Resumen de The red leaf blotch of almond, caused by polystigma amygdalinum, in catalonia: biology and epidemiology
Major biological aspects of the red leaf blotch (RLB) disease of almond, caused by the ascomycete Polystigma amygdalinum, were studied in this PhD Thesis. This disease is one of the most important foliar diseases in most almond-growing regions in the Mediterranean basin and the Middle East, since severe infections may cause a premature defoliation of the tree. Those key aspects were related to i) the epidemiology of P. amygdalinum in two almond-growing regions in Spain, ii) the reliable detection and quantification of the pathogen through quantitative real-time PCR (qPCR), in addition to iii) the search for the molecular and biochemical basis for the differential cultivar susceptibility to the RLB.
The primary inoculum of P. amygdalinum was available in extended periods in the growing season (January to August). The infectivity period in both Spanish almond-growing areas extended from March to mid-June when temperatures are in the range 10 to 20°C. The incubation period was variable among all years studied (2 to 12 weeks), but in general decreased with time in the season. For the detection and quantification of P. amygdalinum, a primer pair was designed based on the ITS region of the fungal rDNA, and this was shown to be highly specific and sensitive, enabling to detect the pathogen accurately in naturally infected leaves showing different stages of RLB development. Detection of a minimum of 12 pg of P. amygdalinum DNA, and seven ascospores in artificially-prepared ascospore suspensions were determined to be the limits of detection. The development of a qPCR-based protocol allowed to quantify the amounts of ascospores on plastic tapes which are commonly used in volumetric air samplers as well as the fungal colonization in infected leaves. We explored the plant defence mechanisms related to the cultivar susceptibility by identifying some relevant physical and chemical strategies involved in the almond tolerance to P. amygdalinum. Thus, we studied the regulation of seven defence-related genes as well as the lignin deposition in two almond cultivars (tolerant ‘Mardía’ and susceptible ‘Tarraco’) with highly differential response to RLB. ‘Mardía’ displayed an up-regulation of the CAD and DFN1 genes at early stages of RLB symptom expression, with further lignin deposition in the fungal-colonized tissues. In contrast, ‘Tarraco’ mainly triggered the up-regulation of HQT and LDOX genes, related to chlorogenic acid and anthocyanin biosynthesis pathways, respectively, while lignin deposition was not clearly noticed.
Our results open a door to future research focused on developing an accurate epidemiological model to predict infection risk events for RLB, by integrating the monitoring of primary inoculum with the aid of volumetric air samplers and the qPCR-based detection method developed in this thesis. In addition, increasing the knowledge on the genetic and molecular bases of the resistance/tolerance to RLB would help in obtaining new almond cultivars with improved tolerant profiles against the RLB. This way, we aim at facing the control of the red leaf blotch of almond in an integrated and sustainable way.
