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 of Monilia Species in Tibet and Characterization of M. yunnanensis in China.

Samples of peach and plum fruits with brown rot symptoms were collected from Tibet in 2019 and 2020, and the causal agent was identified as Monilia yunnanensis, which represents the first characterization of Monilia spp. on peach and plum in Tibet. Morphological investigation showed that some conidia from naturally diseased fruits were larger than those observed in previously isolated M. yunnanensis. Some conidia of M. yunnanensis isolates from Tibet produced more than two, even up to six germ tubes from different parts of each conidium, instead of one or two germ tubes developing from the pointy sides of each conidium. The alignment of ribosomal internal transcribed spacer region sequences revealed that some isolates from Tibet displayed a mutation at the 374th position from adenine (A) to cytosine (C). Although abovementioned differences were observed between isolates from Tibet and other regions, phylogenetic analysis indicated that all of the M. yunnanensis isolates from different stone fruits and different regions in China were clustered together without obvious genetic differentiation. These results revealed that hosts and geographic environments did not play a major role in the evolution of M. yunnanensis.

Cytological Observation of the Infectious Process of Venturia carpophila on Peach Leaves.

Peach scab caused by Venturia carpophila is one of the most destructive fungal diseases of peach worldwide, and it seriously affects peach production. Until now,the infectious process and pathogenesis of V. carpophila on peach have remained unclear. Here we present the infection behavior of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (i.e., light microscopy, confocal laser scanning microscopy, scanning electron microscopy, and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer and then differentiated into thick subcuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the subcuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the subcuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the subcuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further a understanding of V. carpophila pathogenesis.

Genome Sequence of Venturia carpophila, the Causal Agent of Peach Scab.

Venturia carpophila, the causal agent of scab disease on peach, is a host-specific fungus that is widely distributed around the world, including China. In our previous study, samples were collected from 14 provinces in China, and 750 isolates were obtained by single-spore separation. Here, we reported the first highly contiguous whole-genome sequence (35.87 Mb) of the V. carpophila isolate ZJHZ1-1-1, which included 33 contigs with N50 value of 2.01 Mb and maximum contig length of 3.39 Mb. The high-quality genome sequence and annotation resource will be useful to study the fungal biology, pathogen-host interaction, fungicide resistance, characterization of important genes, population genetic diversity, and development of molecular markers for genotyping and species identification.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Sensitivity of Venturia carpophila from China to Five Fungicides and Characterization of Carbendazim-Resistant Isolates.

Peach scab is a fungal disease caused by Venturia carpophila that can significantly reduce peach yield and quality. Fungicide application is the main control measure for peach scab worldwide. To better understand the fungicide-resistance status and devise suitable management strategies, the sensitivity of 135 single-spore V. carpophila isolates to the commonly used fungicides carbendazim, iprodione, propiconazole, azoxystrobin, and boscalid were determined using a microtiter plate test method. Results showed that the mean effective concentrations to cause inhibitions by 50% (EC50) of tested isolates to iprodione, propiconazole, azoxystrobin, and boscalid were 16.287, 0.165, 0.570, and 0.136 µg/ml, respectively. The EC50 values of V. carpophila isolates to four fungicides displayed unimodal frequency distributions, indicating no resistance occurred to these fungicides. On the contrary, bimodal frequency distribution was observed for carbendazim, indicating that V. carpophila developed resistance to carbendazim. Resistance was widely detected from all 14 provinces studied. Molecular analysis showed that the point mutation E198K of the TUB2 gene determined high resistance, whereas E198G conferred moderate resistance. Moderate and high resistances were stable, and the resistant isolates did not show significant fitness penalties. On the contrary, some resistant isolates showed better competitiveness under certain stresses. This is the first report to detect the sensitivity of V. carpophila to fungicides, which enables future monitoring of fungicide resistance and provides basic information to allow the design of suitable peach scab management strategies.

Identification and Characterization of Three Monilinia Species from Plum in China.

In total, 112 Monilinia spp. single-spore isolates were collected from plum fruit (Prunus salicina) symptomatic for brown rot disease from Yunnan, Hubei, and Zhejiang provinces and Chongqing municipality, China between 2012 and 2014. Three distinct colony morphologies (phenotypes) were observed on potato dextrose agar and two isolates per phenotype were selected for further analysis. Colony color, colony shape, conidia size, number of germ tubes per conidia, and pathogenicity on plum were investigated. The ribosomal internal transcribed spacer regions 1 and 2 as well as a polymerase chain reaction-based method that amplified fragments of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and ß-tubulin (TUB2) genes were used to identify the isolates to the species level. The three phenotypes were identified to be three different species: Monilinia fructicola, Monilia mumecola, and Monilia yunnanensis. Phylogenetic analysis based on G3PDH and TUB2 nucleotide sequences revealed that isolates within species clustered together regardless of host or geographical origin, suggesting that these factors did not play an important role for the evolutionary separation of the described species.

Evolutionary analysis revealed the horizontal transfer of the Cyt b gene from Fungi to Chromista.

In this study, the cytochrome b (Cyt b) amino acid sequences were analyzed in 50 organisms covering all 5 kingdoms of eukaryotes. Six conserved domains, i.e., heme bL binding sites, heme bH binding sites, Qo binding sites, Qi binding sites, the interchain domain interface, and the intrachain domain interface were found in all investigated sequences. The topology of the phylogenetic trees was largely consistent with the well recognized taxonomic relationships, indicating that the Cyt b genes originated from a common ancestral gene before the divergence of eukaryotic kingdoms. The eukaryotic Cyt b genes likely originated from an ancient prokaryotic gene in Alphaproteobacteria based on shared conserved domains. We provide evidence that the Cyt b gene of oomycete Pseudoperonospora cubensis was horizontally transferred from a fungus in the order Hypocreales. To our knowledge, this is the first reported evidence of Horizontal gene transfer (HGT) from Fungi to Chromista involving an essential house-keeping gene. Our data suggest that HGT events must be considered when evolutionary trees are constructed only based on Cyt b genes. Additional analysis of thousands of Cyt b sequences from Genbank revealed that introns in mitochondrial Cyt b genes were acquired after the endosymbiosis of alphaproteobacteria in eukaryotic cells.

Colletotrichum Species Associated with Peaches in China.

Colletotrichum is regarded as one of the 10 most important genera of plant pathogens in the world. It causes diseases in a wide range of economically important plants, including peaches. China is the largest producer of peaches in the world but little is known about the Colletotrichum spp. affecting the crop. In 2017 and 2018, a total of 286 Colletotrichum isolates were isolated from symptomatic fruit and leaves in 11 peach production provinces of China. Based on multilocus phylogenetic analyses (ITS, ACT, CAL, CHS-1, GAPDH, TUB2, and HIS3) and morphological characterization, the isolates were identified to be C. nymphaeae, C. fioriniae, and C. godetiae of the C. acutatum species complex, C. fructicola and C. siamense of the C. gloeosporioides species complex, C. karsti of the C. boninense species complex, and one newly identified species, C. folicola sp. nov. This study is the first report of C. karsti and C. godetiae in peaches, and the first report of C. nymphaeae, C. fioriniae, C. fructicola, and C. siamense in peaches in China. C. nymphaeae is the most prevalent species of Colletotrichum in peaches in China, which may be the result of fungicide selection. Pathogenicity tests revealed that all species found in this study were pathogenic on both the leaves and fruit of peaches, except for C. folicola, which only infected the leaves. The present study substantially improves our understanding of the causal agents of anthracnose on peaches in China.

Genetic diversity of Venturia carpophila populations from different hosts and geographic regions in China.

Venturia carpophila, the causal agent of scab disease of peach, mume, and apricot, is widely distributed around the world. Scab of stone fruits is an important disease in China. However, little is known about the population biology and genetic diversity of the V. carpophila. To better understand the genetic diversity and population structure of V. carpophila, 186 single-spore isolates from different hosts and geographic regions were obtained and analyzed by using 31 simple sequence repeat (SSR) markers. This included 156 isolates from peach spanning 14 provinces, 15 isolates from mume and 15 isolates from apricot in Huazhong Agricultural University (HZAU). Diversity analysis with SSR markers showed a low incidence of polymorphisms within mume isolates (32.59% of markers), but a higher incidence of polymorphisms within peach isolates (42.96%) and apricot isolates (57.04%). Within peach isolates, Nei's average gene diversity ranged from 0.07 for Hebei population to 0.18 for Hubei population. AMOVA analysis revealed that 13% of the observed genetic diversity was partitioned among the geographic populations, while 40% of the observed genetic diversity was partitioned among the host populations. Other analyses (PCoA, STRUCTURE, DAPC, MSN, and UPGMA) indicated that the Chinese V. carpophila populations could be clustered into three distinct genetic groups, which correspond to the host boundaries of peach, mume and apricot. The genetic identity of V. carpophila isolates throughout the range is dependent on hosts, but not geographic regions.

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.

Development of a loop-mediated isothermal amplification method for the rapid detection of Venturia carpophila on peach.

BACKGROUND: Peach scab, caused by Venturia carpophila, can significantly reduce both the yield and quality of peach fruit. It is difficult to distinguish peach scab from other peach diseases such as black spot and bacterial spot. An efficient assay is needed to identify V. carpophila in order to develop scientific management strategies. RESULTS: A set of loop-mediated isothermal amplification (LAMP) primers was designed based on the internal transcribed spacer (rDNA-ITS) sequence to detect V. carpophila. Compared with the conventional polymerase chain reaction (PCR) method, the LAMP method not only exhibited higher sensitivity and specificity in the detection of V. carpophila, but also required simpler equipment and less operational time. The minimum detectable concentration of V. carpophila genomic DNA with the LAMP method was 56.6 fg µl-1 , which was 100 times lower than with the conventional PCR method. When eight fungal species including V. carpophila (23 isolates from 14 provinces) and one bacterial species were used with LAMP detection, only V. carpophila showed a color change, from brown to yellowish green, and ladder-like bands in electrophoresis, indicating successful amplification. Moreover, when crude DNAs of peach fruit samples were used in LAMP detection, amplification was observed only from diseased fruits, and not from healthy fruits and the negative control. CONCLUSION: The LAMP assay shows simplicity, rapidity, high sensitivity and specificity, and will be useful in distinguishing scab caused by Venturia carpophila from other diseases with similar symptoms. © 2020 Society of Chemical Industry.

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.

Genetic Mapping and Chromosomal Assignment of Magnaporthe oryzae Avirulence Genes AvrPik, AvrPiz, and AvrPiz-t Controlling Cultivar Specificity on Rice.

ABSTRACT A genetic map including three avirulence (Avr) genes, AvrPik, AvrPiz, and AvrPiz-t, was constructed in a genetic cross of two rice field isolates, 84R-62B and Y93-245c-2. The chromosomal locations of the Avr genes were determined by using selected markers to probe Southern blots of the parental chromosomes that had been separated by contour-clamped homogenous electric fields electrophoresis. Electrophoretic karyotyping showed that both parental isolates 84R-62B and Y93-245c-2 contained seven chromosomes greater than 3.5 megabases (Mb) in size and 84R-62B possessed a small chromosome of approximately 1.6 Mb. The linkage groups containing AvrPiz and AvrPiz-t were assigned to chromosomes 3 and 7, respectively. Some markers from the linkage group that contained AvrPik hybridized with chromosome 1 and the 1.6-Mb chromosome, yet all of the cloned RAPD markers that were closely linked to AvrPik hybridized exclusively to the 1.6-Mb chromosome in 84R-62B, the parent that possesses AvrPik. Thus, we conclude that AvrPik is located on the 1.6-Mb chromosome in 84R-62B.

Frequent gain and loss of introns in fungal cytochrome b genes.

In this study, all available cytochrome b (Cyt b) genes from the GOBASE database were compiled and the evolutionary dynamics of the Cyt b gene introns was assessed. Cyt b gene introns were frequently present in the fungal kingdom and some lower plants, but generally absent or rare in Chromista, Protozoa, and Animalia. Fungal Cyt b introns were found at 35 positions in Cyt b genes and the number of introns varied at individual positions from a single representative to 32 different introns at position 131, showing a wide and patchy distribution. Many homologous introns were present at the same position in distantly related species but absent in closely related species, suggesting that introns of the Cyt b genes were frequently lost. On the other hand, highly similar intron sequences were observed in some distantly related species rather than in closely related species, suggesting that these introns were gained independently, likely through lateral transfers. The intron loss-and-gain events could be mediated by transpositions that might have occurred between nuclear and mitochondria. Southern hybridization analysis confirmed that some introns contained repetitive sequences and might be transposable elements. An intron gain in Botryotinia fuckeliana prevented the development of QoI fungicide resistance, suggesting that intron loss-and-gain events were not necessarily beneficial to their host organisms.

An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides.

BACKGROUND: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self-splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. RESULTS: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5'-3') 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above-mentioned 1166 bp intron, a self-splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. CONCLUSION: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron.

The 1.6Mb chromosome carrying the avirulence gene AvrPik in Magnaporthe oryzae isolate 84R-62B is a chimera containing chromosome 1 sequences.

A genetic map was constructed previously from a cross between Magnaporthe oryzae isolates 84R-62B and Y93-245c-2, and genetic markers closely linked to the cultivar-specific avirulence (Avr) gene, AvrPik, were assigned to a 1.6Mb small chromosome of 84R-62B that is absent from Y93-245c-2. In the present study, the 1.6Mb chromosome was characterized by using contour-clamped homogeneous electric fields (CHEF) electrophoresis and hybridization analysis. CHEF electrophoresis analysis showed that the 1.6Mb chromosome was inherited in Mendelian fashion, and co-segregated with AvrPik. Southern hybridization analysis revealed that the 1.6Mb chromosome carried sequences only distributed to the supernumerary chromosome in M. oryzae isolates, as well as sequences corresponding to those in the supercontig 17 of chromosome 1 in the M. grisea database. Thus, we conclude that the Mendelian 1.6Mb chromosome is a chimera containing sequences from chromosome 1 and from supernumerary chromosomes in M. oryzae.