated control) to 0%.Isolates of Alternaria spp., pestalotia sp. and a non-sporulating isolate also reduced the number of fruit rotting to some extent. Similarly, isolates of Bacillus spp., E purpurascens and Trichoderma spp. reduce d Sclerotinia fruit rot from 100% (inoculated control) to 0%. Isolates of Alternaria spp., Myrothecium verrucaria and Pestalotia sp. were also successful at reducing the level of Sclerotinia fruit rot. It was considered undesirable if potential biological control agents (BCAs) were able to colonize kiwifruit that were to be marketed for human consumption. In order to determine if microorganisms, shown to be effective in preventing Botrytis or Sclerotinia fruit rot, were capable of themselves colonizing fruit, isolations were made from fruit dual inoculated with B. cinerea, S. sclerotiorum and/or one of several BCAs. Strains of the BCAs Bacillus spp., Pseudomonas sp. and E. purpurascens were not found to be saprophytic on fruit. Isolates of Alternaria sp., Bacillus sp., E purpurascens, pestalotia sp., Pseudomonas sp. and T. harzianum significantly inhibited germination and germ tube elongation of B. cinerea conidia in vitro in a nutrient solution, over a 24 h period. For example, the presence of Alternaria alternata A6 spores in a nutrient solution reduced germination of B. cinerea conidia from 100% to 20%. The presence of E purpurascens A77 spores inhibited B. cinerea conidial germ tube elongation from >840 pm (in control conidia) to 27 µm. The presence of any one of the BCAs tested prevented germination of B. cinerea conidia in a non-nutrient water solution, in comparision to germination of up to 86% in controls. A spore or cell suspension of each of the isolates Bacillus sp.M60, E. purpurascens A77 and T. harzianum C65 were spray inoculated onto kiwifruit blossoms produced in vivo in the glasshouse, immediately prior to inoculation of the blossoms with a condial suspension of B. cinerea. Application of the BCAs were completely effective in preventing colonization of blossoms by B- cinerea conidia. The effectiveness of each of the isolates E. purpurascens A77,T. harzianum C65 and either Bacillus sp.M60 or M53 to reduce the viability of sclerotia of B. cinerea and S. sclerotiorum was tested in soil punnets. A spore or cell suspension of each respective BCA was applied to the surface of replicated punnets that were seeded with either B. cinerea or S. sclerotiorum. Following 8 weeks incubation, punnets were harvested and viability of sclerotia assessed. T. harzianum C65 and Bacillus sp. M60 significantly reduced the viability of B. cinerea sclerotia from 8 sclerotia/punnet (control) to 4 sclerotia/punnet. T. harzianum C65 and E. purpurascens A77 caused a significant reduction in apothecia production of S. sclerotiorum, from 2.7 apothecia/punnet (control) to 0.7 apothecia/punnet. Bacillus sp.M8 and E purpurascens A77 were tested for their ability to reduce Botrytis stem end rot and Sclerotinia field rot in a kiwifruit orchard. The isolates tested did not successfully reduce either disease. Possible explanations for this are discussed. In order to monitor the survival of particular isolates of BCAs in the field, a technique was developed to distinguish between individual strains of a BCA species. The polymerase chain reaction (PCR) was utilized to identify DNA polymorphisms within the genome of T. harzianum C65, in comparison with other strains of Trichoderma spp.. A sequence of polymorphic DNA was cloned, sequenced and used as a hybridization probe in southern blotting to enable T. harzianum c65 to be distinguished from other strains of Trichoderma spp.. From the results obtained in this study, it was considered that Bacillus M60, E purpurascens 477 and Pseudomonas M30 were the best isolates for the biological control of Botrytis stem end rot on kiwifruit. Further work to enable application of these isolates as postharvest BCAs is discussed. Of the isolates tested in this study, T. harzianum C65 was considered the best isolate for use against Sclerotinia diseases on kiwifruit. Methods of selecting more effective BCAs against S. sclerotiorum are discussed.

1. When purified turnip ye1low mosaic virus was inoculated mechanically on to Chinese cabbage leaves, using known numbers of virus particles in 0.1 to 1.0 µ1 volumes of inoculum, as few as 10 to 30 particles were required to produce a single local lesion.2. Inoculation of a cotyledon leaf of Chinese cabbage seedlings with turnip yellow mosaic virus produced a rapid transient inhibition in the rate of leaf initiation, so that infected plants developed 0.5 to 1.0 leaf less than healthy plants. 3. The factor that initiated the inhibitory response a t the apical. meristem began moving out of the inoculated cotyledon within 1to 6 hours after inoculation, thus preceding the movement out of the inoculated cotyledon of infectious virus or RNA which was not detectable until about day 5. 4. The transient inhibition of leaf initiation occurred following inoculation with any one of three unrelated viruses, or with infectious turnip ye1low mosaic virus RNA. 5. A factor eluted in an active form from the cut petioles of inoculated 1eaves. 6. It is necessary to inoculate with infectious virus or RNA to initiate the production of the inhibitory factor. 7. No differences were seen in the magnitude or timing of the reduced rate of 1eaf initiation, when the concentration of turnip yellow mosaic virus in the inoculum was varied between 1 µ g/ml and 100 µg/ml. 8. Inoculation of the cotyledons of Chinese cabbage seed1ings with turnip ye1low mosaic virus caused a marked disturbance in the mitotic index a t the apical meristem between 6 and 48 hours. 9. A reduction in the accumulation of starch in the chloroplasts of cell s in the apical meristem occurred at 6 t o 24 hours after inoculation of the cotyledon 1eaf. 10. Abscisic acid applied to the cotyledon in a single 20 µ1 dose, elicited a response that closely paralleled the events that took place when Chinese cabbage seedlings were inoculated with turnip yellow mosaic virus. A decrease in the rate of leaf initiation began 1 t o 2 days after application and the inhibition of leaf initiation was preceded by a disturbance in the mitotic index in the apical meristem. 11. Gibberellic acid applied with the eluate from virus-inoculated leaves, was able to overcome the inhibition of leaf initiation. 12. The leaf inhibition assay in Chinese cabbage seedlings is a sensitive bioassay for abscisic acid. The minimum detectable concentration of 3 x M is comparable to those reported for the Commelina stomata1 closure bioassay which could detect 10 -10 abscisic acid (Ogunkanmi et a1 . 1973). / Note: Whole document restricted due to copyright restrictions but available by request use the feedback form to request access

A detailed study of the leaf blotch disease, caused by Mycosphaerella nubilosa (Cke.) Hansf. on Eucalyptus regnans F. Muell (Mountain Ash) and E. delegatensis R.T. Baker (Syn. E. gigantea Hook. F., Alpine Ash) was made. The taxonomy of the fungus was studied(illustrations provided) and a previously unidentified imperfect state (Colletogloeum nubilosum) was described along with the spermagonial state (Asteromella). The histology and mode of infection process by ascospores and conidia was examined using Scanning Electron Microscopy, and wax and epoxy resin sections. The development of the fungus within the host was studied up to the stage of mature pseudothecia. The fungus was isolated from diseased tissue and Koch's postulates were carried out to prove pathogenicity. Detailed development of the leaf blotch and twig canker symptoms was followed in the field and in glasshouse conditions. The effect of substrate and environmental conditions on growth and sporulation of the fungus was studied. Controlled environment chambers and glasshouses were used. Several day/night temperature combinations (12/6, 18/12, 24/18 and 30/24C) as well as several light levels (50, 75, 150, 200, 300 and 650 μE) were investigated. The optimum temperature for infection was 24/18C and least infection occurred at 12/6C. The optimum light level for infection was 300 to 650 μE while the slowest infection was obtained at 50 and 75 μE. Under constant temperature conditions infection was most severe at 18C for both E. delegatensis and E. regnans. Wetness periods under sprinklers of one to seven days were compared with similar incubation periods in polythene bags. One-day incubation produced very little infection compared with 2, 4 or 7 days. Incubation in polythene bags gave better results than sprinklers. Experiments showed that leaf and stem infection was not affected by nutrient levels. Leaf expansion rates for both the eucalypt species were followed for twelve weeks to determine the optimum leaf size for infection. Fungicide trials in the field and in-vitro suggested that several chemotherapeutants may be effective in controlling the disease. Diseased leaves from field trials were assessed for ascospore discharge to determine fungal viability. From this study it was shown that diseased (inoculated)"plants lost significantly more leaves than controls. If conditions were optimum for infection, tips of inoculated plants were killed, leading to stem distortion, branching and reduced growth. In general, the fungus causes severe damage on susceptible Eucalyptus spp. in sapling stages.

Botrytis cinerea and Sclerotinia sclerotiorum are the two most serious pathogens on kiwifruit in New Zealand. Because of the pesticide regulations in some of the countries to which New Zealand exports fruit, total protection from Botrytis stem end rot with current dicarboximide fungicides is not possible. The aim of this thesis was to investigate biological control measures for Botrytis stem end rot and Sclerotinia diseases of kiwifruit. More than 1000 microorganisms, isolated from the leaves and flowers of kiwifruit during spring and autumn, and selected from BCAs reported to be effective against B. cinerea and./or S. sclerotiorum, were tested in vitro for their antagonistic ability against B. cinerea and S. sclerotiorum. Successful antagonists were those that, in dual culture on agar plates, produced a zone of inhibition, an area of browning of the pathogens, or grew rapidly over the pathogens and inhibited their growth. The fifty most promising isolates from the initial screen were tested on fruit for their ability to reduce Botrytis and Sclerotinia fruit rots. Mature kiwifruit were artificallv wounded and dual inoculated with a spore suspension of one of the fifty test organisms and either a conidial suspension of B. cinerea or a mycelial suspension of S. sclerotiorum. Following 8-12 weeks incubation in a cool store, fruit were assessed for Botrytis or Sclerotinia induced rot. Isolates of Bacillus spp., Epicoccum purpurascens, Pseudomonas sp. and Trichoderma. spp. reduced, Botrytis fruit rot from 92% (inoculated control) to 0%.Isolates of Alternaria spp., pestalotia sp. and a non-sporulating isolate also reduced the number of fruit rotting to some extent. Similarly, isolates of Bacillus spp., E purpurascens and Trichoderma spp. reduce d Sclerotinia fruit rot from 100% (inoculated control) to 0%. Isolates of Alternaria spp., Myrothecium verrucaria and Pestalotia sp. were also successful at reducing the level of Sclerotinia fruit rot. It was considered undesirable if potential biological control agents (BCAs) were able to colonize kiwifruit that were to be marketed for human consumption. In order to determine if microorganisms, shown to be effective in preventing Botrytis or Sclerotinia fruit rot, were capable of themselves colonizing fruit, isolations were made from fruit dual inoculated with B. cinerea, S. sclerotiorum and/or one of several BCAs. Strains of the BCAs Bacillus spp., Pseudomonas sp. and E. purpurascens were not found to be saprophytic on fruit. Isolates of Alternaria sp., Bacillus sp., E purpurascens, pestalotia sp., Pseudomonas sp. and T. harzianum significantly inhibited germination and germ tube elongation of B. cinerea conidia in vitro in a nutrient solution, over a 24 h period. For example, the presence of Alternaria alternata A6 spores in a nutrient solution reduced germination of B. cinerea conidia from 100% to 20%. The presence of E purpurascens A77 spores inhibited B. cinerea conidial germ tube elongation from >840 pm (in control conidia) to 27 µm. The presence of any one of the BCAs tested prevented germination of B. cinerea conidia in a non-nutrient water solution, in comparision to germination of up to 86% in controls. A spore or cell suspension of each of the isolates Bacillus sp.M60, E. purpurascens A77 and T. harzianum C65 were spray inoculated onto kiwifruit blossoms produced in vivo in the glasshouse, immediately prior to inoculation of the blossoms with a condial suspension of B. cinerea. Application of the BCAs were completely effective in preventing colonization of blossoms by B- cinerea conidia. The effectiveness of each of the isolates E. purpurascens A77,T. harzianum C65 and either Bacillus sp.M60 or M53 to reduce the viability of sclerotia of B. cinerea and S. sclerotiorum was tested in soil punnets. A spore or cell suspension of each respective BCA was applied to the surface of replicated punnets that were seeded with either B. cinerea or S. sclerotiorum. Following 8 weeks incubation, punnets were harvested and viability of sclerotia assessed. T. harzianum C65 and Bacillus sp. M60 significantly reduced the viability of B. cinerea sclerotia from 8 sclerotia/punnet (control) to 4 sclerotia/punnet. T. harzianum C65 and E. purpurascens A77 caused a significant reduction in apothecia production of S. sclerotiorum, from 2.7 apothecia/punnet (control) to 0.7 apothecia/punnet. Bacillus sp.M8 and E purpurascens A77 were tested for their ability to reduce Botrytis stem end rot and Sclerotinia field rot in a kiwifruit orchard. The isolates tested did not successfully reduce either disease. Possible explanations for this are discussed. In order to monitor the survival of particular isolates of BCAs in the field, a technique was developed to distinguish between individual strains of a BCA species. The polymerase chain reaction (PCR) was utilized to identify DNA polymorphisms within the genome of T. harzianum C65, in comparison with other strains of Trichoderma spp.. A sequence of polymorphic DNA was cloned, sequenced and used as a hybridization probe in southern blotting to enable T. harzianum c65 to be distinguished from other strains of Trichoderma spp.. From the results obtained in this study, it was considered that Bacillus M60, E purpurascens 477 and Pseudomonas M30 were the best isolates for the biological control of Botrytis stem end rot on kiwifruit. Further work to enable application of these isolates as postharvest BCAs is discussed. Of the isolates tested in this study, T. harzianum C65 was considered the best isolate for use against Sclerotinia diseases on kiwifruit. Methods of selecting more effective BCAs against S. sclerotiorum are discussed.

1. When purified turnip ye1low mosaic virus was inoculated mechanically on to Chinese cabbage leaves, using known numbers of virus particles in 0.1 to 1.0 µ1 volumes of inoculum, as few as 10 to 30 particles were required to produce a single local lesion.2. Inoculation of a cotyledon leaf of Chinese cabbage seedlings with turnip yellow mosaic virus produced a rapid transient inhibition in the rate of leaf initiation, so that infected plants developed 0.5 to 1.0 leaf less than healthy plants. 3. The factor that initiated the inhibitory response a t the apical. meristem began moving out of the inoculated cotyledon within 1to 6 hours after inoculation, thus preceding the movement out of the inoculated cotyledon of infectious virus or RNA which was not detectable until about day 5. 4. The transient inhibition of leaf initiation occurred following inoculation with any one of three unrelated viruses, or with infectious turnip ye1low mosaic virus RNA. 5. A factor eluted in an active form from the cut petioles of inoculated 1eaves. 6. It is necessary to inoculate with infectious virus or RNA to initiate the production of the inhibitory factor. 7. No differences were seen in the magnitude or timing of the reduced rate of 1eaf initiation, when the concentration of turnip yellow mosaic virus in the inoculum was varied between 1 µ g/ml and 100 µg/ml. 8. Inoculation of the cotyledons of Chinese cabbage seed1ings with turnip ye1low mosaic virus caused a marked disturbance in the mitotic index a t the apical meristem between 6 and 48 hours. 9. A reduction in the accumulation of starch in the chloroplasts of cell s in the apical meristem occurred at 6 t o 24 hours after inoculation of the cotyledon 1eaf. 10. Abscisic acid applied to the cotyledon in a single 20 µ1 dose, elicited a response that closely paralleled the events that took place when Chinese cabbage seedlings were inoculated with turnip yellow mosaic virus. A decrease in the rate of leaf initiation began 1 t o 2 days after application and the inhibition of leaf initiation was preceded by a disturbance in the mitotic index in the apical meristem. 11. Gibberellic acid applied with the eluate from virus-inoculated leaves, was able to overcome the inhibition of leaf initiation. 12. The leaf inhibition assay in Chinese cabbage seedlings is a sensitive bioassay for abscisic acid. The minimum detectable concentration of 3 x M is comparable to those reported for the Commelina stomata1 closure bioassay which could detect 10 -10 abscisic acid (Ogunkanmi et a1 . 1973). / Note: Whole document restricted due to copyright restrictions but available by request use the feedback form to request access

A detailed study of the leaf blotch disease, caused by Mycosphaerella nubilosa (Cke.) Hansf. on Eucalyptus regnans F. Muell (Mountain Ash) and E. delegatensis R.T. Baker (Syn. E. gigantea Hook. F., Alpine Ash) was made. The taxonomy of the fungus was studied(illustrations provided) and a previously unidentified imperfect state (Colletogloeum nubilosum) was described along with the spermagonial state (Asteromella). The histology and mode of infection process by ascospores and conidia was examined using Scanning Electron Microscopy, and wax and epoxy resin sections. The development of the fungus within the host was studied up to the stage of mature pseudothecia. The fungus was isolated from diseased tissue and Koch's postulates were carried out to prove pathogenicity. Detailed development of the leaf blotch and twig canker symptoms was followed in the field and in glasshouse conditions. The effect of substrate and environmental conditions on growth and sporulation of the fungus was studied. Controlled environment chambers and glasshouses were used. Several day/night temperature combinations (12/6, 18/12, 24/18 and 30/24C) as well as several light levels (50, 75, 150, 200, 300 and 650 μE) were investigated. The optimum temperature for infection was 24/18C and least infection occurred at 12/6C. The optimum light level for infection was 300 to 650 μE while the slowest infection was obtained at 50 and 75 μE. Under constant temperature conditions infection was most severe at 18C for both E. delegatensis and E. regnans. Wetness periods under sprinklers of one to seven days were compared with similar incubation periods in polythene bags. One-day incubation produced very little infection compared with 2, 4 or 7 days. Incubation in polythene bags gave better results than sprinklers. Experiments showed that leaf and stem infection was not affected by nutrient levels. Leaf expansion rates for both the eucalypt species were followed for twelve weeks to determine the optimum leaf size for infection. Fungicide trials in the field and in-vitro suggested that several chemotherapeutants may be effective in controlling the disease. Diseased leaves from field trials were assessed for ascospore discharge to determine fungal viability. From this study it was shown that diseased (inoculated)"plants lost significantly more leaves than controls. If conditions were optimum for infection, tips of inoculated plants were killed, leading to stem distortion, branching and reduced growth. In general, the fungus causes severe damage on susceptible Eucalyptus spp. in sapling stages.

The fungus Macrophomina phaseolina is the causal agent of several diseases of mungbean (Vigna radiata). One of these diseases, known as charcoal rot, causes spoilage of germinating seed lots, and occurs when seed contaminated with M. phaseolina is used for sprouting. The detection of the pathogen during seed testing prior to export leads to downgrading of the seed and a resultant financial penalty to the grain grower. The aims of this research were: to determine the location of M. phaseolina in diseased and symptomless tissue of mungbean plants; to determine the mode, site and timing of seed colonisation of mungbean; to determine the impact of biotic and abiotic factors on seed colonisation; to conduct an assessment of genotypic variation of M. phaseolina within single plants; to determine modes of transport of inoculum that contribute to foliage infection and seed colonisation of mungbean; and to assess both pre- and post-harvest management strategies in order to reduce or prevent seed colonisation, or to minimise the process of seed transmission. The location of M. phaseolina in mungbean plants was determined through serial sectioning of, and subsequent isolation from, naturally infected host tissue. A large proportion of the mungbean tissue infected by M. phaseolina was found to be symptomless. Moreover, there were large areas of ostensibly pathogen-free tissue separating infection foci, thus indicating a strong likelihood of independent aerial infections. A selection of 14 isolates obtained from serial sectioning were assessed for genotypic variation with six primers using RAPD analysis. Of the 36 bands that were scored, 78% were polymorphic and as a result, 12 distinct genotypes were detected. Polymorphisms were also detected amongst isolates obtained from the same discrete infection area on single plants, which strongly suggests the occurrence of multiple aerial infections. Various methods of controlled inoculation including soil infestation, pod and foliar inoculations, and artificial seed infestation, were used to determine how mungbean seeds are colonised by M. phaseolina. Additionally, most of these inoculation methods were coupled with a series of abiotic treatments (temperature regimes, watering regimes, application of herbicides) that were designed to initiate stress conditions within infected plants, and possibly trigger growth of M. phaseolina from the infection courts and colonise seed. Seed colonisation was established in vitro and in vivo when immature pods were directly inoculated with microsclerotia of M. phaseolina. At least two days exposure at 100% relative humidity (RH) was necessary to establish seed infection in detached mungbean pods that were inoculated with microsclerotia of M. phaseolina. Extensive seed infection was still obtained when one or more days of 100% RH was interrupted by up to three days at low humidity. All except one of the other methods of controlled inoculation failed to produce colonised seed even when combinations of stresses were applied. Only when the bipyridylium herbicide ‘Spray Seed 250’ was applied to plants following the inoculation of mungbean stems within 13 cm of the pods, was seed colonised by M. phaseolina. This result raises the possibility that delayed harvesting of desiccated mungbean crops may promote further colonisation of mungbean tissue, including seed. Very strong evidence for the colonisation of mungbean seeds after deposition of soil-splashed inoculum of M. phaseolina onto pods was obtained through field and laboratory-based studies. Soil-splashed inoculum (most likely microsclerotia) was also found to be the source of inoculum responsible for the development of Macrophomina leaf blight of mungbean at several regional sites. To further investigate this finding, areas of several mungbean crops growing in naturally infested soil were covered in hessian cloth to prevent soil-splash and assessments of the levels of seed colonisation between covered and uncovered areas were made. Although colonisation of seed in the covered areas was significantly lower than in the uncovered areas, covering the soil did not eliminate colonisation of seed. This result suggests that inoculum dispersal, leading to pod infection and subsequent seed colonisation, occurs not only in splashed-soil but also by other means. Soil transported to the extra-floral nectaries of mungbeans by ants was found to contain infective inoculum of M. phaseolina. Furthermore, air-borne debris and dust collected in a trap contained viable microsclerotia of the pathogen. Isolates of M. phaseolina collected from both sources were pathogenic on mungbean seedlings, and suspensions of ant-transported soil and air-borne dust/debris infected mungbean pods and seed. This is the first report of both modes of inoculum dispersal. All three modes undoubtedly contribute to the total level of colonised seed, but their relative importance remains to be determined. Several options for the management of charcoal rot in mungbean seeds were investigated. Application of the fungicide carbendazim to mungbean plants after flowering significantly decreased, but did not prevent, colonisation of mungbean seed by M. phaseolina. Consequently, this method of management holds little promise for mungbean growers. A large number of weeds common in Australian mungbean fields were newly reported as hosts of M. phaseolina. Isolates obtained from the infected, but symptomless weeds were pathogenic on mungbean seedlings, thus indicating a lack of host-specificity toward mungbean. It is strongly suspected that the use of herbicides to control weeds in reduced and zero-tillage farming systems is increasing the risk of infection in subsequent mungbean crops through the build-up of inoculum in soil. This increased risk of infection may be further exacerbated by retaining stubble infested with M. phaseolina on the soil surface, thereby increasing the amount of inoculum that could be splashed onto plant organs. Surface sterilisation, using sodium hypochlorite, significantly reduced colonisation levels in heavily colonised mungbean seed lines, but the process was not enhanced when a partial-vacuum was introduced to the process. In some seed lots, up to 32% of the seed was colonised only on the seed coat (defined here as contamination), whereas the remainder was internally infected. Surface sterilisation also reduced the overall colonisation of 132 commercial lines by approximately one-third. Storage of seed for one month at either 4°C or 15°C significantly reduced colonisation levels in seed, whereas freezing treatments did not. Eradication of M. phaseolina from mungbean seed was possible through thermotherapy. However, the conditions required for eradication also contributed to large increases in abnormal germination levels and large losses in overall germination - an unacceptable trade-off for sprouters. A combination of thermotherapy and surface sterilisation of colonised mungbean seed may provide a more efficient process of seed treatment.

The fungus Macrophomina phaseolina is the causal agent of several diseases of mungbean (Vigna radiata). One of these diseases, known as charcoal rot, causes spoilage of germinating seed lots, and occurs when seed contaminated with M. phaseolina is used for sprouting. The detection of the pathogen during seed testing prior to export leads to downgrading of the seed and a resultant financial penalty to the grain grower. The aims of this research were: to determine the location of M. phaseolina in diseased and symptomless tissue of mungbean plants; to determine the mode, site and timing of seed colonisation of mungbean; to determine the impact of biotic and abiotic factors on seed colonisation; to conduct an assessment of genotypic variation of M. phaseolina within single plants; to determine modes of transport of inoculum that contribute to foliage infection and seed colonisation of mungbean; and to assess both pre- and post-harvest management strategies in order to reduce or prevent seed colonisation, or to minimise the process of seed transmission. The location of M. phaseolina in mungbean plants was determined through serial sectioning of, and subsequent isolation from, naturally infected host tissue. A large proportion of the mungbean tissue infected by M. phaseolina was found to be symptomless. Moreover, there were large areas of ostensibly pathogen-free tissue separating infection foci, thus indicating a strong likelihood of independent aerial infections. A selection of 14 isolates obtained from serial sectioning were assessed for genotypic variation with six primers using RAPD analysis. Of the 36 bands that were scored, 78% were polymorphic and as a result, 12 distinct genotypes were detected. Polymorphisms were also detected amongst isolates obtained from the same discrete infection area on single plants, which strongly suggests the occurrence of multiple aerial infections. Various methods of controlled inoculation including soil infestation, pod and foliar inoculations, and artificial seed infestation, were used to determine how mungbean seeds are colonised by M. phaseolina. Additionally, most of these inoculation methods were coupled with a series of abiotic treatments (temperature regimes, watering regimes, application of herbicides) that were designed to initiate stress conditions within infected plants, and possibly trigger growth of M. phaseolina from the infection courts and colonise seed. Seed colonisation was established in vitro and in vivo when immature pods were directly inoculated with microsclerotia of M. phaseolina. At least two days exposure at 100% relative humidity (RH) was necessary to establish seed infection in detached mungbean pods that were inoculated with microsclerotia of M. phaseolina. Extensive seed infection was still obtained when one or more days of 100% RH was interrupted by up to three days at low humidity. All except one of the other methods of controlled inoculation failed to produce colonised seed even when combinations of stresses were applied. Only when the bipyridylium herbicide ‘Spray Seed 250’ was applied to plants following the inoculation of mungbean stems within 13 cm of the pods, was seed colonised by M. phaseolina. This result raises the possibility that delayed harvesting of desiccated mungbean crops may promote further colonisation of mungbean tissue, including seed. Very strong evidence for the colonisation of mungbean seeds after deposition of soil-splashed inoculum of M. phaseolina onto pods was obtained through field and laboratory-based studies. Soil-splashed inoculum (most likely microsclerotia) was also found to be the source of inoculum responsible for the development of Macrophomina leaf blight of mungbean at several regional sites. To further investigate this finding, areas of several mungbean crops growing in naturally infested soil were covered in hessian cloth to prevent soil-splash and assessments of the levels of seed colonisation between covered and uncovered areas were made. Although colonisation of seed in the covered areas was significantly lower than in the uncovered areas, covering the soil did not eliminate colonisation of seed. This result suggests that inoculum dispersal, leading to pod infection and subsequent seed colonisation, occurs not only in splashed-soil but also by other means. Soil transported to the extra-floral nectaries of mungbeans by ants was found to contain infective inoculum of M. phaseolina. Furthermore, air-borne debris and dust collected in a trap contained viable microsclerotia of the pathogen. Isolates of M. phaseolina collected from both sources were pathogenic on mungbean seedlings, and suspensions of ant-transported soil and air-borne dust/debris infected mungbean pods and seed. This is the first report of both modes of inoculum dispersal. All three modes undoubtedly contribute to the total level of colonised seed, but their relative importance remains to be determined. Several options for the management of charcoal rot in mungbean seeds were investigated. Application of the fungicide carbendazim to mungbean plants after flowering significantly decreased, but did not prevent, colonisation of mungbean seed by M. phaseolina. Consequently, this method of management holds little promise for mungbean growers. A large number of weeds common in Australian mungbean fields were newly reported as hosts of M. phaseolina. Isolates obtained from the infected, but symptomless weeds were pathogenic on mungbean seedlings, thus indicating a lack of host-specificity toward mungbean. It is strongly suspected that the use of herbicides to control weeds in reduced and zero-tillage farming systems is increasing the risk of infection in subsequent mungbean crops through the build-up of inoculum in soil. This increased risk of infection may be further exacerbated by retaining stubble infested with M. phaseolina on the soil surface, thereby increasing the amount of inoculum that could be splashed onto plant organs. Surface sterilisation, using sodium hypochlorite, significantly reduced colonisation levels in heavily colonised mungbean seed lines, but the process was not enhanced when a partial-vacuum was introduced to the process. In some seed lots, up to 32% of the seed was colonised only on the seed coat (defined here as contamination), whereas the remainder was internally infected. Surface sterilisation also reduced the overall colonisation of 132 commercial lines by approximately one-third. Storage of seed for one month at either 4°C or 15°C significantly reduced colonisation levels in seed, whereas freezing treatments did not. Eradication of M. phaseolina from mungbean seed was possible through thermotherapy. However, the conditions required for eradication also contributed to large increases in abnormal germination levels and large losses in overall germination - an unacceptable trade-off for sprouters. A combination of thermotherapy and surface sterilisation of colonised mungbean seed may provide a more efficient process of seed treatment.

Content removed due to copyright restrictions: Taylor, C. H., & Long, P. G. (2000). Review of literature on camellia flower blight caused by ciborinia camelliae. New Zealand Journal of Crop and Horticultural Science, 28(2), 123-138. / Camellias are popular ornamental plants and the most serious pathogen of this plant is camellia flower blight, caused by the fungal pathogen Ciborinia camelliae Kohn. Ascospores of this fungus attack the flowers, turning them brown, rendering infected flowers unattractive. Little is known about the pathogen and control measures are not particularly effective. In this thesis, various aspects of the pathogen's basic and molecular biology and interaction with host species were studied. Surveys of the distribution and spread of C. camelliae within New Zealand determined that the pathogen was present in most regions of the North Island, and north and east coasts of the South Island. Over the distances and time involved, it appeared that the disease was spreading mainly by windborne ascospores rather than human transfer. Sclerotia were germinated out of season to increase the period during which ascospores were available for infection work. Greatest germination was achieved at low temperatures (5°C-10°C) in 24 h darkness. Isolate-specific primers were designed to the ribosomal DNA Internal Transcribed Spacer region to detect the pathogen in planta and distinguish between New Zealand isolates of C. camelliae and other fungal pathogens. Phylogenetic analysis of the ITS region with other Ciborinia, Sclerotinia and Botrytis species showed that C. camelliae was more closely related to S. sclerotiorum than other Ciborinia species. Two inoculation techniques for infecting Camellia petals with ascospores of C. camelliae were developed and tested. Inoculation using airborne ascospores in a settling chamber was a simple and quick method for testing large numbers of species for resistance. Inoculation of ascospores in suspension produced qualitative data, but was more time consuming. Of the four mechanisms of resistance tested, levels of aluminium hyperaccumulation and the presence of phenolic compounds did not correlate with resistance in Camellia species. The large uptake of aluminium, however, did indicate that Camellia species would be good plants for phytoremediation of acid soils. Some resistant species were found to have cell wall modifications and/or lignification of cell walls in response to C. camelliae infection and chitinase activity was found in most resistant Camellia species tested. Further research into these latter two mechanisms is recommended and indicates that the development of resistant Camellia cultivars is possible.