Mutations across Diverse Domains of CjXDR1 Lead to Multidrug Resistance in Clarireedia jacksonii.

Recently, Clarireedia jacksonii has emerged as a significant pathogen threatening turfgrass, and its escalating resistance to multiple drugs often undermines field interventions. This study highlighted the critical role of the fungus-specific transcription factor CjXDR1 (formerly ShXDR1) in regulating multidrug resistance (MDR) in C. jacksonii. This was demonstrated through experiments involving CjXDR1-knockout and CjXDR1-complemented strains. Our sequence analysis revealed five mutations in CjXDR1: G445D, K453E, S607F, D676H, and V690A. All five gain-of-function (GOF) mutations were confirmed to directly contribute to MDR against three different classes of fungicides (propiconazole: demethylation inhibitor, boscalid: succinate dehydrogenase inhibitor, and iprodione: dicarboximide) using the genetic transformation system and in vitro fungicide-sensitivity assay. Comparative transcriptome analysis revealed that CjXDR1 and its GOF mutations led to the overexpression of downstream genes encoding a Phase I metabolizing enzyme (CYP68) and two Phase III transporters (CjPDR1 and CjAtrD) previously reported. Knockout mutants of CYP68, CjPDR1, CjAtrD, and double-knockout mutants of CjPDR1 and CjAtrD exhibited increased sensitivity to all three fungicides tested. Among these, the CYP68-knockout mutants displayed the highest sensitivity to propiconazole, while the CjPDR1 knockout mutant exhibited significantly increased sensitivity to all three fungicides. Double-knockout mutants of CjPDR1 and CjAtrD displayed greater sensitivity than the single knockouts. In conclusion, multiple GOF mutants in CjXDR1 contribute to MDR by upregulating the expression of CjPDR1, CjAtrD, and CYP68. This study enhances our understanding of the molecular mechanisms underlying MDR in plant pathogenic fungi, providing valuable insights into GOF mutation structures and advancing the development of antifungal drugs.

A rapid real-time PCR assay for detecting Microdochium paspali causing sparse leaf patch on seashore paspalum and in environmental samples.

BACKGROUND: Sparse leaf patch (SLP) is one of the most significant diseases affecting seashore paspalum (Paspalum vaginatum Sw.), caused by Microdochium paspali. Fast and accurate detection of this pathogen is crucial for effective disease management. However, conventional culture-based methods are time-consuming and often compromised by the presence of other saprophytic or endophytic fungi. RESULTS: In this study, we developed a real-time fluorescent quantitative (q)PCR method based on the internal transcribed spacer (ITS) region of the ribosomal RNA gene to rapidly detect and quantify M. paspali. The qPCR assay demonstrated the ability to detect all 12 tested isolates of M. paspali, with no cross-reactions observed when tested against 30 isolates of other fungal pathogens from turfgrass samples. The detection limit of the qPCR method was as low as 3.65 × 102 copies µL-1 of M. paspali genomic DNA, and the entire detection process could be completed within 1 h. The fluorescence signal was detectable in the leaf tissues of seashore paspalum without apparent disease symptoms as early as 24 h postinoculation with M. paspali. Moreover, the qPCR method successfully detected M. paspali in both asymptomatic and symptomatic turfgrass samples, including leaf, stem, root and rhizosphere soil, indicating that this assay can significantly enhance the detection of M. paspali. CONCLUSION: The study developed a rapid real-time qPCR assay for the detection of M. paspali causing SLP on seashore paspalum and in environmental samples, which has important implications for early warning and management of SLP. © 2024 Society of Chemical Industry.

Comparative genomics and transcriptome analysis reveals potential pathogenic mechanisms of Microdochium paspali on seashore paspalum.

The sparse leaf patch of seashore paspalum (Paspalum vaginatum Sw.) caused by Microdochium paspali seriously impacts the landscape value of turf and poses a challenge to the maintenance and management of golf courses. Little is known about the genome of M. paspali or the potential genes underlying pathogenicity. In this study, we present a high-quality genome assembly of M. paspali with 14 contigs using the Nanopore and Illumina platform. The M. paspali genome is roughly 37.32 Mb in size and contains 10,365 putative protein-coding genes. These encompass a total of 3,830 pathogen-host interactions (PHI) genes, 481 carbohydrate-active enzymes (CAZymes) coding genes, 105 effectors, and 50 secondary metabolite biosynthetic gene clusters (SMGCs) predicted to be associated with pathogenicity. Comparative genomic analysis suggests M. paspali has 672 species-specific genes (SSGs) compared to two previously sequenced non-pathogenic Microdochium species, including 24 species-specific gene clusters (SSGCs). Comparative transcriptomic analyses reveal that 739 PHIs, 198 CAZymes, 40 effectors, 21 SMGCs, 213 SSGs, and 4 SSGCs were significantly up-regulated during the process of infection. In conclusion, the study enriches the genomic resources of Microdochium species and provides a valuable resource to characterize the pathogenic mechanisms of M. paspali.

Genome Resources for Four Clarireedia Species Causing Dollar Spot on Diverse Turfgrasses.

Dollar spot (DS) is a destructive fungal disease impacting almost all warm- and cool-season turfgrasses worldwide. Multiple fungal species in the genus Clarireedia are causal agents of DS. Here, we present whole-genome assemblies of nine fungal isolates in the genus Clarireedia, including four species (C. paspali, C. hainanense, C. jacksonii, and C. monteithiana) causing DS on seashore paspalum (Paspalum vaginatum Sw.), creeping bentgrass (Agrostis stolonifera L.), and Kentucky bluegrass (Poa pratensis L.) in China. This work provides valuable baseline genomic data to support further research and management of DS pathogens on turfgrasses.

A one-step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing dollar spot on turfgrass.

BACKGROUND: Dollar spot (DS) is one of the most destructive and economically important diseases of cool- and warm-season turfgrasses worldwide. A total of six species causing DS disease in the genus Clarireedia have been described, and four of them have been reported to be distributed countrywide in China. Identification of different species of Clarireedia is a prerequisite for the effective management of DS disease. RESULTS: Here we report a novel polymerase chain reaction (PCR)-based method for the detection and differentiation of the four species of Clarireedia associated with DS on turfgrass in China: C. jacksonii, C. paspali, C. monteithiana and C. hainanense. Species-specific genes were identified for each species by comparative genomics analysis. Four primer pairs were designed and mixed to amplify species-specific PCR fragments with differential sizes for the four species of Clarireedia in a single multiplex PCR assay. No PCR products were generated from the DNA templates of other common fungal pathogens associated with multiple turfgrass diseases. The multiplex PCR method developed can be used for the rapid and accurate detection and differentiation of the four species of Clarireedia from pure cultures as well as from infected turfgrass blades with DS symptoms. CONCLUSION: The study developed a one-step multiplex PCR assay for the detection and differentiation of four species of Clarireedia causing DS on turfgrass in China, which will have important implications for DS management in China and worldwide. © 2022 Society of Chemical Industry.

Distribution and Prevalence of Plant-Parasitic Nematodes of Turfgrass at Golf Courses in China.

We sampled 127 turfgrass soil samples from 33 golf courses in NC, EC, and SC for plant-parasitic nematodes (PPNs). PPNs were extracted from soil samples using the shallow dish method and were identified at the genus or species levels with a combination of morphological and molecular methods. The results revealed 41 species of nematode belonging to 20 genera and 10 families. Nine genera are new records of PPNs associated with turfgrass in China. The PPNs show strong geographical distributions. Of the 20 genera, Helicotylenchus, Paratrichodorus, Hoplolaimus, Meloidogyne, Hemicriconemoides, and Mesocriconema showed higher infestation and frequency, and most of these genera had numbers in soil samples above established damage thresholds. Four golf courses had soil samples with PPNs > 30%, indicating the potential for nematode damage. The biodiversity indices H', SR, J', λ, and H2 showed significant differences among different regions and turfgrass species; H', SR, J', and H2 were significantly higher in EC than in NC and SC, while λ was lowest in EC. Creeping bentgrass had the highest H', SR, J', and H2 and the lowest λ in comparison with seashore paspalum and hybrid bermudagrass. These findings provide baseline information on the occurrence of turfgrass-associated PPNs in China, and have important implications for the effective management of PPNs causing damage on turfgrass.

Clarireedia hainanense: A New Species Is Associated with Dollar Spot of Turfgrass in Hainan, China.

The genus Clarireedia contains multiple species causing dollar spot (DS) on turfgrass worldwide. In November 2020, 119 Clarireedia isolates were obtained from symptomatic seashore paspalum at golf courses in Hainan province and identified to species level based on partial sequence of the internal transcribed spacer (ITS) region. A total of 45 and 22 isolates were identified as C. paspali and C. monteithiana, respectively; the remaining 52 isolates defined a new clade. Isolates from this clade were further selected for phylogenetic, morphological, and biological analyses. Maximum likelihood and Bayesian methods were implemented to obtain phylogenetic trees for partial sequences of the ITS, EF-1α, and McM7 genes. The selected isolates consistently fell into a distinct, well-supported clade within Clarireedia. Morphological and biological characteristics were observed among the different species in Clarireedia. Altogether, this study described a new species, Clarireedia hainanense, which has widespread distribution in Hainan, China. These findings may have important implications for the management of DS disease.