Whole genome resequencing reveals significant genetic differentiation between Exserohilum turcicum populations from maize and sorghum and candidate effector genes related to host specificity (Retracted).

After the manuscript was accepted, inconsistencies in the analyses were detected. These inconsistencies affected the general conclusion of the manuscript. This article was retracted on 27 March 2024.Exserohilum turcicum is a devastating fungal pathogen that infects both maize and sorghum, leading to severe leaf diseases of the two crops. According to host specificity, pathogenic isolates of E. turcicum are divided into two formae speciales, namely E. turcicum f. sp. zeae and E. turcicum f. sp. sorghi. To date, the molecular mechanism underlying the host specificity of E. turcicum is marginally known. In this study, the whole genomes of 60 E. turcicum isolates collected from both maize and sorghum were resequenced, which enabled identification of 147,847 high-quality SNPs in total. Based on the SNPs, all isolates were clustered into four genetic groups that had a close relationship with host source. This observation was validated by the result of principal component analysis. The analysis of population structure revealed that there was obvious genetic differentiation between maize and sorghum host populations. Further analysis showed that 5,431 SNPs, including 612 nonsynonymous SNPs, were completely co-segregated with host source. These nonsynonymous SNPs were located in 539 genes in which 18 genes were predicted to encode secretory proteins, including six putative effector genes. The sequence polymorphism analysis of the six effector genes in 60 isolates indicated that these genes were perfectly co-segregated with host source. All SNVs in the coding regions of these genes were non-synonymous substitutions, suggesting that these genes were subject to strong positive selection pressure. These findings provide new insights into the molecular basis of host specificity in E. turcicum.

Outbreak of Septoria leaf spot caused by Sphaerulina musiva on Populus × euramericana in China.

In June 2023, severe leaf spots were noted in Populus × euramericana cv 'Nanlin95' plantations located in the Nanjing Baguazhou Wetland Park (32°09'16.97″N, 118°48'16.74″E) of Jiangsu Province and Populus × canadensis cv 'Sacrau 79' and Populus × canadensis cv 'Guariento' in the Liyuan Village in Nanyang City (32°53'43.70″N, 112°17'29.12″E) of Henan Province, respectively. The disease incidence in both locations could reach 97.9% (556 out of 568 trees) and 98.9% (2409 out of 2436 trees), respectively. The initial symptoms appear as numerous small and circular spots (1.59 to 3.18 mm in diameter) with gray or tan centers and dark-brown margins on the leaves. As the spots age, they sometimes enlarge, often coalesce, and may extend down the petioles. Diseased leaves and petioles were both surface sterilized with 75% ethanol for 30 seconds. With the aid of a hand lens, pycnidia (brown to black, spherical in profile, 90 to 250 µm diam) were easily picked out in the center of the spots and subsequently transferred into 1 mL sterilized water for preparing the spore suspension plated on KV8 medium amended with 100 mg/liter streptomycin sulfate and 50 mg/liter chloramphenicol. After 12 days of incubation, 86 single-spore isolates were obtained and identified as typical Septoria-like fungi according to morphological features, including slow-growing, gray or black colonies with pink mucilaginous matrix and hyaline, straight or curved conidia (size = 25 to 59 × 3.5 to 4 µm; septa = 1 to 6). Species identification was further validated by PCR amplification and sequencing of the internal transcribed spacer (ITS) region with ITS1/ITS4 primer pairs. Multiple sequence alignments with ClustalW revealed that the obtained ITS sequences of 86 isolates were 100% identical to each other. A BLAST search in GenBank indicated that the selfsame sequences of two representative isolates (isolate BGZ11 of Jiangsu Province, accession no. OR660379; isolate KZB22 of Henan Province, accession no. OR711499) shared 99.8% identity (494 of 495 bp) and 100% identity (504 of 504 bp) with related sequences of Sphaerulina musiva (Peck) Quaedvlieg, Verkley, and Crous (syn. = Septoria musiva Peck) in GenBank (MN275187; KF251619), respectively. Furthermore, we used a S. musiva-specific PCR assay (Abraham et al. 2018) on symptomatic leaf samples collected from the plantation. Each sample consisted of 20 cut-out leaf spots per leaf. Eight of the 10 samples were positive for S. musiva DNA. To confirm pathogenicity, six sterile tissue culture of poplar plants (Populus trichocarpa and Populus × euramericana cv 'Nanlin895') were respectively transplanted into pots and grown in a greenhouse for a week and for a month with an 18-h photoperiod augmented with sodium lamps and a 20°C (day)/16°C (night) temperature regime. Inoculations were conducted by spraying the plants with conidia suspension (106 conidia/mL) (LeBoldus et al. 2010). Control plants were sprayed with distilled water. Leaf spots were developed on the inoculated P. trichocarpa leaves at one week and P. × euramericana cv 'Nanlin895' leaves at 10 days after inoculation while no symptoms were observed on the control plants. The fungus S. musiva was successfully reisolated from all symptomatic leaves fulfilling Koch's postulates. Sphaerulina musiva only causes an endemic leaf spot disease on its natural North American host Populus. deltoides (Feau et al. 2010; Ostry 1987). However, on susceptible Populus species (e.g., P. balsamifera, P. trichocarpa, P. maximowiczii) and hybrids, S. musiva causes not only leaf spots but also severely damaging stem and branch cankers (Jeger et al. 2018; LeBoldus et al. 2009; Sondreli et al. 2020). To our knowledge, this is the first report of S. musiva causing leaf spots on poplar in China. Large-scale timber imports (e.g., cut branches, isolated bark, wood with and without bark) potentially lead to anthropogenic-facilitated transport of this pathogen. This outbreak of Septoria leaf spot underscores the potential threat of this pathogen to P. × euramericana in China, where it is widely planted as a keystone forestry species.

First Report of Leaf Spot Caused by Paramyrothecium foliicola on Peanut in China.

Peanut (Arachis hypogaea) is an important economic and oil crop in China. In September 2022, leaf spots were observed on peanut in Luoyang city, Henan province, China (34°49'N, 112°37'E). The disease occurred on about 30% of the peanut leaves in only one 0.5-acre field. Symptoms appeared primarily as brown spots, that varied in shape, and appeared round, oval or irregular. In addition, some disease patches exhibited a concentric ring pattern. Small pieces (5×5 mm) of five diseased leaves were surface disinfected in 3% NaClO for 2 minutes, rinsed three times in sterile distilled water, dried on sterilized filter paper, and cultured on potato dextrose agar (PDA) at 25°C for 3 days. Five isolates with uniform characteristics were obtained and subcultured by transferring hyphal tips to fresh PDA. The colonies of the isolates were circular and the margins were clean. The colonies showed white coloration, and after 5-7 days of incubation on PDA plates, concentric rings with dark green sporodochia appeared on the surface of the colonies. The conidiophores branched repeatedly. The conidiophore stipes unbranched, hyaline, 10.0 to 23.2×1.5 to 3.3 µm (n=50). The conidia were rod-shaped or long oval and single-celled, measuring 4.6 to 8.6×1.4 to 3.1 µm (n=100). Based on these characteristics, the five isolates were identified as Paramyrothecium foliicola (Lombard et al 2016). Genomic DNA was extracted from the representative isolates LH-1-1 and LH-1-2. The internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), calmodulin (CmdA), and translation elongation factor 1-alpha (tef1) loci were amplified and sequenced using the following primer pairs: ITS1/ITS4 (White et al. 1990), RPB2-5F2/RPB2-7cR (O'Donnell et al. 2007), CAL-228F/CAL-2Rd (Carbone & Kohn 1999), and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. BLASTn analysis revealed that the sequences of ITS (OR352397.1 and OR417392.1), RPB2 (OR413573.1 and OR420678.1), CmdA (OR413572.1 and OR420677.1), and tef1 (OR413574.1 and OR420679.1) had 99 to 100% (553/558 bp, 721/721 bp, 597/598 bp, and 384/389 bp) similarity to P. foliicola (MN593634.1, MN398038.1, OM801785.1, MK335967.1). A phylogenetic tree based on the Maximum Likelihood method also confirmed that the two isolates converge on the same branch as P. foliicola. Pathogenicity tests were performed using leaves of 60-day-old peanut plants (cv. Zhonghua 8). Briefly, uninfected healthy leaves (non-wounded) were inoculated with 30-µl drops containing a spore suspension (5×105 conidia/ml) of LH-1-2, and peanut leaves inoculated with sterile distilled water served as controls. All treatments were incubated in an incubator at 25℃ and high relative humidity with a 12:12 hour light-dark cycle. After 5-7 days, inoculated leaves showed symptoms similar to those observed in the field, while no symptoms were observed on control leaves. The pathogenicity tests were repeated three times. The fungus was reisolated from the infected leaves and identified as P. foliicola based on morphological and molecular characteristics, thus fulfilling Koch's postulates. P. foliicola has previously been reported to cause leaf spot of tomato and mung bean, stem canker of cucumber (Huo et al. 2022; Sun et al.2020; Huo et al. 2021). To our knowledge, this is the first report of P. foliicola causing leaf spot on peanut in the world. Identification of this pathogen will be helpful in monitoring peanut diseases and developing disease control strategies.

Genetic Diversity and Population Genetic Structure of Setosphaeria turcica From Sorghum in Three Provinces of China Using Single Nucleotide Polymorphism Markers.

Setosphaeria turcica is a heterothallic fungus that is the causal agent of northern leaf blight (NLB), which is a devastating foliar disease of sorghum and maize. Despite of its adversary to crop production, little is known about the genetic diversity and population genetic structure of this pathogen from sorghum. In this study, we explored the utilization of single nucleotide polymorphism (SNP) molecular markers and three mating type-specific primers to analyze the genetic diversity, population genetic structure, and mating type distribution of 87 S. turcica isolates that had been collected in sorghum production areas from three provinces, including Henan, Shaanxi, and Shanxi in China. The populations are featured with moderate genetic diversity and relatively equal mating type distribution of MAT1-1 and MAT1-2. The genetic differentiation was significant (p < 0.05) among different populations except those from Henan and Shanxi provinces that showed particularly frequent gene flow between them. Neither the maxinum likelihood phylogenetic tree, nor principal coordinate analysis, nor genetic structure analysis was able to completely separate the three populations. The relatively low genetic distance and high genetic identification were also observed among the three populations. Nevertheless, the genetic variation within populations was the major source of variation as revealed by AMOVA analysis. The findings of this study have improved our current understanding about the genetic diversity, population genetic structure, and the distribution of mating type of S. turcica, which are useful for unraveling the epidemiology of NLB and developing effective disease management strategies.

Genetic Diversity, Population Structure and Mating Type Distribution of Setosphaeria turcica on Corn in Midwestern China.

Setosphaeria turcica is the causal agent of northern corn leaf blight (NCLB), which is a destructive foliar disease of corn around the world. To date, limited information is available on the genetic diversity, population structure, and mating type distribution of the pathogen in the mid-west of China. In this study, based on single nucleotide polymorphism (SNP) markers and mating type-specific primers, we characterized 117 S. turcica isolates collected from Henan, Hebei, Shanxi, and Shaanxi provinces in China. Based on the developed 33 SNP markers, all isolates can be categorized into two genetic groups. Each group consisted of isolates from all four provinces. The Nei's gene diversity of four populations ranged from 0.328 to 0.419 with a mean of 0.391. The analysis of fixation index (Fst) and gene flow (Nm) suggested that low genetic differentiation and high gene flow existed among four geographic populations. The analysis of molecular variance (AMOVA) demonstrated that the principal molecular variance existed within populations (98%) rather than among populations (2%). The analysis of mating type loci revealed that two mating types (MAT1-1 and MAT1-2) were basically in equilibrium in all four populations. These findings advance our understanding of the genetic diversity, population structure and mating type distribution of S. turcica on corn in the mid-west of China and will aid in developing efficient strategies to control NCLB.

Identification of a C2H2 Transcription Factor (PsCZF3) Associated with RxLR Effectors and Carbohydrate-Active Enzymes in Phytophthora sojae Based on WGCNA.

Phytophthora sojae is a destructive soybean pathogen that orchestrates various secreted proteins (effectors) to modulate plant immunity and facilitate infection. Although a number of effectors have been identified and functionally studied in P. sojae, the way these molecules are regulated is marginally known. In this study, we performed a weighted gene correlation network analysis (WGCNA) based on digital RNA-seq, which enabled the identification of a transcription factor (PsCZF3) in P. sojae. This transcription factor is a C2H2-type zinc finger protein that regulates the transcription of 35 RxLR effectors during the early infection stage. Phylogenetic analysis revealed that PsCZF3 is a highly conserved protein across oomycetes, suggesting that this regulation mechanism may broadly exist in oomycete species. In addition, by building a subnetwork of PsCZF3 and correlated genes, we also found that PsCZF3 contributed to the transcriptional regulation of carbohydrate-active enzymes. Our findings suggest that the activation of PsCZF3 facilitates P. sojae infection by up-regulating RxLR effectors and carbohydrate-active enzymes.

Mutations in the Promoter and Coding Regions of Avr3a Cause Gain of Virulence of Phytophthora sojae to Rps3a in Soybean.

Phytophthora sojae threatens soybean production worldwide, and the cultivation of soybean cultivars carrying Rps genes is the most effective way to control this pathogen. However, DNA mutations in the Avr genes of P. sojae can escape recognization of the corresponding Rps genes, leading to the loss of soybean resistance. In this study, we investigated sequence polymorphism and transcript level of the Avr3a gene in Chinese isolates of P. sojae. Twenty-four mutations resulting in five unique Avr3a alleles were discovered in the Avr3a coding region from 32 P. sojae isolates. The Avr3a transcripts were detectable in the isolates containing Avr3a(I), Avr3a(II), Avr3a(III), and Avr3a(IV) but not in the isolates containing Avr3a(V). Promoter and 5'-UTR sequence analysis revealed eight unique mutations in the promoter region of Avr3a(V), suggesting that the mutations could result in the loss of Avr3a(V) transcription. Virulence tests indicated the isolates containing Avr3a(II) and Avr3a(IV) were virulent, suggesting that the mutations in the coding regions of Avr3a(II) and Avr3a(IV) caused the gain of virulence to Rps3a. Based on DNA mutations of Avr3a in virulent alleles, two SNP markers and one PCR-based marker were developed successfully for detecting the virulence of P. sojae isolates to Rps3a. These findings provide new insights into escape mechanisms of Avr3a and effective support for accurate pathotype identification of P. sojae using molecular methods.

Distribution, Pathotypes, and Metalaxyl Sensitivity of Phytophthora sojae from Heilongjiang and Fujian Provinces in China.

Phytophthora sojae causes root and stem rot, one of the most devastating diseases of soybean worldwide. In Heilongjiang and Fujian provinces in China, serious cases of Phytophthora stem and root rot have occurred and caused heavy losses in the past several years. To determine the current population status of this pathogen, we investigated the pathogen's distribution, pathotypes, and metalaxyl sensitivity in both provinces. P. sojae was baited and isolated from 258 soil samples in both provinces using the soybean leaf bait method. The pathotypes of all isolates were characterized on 13 differential soybean cultivars using the hypocotyl slit inoculation method, and the sensitivity of all isolates to metalaxyl was tested in vitro. In all, 75 isolates were recovered from 75 fields in 33 counties; of these, 31 counties were in Heilongjiang Province and 2 counties were in Fujian Province. Thirty-five new pathotypes were identified compared with the previously defined races. Less than 5% of the isolates were virulent to cultivars with individual Rps genes 1a, 1c, or 1k. No metalaxyl-resistant isolates were found; the half maximal effective concentration values of all isolates ranged from 0.04 to 0.22 µg ml-1. These results suggest that effective management of the disease in both provinces can be accomplished through the use of resistant cultivars with Rps genes 1a, 1c, or 1k and the fungicide metalaxyl.

Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9.

Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community. Here, we describe a detailed protocol for CRISPR/Cas9-mediated genome editing in P. sojae, including single guide RNA (sgRNA) design and construction, efficient gene replacement, and mutant-screening strategies. This protocol should be generally applicable for most culturable oomycetes. We also describe an optimized transformation method that is useful for other Phytophthora spp. including P. capsici and P. parasitica. © 2017 by John Wiley & Sons, Inc.

Analysis of polymorphism and transcription of the effector gene Avr1b in Phytophthora sojae isolates from China virulent to Rps1b.

The effector gene Avr1b-1 of Phytophthora sojae determines the efficacy of the resistance gene Rps1b in soybean. The sequences of the Avr1b-1 locus in 34 Chinese isolates of P. sojae were obtained and analysed by polymerase chain reaction (PCR) and inverse PCR. Four different alleles and a complete deletion mutation of the Avr1b-1 gene were identified. Molecular analysis of the deletion breakpoints in the Avr1b-1 locus revealed that an 8-kb DNA sequence containing Avr1b-1 was deleted and a 12.7-kb DNA sequence was inserted at the same locus. A truncated transposase gene was found and five transposable elements were predicted in the inserted sequence, suggesting that the deletion of Avr1b-1 might be attributed to transposon movement. The transcription of Avr1b-1 was analysed in virulent isolates containing four alleles of Avr1b-1 by real-time reverse transcription-PCR. In all virulent isolates, only those isolates containing the second allele transcripted Avr1b-1.

Sequence variants of the Phytophthora sojae RXLR effector Avr3a/5 are differentially recognized by Rps3a and Rps5 in soybean.

The perception of Phytophthora sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies have shown that the Avr3a and Avr5 genes of P. sojae are genetically linked, and the Avr3a gene encoding a secreted RXLR effector protein was recently identified. We now provide evidence that Avr3a and Avr5 are allelic. Genetic mapping data from F(2) progeny indicates that Avr3a and Avr5 co-segregate, and haplotype analysis of P. sojae strain collections reveal sequence and transcriptional polymorphisms that are consistent with a single genetic locus encoding Avr3a/5. Transformation of P. sojae and transient expression in soybean were performed to test how Avr3a/5 alleles interact with soybean Rps3a and Rps5. Over-expression of Avr3a/5 in a P. sojae strain that is normally virulent on Rps3a and Rps5 results in avirulence to Rps3a and Rps5; whereas silencing of Avr3a/5 causes gain of virulence in a P. sojae strain that is normally avirulent on Rps3a and Rps5 soybean lines. Transient expression and co-bombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allele-specific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5.