Abstract
Dieback of fruiting spurs, stems and branches is widespread in walnut (Juglans regia) orchards in Australia, resulting in significant yield loss. Fungal species belonging to the family Botryosphaeriaceae have been implicated as the causative agents in similar diebacks in other countries. Against this background, this research was designed to identify the species of Botryosphaeriaceae present in walnut orchards in Australia and to investigate their pathogenicity and methods of control.
A national survey collected walnut tissues from 14 orchards representing all the major walnut-growing regions in Australia. Botryosphaeriaceae species were recovered from both symptomatic and asymptomatic samples from nine of the orchards. DNA sequencing confirmed the presence of five Botryosphaeriaceae species, namely Diplodia seriata, Dothiorella omnivora, Neofusicoccum macroclavatum, N. parvum and Spencermartinsia viticola. Of the 106 Botryosphaeriaceae isolates recovered, D. seriata and N. parvum were the most prevalent, constituting 53 and 40% respectively.
Pathogenicity studies on detached and attached stems confirmed that Neofusicoccum spp. were the most virulent followed by D. seriata, with the former causing lesions almost three times longer than D. seriata. In addition to inter-species variation, intra-species variation in virulence was observed among the isolates of D. seriata and N. parvum. Both species were re-isolated beyond the dieback lesion; the lesion length as well as pathogen progression beyond the lesion increased over time. Colonisation of N. parvum beyond the lesion edge was greater compared to D. seriata. For N. parvum, the mean colonisation distance beyond the lesion edges was 44 mm in 8 months while D. seriata was re-isolated only within the first 10 mm wood segment beyond the margins of the lesion.
Wound healing was observed in the trunks of the glasshouse plants and 12 months after wounded inoculation, in the absence of any external lesion, both pathogens continued to move through the xylem, causing internal discolouration of the vascular tissues. Pathogen recovery from the side shoots indicated that N. parvum had entered the side shoots and killed more buds than D. seriata.
Both mycelia and conidia were found to be pathogenic, with mycelia showing higher infection rates. There was a strong linear relationship between the dieback length caused by the two types of inocula. Experiments on inoculum dose indicated that as few as two conidia were able to infect wounded tissues. Although wounds were susceptible to infection for more than 4 months post wounding for N. parvum and 1 month for D. seriata, the highest disease incidence occurred in the first week following wounding. The attached and detached stem assays demonstrated that the conidia and mycelia of N. parvum and D. seriata were able to enter non-wounded tissues and cause latent infection. However, severity of the lesions and rate of symptom developments in wounded inoculations confirmed wounds as the major entry points for both species.
Temperature and relative humidity (RH) were found to have a significant effect on the growth of conidia and mycelia of the Botryosphaeriaceae isolates. This study estimated that conidial germination of the prevalent species took place between 3.3 and 37.2 °C, while mycelial growth took place between 8.6 and 35.5 °C. These temperatures are not uncommon during most part of the growing season in the walnut-growing regions of Australia. Conidial germination of N. parvum was less than 4% at 5 °C, and slow at 10 and 15 °C (< 50% germination) at 24 h, but reached 92% at 30 °C. On detached stems, N. parvum isolates were the most virulent at 31.3 °C while the D. seriata isolates were the most virulent at 28.2 °C, positioning N. parvum as a warm climate pathogen in Australia.
Optimum temperature alone was not adequate for germination of conidia; high levels of RH were also required. Maximum conidial germination was recorded at 100% RH, and was diminished at lower levels, with no germination at 84% RH. At 100% RH, 80% of the conidia germinated after 2 h, indicating that a wet period for 2 h at suitable temperature has the risk of triggering disease incidence especially when conidia land on wounded tissues.
Experiments on detached stems of six scion cultivars of J. regia and four rootstock species of Juglans confirmed that none were resistant to D. seriata and N. parvum. Cultivars Lara, Howard and Chandler were less susceptible compared to Serr, Tulare and Vina. Among the rootstocks, Paradox (J. hindsii x J. regia) was the most susceptible to D. seriata and N. parvum compared to the other three rootstocks. The experiment on glasshouse plants of cultivars Chandler, Howard and Lara indicated that Lara was less susceptible to D. seriata and N. parvum compared to Chandler and Howard.
Younger stems of walnut plants were more susceptible to infection by D. seriata and N. parvum than the older stems, in both detached and attached stem assays. Flowers and fruits inoculated with the prevalent species at various stages of development indicated the following pattern in susceptibility to infection; (1) early growth stage: first 6–8 weeks from bloom when flower development and fruit set take place. Premature flower and fruit drop was significantly higher in the inoculated flowers and fruits, indicating high susceptibility; (2) mid-stages of growth: during 8–15 weeks from bloom when the kernels grow and the shell sclerification takes place. Pathogen recovery from inoculated fruits was less compared to the other stages; and (3) later stages of growth post shell sclerification: during 15–22 weeks from bloom (last 5–7 weeks leading to harvest), where susceptibility of the hull increased again, and kernel infection was significantly higher than the mid-stage of growth. This pattern pointed to two critical periods of infection in the phenology of fruit development, with the mid-stage of development being less susceptible to infection.
With respect to dieback management, this research evaluated the inhibitory effect of 10 fungicides on mycelial growth of nine Botryosphaeriaceae isolates of five species and conidial germination of six isolates of the two prevalent species. In vitro experiments demonstrated that nine of the 10 fungicides were effective in reducing mycelial growth and all were effective in inhibiting conidial germination, to varying extents. Tebuconazole, prochloraz manganese chloride, fluazinam, fludioxonil and pyraclostrobin were the most effective at inhibiting mycelial growth whereas pyraclostrobin, fluxapyroxad, fluopyram, penthiopyrad and tebuconazole were the most effective at inhibiting conidial germination. In planta experiments with five fungicides confirmed that preventative applications had greater efficacy than the curative against D. seriata and N. parvum. A field trial with four commercial formulations demonstrated that tebuconazole formulations provided protection against N. parvum for the longest period. The field trial also confirmed the efficacy of pyraclostrobin and the inhibitory effect of fluazinam.
In addition to studies on the potential use of fungicides for dieback management, this study assessed four microbial biocontrol agents (BCAs) for their inhibitory effect on the prevalent species and found them to be more effective as preventative treatments. Bacillus-based products were the most effective followed by Trichoderma spp. and Aureobasidium pullulans. The present study highlighted that as time progressed, the preventative efficacy of the BCAs increased. On detached stems, the Bacillus-based treatments achieved a mean percent disease control of 62–64% on D. seriata and 45–48% on N. parvum, when applied 7 days before pathogen inoculation. The other BCAs that were not effective on inoculations performed 24 h after treatment also showed higher disease control with progression of time and achieved mean percent disease control that varied between 30 and 58%, when applied 7 days before inoculation.
This study increases our understanding of some of the key aspects of pathogenicity and progression of Botryosphaeriaceae species and dieback in Australian walnut orchards. These novel findings have implications for walnut orchard management practices, and in particular cultural practices. Findings on the efficacy of the 10 fungicides and four microbial BCAs assessed in this research open up opportunities for a wider selection of disease management options for sustainable disease control than were previously available to the Australian walnut industry. Collectively the findings from this project can be incorporated into an integrated management strategy that can be developed for reducing the occurrence of Botryosphaeria dieback in Australian walnut orchards.
A national survey collected walnut tissues from 14 orchards representing all the major walnut-growing regions in Australia. Botryosphaeriaceae species were recovered from both symptomatic and asymptomatic samples from nine of the orchards. DNA sequencing confirmed the presence of five Botryosphaeriaceae species, namely Diplodia seriata, Dothiorella omnivora, Neofusicoccum macroclavatum, N. parvum and Spencermartinsia viticola. Of the 106 Botryosphaeriaceae isolates recovered, D. seriata and N. parvum were the most prevalent, constituting 53 and 40% respectively.
Pathogenicity studies on detached and attached stems confirmed that Neofusicoccum spp. were the most virulent followed by D. seriata, with the former causing lesions almost three times longer than D. seriata. In addition to inter-species variation, intra-species variation in virulence was observed among the isolates of D. seriata and N. parvum. Both species were re-isolated beyond the dieback lesion; the lesion length as well as pathogen progression beyond the lesion increased over time. Colonisation of N. parvum beyond the lesion edge was greater compared to D. seriata. For N. parvum, the mean colonisation distance beyond the lesion edges was 44 mm in 8 months while D. seriata was re-isolated only within the first 10 mm wood segment beyond the margins of the lesion.
Wound healing was observed in the trunks of the glasshouse plants and 12 months after wounded inoculation, in the absence of any external lesion, both pathogens continued to move through the xylem, causing internal discolouration of the vascular tissues. Pathogen recovery from the side shoots indicated that N. parvum had entered the side shoots and killed more buds than D. seriata.
Both mycelia and conidia were found to be pathogenic, with mycelia showing higher infection rates. There was a strong linear relationship between the dieback length caused by the two types of inocula. Experiments on inoculum dose indicated that as few as two conidia were able to infect wounded tissues. Although wounds were susceptible to infection for more than 4 months post wounding for N. parvum and 1 month for D. seriata, the highest disease incidence occurred in the first week following wounding. The attached and detached stem assays demonstrated that the conidia and mycelia of N. parvum and D. seriata were able to enter non-wounded tissues and cause latent infection. However, severity of the lesions and rate of symptom developments in wounded inoculations confirmed wounds as the major entry points for both species.
Temperature and relative humidity (RH) were found to have a significant effect on the growth of conidia and mycelia of the Botryosphaeriaceae isolates. This study estimated that conidial germination of the prevalent species took place between 3.3 and 37.2 °C, while mycelial growth took place between 8.6 and 35.5 °C. These temperatures are not uncommon during most part of the growing season in the walnut-growing regions of Australia. Conidial germination of N. parvum was less than 4% at 5 °C, and slow at 10 and 15 °C (< 50% germination) at 24 h, but reached 92% at 30 °C. On detached stems, N. parvum isolates were the most virulent at 31.3 °C while the D. seriata isolates were the most virulent at 28.2 °C, positioning N. parvum as a warm climate pathogen in Australia.
Optimum temperature alone was not adequate for germination of conidia; high levels of RH were also required. Maximum conidial germination was recorded at 100% RH, and was diminished at lower levels, with no germination at 84% RH. At 100% RH, 80% of the conidia germinated after 2 h, indicating that a wet period for 2 h at suitable temperature has the risk of triggering disease incidence especially when conidia land on wounded tissues.
Experiments on detached stems of six scion cultivars of J. regia and four rootstock species of Juglans confirmed that none were resistant to D. seriata and N. parvum. Cultivars Lara, Howard and Chandler were less susceptible compared to Serr, Tulare and Vina. Among the rootstocks, Paradox (J. hindsii x J. regia) was the most susceptible to D. seriata and N. parvum compared to the other three rootstocks. The experiment on glasshouse plants of cultivars Chandler, Howard and Lara indicated that Lara was less susceptible to D. seriata and N. parvum compared to Chandler and Howard.
Younger stems of walnut plants were more susceptible to infection by D. seriata and N. parvum than the older stems, in both detached and attached stem assays. Flowers and fruits inoculated with the prevalent species at various stages of development indicated the following pattern in susceptibility to infection; (1) early growth stage: first 6–8 weeks from bloom when flower development and fruit set take place. Premature flower and fruit drop was significantly higher in the inoculated flowers and fruits, indicating high susceptibility; (2) mid-stages of growth: during 8–15 weeks from bloom when the kernels grow and the shell sclerification takes place. Pathogen recovery from inoculated fruits was less compared to the other stages; and (3) later stages of growth post shell sclerification: during 15–22 weeks from bloom (last 5–7 weeks leading to harvest), where susceptibility of the hull increased again, and kernel infection was significantly higher than the mid-stage of growth. This pattern pointed to two critical periods of infection in the phenology of fruit development, with the mid-stage of development being less susceptible to infection.
With respect to dieback management, this research evaluated the inhibitory effect of 10 fungicides on mycelial growth of nine Botryosphaeriaceae isolates of five species and conidial germination of six isolates of the two prevalent species. In vitro experiments demonstrated that nine of the 10 fungicides were effective in reducing mycelial growth and all were effective in inhibiting conidial germination, to varying extents. Tebuconazole, prochloraz manganese chloride, fluazinam, fludioxonil and pyraclostrobin were the most effective at inhibiting mycelial growth whereas pyraclostrobin, fluxapyroxad, fluopyram, penthiopyrad and tebuconazole were the most effective at inhibiting conidial germination. In planta experiments with five fungicides confirmed that preventative applications had greater efficacy than the curative against D. seriata and N. parvum. A field trial with four commercial formulations demonstrated that tebuconazole formulations provided protection against N. parvum for the longest period. The field trial also confirmed the efficacy of pyraclostrobin and the inhibitory effect of fluazinam.
In addition to studies on the potential use of fungicides for dieback management, this study assessed four microbial biocontrol agents (BCAs) for their inhibitory effect on the prevalent species and found them to be more effective as preventative treatments. Bacillus-based products were the most effective followed by Trichoderma spp. and Aureobasidium pullulans. The present study highlighted that as time progressed, the preventative efficacy of the BCAs increased. On detached stems, the Bacillus-based treatments achieved a mean percent disease control of 62–64% on D. seriata and 45–48% on N. parvum, when applied 7 days before pathogen inoculation. The other BCAs that were not effective on inoculations performed 24 h after treatment also showed higher disease control with progression of time and achieved mean percent disease control that varied between 30 and 58%, when applied 7 days before inoculation.
This study increases our understanding of some of the key aspects of pathogenicity and progression of Botryosphaeriaceae species and dieback in Australian walnut orchards. These novel findings have implications for walnut orchard management practices, and in particular cultural practices. Findings on the efficacy of the 10 fungicides and four microbial BCAs assessed in this research open up opportunities for a wider selection of disease management options for sustainable disease control than were previously available to the Australian walnut industry. Collectively the findings from this project can be incorporated into an integrated management strategy that can be developed for reducing the occurrence of Botryosphaeria dieback in Australian walnut orchards.
Original language | English |
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Qualification | Doctor of Philosophy |
Awarding Institution |
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Supervisors/Advisors |
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Award date | 23 Sept 2024 |
Place of Publication | Australia |
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Publication status | Published - 2024 |