Molecular and Histological Study of Sphaerulina musiva-Populus spp. Interaction

Abstract

Sphaerulina musiva, the causal agent of leaf spot and stem canker, is responsible for critical yield loss of hybrid poplar in agroforestry. This research examined quantification of S. musiva in host tissue, and infection of leaf tissue, plus gene expression between resistant and susceptible poplar genotypes. This study reports the first use of a multiplexed hydrolysis probe qPCR assay for faster and accurate quantification of S. musiva in inoculated stems of resistant, moderately resistant and susceptible genotypes of hybrid poplar at three different time points -1 wpi (weeks post-inoculation ), 3 wpi and 7 wpi. This assay detected significant differences in the level of resistance among the different clones at 3 wpi (p < 0.001) and significant differences among isolates at 1 wpi (p < 0.001), that were not detected by visual phenotyping. Histological and biochemical comparisons were made between resistant and susceptible genotypes inoculated with conidia of S. musiva in order to study the mode of leaf infection and defense response of hybrid poplar. Leaf infection was examined at 48 h, 96 h, 1 wpi, 2 wpi and 3 wpi using scanning electron microscopy (SEM) and fluorescent and laser scanning confocal microscopy. Infection process of S. musiva on Populus spp. was further characterized by transforming S. musiva with red fluorescent protein through Agrobacterium tumefaciens. Results indicated that there was no difference in pre-penetration processes, however, differences were observed in post-penetration between resistant and susceptible genotypes. The host response was also studied by examining the accumulation of hydrogen peroxide (H2O2) using fluorescent microscopy after DAB staining, and a significant difference (p < 0.0001) was observed by 2 wpi. The molecular mechanism underlying host-pathogen interaction was elucidated by studying temporal differentially expressed genes of both the interacting organisms, simultaneously, using RNA-seq. Genes involved in cell wall modification, antioxidants, antimicrobial compounds, signaling pathways, ROS production and necrosis were differentially expressed in the host. In the pathogen, genes involved in CWDE, nutrient limitation, antioxidants, secretory proteins and other pathogenicity genes were differentially expressed. The results from this research provide an improved understanding of poplar resistance/susceptibility to S. musiva.