Transcription factors VviWRKY10 and VviWRKY30 co-regulate powdery mildew resistance in grapevine.

Grapevine (Vitis vinifera) is an economically important fruit crop worldwide. The widely cultivated grapevine is susceptible to powdery mildew caused by Erysiphe necator. In this study, we used CRISPR-Cas9 to simultaneously knock out VviWRKY10 and VviWRKY30 encoding two transcription factors reported to be implicated in defense regulation. We generated 53 wrky10 single mutant transgenic plants and 15 wrky10 wrky30 double mutant transgenic plants. In a 2-yr field evaluation of powdery mildew resistance, the wrky10 mutants showed strong resistance, while the wrky10 wrky30 double mutants showed moderate resistance. Further analyses revealed that salicylic acid (SA) and reactive oxygen species contents in the leaves of wrky10 and wrky10 wrky30 were substantially increased, as was the ethylene (ET) content in the leaves of wrky10. The results from dual luciferase reporter assays, electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated that VviWRKY10 could directly bind to the W-boxes in the promoter of SA-related defense genes and inhibit their transcription, supporting its role as a negative regulator of SA-dependent defense. By contrast, VviWRKY30 could directly bind to the W-boxes in the promoter of ET-related defense genes and promote their transcription, playing a positive role in ET production and ET-dependent defense. Moreover, VviWRKY10 and VviWRKY30 can bind to each other's promoters and mutually inhibit each other's transcription. Taken together, our results reveal a complex mechanism of regulation by VviWRKY10 and VviWRKY30 for activation of measured and balanced defense responses against powdery mildew in grapevine.

Chimeric mutations in grapevine ENHANCED DISEASE RESISTANCE1 improve resistance to powdery mildew without growth penalty.

Grapevine (Vitis vinifera L.) incurs severe quality degradation and yield loss from powdery mildew, a major fungal disease caused by Erysiphe necator. ENHANCED DISEASE RESISTANCE1 (EDR1), a Raf-like mitogen-activated protein kinase kinase kinase, negatively regulates defense responses against powdery mildew in Arabidopsis (Arabidopsis thaliana). However, little is known about the role of the putatively orthologous EDR1 gene in grapevine. In this study, we obtained grapevine VviEDR1-edited lines using CRISPR/Cas9. Plantlets containing homozygous and bi-allelic indels in VviEDR1 developed leaf lesions shortly after transplanting into the soil and died at the seedling stage. Transgenic plants expressing wild-type VviEDR1 and mutant Vviedr1 alleles as chimera (designated as VviEDR1-chi) developed normally and displayed enhanced resistance to powdery mildew. Interestingly, VviEDR1-chi plants maintained a spatiotemporally distinctive pattern of VviEDR1 mutagenesis: while almost no mutations were detected from terminal buds, ensuring normal function of the apical meristem, mutations occurred in young leaves and increased as leaves matured, resulting in resistance to powdery mildew. Further analysis showed that the resistance observed in VviEDR1-chi plants was associated with callose deposition, increased production of salicylic acid and ethylene, H2O2 production and accumulation, and host cell death. Surprisingly, no growth penalty was observed with VviEDR1-chi plants. Hence, this study demonstrated a role of VviEDR1 in the negative regulation of resistance to powdery mildew in grapevine and provided an avenue for engineering powdery mildew resistance in grapevine.

Application of bulk segregant RNA-Seq (BSR-Seq) and allele-specific primers to study soybean powdery mildew resistance.

BACKGROUND: Powdery mildew (PM) is one of the important soybean diseases, and host resistance could practically contribute to soybean PM management. To date, only the Rmd locus on chromosome (Chr) 16 was identified through traditional QTL mapping and GWAS, and it remains unclear if the bulk segregant RNA-Seq (BSR-Seq) methodology is feasible to explore additional PM resistance that might exist in other varieties. RESULTS: BSR-Seq was applied to contrast genotypes and gene expressions between the resistant bulk (R bulk) and the susceptible bulk (S bulk), as well as the parents. The ∆(SNP-index) and G' value identified several QTL and significant SNPs/Indels on Chr06, Chr15, and Chr16. Differentially expressed genes (DEGs) located within these QTL were identified using HISAT2 and Kallisto, and allele-specific primers (AS-primers) were designed to validate the accuracy of phenotypic prediction. While the AS-primers on Chr06 or Chr15 cannot distinguish the resistant and susceptible phenotypes, AS-primers on Chr16 exhibited 82% accuracy prediction with an additive effect, similar to the SSR marker Satt431. CONCLUSIONS: Evaluation of additional AS-primers in the linkage disequilibrium (LD) block on Chr16 further confirmed the resistant locus, derived from the resistant parental variety 'Kaohsiung 11' ('KS11'), not only overlaps with the Rmd locus with unique up-regulated LRR genes (Glyma.16G213700 and Glyma.16G215100), but also harbors a down-regulated MLO gene (Glyma.16G145600). Accordingly, this study exemplified the feasibility of BSR-Seq in studying biotrophic disease resistance in soybean, and showed the genetic makeup of soybean variety 'KS11' comprising the Rmd locus and one MLO gene.

Vibrational spectroscopic profiling of biomolecular interactions between oak powdery mildew and oak leaves.

Oak powdery mildew, caused by the biotrophic fungus Erysiphe alphitoides, is a prevalent disease affecting oak trees, such as English oak (Quercus robur). While mature oak populations are generally less susceptible to this disease, it can endanger young oak seedlings and new leaves on mature trees. Although disruptions of photosynthate and carbohydrate translocation have been observed, accurately detecting and understanding the specific biomolecular interactions between the fungus and the leaves of oak trees is currently lacking. Herein, via hybrid Raman spectroscopy combined with an advanced artificial neural network algorithm, the underpinning biomolecular interactions between biological soft matter, i.e., Quercus robur leaves and Erysiphe alphitoides, are investigated and profiled, generating a spectral library and shedding light on the changes induced by fungal infection and the tree's defence response. The adaxial surfaces of oak leaves are categorised based on either the presence or absence of Erysiphe alphitoides mildew and further distinguishing between covered or not covered infected leaf tissues, yielding three disease classes including healthy controls, non-mildew covered and mildew-covered. By analysing spectral changes between each disease category per tissue type, we identified important biomolecular interactions including disruption of chlorophyll in the non-vein and venule tissues, pathogen-induced degradation of cellulose and pectin and tree-initiated lignification of cell walls in response, amongst others, in lateral vein and mid-vein tissues. Via our developed computational algorithm, the underlying biomolecular differences between classes were identified and allowed accurate and rapid classification of disease with high accuracy of 69.6% for non-vein, 73.5% for venule, 82.1% for lateral vein and 85.6% for mid-vein tissues. Interfacial wetting differences between non-mildew covered and mildew-covered tissue were further analysed on the surfaces of non-vein and venule tissue. The overall results demonstrated the ability of Raman spectroscopy, combined with advanced AI, to act as a powerful and specific tool to probe foliar interactions between forest pathogens and host trees with the simultaneous potential to probe and catalogue molecular interactions between biological soft matter, paving the way for exploring similar relations in broader forest tree-pathogen systems.

Identification of Gene Responsible for Conferring Resistance against Race KN2 of Podosphaera xanthii in Melon.

Powdery mildew caused by Podosphaera xanthii is a serious fungal disease which causes severe damage to melon production. Unlike with chemical fungicides, managing this disease with resistance varieties is cost effective and ecofriendly. But, the occurrence of new races and a breakdown of the existing resistance genes poses a great threat. Therefore, this study aimed to identify the resistance locus responsible for conferring resistance against P. xanthii race KN2 in melon line IML107. A bi-parental F2 population was used in this study to uncover the resistance against race KN2. Genetic analysis revealed the resistance to be monogenic and controlled by a single dominant gene in IML107. Initial marker analysis revealed the position of the gene to be located on chromosome 2 where many of the resistance gene against P. xanthii have been previously reported. Availability of the whole genome of melon and its R gene analysis facilitated the identification of a F-box type Leucine Rich Repeats (LRR) to be accountable for the resistance against race KN2 in IML107. The molecular marker developed in this study can be used for marker assisted breeding programs.

Wheat Transcriptional Corepressor TaTPR1 Suppresses Susceptibility Genes TaDND1/2 and Potentiates Post-Penetration Resistance against Blumeria graminis forma specialis tritici.

The obligate biotrophic fungal pathogen Blumeria graminis forma specialis tritici (B.g. tritici) is the causal agent of wheat powdery mildew disease. The TOPLESS-related 1 (TPR1) corepressor regulates plant immunity, but its role in regulating wheat resistance against powdery mildew remains to be disclosed. Herein, TaTPR1 was identified as a positive regulator of wheat post-penetration resistance against powdery mildew disease. The transient overexpression of TaTPR1.1 or TaTPR1.2 confers wheat post-penetration resistance powdery mildew, while the silencing of TaTPR1.1 and TaTPR1.2 results in an enhanced wheat susceptibility to B.g. tritici. Furthermore, Defense no Death 1 (TaDND1) and Defense no Death 2 (TaDND2) were identified as wheat susceptibility (S) genes facilitating a B.g. tritici infection. The overexpression of TaDND1 and TaDND2 leads to an enhanced wheat susceptibility to B.g. tritici, while the silencing of wheat TaDND1 and TaDND2 leads to a compromised susceptibility to powdery mildew. In addition, we demonstrated that the expression of TaDND1 and TaDND2 is negatively regulated by the wheat transcriptional corepressor TaTPR1. Collectively, these results implicate that TaTPR1 positively regulates wheat post-penetration resistance against powdery mildew probably via suppressing the S genes TaDND1 and TaDND2.

Genome-wide identification of the CYP82 gene family in cucumber and functional characterization of CsCYP82D102 in regulating resistance to powdery mildew.

The cytochrome P450 (CYP450) gene family plays a vital role in basic metabolism, hormone signaling, and enhances plant resistance to stress. Among them, the CYP82 gene family is primarily found in dicots, and they are typically activated in response to various specific environmental stresses. Nevertheless, their roles remain considerably obscure, particularly within the context of cucumber. In the present study, 12 CYP82 subfamily genes were identified in the cucumber genome. Bioinformatics analysis included gene structure, conserved motif, cis-acting promoter element, and so on. Subcellular localization predicted that all CYP82 genes were located in the endoplasmic reticulum. The results of cis element analysis showed that CYP82s may significantly affect the response to stress, hormones, and light exposure. Expression patterns of the CYP82 genes were characterized by mining available RNA-seq data followed by qRT-PCR (quantitative real-time polymerase chain reaction) analysis. Members of CYP82 genes display specific expression profiles in different tissues, and in response to PM and abiotic stresses in this study, the role of CsCYP82D102, a member of the CYP82 gene family, was investigated. The upregulation of CsCYP82D102 expression in response to powdery mildew (PM) infection and treatment with methyl jasmonate (MeJA) or salicylic acid (SA) was demonstrated. Further research found that transgenic cucumber plants overexpressing CsCYP82D102 display heightened resistance against PM. Wild-type (WT) leaves exhibited average lesion areas of approximately 29.7% at 7 dpi upon powdery mildew inoculation. In contrast, the two independent CsCYP82D102 overexpression lines (OE#1 and OE#3) displayed significantly reduced necrotic areas, with average lesion areas of approximately 13.4% and 5.7%. Additionally, this enhanced resistance is associated with elevated expression of genes related to the SA/MeJA signaling pathway in transgenic cucumber plants. This study provides a theoretical basis for further research on the biological functions of the P450 gene in cucumber plants.

A method for phenotypic evaluation of grapevine resistance in relation to phenological development.

Fungus-resistant grapevine cultivars, so called PIWIs, are characterized by increased resistance to powdery mildew and downy mildew. However, in order to maintain the durability of resistance in these new grape cultivars, targeted fungicide treatments are recommended. For ideal schedule of these treatments, it is necessary to recognize the most sensitive organs of the grape. This study introduces a method for phenotypic evaluation of Plasmopara viticola resistance in grape clusters under controlled and standardized conditions during phenological development over the entire season. The approach was validated with the traditional cultivar Pinot Noir and the PIWIs Cabernet Cortis (Rpv3.3, Rpv10), Solaris (Rpv3.3, Rpv10) and Souvignier Gris (Rpv3.2). All cultivars were susceptible during the early stages of development up to flowering, and resistance levels increased as phenological development progressed. Cabernet Cortis and Solaris clusters were susceptible until fruit development (BBCH 71-73) when they became almost completely resistant. No differences between Souvignier Gris and Pinot Noir were detected until berries were pea-sized (BBCH 75) when P. viticola resistance of Souvignier Gris clusters increased significantly. Ontogenetic resistance in Pinot Noir was detected at berry touch (BBCH 77-79) and clusters of this cultivar were almost completely resistant at the beginning of ripening (BBCH 81-83). These results indicate that the approach presented is suitable for determining the resistance of grape cultivars at different stages of development. Consequently, in the future, fungicide applications can be adjusted more precisely to the resistance level of a grape cultivar during the growing season.

DNA barcoding and scanning electron microscopy reveals Erysiphe ahmadii sp. nov. and a new record Erysiphe populicola (Erysiphaceae, Helotiales) on salicaceous hosts from Pakistan.

A new species of powdery mildew fungus Erysiphe ahmadii and a new record, Erysiphe populicola, on Salicaceae are described from Pakistan. In addition to light microscopy, scanning electron microscopy is also done to clearly demonstrate the surface characters of chasmothecia. E. ahmadii sp. nov. is characterized by large conidia ((-26)29-35(-37) × (-16)17-21(-23) µm), long chasmothecial appendages (198-286 µm) and small conidiophores. The novelty is confirmed by analyzing the genetic variation of internal transcribed spacer region (ITS1-5.8S-ITS2) of the ribosomal DNA gene, a universal fungal marker. E. populicola is characterized for the first time using molecular phylogenetic markers. Detailed descriptions along with scanning electron microscopy (SEM) photographs are provided in this paper. RESEARCH HIGHLIGHTS: Powdery mildews are obligate biotrophic pathogens of plants. Erysiphe ahmadii, a new powdery mildew fungus on willow trees, is described. First reference sequence of Erysiphe populicola is also generated. Both taxa are discussed in detail using macro- and micro-morphological and DNA barcoding techniques.

Powdery mildew caused by Erysiphe corylacearum: An emerging problem on hazelnut in Italy.

Erysiphe corylacearum has recently been reported in northern Italy (Piedmont) and other European countries as the causal agent of a new emerging powdery mildew on hazelnut. This disease is much more dangerous than the common hazelnut powdery mildew caused by Phyllactinia guttata as it significantly reduces yield and quality of hazelnuts. This study aimed to perform morphological and molecular characterization of the fungal isolates from powdery mildew-infected plants in the Piedmont Italian region. Additionally, genetic diversity studies and pathogenicity tests were conducted. Thirty-six fungal isolates originating from symptomatic hazelnut plants exhibiting specific powdery mildew symptoms on the superior leaf side were identified morphologically as E. corylacearum. Single- and multilocus sequence typing of five loci (ITS, rpb2, CaM, GAPDH and GS) assigned all isolates as E. corylacearum. Multilocus and GAPDH phylogenetic studies resulted in the most efficient characterization of E. corylacearum. Studied fungal isolates were able to cause new emerging powdery mildew disease by fulfilling Koch's postulates. The emergence of powdery mildew disease in Italy revealed the E. corylacearum subgrouping, population expansion, and high nucleotide similarity with other recently identified E. corylacearum hazelnut isolates. To contain this harmful disease and inhibit the fungus spread into new geographical zones, it will be necessary to implement more rigorous monitoring in neighboring hazelnut plantations near infected hazelnuts, use sustainable fungicides and search for new biocontrol agents.

WRR4B contributes to a broad-spectrum disease resistance against powdery mildew in Arabidopsis.

Oidium heveae HN1106, a powdery mildew (PM) that infects rubber trees, has been found to trigger disease resistance in Arabidopsis thaliana through ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-, PHYTOALEXIN DEFICIENT 4 (PAD4)- and salicylic acid (SA)-mediated signalling pathways. In this study, a typical TOLL-INTERLEUKIN 1 RECEPTOR, NUCLEOTIDE-BINDING, LEUCINE-RICH REPEAT (TIR-NB-LRR)-encoding gene, WHITE RUST RESISTANCE 4 (WRR4B), was identified to be required for the resistance against O. heveae in Arabidopsis. The expression of WRR4B was upregulated by O. heveae inoculation, and WRR4B positively regulated the expression of genes involved in SA biosynthesis, such as EDS1, PAD4, ICS1 (ISOCHORISMATE SYNTHASE 1), SARD1 (SYSTEMIC-ACQUIRED RESISTANCE DEFICIENT 1) and CBP60g (CALMODULIN-BINDING PROTEIN 60 G). Furthermore, WRR4B triggered self-amplification, suggesting that WRR4B mediated plant resistance through taking part in the SA-based positive feedback loop. In addition, WRR4B induced an EDS1-dependent hypersensitive response in Nicotiana benthamiana and contributed to disease resistance against three other PM species: Podosphaera xanthii, Erysiphe quercicola and Erysiphe neolycopersici, indicating that WRR4B is a broad-spectrum disease resistance gene against PMs.

Alternative products to control powdery mildew in soybeans culture in field

The occurrence of powdery mildew (Microsphaera diffusa) in soybean (Glycine max L.) has increased in the last harvests. In order to study the efficiency of powdery mildew control due to the application of alternative products and conventional fungicide, trials were conducted in Ponta Grossa, PR, Brazil, during the 2013/2014 and 2014/2015 growing seasons. The design used was randomized blocks with four replications. The treatments for the experiments were: 1 - control; 2 - acibenzolar-S-methyl (Bion 500 WG®); 3 - calcium (Max Fruit®); 4 - Micronutrients: copper, manganese and zinc (Wert Plus®); 5 - Micronutrients: manganese, zinc and molybdenum (V6®); 6 - NK fertilizer (Hight Roots®); 7 - Ascophyllum nodosum (Acadian®) and 8 - fungicide (azoxystrobin + cyproconazole) (Priori XTRA®) with the addition of the adjuvant. Four applications of alternative products (phenological stages V3, V6, R1 and R5.1) and two of fungicide (phenological stages R1 and R5.1) were carried out. The parameters evaluated were powdery mildew severity and productivity. The severity data made it possible to calculate the area under the disease progress curve (AUDPG). Alternative products didn't reduce powdery mildew in the two harvests. The conventional fungicide treatment was the only one that controlled powdery mildew and didn't reduce the productivity in both experiments.

Danos causados pela infecção de oídio em diferentes estádios fenológicos da soja / Damages caused by infection of powdery mildews in different phenological stages of soybeans

Ainda não há estudos precisos que quantifiquem os prejuízos decorrentes de infecção por oídio e/ou outras doenças foliares, para a maioria das culturas de importância econômica no Brasil. O objetivo foi quantificar as perdas causadas por oídio (Microsphaera diffusa) infectando a cultura da soja em diferentes estádios fenológicos e relacioná-las ao desenvolvimento e produtividade da cultura. O experimento foi desenvolvido em ambiente protegido, e os tratamentos foram testemunha controlada, testemunha sem controle, infecção iniciada em R1 ­ R2, infecção iniciada em R ­ R, infecção iniciada em R ­ R e infecção iniciada em R ­ R. A avaliação foi feita 345.15.25.35.4 semanalmente, considerando a porcentagem da área foliar infectada. Os resultados mostraram que, no tratamento em que houve infecções iniciadas em R1-R2 e R3-R4, a porcentagem de área foliar afetada foi maior (41% e 38%, respectivamente), com consequente menor produtividade (1.186,6 e 1.309,5 kg.ha-1 respectivamente). No tratamento em que a infecção ocorreu em R ­ R, houve 5.35.4 a menor média de área foliar afetada pela doença (24%) e a produtividade teve queda de 26%. Os resultados mostraram que as perdas de produtividade pelo oídio na cultivar Embrapa 48 variaram ao redor de 26 a 50%, e que a recomendação oficial para o início de controle do oídio da soja, quando esta se apresentar entre 40 e 50% de severidade, deve ser questionada e outros trabalhos neste âmbito devem ser desenvolvidos para determinação das perdas ocasionadas por esta doença na cultura.

At present there are no precise studies quantifying the damages caused by powdery mildews and other foliar diseases for the majority of economically important crops in Brazil. The objective of the present study was to quantify the losses caused by powdery mildews (Microsphaera diffusa) in soybeans in different phenological stages, and to correlate them with the development and yield of the crop. The trials was carried out in the greenhouse and the treatments were: controlled check, noncontrolled check, infection initiated at stage R1-R2, infection initiated at stage R3-R4, infection initiated at stage R5.1-R5.2, infection initiated at stage R5.3-R5.4. The evaluation was done weekly considering the percentage of infected leaf area. The results showed that for the infection beginning at stages R1-R2 and R3-R4 the percentage of affected leaf area was higher (41% and 38%), with consequently lower yields (1,200 and 1,240 kg ha-1). When the infection occurred later at stage R5.3-R5.4, a lower affected leaf area (24%) was observed, and the yield decreased 26%. The results showed that the loss of yield by powdery mildew in cultivar Embrapa 48 ranged from around 26 to 50%, and that the official recommendation for the beginning of control of powdery mildew of soybean, where it presents between 40 and 50% of severity, should be questioned, and other work in this area should be undertaken to determine the loss caused by this disease in the crop.