The velvet family proteins mediate low resistance to isoprothiolane in Magnaporthe oryzae.

Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Through IPT adaptation on PDA medium, we obtained a variety of IPT-resistant mutants. Based on their EC50 values to IPT, the resistant mutants were mainly divided into three distinct categories, i.e., low resistance (LR, 6.5 ≤ EC50 < 13.0 µg/mL), moderate resistance 1 (MR-1, 13.0 ≤ EC50 < 25.0 µg/mL), and moderate resistance 2 (MR-2, 25.0 ≤ EC50 < 35.0 µg/mL). Molecular analysis of MoIRR (Magnaporthe oryzae isoprothiolane resistance related) gene demonstrated that it was associated only with the moderate resistance in MR-2 mutants, indicating that other mechanisms were associated with resistance in LR and MR-1 mutants. In this study, we mainly focused on the characterization of low resistance to IPT in M. oryzae. Mycelial growth and conidial germination were significantly reduced, indicating fitness penalties in LR mutants. Based on the differences of whole genome sequences between parental isolate and LR mutants, we identified a conserved MoVelB gene, encoding the velvet family transcription factor, and genetic transformation of wild type isolate verified that MoVelB gene was associated with the low resistance. Based on molecular analysis, we further demonstrated that the velvet family proteins VelB and VeA were indispensable for IPT toxicity and the deformation of the VelB-VeA-LaeA complex played a vital role for the low IPT-resistance in M. oryzae, most likely through the down-regulation of the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT.

Identification, Genetic Diversity, and Chemical Control of Xanthomonas arboricola pv. pruni in China.

Peach bacterial spot caused by Xanthomonas arboricola pv. pruni has become widespread in most peach-producing areas of China and has caused devastating losses to the peach industry. However, little is known about the population biology and epidemiology of X. arboricola pv. pruni in China, thus no effective management strategy is available. Altogether, 321 symptomatic samples of peach bacterial spot from 12 provinces in China were collected from which 612 bacterial isolates were obtained. Based on 16S rDNA sequence comparison in GenBank, the obtained isolates were identified as Pantoea spp. (514) and Xanthomonas spp. (98). The pathogenicity test demonstrated that the causal agent of the peach bacterial spot was the Xanthomonas spp. instead of the Pantoea spp. Based on morphological observation, physiological and biochemical characterization, and molecular identification, the Xanthomonas spp. were further identified to be X. arboricola pv. pruni. Then, 41 X. arboricola pv. pruni isolates representing different populations were selected and analyzed with repetitive element sequence based-PCR and intersimple sequence repeat markers to understand the genetic diversity and population structure along with four X. arboricola pv. pruni isolates from plum and three isolates of X. arboricola pv. juglandis as comparison. A total of 98 polymorphic alleles were identified, with a mean value of percentage of polymorphic loci of 14. Genetic diversity and phylogenetic analysis revealed the profound heterogeneity between X. arboricola pv. juglandis and X. arboricola pv. pruni, moderate genetic differentiation within X. arboricola pv. pruni, and obvious host specificity but weak geographical differentiation in X. arboricola population. Finally, the efficiency of bactericides on X. arboricola pv. pruni was evaluated in vitro and in vivo. The parallel repeated field trials in two orchards demonstrated that 80% Mancozeb (1:800) and 47% Kocide (1:800, 1:1,500, and 1:2,000) had excellent control efficacies for X. arboricola pv. pruni, especially as the control efficacy of Kocide could even reach 90%. This study conducted a systematic investigation for the occurrence, population variance, and chemical control of X. arboricola pv. pruni. It improved the understanding of the pathogen populations of peach bacterial spot in China and provided solid theoretical and practical guidance for X. arboricola pv. pruni control.

Transcriptomic Analysis of Resistant and Wild-Type Isolates Revealed Fludioxonil as a Candidate for Controlling the Emerging Isoprothiolane Resistant Populations of Magnaporthe oryzae.

The point mutation R343W in MoIRR, a putative Zn2Cys6 transcription factor, introduces isoprothiolane (IPT) resistance in Magnaporthe oryzae. However, the function of MoIRR has not been characterized. In this study, the function of MoIRR was investigated by subcellular localization observation, transcriptional autoactivation test, and transcriptomic analysis. As expected, GFP-tagged MoIRR was translocated in the nucleus, and its C-terminal could autonomously activate the expression of reporter genes HIS3 and α-galactosidase in absence of any prey proteins in Y2HGold, suggesting that MoIRR was a typical transcription factor. Transcriptomic analysis was then performed for resistant mutant 1a_mut (R343W), knockout transformant ΔMoIRR-1, and their parental wild-type isolate H08-1a. Upregulated genes in both 1a_mut and ΔMoIRR-1 were involved in fungicide resistance-related KEGG pathways, including the glycerophospholipid metabolism and Hog1 MAPK pathways. All MoIRR deficiency-related IPT-resistant strains exhibited increased susceptibility to fludioxonil (FLU) that was due to the upregulation of Hog1 MAPK pathway genes. The results indicated a correlation between FLU susceptibility and MoIRR deficiency-related IPT resistance in M. oryzae. Thus, using a mixture of IPT and FLU could be a strategy to manage the IPT-resistant populations of M. oryzae in rice fields.

Recombinase Polymerase Amplification/Cas12a-Based Identification of Xanthomonas arboricola pv. pruni on Peach.

Peach bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap) is a devastating disease worldwide and frequently causes massive economic losses. In recent years, it has become a pandemic outbreak in most peach production areas of China, especially on precocious peaches in the middle reach of the Yangtze River. Rapid, user-friendly detection is extremely important to make the correct diagnosis and develop suitable control strategies. In this study, we described a recombinase polymerase amplification (RPA)/Cas12a-based system that combines RPA and CRISPR/Cas12a for Xap identification. A total of three crRNAs were designed to target a highly conserved ABC transporter ATP-binding protein-encoding gene ftsX to make specific detection of Xap. Results showed that crRNA 2 and crRNA 3 could get consistent detection for Xap. To realize the visualization of detection results, we additionally introduced FQ-reporter and FB-reporter. The developed method was highly sensitive and could detect as low as 10-18 M Xap gDNA with a mini-UV torch, corresponding to 1.63 copies/µl or 8.855 fg/µl gDNA of Xap, while with lateral flow strips, the sensitivity was 10-17 M. In addition, this method could specifically detect Xap from other closely related bacteria or pathogens associated with peach diseases. Furthermore, this method could make correct identification for Xap with crude DNA using NaOH-based extraction (3 min) directly from diseased peach samples. Considering that the developed method could get results within 2 h and could be performed at 37°C (body temperature), it is promising to be applied for Xap diagnosis and monitoring in fields.

A Putative Zn2Cys6 Transcription Factor Is Associated With Isoprothiolane Resistance in Magnaporthe oryzae.

Isoprothiolane (IPT), a systemic fungicide, has been applied to control rice blast since the 1970s. Although resistance to IPT has been observed, the mechanism of resistance still has not been fully elucidated. In this study, nucleotide polymorphisms were detected between two IPT-resistant mutants generated in the lab, and their parental wild type isolates using a whole-genome sequencing approach. In the genomes of the two resistant mutants, single point mutations were identified in a gene encoding a Zn2Cys6 transcription factor-like protein. Notably, either knocking out the gene or replacing the wild type allele with the mutant allele (R343W) in a wild type isolate resulted in resistance to IPT, indicating that the gene is associated with IPT resistance, and thus was designated as MoIRR (Magnaporthe oryzae isoprothiolane resistance related). Along with point mutations R343W in mutant 1a_mut, and R345C in 1c_mut, a 16 bp insertion in 6c_mut was also located in the Fungal_TF_MHR domain of MoIRR, revealing that this domain may be the core element for IPT resistance. In addition, IPT-resistant mutants and transformants showed cross-resistance with iprobenfos (IBP), which was consistent with previous observations. These results indicated that MoIRR is strongly connected to resistance to choline biosynthesis inhibitor (CBI), and further work should focus on investigating downstream effects of MoIRR.