Search for a command to run...
Grapevine trunk diseases (GTDs) are considered a “disease complex” caused by multiple co-existing pathogens that accelerate vine decline and reduce yield. In Australia, Botryosphaeria dieback (BD) is considered one of the major GTDs, with Diplodia seriata and Neofusicoccum parvum as the most prevalent and virulent causal organisms. However, recent microbial profiling of vineyards and nurseries revealed a higher prevalence of pathogens associated with Petri disease (PD), such as Phaeomoniella chlamydospora, Phaeoacremonium minimum, and Cadophora luteo-olivacea, than BD pathogens. Several studies have shown the frequent co-occurrence of both BD and PD pathogens in the same host tissue, especially in nurseries and young vineyards. Given this increasing prevalence of PD pathogens in Australian vineyards and the frequent co-occurrence of BD and PD pathogens in grapevines, it is critical to investigate their interactions and how they may influence symptom development and disease progression. <br/><br/>This study investigated, in vitro, the antagonistic and synergistic interactions of the GTD pathogens both within (BD x BD and PD x PD) and between (BD x PD) groups. These interactions were assessed through mycelial growth and morphological changes. Within BD, N. parvum displayed antagonism with D. seriata through deadlock interaction and formation of a barrage zone. Within PD, Pm. minimum consistently reduced the mycelial growth of Pa. chlamydospora, along with the concurrent overgrowth by the mycelium of Pm. minimum. Between pathogen groups (BD x PD), a significant reduction in mycelial growth of PD pathogens (Pm. minimum, Pa. chlamydospora, C. luteo-olivacea) was observed when co-inoculated with N. parvum. In contrast, N. parvum showed a notable increase in its mycelial growth when paired with PD pathogens, suggesting possible synergistic effects in vitro.<br/><br/>Optimised singleplex hydrolysis probe-based quantitative real-time (qPCR) assays for BD pathogens were developed, while established assays were used for PD pathogens. The singleplex assay with primers and a probe set for N. parvum was specific at an annealing temperature of 61°C for 30 s, without amplifying non-target species. Similarly, the singleplex assay for D. seriata showed specific amplification at 64°C, 15 s. For both assays, the qPCR conditions to generate standard curves were within the acceptable range for an optimised qPCR reaction. Furthermore, the developed duplex qPCR assay for simultaneous detection of the pathogens was suitable and robust for D. seriata but suboptimal for N. parvum. <br/><br/>The in vivo interactions of BD and PD pathogens were investigated using qPCR assays following simultaneous and sequential inoculations on detached canes and potted vines. Within BD, N. parvum notably decreased the mean DNA copies of D. seriata, along with a significant reduction in the mean lesion length of their co-inoculation compared to N. parvum alone. Within PD, Pm. minimum decreased the average DNA copies of both Pa. chlamydospora and C. luteo-olivacea on canes. This antagonistic effect was also evident in potted vines, where Pm. minimum reduced the DNA copies of Pa. chlamydospora when co-inoculated. However, the mean lesion length of their co-inoculation did not differ significantly from either of the single inoculations. Between pathogen groups, all PD pathogens (Pm. minimum, Pa. chlamydospora, C. luteo-olivacea) showed a general reduction in the average DNA copies when co-inoculated with BD pathogens (N. parvum and D. seriata), while both BD pathogens showed increased DNA copies compared to their single inoculation on canes. In potted vines, N. parvum reduced the DNA copies of Pm. minimum in simultaneous inoculation, although the mean lesion length of their co-inoculation did not differ significantly from either of the single inoculations. Overall, sequential inoculations revealed that the pathogens inoculated first showed a competitive advantage towards the pathogen inoculated second. <br/><br/>N. parvum showed the strongest antagonistic potential among the different pathogen combinations evaluated in the study, both in vitro and in vivo. The role of secondary metabolites (SMs) in mediating this antagonistic effect of N. parvum against Pm. minimum was investigated through targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics, along with phytotoxicity bioassays in single and co-culture conditions. The upregulation of (R)-mellein in the co-culture extracts compared to N. parvum single extract was positively correlated with the highest percentage of leaf necroses. Consequently, the production of the metabolites associated with the virulence and pathogenicity of Pm. minimum, such as isosclerone and scytalone, were downregulated and below the limit of detection (LOD) in the same co-culture conditions. This upregulation of (R)-mellein during fungal antagonism between N. parvum and Pm. minimum may enhance the overall virulence of the pathogen complex on grapevine tissue. Further investigation into the role of SMs between N. parvum and Pm. minimum in planta is strongly recommended to confirm the antagonistic-mediated interaction between these pathogens and how it may influence disease severity in the host. <br/><br/>In summary, this research provides compelling evidence of antagonistic and synergistic interactions between BD and PD pathogens. To my knowledge, this study is the first to report a multidisciplinary approach that combines cultural, molecular, and metabolomics methods to investigate interactions among key GTD pathogens. Their interactions may influence pathogen abundance, symptom development, and eventually, disease progression in grapevine, which are critical for nursery and vineyard management and biosecurity purposes in Australian viticulture.