Recyclable N-Heterocyclic Carbene Porous Coordination Polymers with Two Distinct Metal Sites for Transformation of CO2 to Cyclic Carbonates.

Single-component catalysts with integrated multiple reactive centers could work in concert to achieve enhanced activity tailored for specific catalytic reactions, but they remain underdeveloped. Herein, we report the construction of heterogeneous bimetallic porous coordination polymers (PCPs) containing both porphyrin and N-heterocyclic carbene (NHC) metal sites via the coordinative assembly of the NHC functionalities. Three heterobimetallic PCPs (TIPP-Zn-Pd, TIPP-Cu-Pd and TIPP-Ni-Pd) have been prepared to verify this facile synthetic strategy for the first time. In order to establish a cooperative action toward the catalytic CO2 cycloaddition with epoxides, an additional tetraalkylammonium bromide functionality has also been incorporated into these polymeric structures through the N-substituent of the NHC moieties. The resulting heterogeneous bimetallic catalyst TIPP-Zn-Pd exhibits the best catalytic performance in CO2 cycloaddition with styrene oxide (SO) under solvent-free conditions at atmospheric pressure and is applicable to a wide range of epoxides. More importantly, TIPP-Zn-Pd works smoothly and is recyclable in the absence of a cocatalyst under 1.0 MPa of CO2 at 60 °C. This indicates that TIPP-Zn-Pd is quite competitive with the reported heterogeneous catalysts, which typically require a high reaction temperature above 100 °C under cocatalyst-free conditions. Thus, this work provides a new approach to design heterogeneous bimetallic PCP catalysts for high-performance CO2 fixation under mild reaction conditions.

Valsa mali PR1-like protein modulates an apple valine-glutamine protein to suppress JA signaling-mediated immunity.

Apple Valsa canker (AVC) is a devastating disease of apple (Malus × domestica), caused by Valsa mali (Vm). The Cysteine-rich secretory protein, Antigen 5, and Pathogenesis-related protein 1 (CAP) superfamily protein PATHOGENESIS-RELATED PROTEIN 1-LIKE PROTEIN c (VmPR1c) plays an important role in the pathogenicity of Vm. However, the mechanisms through which it exerts its virulence function in Vm-apple interactions remain unclear. In this study, we identified an apple valine-glutamine (VQ)-motif-containing protein, MdVQ29, as a VmPR1c target protein. MdVQ29-overexpressing transgenic apple plants showed substantially enhanced AVC resistance as compared with the wild type. MdVQ29 interacted with the transcription factor MdWRKY23, which was further shown to bind to the promoter of the jasmonic acid (JA) signaling-related gene CORONATINE INSENSITIVE 1 (MdCOI1) and activate its expression to activate the JA signaling pathway. Disease evaluation in lesion areas on infected leaves showed that MdVQ29 positively modulated apple resistance in a MdWRKY23-dependent manner. Furthermore, MdVQ29 promoted the transcriptional activity of MdWRKY23 toward MdCOI1. In addition, VmPR1c suppressed the MdVQ29-enhanced transcriptional activation activity of MdWRKY23 by promoting the degradation of MdVQ29 and inhibiting MdVQ29 expression and the MdVQ29-MdWRKY23 interaction, thereby interfering with the JA signaling pathway and facilitating Vm infection. Overall, our results demonstrate that VmPR1c targets MdVQ29 to manipulate the JA signaling pathway to regulate immunity. Thus, this study provides an important theoretical basis and guidance for mining and utilizing disease-resistance genetic resources for genetically improving apples.

Rapid and Accurate Detection of Marssonina coronariae in Apple Trees using Loop-Mediated Isothermal Amplification (LAMP).

Marssonina blotch of apple is a well-known plant disease caused by Marssonina coronariae, which can cause severe economic consequences. Due to the importance of early diagnosis for effective plant disease management, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay that could rapidly detect M. coronariae in apple plants. The ribosomal DNA internal transcribed spacer (rDNA-ITS) sequence of M. coronariae was selected as the target for primer design. Our results showed optimal conditions for the LAMP reaction at 62℃ for 50 min, as indicated by color change and gel electrophoresis. The LAMP assay demonstrated specific discriminatory capability in differentiating M. coronariae from other pathogenic fungi in apple plants. In addition, the sensitivity tests revealed a detection limit of 100 fg µL-1 genomic DNA and 100 spores of M. coronariae for the LAMP assay. Finally, we successfully applied the LAMP assay to detect M. coronariae in apple leaf samples from the field. In general, our study provided a straightforward and efficient method for rapid diagnosis of apple blotch caused by M. coronariae, which could be applied in field condition and early occurrence of disease caused by M. coronariae could be detected.

Exploring the efficacy of carvacrol as a biocontrol agent against pear Valsa canker.

Valsa canker, a fungal disease caused by Valsa pyri, poses a significant threat to the pear industry. Currently, chemical control serves as the primary method to control valsa canker. However, the emergence of resistance can pose a challenge to its effectiveness. Biopesticides are a relatively new option for disease control, but there is limited research on their effects on pear Valsa canker. To determine the effectiveness of different biopesticides, we selected 10 common biopesticides to test their inhibition efficacy and impacts on mycelial growth rate and conidial germination. Results showed that carvacrol had very good antifungal activity; therefore its inhibition mechanisms were further investigated. Electron microscopy and transcriptome data analysis were utilized to examine how carvacrol impeded V. pyri by inducing mycelium deformation, wrinkling, and rupture. Carvacrol also affected plant hormones, thus improving plant resistance to the disease. This study lays the groundwork for the utilization of 10 distinct biopesticides to control V. pyri while elucidating how carvacrol harms the pathogen and prompts the plant defense control mechanism.

Inhibitory effects of Bacillus vallismortis T27 against apple Valsa canker caused by Valsa mali.

Apple Valsa canker caused by the pathogenic fungus Valsa mali, are one of the most destructive diseases of woody plants worldwide. One rhizosphere microbe strain, designated as T27 and subsequently identified as Bacillus vallismortis based on morphological and phylogenetic analyses, was studied as a potential biocontrol agent. Inoculation assay showed the B. vallismortis T27 suppressed the mycelial growth of V. mali with 81.33% antifungal effect on dual culture plates and caused hyphal deformities, wrinkles. The T27 fermentation broth significantly suppress the fungi's ability to acidify the surrounding environment. The addition of T27 cell-free supernatant (CFS) caused the pH of the fungal culture medium to increase from 3.60 to 5.10. B. vallismortis T27 showed the presence of Surfactin, IturinA and Bacilysin antimicrobial biosynthetic genes by the PCR assay. In addition, the B. vallismortis T27 was able to promote plant growth by producing siderophores and solubilizing phosphorus. The application of 2% fermentation broth of T27 resulted in a significant increase of 55.99% in the height of tomato plants and a 33.03% increase in the fresh weight of tomatoes. Under laboratory and field conditions, the B. vallismortis T27 exhibited strong antifungal activities on detached twigs and intact plants. The treatment of T27 resulted in a 35.9% reduction in lesion area on detached twigs. Furthermore, when applied to intact plants, T27 demonstrated a scar healing rate of 85.7%, surpassing the 77.8% observed in the treatment with tebuconazole. Comparative transcriptome analysis showed down-regulation of the genes associated with the fungal cell wall and cell membrane's synthesis and composition during V. mali treated with the B. vallismortis T27. In addition, gene transcription level analysis under treatment with B. vallismortis T27 revealed a significant increase in the expression levels of genes associated with diterpene biosynthesis, alanine, aspartic acid and glutamate metabolism, and plant hormone signaling in the apple, consistent with qRT-PCR and RNA-seq results. In this study, B. vallismortis T27 isolated from rhizosphere soil and identified as a novel biological control agent against apple Valsa canker. It exhibited effectively control over Valsa canker through multiple mechanisms, including disrupting the fungal cell membrane structure, altering the fungal growth environment, activating the plant MAPK pathway, and inducing upregulation of plant terpene biosynthetic genes. These findings highlight the potential of B. vallismortis T27 as a promising and multifaceted approach for managing apple Valsa canker.

VmPacC-mediated pH regulation of Valsa mali confers to host acidification identified by comparative proteomics analysis.

Apple valsa canker caused by the Ascomycete fungus Valsa mali is one of the most serious diseases of apple, resulting in huge economic losses in the apple-growing area of China. Previous study found that the pathogen could acidify the infected tissues to make lower ambient pH (from 6.0 to 3.5) for their successfully colonization. The pH signaling transcription factor VmPacC is required for acidification of its environment and for full virulence in V. mali. It is known that the functional cooperation of proteins secreted by V. mali plays pivotal role in its successful colonization of host plants. In this study, we used tandem mass tag (TMT) labeling coupled with LC-MS/MS-based quantitative proteomics to analyze the VmPacC-mediated pH regulation in V. mali, focusing on differentially expressed proteins (DEPs). We identified 222 DEPs specific to VmPacC deletion, and 921 DEPs specific to different pH conditions (pH 6.0 and 3.4). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that these DEPs were mainly involved in pathways associated with carbon metabolism, biosynthesis of antibiotics, citrate cycle (TCA cycle), glycolysis/gluconeogenesis, glutathione metabolism, ribosomes, and pentose phosphate pathways. Additionally, we identified 119 DEPs that were shared among the VmPacC deletion mutant and different pH conditions, which were mainly related to energy metabolism pathways, providing the energy required for the hyphal growth and responses to environmental stresses. A protein-protein interaction (PPI) network analysis indicated that most of the shared proteins were mapped to an interaction network with a medium confidence score of 0.4. Notably, one uncharacterized protein (KUI69106.1), and two known proteins (heat shock protein 60 (KUI73579.1), aspartate aminotransferase (KUI73864.1)) located in the core of the network were highly connected (with ≥ 38 directed edges) with the other shared DEPs. Our results suggest that VmPacC participates in the pathogen's regulation to ambient pH through the regulation of energy metabolism pathways such as the glycolysis/gluconeogenesis pathway and TCA cycle. Finally, we proposed a sophisticated molecular regulatory network to explain pH decrease in V. mali. Our study, by providing insights into V. mali regulating pH, helps to elucidate the mechanisms of host acidification during pathogen infection.

VmPma1 contributes to virulence via regulation of the acidification process during host infection in Valsa mali.

Valsa mali is a destructive phytopathogenic fungus that mainly infects apple and pear trees. Infection with V. mali results in host tissue acidification via the generation of citric acid, which promote invasion. Here, two plasma membrane H+-ATPases, VmPma1 and VmPma2, were identified in V. mali. The VmPma1 deletion mutant (∆VmPma1) displayed higher intracellular acid accumulation and a lower growth rate compared to the wild type. In contrast, the VmPma2 deletion mutant (∆VmPma2) showed no obvious phenotypic differences. Meanwhile, loss of VmPma1, but not VmPma2, in V. mali led to a significant decrease in growth under acidic or alkaline conditions compared with WT. More importantly, ∆VmPma1 showed a greater reduction in ATPase hydrolase activity and acidification of the external environment, more sensitivity to abiotic stress, and weaker pathogenicity than ∆VmPma2. This evidence indicates that VmPma1 is the main gene of the two plasma membrane H+-ATPases. Transcriptomic analysis indicated that many metabolic processes regulated by VmPma1 are strictly pH-regulated. Besides, we identified two genes (named VmAgn1p and Vmap1) that contribute to the pathogenicity of V. mali by differentially regulating external acidification capacity. Overall, our findings show that VmPma1 plays a pivotal role in pathogenicity by affecting the acidification of V. mali.

Transcriptome Analysis of Early Senescence in the Post-Anthesis Flag Leaf of Wheat (Triticum aestivum L.).

Flag leaf senescence is an important determinant of wheat yield, as leaf senescence occurs in a coordinated manner during grain filling. However, the biological process of early senescence of flag leaves post-anthesis is not clear. In this study, early senescence in wheat was investigated using a high-throughput RNA sequencing technique. A total of 4887 differentially expressed genes (DEGs) were identified, and any showing drastic expression changes were then linked to particular biological processes. A hierarchical cluster analysis implied potential relationships between NAC genes and post-anthesis senescence in the flag leaf. In addition, a large set of genes associated with the synthesis; transport; and signaling of multiple phytohormones (JA, ABA, IAA, ET, SA, BR, and CTK) were expressed differentially, and many DEGs related to ABA and IAA were identified. Our results provide insight into the molecular processes taking place during the early senescence of flag leaves, which may provide useful information in improving wheat yield in the future.

VmMon1-Ccz1 Complex Is Required for Conidiation, Autophagy, and Virulence in Valsa mali.

Apple Valsa canker caused by Valsa mali is a serious disease in eastern Asia, especially in China. In our previous proteomics study, monensin sensitivity 1 protein in Valsa mali (VmMon1) was identified to be significantly upregulated during V. mali infection. It was reported Mon1 protein formed a heterodimer called MC (Mon1-Ccz1) complex with caffeine, calcium, and zinc sensitivity 1 protein (Ccz1) in yeast. However, Ccz1 had not been identified in plant-pathogenic fungi such as Fusarium graminearum and Magnaporthe oryzae. Here, we identified a Ccz1 ortholog VmCcz1 in V. mali, by using DELTA-BLAST. The interaction of VmMon1 and VmCcz1 were verified using yeast two-hybrid assay, bimolecular fluorescence complementation, and co-immunoprecipitation assays. Further yeast three-hybrid screenings determined that VmRab7 (Ras-related protein in V. mali) interacted with the MC complex. Targeted gene deletion showed that the ∆VmMon1 and ∆VmCcz1 mutants were defective in vegetative growth, conidiation, and pathogenicity. In addition, both mutants were more sensitive to osmotic and oxidative stresses and intracellular protein transport inhibitors. Cytological examination revealed that the ∆VmMon1 and ∆VmCcz1 mutants were impaired in vacuole fusion and autophagy. More importantly, expression of pectinase genes decreased in both mutants compared with those of the wild type during infection. Overall, our study identified Mon1 and Ccz1 genes in V. mali and provided evidence that VmMon1 and VmCcz1 are critical components that modulate vacuole fusion and autophagy, thereby affecting the development, conidiation, and pathogenicity of V. mali. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Valsa mali secretes an effector protein VmEP1 to target a K homology domain-containing protein for virulence in apple.

The K homology (KH) repeat is an RNA-binding motif that exists in various proteins, some of which participate in plant growth. However, the function of KH domain-containing proteins in plant defence is still unclear. In this study, we found that a KH domain-containing protein in apple (Malus domestica), HEN4-like (MdKRBP4), is involved in the plant immune response. Silencing of MdKRBP4 compromised reactive oxygen species (ROS) production and enhanced the susceptibility of apple to Valsa mali, whereas transient overexpression of MdKRBP4 stimulated ROS accumulation in apple leaves, indicating that MdKRBP4 is a positive immune regulator. Additionally, MdKRBP4 was proven to interact with the VmEP1 effector secreted by V. mali, which led to decreased accumulation of MdKRBP4. Coexpression of MdKRBP4 with VmEP1 inhibited cell death and ROS production induced by MdKRBP4 in Nicotiana benthamiana. These results indicate that MdKRBP4 functions as a novel positive regulatory factor in plant immunity in M. domestica and is a virulence target of the V. mali effector VmEP1.

A fungal extracellular effector inactivates plant polygalacturonase-inhibiting protein.

Plant pathogens degrade cell wall through secreted polygalacturonases (PGs) during infection. Plants counteract the PGs by producing PG-inhibiting proteins (PGIPs) for protection, reversibly binding fungal PGs, and mitigating their hydrolytic activities. To date, how fungal pathogens specifically overcome PGIP inhibition is unknown. Here, we report an effector, Sclerotinia sclerotiorum PGIP-INactivating Effector 1 (SsPINE1), which directly interacts with and functionally inactivates PGIP. S. sclerotiorum is a necrotrophic fungus that causes stem rot diseases on more than 600 plant species with tissue maceration being the most prominent symptom. SsPINE1 enhances S. sclerotiorum necrotrophic virulence by specifically interacting with host PGIPs to negate their polygalacturonase-inhibiting function via enhanced dissociation of PGIPs from PGs. Targeted deletion of SsPINE1 reduces the fungal virulence. Ectopic expression of SsPINE1 in plant reduces its resistance against S. sclerotiorum. Functional and genomic analyses reveal a conserved virulence mechanism of cognate PINE1 proteins in broad host range necrotrophic fungal pathogens.

A Valsa mali Effector Protein 1 Targets Apple (Malus domestica) Pathogenesis-Related 10 Protein to Promote Virulence.

To successfully colonize the plants, the pathogenic microbes secrete a mass of effector proteins which manipulate host immunity. Apple valsa canker is a destructive disease caused by the weakly parasitic fungus Valsa mali. A previous study indicated that the V. mali effector protein 1 (VmEP1) is an essential virulence factor. However, the pathogenic mechanism of VmEP1 in V. mali remains poorly understood. In this study, we found that the apple (Malus domestica) pathogenesis-related 10 proteins (MdPR10) are the virulence target of VmEP1 using a yeast two-hybrid screening. By bimolecular fluorescence (BiFC) and coimmunoprecipitation (Co-IP), we confirmed that the VmEP1 interacts with MdPR10 in vivo. Silencing of MdPR10 notably enhanced the V. mali infection, and overexpression of MdPR10 markedly reduced its infection, which corroborates its positive role in plant immunity against V. mali. Furthermore, we showed that the co-expression of VmEP1 with MdPR10 compromised the MdPR10-mediated resistance to V. mali. Taken together, our results revealed a mechanism by which a V. mali effector protein suppresses the host immune responses by interfering with the MdPR10-mediated resistance to V. mali during the infection.

Control of stripe rust of wheat using indigenous endophytic bacteria at seedling and adult plant stage.

Stripe rust (caused by Puccinia striiformis tritici) is one of the most devastating diseases of wheat. The most effective ways to control stripe rust are the use of resistant cultivars and the timely use of an appropriate dose of fungicide. However, the changing nature of rust pathogen outwits the use of resistant cultivars, and the use of a fungicide is associated with environmental problems. To control the disease without sacrificing the environment, we screened 16 endophytic bacteria, which were isolated from stripe rust-resistant wheat cultivars in our previous study, for their biocontrol potential. A total of 5 bacterial strains Serratia marcescens 3A, Bacillus megaterium 6A, Paneibacillus xylanexedens 7A, Bacillus subtilis 11A, and Staphyloccus agentis 15A showed significant inhibition of Puccinia striiformis f. sp. tritici (Pst) urediniospores germination. Two formulations i.e., fermented liquid with bacterial cell (FLBC) and fermented liquid without bacterial cells (FL) of each bacterial strain, were evaluated against the urediniospores germination. Formulations of five selected endophytic bacteria strains significantly inhibited the uredinioospores germination in the lab experiments. It was further confirmed on seedlings of Pakistani susceptible wheat cultivar Inqilab-91 in the greenhouse, as well as in semi-field conditions. FLBC and FL formulations applied 24 h before Pst inoculation (hbi) displayed a protective mode. The efficacy of FLBC was between 34.45 and 87.77%, while the efficacy of FL was between 39.27 and 85.16% when applied 24 hbi. The inoculated wheat cultivar Inqilab-91 was also tested under semi-field conditions during the 2017-2018 cropping season at the adult plant stage. The strains Bacillus megaterium 6A and Paneibacillus xylanexedens 7A alone significantly reduced the disease severity of stripe rust with the efficacy of 65.16% and 61.11% for the FLBC in protective effect, while 46.07% and 44.47% in curative effect, respectively. Inoculated seedlings of Inqilab-91 showed higher activities of antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL). The treated seedlings also showed higher expressions of pathogenesis-related (PR) protein genes, antifungal protein (PR-1), ß-1,3-endoglucanases (PR-2), endochitinases (PR-4), peroxidase (PR-9), and ribonuclease-like proteins (PR-10). These results indicated that endophytic bacteria have the biocontrol potential, which can be used to manage stripe rust disease. High production antioxidant enzymes, as well as high expression of PR protein genes, might be crucial in triggering the host defense mechanism against Pst.

A Real-Time Loop-Mediated Isothermal Amplification for Detection of the Wheat Dwarf Virus in Wheat and the Insect Vector Psammotettix alienus.

Wheat dwarf virus (WDV; genus Mastrevirus, family Geminiviridae) is an economically important and widespread pathogen of cereal crops. It causes huge yield loss in wheat because of the unavailability of resistant varieties and rapid transmission by the vector leafhopper, Psammotettix alienus (Dahlb). To monitor and forecast this viral disease, an early diagnosis method is required for WDV detection in both infected plants and the virus vectors. In this study, we developed a real-time loop-mediated isothermal amplification (LAMP) assay for WDV detection. The positive sample could be detected within 28 to 32 min by following a simple, cost-effective procedure. The real-time LAMP assay showed a sensitivity of 2.7 × 105-6 copies/µl for detection and a high specificity for WDV amplification, with a similar accuracy to quantitative PCR. Furthermore, a closed-tube dye method facilitates the inspection of the LAMP reaction and avoids cross-contamination in the detection of the virus. This valuable detection assay could serve as an important tool for diagnosis and forecasting wheat dwarf disease intensity in the field.

Development and Application of a LAMP Assay for the Detection of the Latent Apple Tree Pathogen Valsa mali.

Valsa mali, the causal agent of apple Valsa canker, produces cankers, resulting in the death of infected tissues and eventually the entire tree. Because of the long latent period of the disease, it is necessary to develop a rapid, sensitive, and reliable field-based assay to effectively diagnose apple Valsa canker when the plant is still symptomless. Loop-mediated isothermal amplification (LAMP) is a novel detection method that synthesizes a large amount of DNA and produces the visible byproduct (magnesium pyrophosphate) without conventional thermal cycling. Six LAMP primers were designed to target a species-specific region of the elongation factor-1α sequence, which can be completed at 61°C in 60 min. A positive result is indicated by color change after the intercalating dye SYBR Green I is added. The specificity of the LAMP was validated with DNA from 45 representative isolates of V. mali and closely related species V. malicola, V. leucostoma, and V. sordida. The sensitivity of the LAMP was determined to be 1 ng of DNA or as few as 10 spores. Because the assay does not require expensive equipment or specialized techniques, the LAMP-based diagnostic method can be applied under field conditions to more precisely and efficiently access disease pressure in apple orchards.

LaeA Controls Virulence and Secondary Metabolism in Apple Canker Pathogen Valsa mali.

Apple Valsa canker is a destructive disease caused by the ascomycete Valsa mali and poses a serious threat to apple production. Toxins synthesized by secondary metabolite biosynthetic gene clusters (SMBGCs) have been proven to be crucial for pathogen virulence. A previous study showed that V. mali genome contains remarkably expanded SMBGCs and some of their genes were significantly upregulated during infection. In this study, we focus on LaeA, a known regulator of secondary metabolism, for its role in SMBGC regulation, toxin production, and virulence of V. mali. Deletion of VmLaeA led to greatly reduced virulence with lesion length reduced by 48% on apple twigs. Toxicity tests proved that toxicity of secondary metabolites (SMs) produced by VmLaeA deletion mutant (ΔVmlaeA) was markedly decreased in comparison with wild-type (WT). Transcriptomic and proteomic analyses of WT and ΔVmlaeA indicated that a portion of transporters and about half (31/60) SMBGCs are regulated by VmLaeA. Function analysis of eight gene clusters including PKS7, PKS11, NRPS14, PKS16, PKS23, PKS31, NRPS/PKS33, and PKS39 that were differentially expressed at both transcriptional and translational levels showed that four of them (i.e., PKS11, PKS16, PKS23, and PKS31) were involved in pigment production and NRPS14 contributed to virulence. Our findings will provide new insights and gene resources for understanding the role of pathogenicity-related toxins in V. mali.

VmPacC Is Required for Acidification and Virulence in Valsa mali.

The role of the transcription factor PacC has been characterised in several pathogenic fungi, and it affects virulence via several mechanisms. In this study, we examined the role of the PacC homolog VmPacC in Valsa mali, the causal agent of apple canker disease. We found that the expression of VmPacC was up-regulated in neutral and alkaline pH and during infection. At pH 6-10, the radial growth of a VmPacC deletion mutant decreased compared to wild-type. In addition, the sensitivity to oxidative stress of the VmPacC deletion mutant was impaired, as its growth was more severely inhibited by H2O2 than that of the wild-type. The lesion size caused by the VmPacC deletion mutant was smaller than that of the wild-type on apple leaves and twigs. Interestingly, expression of pectinase genes increased in deletion mutant during infection. To further confirm the negative regulation, we generated dominant activated C-27 allele mutants that constitutively express VmPacC. The pectinase activity of activated mutants was reduced at pH 4. We further observed that V. mali can acidify the pH during infection, and that the capacity for acidification was impaired after VmPacC deletion. Furthermore, VmPacC is involved in the generation of citric acid, which affects virulence. These results indicate that VmPacC is part of the fungal responses to neutral and alkaline pH and oxidative stress. More importantly, VmPacC is required for acidification of its environment and for full virulence in V. mali.

Transcription factor VmSeb1 is required for the growth, development, and virulence in Valsa mali.

Transcription factor Seb1 contains two C2H2 zinc finger motifs which are similar to the Msn2/4 of Saccharomyces cerevisiae. The homologous proteins of Seb1 function to regulate the response to various stresses or decomposing and utilizing pectin in some fungi. In this study, we characterized a homologue of Seb1 gene, VmSeb1, in Valsa mali, which causes a highly destructive bark disease on apple. VmSeb1 deletion mutant showed a drastic reduction in growth rate in vitro. It is also important for conidiation because VmSeb1 deletion mutant formed more pycnidia on PDA medium. Deletion mutant of VmSeb1 increased melanin genes expression. In addition, the sensitivity to oxidative stress increased and cell wall inhibitor in VmSeb1 deletion mutant, as its growth was more severely inhibited by H2O2 and Congo red than that in the wild-type. The virulence assay showed that the lesion length caused by the VmSeb1 deletion mutant was smaller compared to wild-type on detached apple twigs. However, expression of pectinase genes and pectinase activity in deletion mutant were the same as those of the wild-type during infection. These results indicate that VmSeb1 plays important roles in growth, asexual development, response to oxidative stress, maintenance of cell wall integrity, and virulence. However, VmSeb1 is not involved in the regulation of pectinase genes expression in V. mali.

Sclerotinia sclerotiorum: An Evaluation of Virulence Theories.

Oxalic acid production in Sclerotinia sclerotiorum has long been associated with virulence. Research involving UV-induced, genetically undefined mutants that concomitantly lost oxalate accumulation, sclerotial formation, and pathogenicity supported the conclusion that oxalate is an essential pathogenicity determinant of S. sclerotiorum. However, recent investigations showed that genetically defined mutants that lost oxalic acid production but accumulated fumaric acid could cause disease on many plants and substantiated the conclusion that acidic pH, not oxalic acid per se, is the necessary condition for disease development. Critical evaluation of available evidence showed that the UV-induced mutants harbored previously unrecognized confounding genetic defects in saprophytic growth and pH responsiveness, warranting reevaluation of the conclusions about virulence based on the UV-induced mutants. Furthermore, analyses of the evidence suggested a hypothesis for the existence of an unrecognized regulator responsive to acidic pH. Identifying the unknown pH regulator would offer a new avenue for investigating pH sensing/regulation in S. sclerotiorum and novel targets for intervention in disease control strategies.

Combining Single Nucleotide Polymorphism Genotyping Array with Bulked Segregant Analysis to Map a Gene Controlling Adult Plant Resistance to Stripe Rust in Wheat Line 03031-1-5 H62.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most devastating diseases of wheat worldwide. Growing resistant cultivars is considered the best approach to manage this disease. In order to identify the resistance gene(s) in wheat line 03031-1-5 H62, which displayed high resistance to stripe rust at adult plant stage, a cross was made between 03031-1-5 H62 and susceptible cultivar Avocet S. The mapping population was tested with Chinese P. striiformis f. sp. tritici race CYR32 through artificial inoculation in a field in Yangling, Shaanxi Province and under natural infection in Tianshui, Gansu Province. The segregation ratios indicated that the resistance was conferred by a single dominant gene, temporarily designated as YrH62. A combination of bulked segregant analysis (BSA) with wheat 90K single nucleotide polymorphism (SNP) array was used to identify molecular markers linked to YrH62. A total of 376 polymorphic SNP loci identified from the BSA analysis were located on chromosome 1B, from which 35 kompetitive allele-specific PCR (KASP) markers selected together with 84 simple sequence repeat (SSR) markers on 1B were used to screen polymorphism and a chromosome region associated with rust resistance was identified. To saturate the chromosomal region covering the YrH62 locus, a 660K SNP array was used to identify more SNP markers. To develop tightly linked markers for marker-assisted selection of YrH62 in wheat breeding, 18 SNPs were converted into KASP markers. A final linkage map consisting of 15 KASP and 3 SSR markers was constructed with KASP markers AX-109352427 and AX-109862469 flanking the YrH62 locus in a 1.0 cM interval. YrH62 explained 63.8 and 69.3% of the phenotypic variation for disease severity and infection type, respectively. YrH62 was located near the centromeric region of chromosome 1BS based on the positions of the SSR markers in 1B deletion bins. Based on the origin, responses to P. striiformis f. sp. tritici races, and marker distances, YrH62 is likely different from the other reported stripe rust resistance genes/quantitative trait loci on 1B. The gene and tightly linked KASP markers will be useful for breeding wheat cultivars with resistance to stripe rust.