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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.

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.

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

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.

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.

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), β-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.

Wheat dwarf virus (WDV; genus

A spectrophotometric technique is developed to simultaneously quantify nitrate and nitrite in plasma treated water. The measurement is based on examining the inflection points (wavelengths) in the derivative absorbance of the nitrate or nitrite solution. At the inflection points of the pure nitrate solution, the derivative absorbance is zero and independent of the nitrate’s concentration, and thus the nitrite’s concentration in a mixed nitrate and nitrite solution can be obtained by using the Beer’s law at these points. The nitrate’s concentration can also be achieved from the inflection points of nitrite in the same manner. The relation between the tested substance’s (nitrate or nitrite) concentration and the second- or the third-order absorbances is obtained at these inflection points. Test measurements for mixed aqueous solutions of nitrate and nitrite with or without hydrogen peroxide confirm the reliability of this technique. We applied this technique to quantify the nitrate and nitrite generated in air plasma treated aqueous solutions. The results indicate that both nitrate and nitrite concentrations increase with the plasma treatment time, and the nitrite species is found to be generated prior to the nitrate species in the air plasma treated aqueous solution. Moreover, the production rate of total nitrogen species is independent of the solutions’ pH value. These results are relevant to diverse applications of plasma activated solutions in materials processing, biotechnology, medicine and other fields.

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, a homothallic plant pathogen, undergoes sexual reproduction via haploid selfing (equivalent to clonal reproduction), and produces long-lasting surviving vegetative structures called sclerotia, enhancing clonal persistence and spread. Thus it is not surprising to detect clones of the species. Whether outcrossing can occur in the homothallic S. sclerotiorum remains unanswered. Early studies showed that S. sclerotiorum has a clonal population structure, consistent with its life history traits. However, recent studies using polymorphic and co-dominant molecular markers showed frequent genetic recombination, suggesting outcrossing. This review focuses on recent developments in population genetics studies related to detecting recombination, random association of alleles and dynamic mating type (MAT) alleles in Sclerotinia. Despite frequent reports of random association of alleles, the mechanisms for outcrossing in a homothallic species remain elusive. Recent intriguing findings are: the MAT genes in Sclerotinia are subject to inversion or deletion in every meiotic generation, the MAT gene deletion is related to ascospore dimorphism and mating type switching in S. trifoliorum, and ascospore dimorphism was also observed in S. sclerotiorum. Determining the nature of the dimorphic ascospores and their prevalence in relation to environmental cues could significantly advance our understanding how S. sclerotiorum populations behave in nature.

The interactions between discharge plasmas and an aqueous solutions can enable the production of reactive species and charge transfer at the plasma-liquid interface, forming the plasma electrochemical system (PES). The PES are promising for diverse applications, such as nanomaterials synthesis, due to the activation of the solution chemistry by the plasma. In this paper, we investigate the influence of the solution’s pH value on the formation of silver nanoparticles (AgNPs) in a direct current (DC) PES. Dual argon DC plasmas are generated in an H-type electrochemical cell containing an aqueous solution of silver nitrate with pH values in the range of 1.99–10.71. By this design, the solution acts as a cathode at one end of the H-type cell, and as an anode at the other end. The results show that the AgNPs are formed at the anode except for the solution with the pH value of 1.99. However, at the cathode, the AgNPs only appear in the solution with the pH value of 10.71. We find that the solvated electrons and hydrogen peroxide produced by the plasma-liquid interactions are responsible for the Ag+ reduction at the solution anode and the solution cathode, respectively.

Apple Valsa canker, caused by Valsa mali, is one of the most serious fungal diseases in apple-producing regions in east Asia. One endophyte strain, designated as D4 and subsequently identified as Bacillus velezensis based on morphological and phylogenetic analyses, was studied as a potential biocontrol agent. Challenge inoculation assay showed the strain D4 suppressed the mycelial growth of V. mali on dual culture plates and caused hyphal deformities, wrinkles, and ruptures. Furthermore, the strain D4 was able to promote plant growth by producing siderophores and solubilizing phosphorus. Under laboratory and field conditions, the strain D4 exhibited strong antifungal activities on detached twigs and intact plants. Re-isolation of endophytes confirmed the strain D4 could completely colonize the plants. Comparative transcriptome analysis showed downregulation of the genes associated with the fungal cell wall’s synthesis and composition, cell membrane, and transport and energy metabolism during V. mali treatment with the D4 strain. Furthermore, the global gene expression patterns revealed that host genes mainly involved in salicylic acid, ethylene, jasmonic acid signaling and brassinosteroid synthesis pathways were upregulated during apple treatment with the strain D4. qRT-PCR conducted on a subset of 13 genes was consistent with the results of RNA-seq data. The present study explores a potential biocontrol agent for the control of apple Valsa canker and sheds light on the control mechanisms of how the strain D4 impairs the pathogen and triggers the plant’s defense.

Mitogen-activated protein kinases (MAPKs) play critical roles in the regulation of different developmental processes and hydrolytic enzyme production in many fungal plant pathogens. In this study, an FUS3/KSS1-related MAPK gene, VmPmk1, was identified and characterized in Valsa mali, which causes a highly destructive canker disease on apple. VmPmk1 deletion mutant showed a significant reduction in growth rate in vitro, and could not produce pycnidium, indicating that the MAPK gene is important for growth and asexual development. Also, VmPmk1 played a significant role in response to oxidative stress and in the maintenance of cell wall integrity. More importantly, when deletion mutant was inoculated onto detached apple leaves and twigs, an obvious decrease in lesion size was observed. Furthermore, expression of many cell wall degrading enzyme (CWDE) genes declined in the VmPmk1 deletion mutant during infection. VmPmk1 deletion mutant also showed a significant reduction in activities of CWDEs in both induced media and infection process. Finally, the determination of immunogold labeling of pectin demonstrated that the capacity of degradation pectin was attenuated due to the deletion of VmPmk1. These results indicated that VmPmk1 plays important roles in growth, asexual development, response to oxidative stress, and maintenance of cell wall integrity. More importantly, VmPmk1 is involved in pathogenicity of V. mali mainly by regulating CWDE genes expression.

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.

Summary The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV-induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate-minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate-minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate-minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate-minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases.

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.

Velvet protein family members are important fungal‐specific regulators which are involved in conidial development, secondary metabolism and virulence. To gain a broader insight into the physiological functions of the velvet protein family of Valsa mali, which causes a highly destructive canker disease on apple, we conducted a functional analysis of two velvet protein family members (VmVeA and VmVelB) via a gene replacement strategy. Deletion mutants of VmVeA and VmVelB showed increased melanin production, conidiation and sensitivity to abiotic stresses, but exhibited reduced virulence on detached apple leaves and twigs. Further studies demonstrated that the regulation of conidiation by VmVeA and VmVelB was positively correlated with the melanin synthesis transcription factor VmCmr1. More importantly, transcript levels of pectinase genes were shown to be decreased in deletion mutants compared with those of the wild‐type during infection. However, the expression of other cell wall‐degrading enzyme genes, including cellulase, hemi‐cellulase and ligninase genes, was not affected in the deletion mutants. Furthermore, the determination of pectinase activity and immunogold labelling of pectin demonstrated that the capacity for pectin degradation was attenuated as a result of deletions of VmVeA and VmVelB. Finally, the interaction of VmVeA with VmVelB was identified through co‐immunoprecipitation assays. VmVeA and VmVelB play critical roles in conidiation and virulence, probably via the regulation of the melanin synthesis transcription factor VmCmr1 and their effect on pectinase gene expression in V. mali, respectively.

Stripe rust is a devastating disease in common wheat (Triticum aestivum) worldwide. Growing cultivars with adult-plant resistance (APR) is an environmental friendly approach that provides long-term protection to wheat from this disease. Wheat cultivar Yaco“S” showed a high level of APR to stripe rust in the field from 2008 to 2014. The objective of this study was to detect the major quantitative trait loci (QTL) for APR to stripe rust in Yaco“S”. One hundred and eighty-four F2:3 lines were developed from a cross between Yaco“S” and susceptible cultivar Mingxian169. Illumina 90K and 660K single nucleotide polymorphism (SNP) chips were implemented to bulked pools and their parents to identify SNPs associated with the major QTL. A high-density linkage map was constructed using simple sequence repeat (SSR) and SNP markers. Inclusive composite interval mapping detected a major effect QTL Qyryac.nwafu-2BS conferring stable resistance to stripe rust in all tested environments. Qyryac.nwafu-2BS were mapped to a 1.3 cm interval and explained 17.3–51.9% of the phenotypic variation. Compared with stripe rust resistance genes previously mapped to chromosome 2B, Qyryac.nwafu-2BS is likely a new APR gene to stripe rust. Combining SNP iSelect assay and kompetitive allele specific PCR technology, we found that the APR gene could be rapidly and accurately mapped and it is useful for improving stripe rust resistance in wheat breeding.

The necrotrophic fungal pathogen Sclerotinia trifoliorum exhibits ascospore dimorphism and unidirectional mating type switching - self-fertile strains derived from large ascospores produce both self-fertile (large-spores) and self-sterile (small-spores) offsprings in a 4:4 ratio. The present study, comparing DNA sequences at MAT locus of both self-fertile and self-sterile strains, found four mating type genes (MAT1-1-1, MAT1-1-5, MAT1-2-1 and MAT1-2-4) in the self-fertile strain. However, a 2891-bp region including the entire MAT1-2-1 and MAT1-2-4 genes had been completely deleted from the MAT locus in the self-sterile strain. Meanwhile, two copies of a 146-bp direct repeat motif flanking the deleted region were found in the self-fertile strain, but only one copy of this 146-bp motif (a part of the MAT1-1-1 gene) was present in the self-sterile strain. The two direct repeats were believed to be responsible for the deletion through homologous intra-molecular recombination in meiosis. Tetrad analyses showed that all small ascospore-derived strains lacked the missing DNA between the two direct repeats that was found in all large ascospore-derived strains. In addition, heterokaryons at the MAT locus were observed in field isolates as well as in laboratory derived isolates.

The fungal plant pathogens Sclerotinia sclerotiorum and S. trifoliorum are morphologically similar, but differ considerably in host range. In an effort to elucidate mechanisms of the host range difference, transcriptomes of the 2 species at vegetative growth stage were compared to gain further insight into commonality and uniqueness in gene expression and pathogenic mechanisms of the 2 closely related pathogens. A total of 23133 and 21043 unique transcripts were obtained from S. sclerotiorum and S. trifoliorum, respectively. Approximately 43% of the transcripts were genes with known functions for both species. Among 1411 orthologous contigs, about 10% (147) were more highly (>3-fold) expressed in S. trifoliorum than in S. sclerotiorum, and about 12% (173) of the orthologs were more highly (>3-fold) expressed in S. sclerotiorum than in S. trifoliorum. The expression levels of genes on the supercontig 30 have the highest correlation coefficient value between the 2 species. Twenty-seven contigs were found to be new and unique for S. trifoliorum. Additionally, differences in expressed genes involved in pathogenesis like oxalate biosynthesis and endopolygalacturonases were detected between the 2 species. The analyses of the transcriptomes not only discovered similarities and uniqueness in gene expression between the 2 closely related species, providing additional information for annotation the S. sclerotiorum genome, but also provided foundation for comparing the transcriptomes with host-infecting transcriptomes.

The plant-specific NAC domain proteins have been shown previously to play important roles in both abiotic and biotic stresses or in diverse developmental processes. In this research, the full-length cDNA sequence of a novel NAC (for N AM, A TAF1/2, C UC2) transcription factor gene, designated as TaNAC8, was isolated from wheat (Triticum aestivum) using in silico cloning, reverse transcription polymerase chain reaction and 3’ rapid amplification of cDNA ends PCR methods. TaNAC8 was highly homologous to the rice OsNAC8 gene and predicted to encode a protein of 481 amino acids. The encoded TaNAC8 protein contained a NAC domain in the N-terminus and a transmembrane helices motif in the C-terminus. Using the yeast one-hybrid assay, we detected that the C-terminal region of the TaNAC8 protein had transcriptional activity. TaNAC8 was expressed strongly in the developing wheat seeds, but weakly in stems and flowers. The expression of TaNAC8 in leaves was induced by the infection of the stripe rust pathogen (Puccinia striiformis f. sp. tritici) in the incompatible interaction at 24 h post inoculation, and also by treatments with methyl jasmonate and ethylene. However, salicylic acid and abscisic acid had no any significant effect on the gene expression. Several environmental stimuli, including high salinity, PEG (polyethylene glycol) treatment, and low-temperature, also enhanced the TaNAC8 expression. These results suggest that the novel TaNAC8 protein functions as a transcriptional activator involved in wheat defense responses to abiotic and biotic stresses.

The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV-induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate-minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate-minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate-minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate-minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases.

This manuscript reports two new genes ( Yr64 and Yr65 ) for effective resistance to stripe rust and usefulness of their flanking SSR markers for marker-assisted selection. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide and resistance is the best control strategy. Durum wheat accessions PI 331260 and PI 480016 were resistant to all tested Pst races. To transfer the resistance genes to common wheat and map them to wheat chromosomes, both accessions were crossed with the stripe rust-susceptible spring wheat ‘Avocet S’. Resistant F3 plants with 42 chromosomes were selected cytologically and by rust phenotype. A single dominant gene for resistance was identified in segregating F4 lines from each cross. F6 populations for each cross were developed from single F5 plants and used for genetic mapping. Different genes from PI 331260 and PI 480016 were mapped to different loci in chromosome 1BS using simple sequence repeat markers. The gene from PI 331260 was flanked by Xgwm413 and Xgdm33 in bin 1BS9-0.84-1.06 at genetic distances of 3.5 and 2.0 cM; and the gene from PI 480016 was flanked by Xgwm18 and Xgwm11 in chromosome bin C-1BS10-0.50 at 1.2 and 2.1 cM, respectively. Chromosomal locations and race and allelism tests indicated that the two genes are different from previously reported stripe rust resistance genes, and therefore are named as Yr64 from PI 331260 and Yr65 from PI 480016. These genes and their flanking markers, and selected common wheat lines with the genes should be valuable for diversifying resistance genes used in breeding wheat cultivars with stripe rust resistance.

In reverse engineering applications, there is a problem of precise alignment from measured data to a CAD model. This paper presents a new method where the measurement points can be precisely aligned with its corresponding CAD model. Initially, the measured data is aligned approximately through a least-square method with the minimum distance principle. Based on this initial alignment result, the precise alignment is completed through the Differential Evolution (DE) algorithm and the Simplex method. The experimental results show that the alignment algorithm proposed in this paper possesses an accuracy of 0.055m and spending less processing time than the genetic algorithm. Therefore, the proposed method is effective for the measured data alignment to the original surface model. DOI: http://dx.doi.org/10.11591/telkomnika.v11i3.2310

Agrobacterium-mediated transformation (AMT) was used to identify potential virulence factors in Sclerotinia sclerotiorum. Screening AMT transformants identified two mutants showing significantly reduced virulence. The mutants showed growth rate, sclerotial formation, and oxalate production similar to that of the wild type. The mutation was due to a single T-DNA insertion at 212 bp downstream of the Cu/Zn superoxide dismutase (SOD) gene (SsSOD1, SS1G_00699). Expression levels of SsSOD1 were significantly increased under oxidative stresses or during plant infection in the wild-type strain but could not be detected in the mutant. SsSOD1 functionally complemented the Cu/Zn SOD gene in a Δsod1 Saccharomyces cerevisiae mutant. The SOD mutant had increased sensitivity to heavy metal toxicity and oxidative stress in culture and reduced ability to detoxify superoxide in infected leaves. The mutant also had reduced expression levels of other known pathogenicity genes such as endo-polygalacturanases sspg1 and sspg3. The functions of SsSOD1 were further confirmed by SsSOD1-deletion mutation. Like the AMT insertion mutant, the SsSOD1-deletion mutant exhibited normal growth rate, sclerotial formation, oxalate production, increased sensitivity to metal and oxidative stress, and reduced virulence. These results suggest that SsSOD1, while not being required for saprophytic growth and completion of the life cycle, plays critical roles in detoxification of reactive oxygen species during host–pathogen interactions and is an important virulence factor of Sclerotinia sclerotiorum.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most damaging diseases of wheat worldwide. It is essential to identify new genes for effective resistance against the disease. Durum wheat PI 480148, originally from Ethiopia, was resistant in all seedling tests with several predominant Pst races in the US under controlled greenhouse conditions and at multiple locations subject to natural infection for several years. To map the resistance gene(s) and to transfer it to common wheat, a cross was made between PI 480148 and susceptible common wheat genotype Avocet S (AvS). Resistant F3 plants with 42 chromosomes were selected cytologically and by testing with Pst race PST-100. A total of 157 F4 plants from a single F3 plant with 2n = 42 tested with PST-100 segregated in a 3 resistant: 1 susceptible ratio, indicating that a single dominant gene from PI 480148 conferred resistance. Using the F3:4 population and the resistance gene-analog polymorphism (RGAP) and simple sequence repeat (SSR) markers, the gene was mapped to the long arm of chromosome 2B. SSR marker Xwmc441 and RGAP marker XLRRrev/NLRRrev 350 flanked the resistance gene by 5.6 and 2.7 cM, respectively. The effective resistance of the gene to an Australian Pst isolate virulent to Yr5, which is also located on 2BL and confers resistance to all US Pst races, together with an allelism test of the two genes, indicated that the gene from PI 480148 is different from Yr5 and should be a new and useful gene for resistance to stripe rust. Resistant common wheat lines with plant types similar to AvS were selected for use in breeding programs.

The methionine salvage pathway is conserved from prokaryotes to high eukaryotes. The reaction catalyzed by aci-reductone-dioxygenase (ARD) represents a branch point in the methionine salvage pathway. A novel aci-reductone-dioxygenase gene, designed as TaARD, was identified in a subtraction library constructed with RNA isolated from wheat leaves infected with the stripe rust pathogen. TaARD was predicted to encode a 197 amino acid protein that belongs to the cupin superfamily. In transient expression assays with onion epidermal cells, the TaARD-GFP fusion protein localized to the nucleus and cytoplasm. Southern blot analysis showed that the wheat genome had multiple copies of TaARD. Quantitative real-time RT-PCR (qRT-PCR) analyses revealed that the TaARD transcript was induced in wheat leaves infected with a compatible stripe rust strain. However, its expression was reduced or suppressed in incompatible interactions and by ABA, ethephon (ET), or salicylic acid (SA) treatments. With methyl jasmonate (MeJA) treatment, TaARD transcript level was suppressed in the first 6 h but increased afterwards. The expression of TaARD also was inhibited by wounding and environmental stimuli, including high salinity and low temperature. Because of the role of ARD in the methionine salvage pathway, these results suggest that TaARD may be involved in ethylene synthesis and ethylene signaling in response to biotic and abiotic stresses.

Background Puccinia striiformis f. sp. tritici is an obligate biotrophic pathogen that causes leaf stripe rust on wheat. Although it is critical to understand molecular mechanisms of pathogenesis in the wheat stripe rust fungus for developing novel disease management strategies, little is known about its genome and gene functions due to difficulties in molecular studies with this important pathogen. To identify genes expressed during early infection stages, in this study we constructed a cDNA library with RNA isolated from urediniospores of P. striiformis f. sp. tritici germinated for 10 h. Results A total of 4798 ESTs were sequenced from the germinated urediniospore library and assembled into 315 contigs and 803 singletons. About 23.9% and 13.3% of the resulting 1118 unisequences were homologous to functionally characterized proteins and hypothetical proteins, respectively. The rest 62.8% unisequences had no significant homologs in GenBank. Several of these ESTs shared significant homology with known fungal pathogenicity or virulence factors, such as HESP767 of the flax rust and PMK1, GAS1, and GAS2 of the rice blast fungus. We selected six ESTs (Ps28, Ps85, Ps87, Ps259, Ps261, and Ps159) for assaying their expression patterns during urediniospore germination and wheat infection by quantitative real-time PCR. All of them had the highest transcript level in germinated urediniospores and a much less transcript level in un-germinated urediniospores and infected wheat tissues (1–7 dpi). The transcript level of Ps159 increased at later infection stages (6–7 dpi). Our data indicated that these genes were highly expressed in germinated urediniospores and may play important roles in fungal-plant interactions during early infection stages in the wheat stripe rust fungus. Conclusion Genes expressed in germinated urediniospores of P. striiformis f. sp. tritici were identified by EST analysis. Six of them were confirmed by quantitative real-time PCR assays to be highly expressed in germinated urediniospores.