Wheat powdery mildew caused by Blumeria graminis f. sp tritici (Bgt), an obligate biotrophic fungal pathogen, is a destructive disease world-wide, particularly severe in China. However, the molecular mechanisms underlying virulence variation and pathogenesis of Chinese Bgt isolates remain poorly understood. Here, we constructed a chromosome-level genome assembly (136.19 Mb) of Chinese isolate 21-2, with repetitive element expansion (predominantly retrotransposons), by integrating Illumina, PacBio, Nanopore, and Hi-C sequencing technologies. The genome was predicted to contain 9215 protein-coding genes, of which 1569 were assessed as pathogenicity-related genes. This included 998 effectors, 371 involved in pathogen-host interactions (PHI), 223 CAZymes-encoding with plant cell wall degrading capacity, and 79 lipase-coding implicated in pathogenic infection. Compared to Swiss isolate 96224, isolate 21-2 displayed a distinct virulence pattern on 21 wheat differential lines. Comparative genomics analysis revealed that variations in composition and sequence of effector genes between the two isolates resulted in different virulence spectra (e.g., AvrPm1a, AvrPm3a 2/f2, AvrPm3b2/c2, AvrPm3d3 ). Transcriptome analysis of 21-2 revealed 64 effector genes exhibiting preferential expression during haustorial development, suggesting their potential involvement in pathogenesis. Among them, one can bind a defence-related protein of wheat and may play a key role in suppressing host immune responses and promoting disease progression. This study provides comprehensive genomic and transcriptomic insights into Chinese Bgt isolate 21-2, and reveals virulence determinants and their variants fundamental to pathogenesis. Future functional analysis of such genes will enhance our understanding of pathogenic mechanisms of powdery mildews.

Cereal powdery mildews, which are caused by Blumeria graminis, are economically important diseases that are distributed throughout the world. To successfully evade the host defence mechanism, the wheat powdery mildew pathogen known as B. graminis f. sp. tritici (Bgt) secretes an array of effectors into plant cells to interfere with host immunity and promote fungal invasion and colonisation during the infection process. However, little is known about the functions of the vast majority of these effectors in immune manipulation. In this study, we identified an effector-coding gene known as BgtE-20069a from Bgt. This gene encodes a short protein carrying an N-terminal signal peptide with a secretory function and is highly upregulated in the early stage of Bgt infection in wheat. We observed that transient expression of BgtE-20069a in Nicotiana benthamiana suppressed programmed cell death (PCD) induced by both the proapoptotic protein Bax and the elicitor PAMP INF1 from Phytophthora infestans. The mature form of BgtE-20069a (which lacks a signal peptide) is localised to the cytoplasm and nucleus of plant cells. Moreover, the knockdown of BgtE-20069a resulted in reduced virulence towards wheat, with significantly decreased conidia production and a decreased haustorial formation rate being observed. Together, these results suggest that BgtE-20069a is a vital virulence factor that is required for Bgt infection in wheat; moreover, the results indicate that it can suppress plant immunity and increase Bgt virulence. Our findings broaden the current understanding of the role of effectors in promoting Bgt infection by manipulating host immunity, thereby providing new insights into the molecular mechanism of Bgt pathogenesis.

The discovery of natural bioactive compounds from endophytes or medicinal plants against plant diseases is an attractive option for reducing the use of chemical fungicides. In this study, three compounds, indole-3-carbaldehyde, indole-3-carboxylic acid (3-ICA), and jasmonic acid (JA), were isolated from the EtOAc extract of the culture filtrate of the endophytic fungus Lasiodiplodia pseudotheobromae LPS-1, which was previously isolated from the medicinal plant, Ilex cornuta. Some experiments were conducted to further determine the antifungal activity of these compounds on wheat powdery mildew. The results showed that JA was much more bioactive than indole-3-carbaldehyde and 3-ICA against Blumeria graminis, and the disease severity caused by B. graminis decreased significantly with the concentration increase of JA treatment. The assay of the interaction of 3-ICA and JA indicated that there was a significant synergistic effect between the two compounds on B. graminis in each of the ratios of 3-ICA to JA (3-ICA:JA) ranging from 1:9 to 9:1. When the compound ratio of 3-ICA to JA was 2:8, the synergistic coefficient was the highest as 22.95. Meanwhile, a histological investigation indicated that, under the treatment of JA at 500 μg/ml or 3-ICA:JA (2:8) at 40 μg/ml, the appressorium development and haustorium formation of B. graminis were significantly inhibited. Taken together, we concluded that JA plays an important role in the infection process of B. graminis and that 3-ICA as a synergist of JA enhances the antagonism against wheat powdery mildew.

Context Cytokinin response regulators (RRs) are important components of the two component signal systems that are involved in the regulation of plant growth and development, and in the response to abiotic stresses. Plant cytokinin response regulators (RR) were divided into type A and type B. A-type RR proteins act as negative feedback to regulate cytokinin signals, while B-type RRs could regulate A-type RR gene expression, and B-type RR genes have proved to play important roles in regulating cytokinin signal transduction in various biological processes. Aims We aimed to explore and analyse B-type RR genes in wheat in a preliminary fashion. Methods Using bioinformatics methods, wheat type B RR genes were identified, and type B Triticum aestivum RR (TaRR) genes were analysed using quantitative real-time polymerase chain reaction. In order to further analyse the function of TaRR, staining experiments were performed. Key results Twenty-nine B-type TaRR genes were identified in the wheat genome, divided into three groups according to their phylogenetic relationships. Chromosome mapping showed that 29 TaRRs were evenly distributed on 12 chromosomes, while there were no genes located on the other nine chromosomes, which may have experienced gene loss during evolution. The polymerase chain reaction results showed that TaRRs were significantly up-regulated under polyethylene glycol treatments. Under sodium chloride stress, TaRRs were up-regulated to varying degrees, reaching the maximum at 24 h. The study also found that the expression pattern of TaRRs was different in the root and leaf under different abiotic stresses. In addition, staining experiments also showed that TaRR5.1-6A could induce the self-defence function of leaves. Conclusions These results form the basis for further exploring the role of B-type TaRR genes in plant response to drought stress and salt stress. Implications This study lays the molecular biology foundation for the functional study of the B-type TaRR genes.

Bacillus amyloliquefaciens strain EA19 is an endophyte isolated from Erigeron annuus with antifungal activity against Blumeria graminis f. sp. tritici , Magnaporthe oryzae , and Fusarium graminearum . The genome sequence of this strain is 3.96 Mb and contains 3,421 coding sequences, which will facilitate an understanding of the mechanisms of biocontrol.

Conidia of the obligate biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt) play a vital role in its survival and rapid dispersal. However, little is known about the genetic basis for its asexual reproduction. To uncover the primary metabolic and regulatory events during conidiation, we sequenced the transcriptome of Bgt epiphytic structures at 3 (vegetative hyphae growth), 4 (foot cells initiation), and 5 (conidiophore erection) days post-inoculation (dpi). RNA-seq analyses identified 556 and 404 (combined 685) differentially expressed genes (DEGs) at 4 and 5 dpi compared with their expression levels at 3 dpi, respectively. We found that several genes involved in the conversion from a variety of sugars to glucose, glycolysis, the tricarboxylic acid cycle (TAC), the electron transport chain (ETC), and unsaturated fatty acid oxidation were activated during conidiation, suggesting that more energy supply is required during this process. Moreover, we found that glucose was converted into glycogen, which was accumulated in developing conidiophores, indicating that it could be the primary energy storage molecule in Bgt conidia. Clustering for the expression profiles of 91 regulatory genes showed that calcium (Ca2+), H2O2, and phosphoinositide (PIP) signaling were involved in Bgt conidiation. Furthermore, a strong accumulation of H2O2 in developing conidiophores was detected. Application of EGTA, a Ca2+ chelator, and trifluoperazine dihydrochloride (TFP), a calmodulin (CaM) antagonist, markedly suppressed the generation of H2O2, affected foot cell and conidiophore development and reduced conidia production significantly. These results suggest that Ca2+ and H2O2 signaling play important roles in conidiogenesis and a crosslink between them is present. In addition to some conidiation-related orthologs known in other fungi, such as the velvet complex components, we identified several other novel B. graminis-specific genes that have not been previously found to be implicated in fungal conidiation, reflecting a unique molecular mechanism underlying asexual development of cereal powdery mildews.

Deployment of cultivars with genetic resistance is an effective approach to control the diseases of powdery mildew (PM) and yellow rust (YR). Chinese wheat cultivar XK0106 exhibits high levels of resistance to both diseases, while cultivar E07901 has partial, adult plant resistance (APR). The aim of this study was to map resistance loci derived from the two cultivars and analyze their effects against PM and YR in a range of environments. A doubled haploid population (388 lines) was used to develop a framework map consisting of 117 SSR markers, while a much higher density map using the 90K Illumina iSelect SNP array was produced with a subset of 80 randomly selected lines. Seedling resistance was characterized against a range of PM and YR isolates, while field scores in multiple environments were used to characterize APR. Composite interval mapping (CIM) of seedling PM scores identified two QTLs (QPm.haas-6A and QPm.haas-2A), the former being located at the Pm21 locus. These QTLs were also significant in field scores, as were Qpm.haas-3A and QPm.haas-5A. QYr.haas-1B-1 and QYr.haas-2A were identified in field scores of YR and were located at the Yr24/26 and Yr17 chromosomal regions respectively. A second 1B QTL, QYr.haas-1B-2 was also identified. QPm.haas-2A and QYr.haas-1B-2 are likely to be new QTLs that have not been previously identified. Effects of the QTLs were further investigated in multiple environments through the testing of selected lines predicted to contain various QTL combinations. Significant additive interactions between the PM QTLs highlighted the ability to pyramid these loci to provide higher level of resistance. Interactions between the YR QTLs gave insights into the pathogen populations in the different locations as well as showing genetic interactions between these loci.

Summary There is a large diversity of genetically defined resistance genes in bread wheat against the powdery mildew pathogen Blumeria graminis (B. g.) f. sp. tritici. Many confer race‐specific resistance to this pathogen, but until now only the mildew avirulence gene AvrPm3a2/f2 that is recognized by Pm3a/f was known molecularly. We performed map‐based cloning and genome‐wide association studies to isolate a candidate for the mildew avirulence gene AvrPm2. We then used transient expression assays in Nicotiana benthamiana to demonstrate specific and strong recognition of AvrPm2 by Pm2. The virulent AvrPm2 allele arose from a conserved 12 kb deletion, while there is no protein sequence diversity in the gene pool of avirulent B. g. tritici isolates. We found one polymorphic AvrPm2 allele in B. g. triticale and one orthologue in B. g. secalis and both are recognized by Pm2. AvrPm2 belongs to a small gene family encoding structurally conserved RNase‐like effectors, including Avra13 from B. g. hordei, the cognate Avr of the barley resistance gene Mla13. These results demonstrate the conservation of functional avirulence genes in two cereal powdery mildews specialized on different hosts, thus providing a possible explanation for successful introgression of resistance genes from rye or other grass relatives to wheat.

•208 endophytic fungal isolates were collected from stems (83), leaves (121) and flowers (4) of 26 medicinal plants.•Fifteen endophytic fungi exhibited antifungal activity.•Strain of L. pseudotheobromae has strong biological control of wheat powdery mildew.•The first report of L. pseudotheobromae having antifungal activity against plant pathogens.

Blumeria graminis f. sp. tritici (Bgt) is an obligate parasite that only infects living tissues of wheat (Triticum aestivum L.). Long term preservation of Bgt is useful for genetic studies but it is challenging due to difficulty of artificial cultivation. In this study a simple protocol was developed in which desiccating the conidia using crystals (3–6 mm in diameter) of silica gel allowed viable storage for 12 months. The conidia were mixed with silica gel, dried for 5 h at 23 °C, and then stored at −80 °C. The preserved Bgt isolates still maintained their viability after 12 months and successfully infected detached leaves of wheat seedlings. Analysis of 20 Bgt isolates revealed that the cryopreservation process had no effect on two key phenotypic variants: virulence against 12 near-isogenic wheat cultivars and their sensitivity to the fungicide triadimefon. DNA sequencing analysis confirmed that the nucleotide sequences of the 1.4 α-demethylase inhibitor (cyp51), chitin synthase 1 (chs1), and β-tubulin (tub2) genes had not changed and still contained the corresponding single nucleotide mutations. These results indicate that the silica gel-based desiccation protocol is suitable for the long term preservation of large numbers of Bgt isolates.

A broad survey was conducted and infected heads of wheat were collected from 33 counties in Sichuan,Chongqin,Hubei,Anhui,Jiangsu and Henan Provinces in 2008.Totally 433 Fusarium single spore isolates were obtained and the population and mycotoxin chemotypes had been studied through PCR assay.Four Fusarium species,F.asiaticum,F.graminearum,F.avenaceum and F.meridionale,were detected in Sichuan,while two species F.asiaticum and F.graminearum were detected in Chongqing,Hubei,Anhui and Jiangsu.In Henan Province,only F.graminearum was found.Chemotype detection results showed that Nivalenol was the main chemotype in Sichuan and Chongqing,while Deoxynivalenol was the major chemotype in Hubei,Henan,Anhui and Jiangsu.After further divided the DON chemotype strains into 3-AcDON and 15-AcDON,the results showed that 3-AcDON isolates mainly lied in Sichuan,Hubei and Jiangsu,both 3-AcDON and 15-AcDON isolates mixed in Anhui,and all the isolates in Henan belonged to 15-AcDON.The results indicated that F.asiaticum was the dominant specie in Sichan,Chongqing,Hubei and Jiangsu Province.It also showed that there was a clear geographical distribution of DON and NIV produced by Fusarium species.NIV was the dominant chemotype in wheat producing region belonged to the upper reaches of Yangtze River while DON was the dominant chemotype in the lower reaches.There were some relationship between DON chemotype and Fusarium species.

BACKGROUND Physcion is a key active ingredient of the ethanol extract from roots of Chinese rhubarb (Rheum officinale Baill.) that has been commercialised in China for controlling powdery mildews. The biological mechanism of action of physcion against the barley powdery mildew pathogen was studied using bioassay and microarray methods. RESULTS Bioassay indicated that physcion did not directly affect conidial germination of Blumeria graminis Speer f. sp. hordei Marchal, but significantly inhibited conidial germination in vivo. Challenge inoculation indicated that physcion induced localised resistance rather than systemic resistance against powdery mildew. Gene expression profiling of physcion-treated barley leaves detected four upregulated and five downregulated genes (ratio >or= 2.0 and P-value < 0.05) by using an Affymetrix Barley GeneChip. The five upregulated probe sequences blasted to the same barley leaf-specific thionin gene, with significant changes varying from 4.26 to 19.91-fold. All downregulated genes were defence-related, linked to peroxidase, oxalate oxidase, bsi1 protein and a pathogenesis-related protein. These changes varied from - 2.34 to - 2.96. Quantitative real-time PCR data confirmed that physcion enhanced the gene expression of leaf-specific thionin of barley. CONCLUSION Results indicated that physcion controls powdery mildew mainly through changing the expression of defence-related genes, and especially enhancing expression of leaf-specific thionin in barley leaves.

The ethanol extract from rhubarb was commercialized in China (Veguard, Physcion, 5 g/liter AS) for control of cucumber powdery mildew (Sphaerotheca fuliginea) and cucumber downy mildew (Pseudoperonospora cubensis). To provide the basic data for the risk assessment of resistance to this product, physcion was selected to represent the active ingredients to establish baseline sensitivity of powdery mildew and downy mildew populations. For powdery mildew, 262 isolates of S. fuliginea from nine regions and, for downy mildew populations, 116 isolates of P. cubensis from six regions were collected in China during 2004 and 2005 and tested for sensitivity. In addition, the sensitivity of a powdery mildew isolate was monitored for 15 asexual generations under selection pressure with physcion. The results showed that there was no significant difference among regions in the frequency distribution of baseline sensitivity to this ingredient for either cucumber powdery mildew isolates or cucumber downy mildew isolates. Baseline sensitivity was distributed as a normal unimodal curve with a mean median (50%) effective concentration (EC50) of 0.304 μg/ml for powdery mildew population and mean EC50 of 0.501 μg/ml for downy mildew population. The variation of sensitivity to physcion was low because the range factor (maximum EC50/minimum EC50 of isolates within population) varied from 1.63 to 3.42 among powdery mildew populations and from 1.70 to 2.38 among downy mildew populations. The powdery mildew isolate XZ4 did not decrease sensitivity under the selection pressure of physcion at the dose of EC70 for 15 generations.