Identifying the critical inocula sources for systemic downy mildew of opium poppy

Opium poppy is grown commercially for the extraction of medicinal alkaloid compounds. Australia is currently one of world‚ÄövÑv¥s largest producers of licit opiates. The majority of Australian poppy production occurs in Tasmania. Downy mildew is a major disease affecting Australian poppy crops. The first records of downy mildew in Australia were formally reported in 1996 with plants showing localized leaf necrosis due to infection with Peronospora meconopsidis. In the mid-2010s, new symptoms in Tasmanian field crops were found with poppies showing stunted growth and chlorotic and distorted leaves which could lead to early plant death. Peronospora somniferi was identified to cause these new systemic symptoms. Spread of systemic downy mildew in opium poppy can occur via primary inocula (oospores) within soil and seed and by secondary inocula (conidia) moved by air. The major aim of this thesis was to identify the critical inocula sources that drive systemic downy mildew disease epidemics in opium poppy. This will aid development of management strategies to control the disease and to maintain the sustainability of Australian poppy industry. To identify the relative impact of primary and secondary infection on disease progress and alkaloid yield, two field trials were conducted in consecutive seasons. Half of the plots were treated with fungicide to minimize secondary spread and the other half were left untreated. Incidence of visual systemic symptoms in non-treated plots relative to treated plots over time was not statistically significant. Regardless of whether plots were treated or not, increases in latent infection were observed over time in the field crops. Downy mildew sporulation observed in potted trap plants confirmed the occurrence of secondary spread during both seasons. However, there was no significant increase in trap plant disease incidence over time, suggesting that the level of secondary spread observed was not sufficient to drive the disease epidemics. Additionally, capsule biomass and alkaloid yield were higher in fungicide treated plots but the increases were not significant in either season. This study suggested that systemic downy mildew in field poppy crops was predominantly driven by soil-borne primary inocula (oospores). Simple sequence repeat (SSR) markers were developed to evaluate the genetic variation of P. somniferi in field populations. In total, 102 SSRs were tested and 34 successfully amplified P. somniferi. From the 34 SSRs amplified, seven SSR markers were informative. Sanger sequencing demonstrated that one of the seven markers was perfect while four of seven markers were imperfect, and two of seven markers exhibited non-SSR locus variation. Genotyping of five DNA samples from individual leaves showed that only 18 of 30 leaves were infected with a single multi-locus genotype. Thus, a DNA extraction from a single leaf lesion couldn‚ÄövÑv¥t be assumed to consist of single multi-locus genotype to be used in field population studies. Temporal change in the genetic structure of P. somniferi in the field trials was examined using the developed polymorphic SSR markers. In each season, allelic diversity increased with time in both field and trap plants in non-treated plots, relative to the treated plots. Analysis of molecular variance (AMOVA) demonstrated significant (p = 0.001) temporal genetic variance (˜ìvâ = 0.01) in 2020/21. Additionally, private alleles were recovered only from the non-treated field plant population at 124 days after sowing (DAS) in 2020/21. In the 2019/20 season, private alleles were recovered from both treated and non-treated field plant populations at 58DAS and from the non-treated trap plant population at 124DAS. These indicated the potential for secondary inocula blowing in from neighbouring fields. However, the low frequency of private alleles suggests that the external source of conidia may not be sufficient to drive the disease epidemics. In addition, the AMOVA analysis demonstrated significant genetic variation (˜ìvâ = 0.02; p = 0.012) between treatment populations within the 2019/20 season. UPGMA clustering did not support differentiation between individual sub-populations. Therefore, the genetic structure of P. somniferi in field populations may be mostly related to the initial primary infections. Hence, this study reconfirmed the importance of oospore infection from the field trials based on visual disease assessment. The final studies examined the impact of soil type and plant development stage on disease spread under controlled conditions. Soil from three Tasmanian poppy growing areas were artificially infested with infected leaf material or left non-infested to identify the relative transmissibility of P. somniferi. Soil textures were classified as coarse sand, sandy clay loam, and clay. Coarse sand and clay soils demonstrated a higher reduction in plant emergence (~32-43 %) and height (34-58 %) than the sandy clay loam soil. The highest percentage of plants with systemic downy mildew symptoms were found in sandy clay loam, though the incidence was low (~3.6 %). This suggests that soil type, specifically sandy and clay soils, appear to influence soil-borne P. somniferi transmission to germinating poppy seedlings. A greenhouse trial was conducted to identify the optimal plant development stage/s for secondary infection via air-borne conidia. Separate plants at four plant development stages (10-leaf, large rosette, flower, and capsule stages) were inoculated at different times with P. somniferi conidia and were assessed for infection until crop maturity. A chlorotic lesion was found initiated at the 10-leaf stage, but it did not develop further over time. At 10-leaf stage, conidial germination was observed via histological examination in 2/3 plants. Histological observations were further confirmed via PCR results. The statistical analysis on PCR results showed that significantly higher P. somniferi incidence (77.8%) was observed at the 10-leaf stage relative to other plant development stages tested. This suggests that the 10-leaf stage is the most susceptible development stage to secondary infection via air-borne conidia. However, all tested plant development stages were susceptible to secondary infection. Overall, the above studies identified primary inocula sources are the most important in driving epidemics of systemic downy mildew in opium poppy. Therefore, developing strategies focused on soil-borne oospores, rather than air-borne conidia, is important to improve the efficacy of control measures in commercial poppy fields. Possible suggestions are extending crop rotation periods and removal of almost all crop residues prior to and after cropping.