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Strawberries are an extremely perishable fruit mainly due to their soft texture and sensitivity to fungal infection. The fungal pathogen Botrytis cinerea is responsible for grey mould on strawberries and is the main causal agent of postharvest decay and subsequent economic loss. As an alternative to fungicides, manipulation of plant nutrition, such as calcium and boron, has been suggested as a means of disease management. This project investigated the effects of calcium and boron application on fruit quality and grey mould development in strawberry. The effect of calcium on fruit quality, grey mould development and leaf blight in strawberry cultivars ‘Aromas’ and ‘Selva’ was investigated through preharvest and postharvest applications. To determine the effect of preharvest application, calcium sulphate in 0.25X strength Hoagland’s solution was applied at 0, 100, 300 and 500 ppm Ca through fertigation. Fully-ripened fruit were harvested and evaluated for postharvest quality at harvest and then after storage at 10⁰C, 90±5% RH for 2 to 10 days. Although fruit firmness of both cultivars declined slightly during storage, this was not affected by preharvest calcium application. Similarly, preharvest calcium treatment had no effect on the external appearance, pH, soluble solids content (SSC) or titratable acidity (TA). No grey mould development was observed on fruit at harvest when flowers were inoculated with a conidia suspension of B. cinerea (10⁴ conidia per mL). However, fruit harvested from plants that received calcium at any concentration had less incidence of grey mould during storage at 10⁰C, 90±5% RH for 14 days than fruit harvested from plants that received no calcium for both cultivars. For ‘Aromas’, 79% and 51% of fruit, and for ‘Selva’, 69% and 43% of fruit, showed rot when treated with 0 and 500 ppm Ca, respectively. The shelf life of ‘Aromas’ and ‘Selva’ increased by about 8% when plants received 500 ppm Ca in comparison with plants that received 0 ppm Ca. After 7 days of incubation at 22 to 24⁰C, there was no difference between blight lesions on wound-inoculated detached leaves from different calcium treatments for either cultivar. However, the lesions on ‘Selva’ were smaller than on ‘Aromas’. The calcium levels in leaves from plants that received calcium at any concentration were adequate for strawberry growing and significantly higher (P < 0.05) than in leaves from plants that received 0 ppm Ca. However, calcium treatment did not ensure transfer of calcium to fruit tissues. Calcium lactate and calcium chloride were used as postharvest calcium treatments at 1500, 3000 and 4500 ppm Ca. Fruit of ‘Selva’ were dipped in calcium solution for 5 min and wound-inoculated with B. cinerea (10⁶ conidia per mL). Calcium lactate and calcium chloride at 3000 and 4500 ppm Ca, respectively, were most effective in delaying Botrytis rot development on ‘Selva’ after 7 days of storage at 10⁰C, 90±5% RH. Storage for least 24 h after calcium dips prior to inoculation was required to delay the development of fruit rot. Fruit harvested early in the season seemed to be less susceptible to grey mould than those harvested later. However, calcium treatment tended to be more effective when applied to late-season fruit. Preharvest boron treatment, applied as for calcium but at 0, 0.25, 0.5 and 1.0 ppm B, had no effect on fruit firmness of either cultivar. However, firmness of ‘Aromas’ fruit was slightly greater than ‘Selva’ fruit for all treatments. The amount of boron applied had no effect on the external appearance, pH, SSC or TA for either cultivar after storage of fruit for up to 10 days. Application of boron had no effect on fruit grey mould development in either cultivar. Furthermore, boron had minimal effect on the incidence of blight on woundinoculated detached leaves of ‘Aromas’ 7 days after inoculation. However, blight lesion diameters on ‘Selva’ leaves in the 1.0 ppm B treatment (8.0 mm) were significantly smaller (P < 0.001) than in the 0 ppm B treatment (13.0 mm). Phytotoxicity was observed in boron treatments even at the level considered optimum for strawberry growing. Severity increased with increasing boron concentration but no consistent effect on flower death or flower abortion was observed. In conclusion, strawberry is sensitive to boron toxicity. Calcium may enhance fruit firmness and, consequently, delay grey mould development if calcium penetrates the fruit. Postharvest calcium treatment tended to be more effective in delaying development of grey mould when applied to late-season fruit. Calcium lactate is a potential alternative to calcium chloride for reducing decay caused by B. cinerea in strawberry without providing undesirable bitterness. This finding may provide a basis for application in industry. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1331382 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine 2008

<p>Pesticides are designed to control or eliminate pests such as insects, rodents, weeds,<br /> bacteria, and fungi. They are used at a global scale for agricultural produce. Although<br /> pesticides play a significant role in increasing food production and eliminating diseases,<br /> exposure to pesticides may be harmful to non-target organisms. As a result concern over<br /> safety and resistance to pesticides has increased and there is pressure to reduce use and<br /> search for more environmentally and toxicologically safe and efficacious pesticides. Most<br /> pesticides currently in use are synthetic / therefore an alternative to synthetic pesticides is<br /> the use of naturally occurring products/ botanicals with pesticidal properties.</p> <p>Two plants indigenous to South African with pesticidal properties were chosen for this<br /> study. Dicerothamnus rhinocerotis (D. rhinocerotis) and Galenia africana (G. africana)<br /> have potential antifungal properties thus, may have potential use on agricultural produce<br /> as fungicides. Galenia africana and D. rhinocerotis extracts inhibit growth of B. cinerea<br /> (a fungal pathogen) at concentrations greater than 31.25 mg/ml and 125mg/ml<br /> respectively. A major consideration in approving pesticides for use is whether they pose<br /> an unreasonable risk to humans and to the environment. Toxicity studies are required to<br /> determine the safety of the plant extracts.</p> <p>The purpose of this study was to evaluate potential toxicity of ethanol extracts of D.<br /> rhinocerotis and G. africana, which is important when designing practices to reduce or<br /> eliminate excess exposure to them. Natural plant products with pesticidal properties could<br /> provide an alternative to synthetic pesticides and may thus effectively reduce resistance<br /> levels.<br /> <br /> <br /> &nbsp / </p>

<p>Pesticides are designed to control or eliminate pests such as insects, rodents, weeds,<br /> bacteria, and fungi. They are used at a global scale for agricultural produce. Although<br /> pesticides play a significant role in increasing food production and eliminating diseases,<br /> exposure to pesticides may be harmful to non-target organisms. As a result concern over<br /> safety and resistance to pesticides has increased and there is pressure to reduce use and<br /> search for more environmentally and toxicologically safe and efficacious pesticides. Most<br /> pesticides currently in use are synthetic / therefore an alternative to synthetic pesticides is<br /> the use of naturally occurring products/ botanicals with pesticidal properties.</p> <p>Two plants indigenous to South African with pesticidal properties were chosen for this<br /> study. Dicerothamnus rhinocerotis (D. rhinocerotis) and Galenia africana (G. africana)<br /> have potential antifungal properties thus, may have potential use on agricultural produce<br /> as fungicides. Galenia africana and D. rhinocerotis extracts inhibit growth of B. cinerea<br /> (a fungal pathogen) at concentrations greater than 31.25 mg/ml and 125mg/ml<br /> respectively. A major consideration in approving pesticides for use is whether they pose<br /> an unreasonable risk to humans and to the environment. Toxicity studies are required to<br /> determine the safety of the plant extracts.</p> <p>The purpose of this study was to evaluate potential toxicity of ethanol extracts of D.<br /> rhinocerotis and G. africana, which is important when designing practices to reduce or<br /> eliminate excess exposure to them. Natural plant products with pesticidal properties could<br /> provide an alternative to synthetic pesticides and may thus effectively reduce resistance<br /> levels.<br /> <br /> <br /> &nbsp / </p>

Strawberries are an extremely perishable fruit mainly due to their soft texture and sensitivity to fungal infection. The fungal pathogen Botrytis cinerea is responsible for grey mould on strawberries and is the main causal agent of postharvest decay and subsequent economic loss. As an alternative to fungicides, manipulation of plant nutrition, such as calcium and boron, has been suggested as a means of disease management. This project investigated the effects of calcium and boron application on fruit quality and grey mould development in strawberry. The effect of calcium on fruit quality, grey mould development and leaf blight in strawberry cultivars ‘Aromas’ and ‘Selva’ was investigated through preharvest and postharvest applications. To determine the effect of preharvest application, calcium sulphate in 0.25X strength Hoagland’s solution was applied at 0, 100, 300 and 500 ppm Ca through fertigation. Fully-ripened fruit were harvested and evaluated for postharvest quality at harvest and then after storage at 10⁰C, 90±5% RH for 2 to 10 days. Although fruit firmness of both cultivars declined slightly during storage, this was not affected by preharvest calcium application. Similarly, preharvest calcium treatment had no effect on the external appearance, pH, soluble solids content (SSC) or titratable acidity (TA). No grey mould development was observed on fruit at harvest when flowers were inoculated with a conidia suspension of B. cinerea (10⁴ conidia per mL). However, fruit harvested from plants that received calcium at any concentration had less incidence of grey mould during storage at 10⁰C, 90±5% RH for 14 days than fruit harvested from plants that received no calcium for both cultivars. For ‘Aromas’, 79% and 51% of fruit, and for ‘Selva’, 69% and 43% of fruit, showed rot when treated with 0 and 500 ppm Ca, respectively. The shelf life of ‘Aromas’ and ‘Selva’ increased by about 8% when plants received 500 ppm Ca in comparison with plants that received 0 ppm Ca. After 7 days of incubation at 22 to 24⁰C, there was no difference between blight lesions on wound-inoculated detached leaves from different calcium treatments for either cultivar. However, the lesions on ‘Selva’ were smaller than on ‘Aromas’. The calcium levels in leaves from plants that received calcium at any concentration were adequate for strawberry growing and significantly higher (P < 0.05) than in leaves from plants that received 0 ppm Ca. However, calcium treatment did not ensure transfer of calcium to fruit tissues. Calcium lactate and calcium chloride were used as postharvest calcium treatments at 1500, 3000 and 4500 ppm Ca. Fruit of ‘Selva’ were dipped in calcium solution for 5 min and wound-inoculated with B. cinerea (10⁶ conidia per mL). Calcium lactate and calcium chloride at 3000 and 4500 ppm Ca, respectively, were most effective in delaying Botrytis rot development on ‘Selva’ after 7 days of storage at 10⁰C, 90±5% RH. Storage for least 24 h after calcium dips prior to inoculation was required to delay the development of fruit rot. Fruit harvested early in the season seemed to be less susceptible to grey mould than those harvested later. However, calcium treatment tended to be more effective when applied to late-season fruit. Preharvest boron treatment, applied as for calcium but at 0, 0.25, 0.5 and 1.0 ppm B, had no effect on fruit firmness of either cultivar. However, firmness of ‘Aromas’ fruit was slightly greater than ‘Selva’ fruit for all treatments. The amount of boron applied had no effect on the external appearance, pH, SSC or TA for either cultivar after storage of fruit for up to 10 days. Application of boron had no effect on fruit grey mould development in either cultivar. Furthermore, boron had minimal effect on the incidence of blight on woundinoculated detached leaves of ‘Aromas’ 7 days after inoculation. However, blight lesion diameters on ‘Selva’ leaves in the 1.0 ppm B treatment (8.0 mm) were significantly smaller (P < 0.001) than in the 0 ppm B treatment (13.0 mm). Phytotoxicity was observed in boron treatments even at the level considered optimum for strawberry growing. Severity increased with increasing boron concentration but no consistent effect on flower death or flower abortion was observed. In conclusion, strawberry is sensitive to boron toxicity. Calcium may enhance fruit firmness and, consequently, delay grey mould development if calcium penetrates the fruit. Postharvest calcium treatment tended to be more effective in delaying development of grey mould when applied to late-season fruit. Calcium lactate is a potential alternative to calcium chloride for reducing decay caused by B. cinerea in strawberry without providing undesirable bitterness. This finding may provide a basis for application in industry. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1331382 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine 2008

Philosophiae Doctor - PhD / Pesticides are designed to control or eliminate pests such as insects, rodents, weeds, bacteria, and fungi. They are used at a global scale for agricultural produce. Although pesticides play a significant role in increasing food production and eliminating diseases, exposure to pesticides may be harmful to non-target organisms. As a result concern over safety and esistance to pesticides has increased and there is pressure to reduce use and search for more environmentally and toxicologically safe and efficacious pesticides. Most pesticides currently in use are synthetic; therefore an alternative to synthetic pesticides is the use of naturally occurring products/ botanicals with pesticidal properties. Two plants indigenous to South African with pesticidal properties were chosen for this study. Dicerothamnus rhinocerotis (D. rhinocerotis) and Galenia africana (G. africana) have potential antifungal properties thus, may have potential use on agricultural produce as fungicides. Galenia africana and D. rhinocerotis extracts inhibit growth of B. cinerea (a fungal pathogen) at concentrations greater than 31.25 mg/ml and 125mg/ml respectively. A major consideration in approving pesticides for use is whether they pose an unreasonable risk to humans and to the environment. Toxicity studies are required to determine the safety of the plant extracts. The purpose of this study was to evaluate potential toxicity of ethanol extracts of D. rhinocerotis and G. africana, which is important when designing practices to reduce or eliminate excess exposure to them. Natural plant products with pesticidal properties could provide an alternative to synthetic pesticides and may thus effectively reduce resistance levels. This first objective of this study was to assess the cytotoxicity of D. rhinocerotis and G. africana on human cell cultures. Human whole blood and the human breast adenocarcinoma cell line (MCF-7) were treated with varying concentrations of the plant extracts and cytotoxicity determined. Cytotoxicity was measured using several biomarkers. Inhibiotory concentration for a 50% effect (IC50) and no observable effect level (NOEL) values were obtained for D. rhinocerotis and G. africana. The results showed that extracts of D. rhinocerotis and G. africana had cytotoxic effects on the cell cultures. The second objective of this study was to determine the ecotoxicity of D. rhinocerotis and G. africana. A series of acute toxicity tests, with effective concentration for a 50% effect (EC50) and lethal concentration for a 50% effect (LC50) as biomarkers, were conducted to estimate the potential environmental effect of the two plants. The tests were carried out using Vibrio fischeri, Selenastrum capricornutum, Daphnia pulex, and Poecilia reticulata as bioindicators. Results obtained showed that G. africana had higher toxicity units than D. rhinocerotis, thus showing that G. africana is more toxic to the aquatic species used as compared to D. rhinocerotis. The third objective of this study was to investigate the immunomodulatory effects of the two plant extracts. This was achieved by using mouse spleen cell cultures. Exposure of pesticides to the immune system may result in alteration of the normal immune functions. The cytokines IFN-γ and IL-4 were used as biomarkers to determine the T-cell activity of the immune system when exposed to the two botanical products. The results obtained showed that both D. rhinocerotis and G. africana decrease of the cytokines interferongamma (IFN-γ) and interleukin-4 (IL-4), thus may have immunotoxic effects. The fourth objective was to investigate the hepatotoxicity of the two plant extracts. Injury to the liver was investigated using a range of clinical biochemical tests that monitor liver enzyme activity and metabolic activity. Primary liver cell cultures were exposed to the plant products in question, after which the biochemical tests were carried out. The enzymes that were monitored were alanine aminotrasferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). The results obtained showed that both of D. rhinocerotis and G. africana may have effects on the liver, as shown by the increased levels of enzymes released from cells upon exposure to plant extracts. The final objective of this study was to investigate the effect of the two plants on the male reproductive system. Injury to the male reproductive system was investigated using testicular cell cultures. Primary cell cultures were stimulated with luteinizing-hormone (LH) and exposed to the plant extracts. LH results in the production of testosterone, thus testosterone was used as a biomarker for assessing reproductive toxicity. The results obtained showed that both of D. rhinocerotis and G. africana have effects on the male reproductive system, as shown by the decreased testosterone secretion. Botanicals provide a simple, inexpensive and environmentally friendly (non-pollution and lesser toxicological concerns) alternative for pest control. However, motivation for the commercial use of botanicals as pesticides requires validating the efficacy of the plant as a pesticide, and also assessing its effects on human health and the environment. An important component of this evaluation involves toxicity studies, which enables cautions of dangerous practices and toxic effects of the plants to be issued. / South Africa

Doctor Educationis / The fungus Botrytis cinerea is an opportunistic pathogen on a wide variety of crops, causing a disease known as grey mould through infections via wounds or dead plant parts. Synthetic fungicides for controlling this disease are fast becoming ineffective due to the development of resistance. This, coupled with consumers' world wide becoming increasingly conscious of potential environmental and health problems associated with the build-up of toxic chemicals, (particularly in food products), have resulted in pressure to reduce the use of chemical pesticide volumes as well as its residues. An emerging alternative to random chemical synthesis is the study and exploitation of naturally occurring products with fungicidal properties. One group of compounds known as strobilurins produced by Strobilurus species, woodland basidiomycete fungi, is a good example of this phenomenon. Plants produce an enormous array of secondary metabolites, and it is commonly reasoned that a significant part of this chemical diversity serves to protect plants against plant pathogens. A problem with plant-produced compounds as potential fungicides is that in the natural state, they are generally only weakly active compared to synthetic fungicides. There have been reports on the uses of mixtures of synthetic fungicides for the control of plant pathogenic fungi. When utilized in two-way mixtures, such fungicides may maintain or enhance the level of control of a pathogen at reduced rates for both components utilized in combinations, or alone at normal rates. These studies provide an important precedent for the idea of synergism. For this study, we hypothesize that the addition of plant extracts may enhance the antifungal efficacy of the synthetic strobilurin fungicide, kresoxim-rnethyl against B. cinerea. We selected South African medicinal plant species such as Artemesia afra, Elyptropappus rhinocerotis, Galenia africana, Hypoxis hemerocallidea, Siphonochilus aetheopicus, Sutherlundia frutescence, Tulbaghia violacea and Tulbaghia alliaceae for this study. For the in vitro study, indigenous medicinal plant extracts were prepared at twofold dilution concentrations and combined with kresoxim-rnethyl at concentrations of 0.25 and 0.5% (w/v). The B. cinerea mycelial plug assays showed potent antifungal inhibitory effects with the plant extract and kresoxim-rnethyl mixtures. Further analyses of the mixtures indicate synergistic effects between the fungicide and plant extracts. I surmise that these in vitro effects are also achievable in vivo. Combinations of these agents represent an attractive avenue for the development of new management strategies for controlling B. cinerea in the future. A second study was conducted to analyse the final dose rates for synergistic reactions for combinations of kresoxim-methyl and medicinal plant extracts against B. cinerea in vivo. A series of two-fold concentrations of medicinal plant extracts were combined with kresoxim-methyl to conduct decay inhibition studies on Granny Smith apples. Synergistic effects were observed for many of the kresoxim-methyl and plant extract combinations. I, therefore, came to the conclusion that indigenous South African plant species produce modulators that potentiate the activity of fungicides. Whether these synergistic effects are due to the inhibition of fungal multi-drug resistant pumps require further studies at the molecular level. However, these inhibitory effects are likely to be advantageous for developing fungicide formulations and application strategies with low toxicity effects on the environment. This approach not only makes it possible to reduce fungicide concentrations while maintaining adequate decay control, but also ensures a reduction of the chemical residue on the fruit.

Philosophiae Doctor - PhD / Pesticides are designed to control or eliminate pests such as insects, rodents, weeds, bacteria, and fungi. They are used at a global scale for agricultural produce. Although pesticides playa significant role in increasing food production and eliminating diseases, exposure to pesticides may be harmful to non-target organisms. As a result concern over safety and resistance to pesticides has increased and there is pressure to reduce use and search for more environmentally and toxicologically safe and efficacious pesticides. Most pesticides currently in use are synthetic; therefore an alternative to synthetic pesticides is the use of naturally occurring products! botanicals with pesticidal properties. Two plants indigenous to South African with pesticidal properties were chosen for this study. Dicerothamnus rhinocerotis (D. rhinocerotis) and Galenia africana (G. africana) have potential antifungal properties thus, may have potential use on agricultural produce as fungicides. Galenia africana and D. rhinocerotis extracts inhibit growth of B. cinerea (a fungal pathogen) at concentrations greater than 31.25 mg/ml and 125mg/ml respectively. A major consideration in approving pesticides for use is whether they pose an unreasonable risk to humans and to the environment. Toxicity studies are required to determine the safety of the plant extracts. The purpose of this study was to evaluate potential toxicity of ethanol extracts of D. rhinocerotis and G. africana, which is important when designing practices to reduce or eliminate excess exposure to them. Natural plant products with pesticidal properties could provide an alternative to synthetic pesticides and may thus effectively reduce resistance levels. This first objective of this study was to assess the cytotoxicity of D. rhinocerotis and G. africana on human cell cultures. Human whole blood and the human breast adenocarcinoma cell line (MCF-7) were treated with varying concentrations of the plant extracts and cytotoxicity determined. Cytotoxicity was measured using several biomarkers. Inhibiotory concentration for a 50% effect (ICso) and no observable effect level (NOEL) values were obtained for D. rhinocerotis and G. africana. The results showed that extracts of D. rhinocerotis and G. africana had cytotoxic effects on the cell cultures. The second objective of this study was to determine the ecotoxicity of D. rhinocerotis and G. africana. A series of acute toxicity tests, with effective concentration for a 50% effect (ECso) and lethal concentration for a 50% effect (LCso) as biomarkers, were conducted to estimate the potential environmental effect of the two plants. The tests were carried out using Vibrio fischeri, Selenastrum capricornutum, Daphnia pulex, and Poecilia reticulata as bioindicators. Results obtained showed that G. africana had higher toxicity units than D. rhinocerotis, thus showing that G. africana is more toxic to the aquatic species used as compared to D. rhinocerotis. The third objective of this study was to investigate the immunomodulatory effects of the two plant extracts. This was achieved by using mouse spleen cell cultures. Exposure of pesticides to the immune system may result in alteration of the normal immune functions. The cytokines IFN-y and IL-4 were used as biomarkers to determine the T-cell activity of the immune system when exposed to the two botanical products. The results obtained showed that both D. rhinocerotis and G. africana decrease of the cytokines interferon gamma (IFN-y) and interleukin-4 (IL-4), thus may have immunotoxic effects. The fourth objective was to investigate the hepatotoxicity of the two plant extracts. Injury to the liver was investigated using a range of clinical biochemical tests that monitor liver enzyme activity and metabolic activity. Primary liver cell cultures were exposed to the plant products in question, after which the biochemical tests were carried out. The enzymes that were monitored were alanine aminotrasferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). The results obtained showed that both of D. rhinocerotis and G. africana may have effects on the liver, as shown by the increased levels of enzymes released from cells upon exposure to plant extracts. The final objective of this study was to investigate the effect of the two plants on the male reproductive system. Injury to the male reproductive system was investigated using testicular cell cultures. Primary cell cultures were stimulated with luteinizing-hormone (LH) and exposed to the plant extracts. LH results in the production of testosterone, thus testosterone was used as a biomarker for assessing reproductive toxicity. The results obtained showed that both of D. rhinocerotis and G. africana have effects on the male reproductive system, as shown by the decreased testosterone secretion. Botanicals provide a simple, inexpensive and environmentally friendly (non-pollution and lesser toxicological concerns) alternative for pest control. However, motivation for the commercial use of botanicals as pesticides requires validating the efficacy of the plant as a pesticide, and also assessing its effects on human health and the environment. An important component of this evaluation involves toxicity studies, which enables cautions of dangerous practices and toxic effects of the plants to be issued.

Nitrogen (N) fertilization is known to influence the susceptibility of many plants to a variety of diseases. In the case of diseases caused by Botrytis cinerea, the role of N fertilization appears to be variable, with high levels either fostering or reducing severity depending on the studies. To test whether this variability could be due to possible differences in the host plants, inoculum pressure or in the behavior of different strains of the pathogen, studies were carried out to investigate the effect of different N fertilization regimes on the susceptibility of tomato and lettuce to six isolates of B. cinerea. Possible epidemiological effects of N fertilization through the sporulation of the pathogen and on the pathogenicity of resulting secondary inoculum were also investigated on tomato. Plants were grown in a soil-less drip-irrigation system. Differential N nutrition ranging from 0.5 to 30 mM NO3- was applied for the last four weeks prior to inoculation on the leaves (lettuce) or on leaf pruning wounds (tomato) and incubation of the plants in conditions conducive to disease development. On the tomato stems, disease onset was delayed and overall severity was lower for all isolates on plants with higher N inputs, regardless of inoculum concentration. However, the rate of stem lesion expansion was differentially affected depending on the strains, decreasing with increasing N fertilization levels for the more aggressive isolates, while increasing for the less aggressive isolates.In contrast with tomato, high N fertilization increased disease severity on lettuce for all isolates tested. On tomato plant tissue, sporulation of B. cinerea decreased significantly with increasing N fertilization up to 15-30 mM NO3- and the pathogenicity of the spores was significantly influenced by the nutritional status of their production substrate. It was highest for spores produced on plants with very low or very high N fertilization (0.5 or 30 mM NO3-) and lowest for those from plants with moderate levels of N fertilization. Plant fertilization also strongly affected the efficacy of two biocontrol agents (Trichoderma atroviride and Microdochium dimerum) to protect pruning wounds of tomato against B. cinerea. The highest levels of protection were obtained with high N fertilization and related to a delay in symptom development on the stems, sometimes associated with a slowdown in lesion expansion. Histological studies showed that the decrease in disease severity at high N fertilization was associated to structural alteration of Botrytis mycelial cells. In the presence of a biocontrol agent, the effect on the pathogen was further associated to vacuolisation, glycogen deposition and mycelial cell death. Hypotheses to explain these results are discussed in light of the possible physiological effects of nitrogen fertilization on nutrient availability for the pathogen in the host tissue and of possible production of defense metabolites by the plant. These results also open new possibilities for including the manipulation of N fertilization as a tool for the integrated protection of vegetable crops

Waxflower (Chamelaucium spp. and hybrids) is the singlemost important Australian export cut-flower. The major problem in waxflower trading is flower abscission after harvest. While several factors are involved, ethylene production resulting from preharvest infection with the fungus Botrytis cinerea is the most important cause. The general objectives of this study were to investigate the biology of Botrytis infecting waxflower flowers and potential elicitation of host defence against this pathogen. Effects of anti-ethylene and S-carvone treatments on Botrytis-induced flower abscission were also evaluated. Infection of flowers by Botrytis was studied on two waxflower cvs. Mullering Brook and My Sweet Sixteen using light and electron microscopy. Conidial germination and protoappressorial formation occurred within 8 h post-inoculation (hpi). Infection of most floral organs, including petals, anthers and filaments, stigma, and hypanthium, was within 24 hpi. Infection cushions on stamen bases were formed at 36 hpi by saprophytic hyphae that originated from anthers. This infection route probably gives rise to the typical tan-coloured Botrytis symptoms that appear to radiate from this part of the flower. Subcuticular hyphae were present at very high density near stamen bases. They evidently resulted at multiple penetrations from single infection cushions. Flower abscission occurred at 72 hpi. At this time, floral tube tissues remained uninfected. This temporal pattern infers the possible transmission of a signal (e.g. ethylene) upon Botrytis infection (6–36 hpi) that intiates a defence response of shedding infected flowers (72 hpi). Susceptibility of waxflower before and after harvest to B. cinerea under various environmental conditions (laboratory, greenhouse, and field) was investigated. Flowers, either on plants or on cut stems showed similar susceptibility to B. cinerea and abscised under cool temperatures (~20 ºC) and high humidity (>95% RH) conditions following infection. Compared to cv. Mullering Brook, cv. My Sweet Sixteen was somewhat more resistant to B. cinerea infection under field conditions. Constitutive and inducible antifungal compounds in waxflower flower tissues were screened in cvs. CWA Pink, Stephan’s Delight, Mullering Brook and My Sweet Sixteen using thin layer chromatography bioassays with isolates of B. cinerea and Alternaria alternata (pathogenic) and Cladosporium cladosporioides (non-pathogenic). Common inhibition zone observed at Rf 0.28–0.38, 0.46–0.56 and 0.67–0.76 contained phenolic compounds. There were at least five (cv. Mullering Brook) and one (cv. My Sweet Sixteen) inducible antifungal phenolic compounds as judged by increases in inhibition area as a result of B. cinerea infection and methyl jasmonate treatment. The total areas of B. cinerea- and MeJA-induced inhibition zones were approximately 2.0- and 2.5-folds greater, respectively, than zones from control flowers. Preharvest sprays of three different known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH), and silicon (Si), were applied to waxflower cvs. Mullering Brook and My Sweet Sixteen plants. BTH or Si sprays generally had no significant effect on postharvest Botrytis severity on either cultivar. MeJA sprays did not reduce B. cinerea on cv. Mullering Brook. MeJA slightly suppressed B. cinerea on cv. My Sweet Sixteen at 500 and 750 µM. Overall, field applications of these host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut sprigs. Moreover, they did not appreciably suppress B. cinerea or associated postharvest floral abscission. The efficacy of combined elicitor treatments and combined pre- and postharvest MeJA treatments were assessed. Preharvest foliar applications of MeJA (1000 µM; 2 or 4 times), MeJA (1000 µM) combined with BTH (150 mg/L), and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress postharvest B. cinerea development and flower abscission from harvested sprigs. A pre- plus post-harvest 1000 µM MeJA spray treatment consistently but only slightly suppressed B. cinerea infection on flowers from both pot- and field-grown plants. Pre- and post-harvest MeJA treatments reduced B. cinerea development, but increased flower abscission. Combined MeJA and anti-ethylene treatments were then screened for potential to suppress B. cinerea while preventing flower abscission. However, the combined MeJA and 1-MCP treatment reduced neither Botrytis disease nor flower abscission on sprigs from pot- and field-grown plants. The combined MeJA and STS treatment reduced disease severity for up to 6 days on sprigs harvested from pot-grown plants but tended to increase Botrytis severity on sprigs from field-grown plants 6 days after inoculation. Antifungal effects of the essential oil S-carvone against B. cinerea germination and mycelial growth were demonstrated in vitro. Inhibition increased with increasing S-carvone concentrations from 0.64 mM to 5.08 mM. However, in planta, S-carvone concentrations in this range did not affect either Botrytis disease levels or flower abscission on cut waxflower flowers.