Rhizoctonia solani anastomosis group 3 (AG-3) is the main causative agent of the soil-borne disease known as potato black scurf, which poses a huge threat to potato production. Rapid and accurate identification of R. solani AG-3 isolates in soil and potato seed tubers prior to planting is essential for good production. In this study, a multiplex PCR assay was established for the detection of R. solani AG-3. Two pairs of target-specific primers were designed from sequences for endopolygalacturonase and pyridoxine biosynthesis genes downloaded from GenBank. The main factors influencing PCR amplification, such as annealing temperature and primer concentration, were optimized. Results show that the proposed multiplex PCR assay is highly sensitive and specific for the target genes in the pathogen even when the DNA concentration is reduced to 20 fg/μL. The resulting calibration plot shows a linear relationship between electrophoretic band peaks and genomic DNA concentration (R² = 0.98). The primer specificity was confirmed by applying them to other R. solani AG groups and plant pathogen species on which no amplicons were produced. Using the primers, we successfully detected small amounts of R. solani AG-3 present in soil and potato tuber samples. Taken together, the detection assay developed in this study has high sensitivity, strong specificity, and accuracy and can be used to detect and identify soil and potato seed tubers infected with Rhizoctonia solani AG-3.

Effectors, a group of small proteins secreted by pathogens, play a central role in antagonistic interactions between plant hosts and pathogens. The evolution of effector genes threatens plant disease management and sustainable food production, but population genetic analyses to understand evolutionary mechanisms of effector genes are limited compared to molecular and functional studies. Here we investigated the evolution of the Avr1 effector gene from 111 Phytophthora infestans isolates collected from six areas covering three potato cropping regions in China using a population genetic approach. High genetic variation of the effector gene resulted from diverse mechanisms including base substitution, pre-termination, intragenic recombination and diversifying selection. Nearly 80% of the 111 sequences had a point mutation in the 512th nucleotide (T512G), which generated a pre-termination stop codon truncating 38 amino acids in the C-terminal, suggesting that the C-terminal may not be essential to ecological and biological functions of P. infestans. A significant correlation between the frequency of Avr1 sequences with the pre-termination and annual mean temperature in the collection sites suggests that thermal heterogeneity might be one of contributors to the diversifying selection, although biological and biochemical mechanisms of the likely thermal adaptation are not known currently. Our results highlight the risk of rapid adaptation of P. infestans and possibly other pathogens as well to host resistance, and the application of eco-evolutionary principles is necessary for sustainable disease management in agricultural ecosystems.

Climate change is considered a major threat to society and nature. UV irradiation is the most important environmental genotoxic agent. Thus, how elevated UV irradiation may influence human health and ecosystems has generated wide concern in the scientific community, as well as with policy makers and the public in general. In this study, we investigated patterns and mechanisms of UV adaptation in natural ecosystems by studying a gene-specific variation in the potato late blight pathogen, Phytophthora infestans. We compared the sequence characteristics of radiation sensitive 23 (RAD23), a gene involved in the nucleotide excision repair (NER) pathway and UV tolerance, in P. infestans isolates sampled from various altitudes. We found that lower genetic variation in the RAD23 gene was caused by natural selection. The hypothesis that UV irradiation drives this selection was supported by strong correlations between the genomic characteristics and altitudinal origin (historic UV irradiation) of the RAD23 sequences with UV tolerance of the P. infestans isolates. These results indicate that the RAD23 gene plays an important role in the adaptation of P. infestans to UV stress. We also found that different climatic factors could work synergistically to determine the evolutionary adaptation of species, making the influence of climate change on ecological functions and resilience more difficult to predict. Future attention should aim at understanding the collective impact generated by simultaneous change in several climate factors on species adaptation and ecological sustainability, using state of the art technologies such as experimental evolution, genome-wide scanning, and proteomics.

Protein kinases and phosphatases catalyze the phosphorylation and dephosphorylation of their protein substrates, respectively, and these are important mechanisms in cellular signal transduction. The rice blast fungus Magnaporthe oryzae possesses 6 protein phosphatases of type 2C class, including MoPtc1, 2, 5, 6, 7 and 8. However, only very little is known about the roles of these phosphatases in filamentous fungi. Here in, we deployed genetics and molecular biology techniques to identify, characterize and establish the roles of MoPtc5 and MoPtc7 in M. oryzae development and pathogenicity. We found that during pathogen-host interaction, MoPTC7 is differentially expressed. Double deletion of MoPTC7 and MoPTC5 suppressed the fungal vegetative growth, altered its cell wall integrity and reduced its virulence. The two genes were found indispensable for stress tolerance in the phytopathogen. We also demonstrated that disruption of any of the two genes highly affected appressorium turgor generation and Mps1 and Osm1 phosphorylation levels. Lastly, we demonstrated that both MoPtc5 and MoPtc7 are localized to mitochondria of different cellular compartments in the blast fungus. Taken together, our study revealed synergistic coordination of M. oryzae development and pathogenesis by the type 2C protein phosphatases.

Rice blast caused by Magnaporthe oryzae pathotype is the worst disease that leads to serious food insecurity globally. Understanding rice blast disease pathogenesis is therefore essential for the development of a blast disease mitigation strategy. Reverse phosphorylation mediated by phosphatases performs a vital function in the activation of diverse biological mechanisms within eukaryotic. However, little has been reported on the roles of PP2Cs in the virulence of blast fungus. In this current work, we deployed functional genomics and biochemical approaches to characterize type 2C protein phosphatase MoPtc6 in blast fungus. Deletion of MoPTC6 led to a drastic reduction in conidiophore development, conidia production, hyphal growth, and stress tolerance. Western blotting assay demonstrated that the phosphorylation level of MoOsm1 was decreased while MoMps1 was increased in the MoPtc6 deletion mutant, and comparative transcriptome assay revealed a higher number of expressed genes between mutant and wild type. Localization assay confirmed that MoPtc6 is sub-localized in the cytoplasm of mycelia, spores, and in the appressoria of M. oryzae. Furthermore, disruption of MoPTC6 impaired appressoria turgor pressure and glycogen utilization; more findings revealed attenuation of hyphal penetration and virulence upon deletion of MoPTC6. Generally, present findings suggested the role of MoPtc6 in the growth and virulence of M. oryzae.