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Apple Valsa canker is considered one of the most serious apple diseases. Dopamine is a catecholamine with key physiological functions in plants. Tyrosine decarboxylase (TYDC) is not only involved in the synthesis of dopamine in plants but may also play an important role in the resistance of plants to pathogen infection. In this study, we show that 100-μM exogenous dopamine application and Malus domestica TYDC (MdTYDC) overexpression (or overexpressing, OE) enhances the resistance of apple to Valsa mali infection, likely because the increased dopamine content reduces the accumulation of H2O2 and increases the accumulation of phenolic compounds and salicylic acid (SA) in dopamine-treated and OE apple plants. The activity of chitinase and β-1, 3-glucanase and the expression of SA-related genes were induced more strongly by V. mali in dopamine-treated and OE apples. The dopamine content was significantly higher in dopamine-treated and OE apples than in their respective controls under both normal and inoculated conditions (P < 0.05). Overall, these findings indicate that the application of exogenous dopamine and the OE of MdTYDC may enhance the resistance of apples to V. mali infection by altering the dopamine content, which improves antioxidant capacity, promotes the accumulation of phenolic compounds and SA, and enhances the activity of disease resistance-related proteins.

Apple valsa canker (AVC), caused by Valsa mali, is one of the most important diseases of apple trees in China. AVC occurred severely along with cold winter or cold spring. However, the effect of lower temperature on V. mali is poorly understood. This study evaluated the influence of lower temperature pretreatment of V. mali on the infection of apple twigs and leaves. The results showed that exposing V. mali to lower temperatures (between –10°C and 10°C) for more than 18 h significantly increased the disease severity of apple leaves and twigs, with a higher lesion area ratio (LAR), lesion length, and disease incidence (DI) than that at 25°C. In addition, cold treatment ranging from –5°C to 10°C promoted colony growth. Meanwhile, the relative expression of four cell wall degrading enzyme (CWDE)-related genes pretreated at –5°C and 5°C were significantly higher than that at 25°C. The results indicated that the virulence of V. mali mycelium is sensitive to lower temperatures. After sensing lower temperature changes, V. mali can adjust its infection of apple trees by regulating the expression of pathogenicity gene and growth rate. Spring has very frequent temperature changes, and V. mali is highly invasive in this season. Therefore, more attention should be paid in spring to protecting apple trees from infection of V. mali, by reducing pruning wound formation in spring and applying protective agents to pruning wounds in time.

In Rosaceae , tandem duplication caused the drastic expansion of CNGC gene family Group I. The members MdCN11 and MdCN19 negatively regulate Valsa canker resistance. Apple (Malus domestica) and pear (Pyrus bretschneideri and P. communis) are important fruit crops in Rosaceae family but are suffering from threats of Valsa canker. Cyclic nucleotide-gated ion channels (CNGCs) take crucial roles in plant immune responses. In the present study, a total of 355 CNGCs was identified from 8 Rosaceae plants. Based on phylogenetic analysis, 540 CNGCs from 18 plants (8 in Rosaceae and 10 others) could be divided into four groups. Group I was greatly expanded in Rosaceae resulted from tandem duplications. A large number of cis-acting regulatory elements (cis-elements) responsive to signals from multiple stresses and hormones were identified in the promoter regions of CNGCs in Malus spp. and Pyrus spp. Expressions of most Group I members were obviously up-regulated in Valsa canker susceptible varieties but not in the resistant ones. Furthermore, overexpression of the MdCN11 and MdCN19 in both apple fruits and ‘Duli’ (P. betulifolia) suspension cells compromised Valsa canker resistance. Overexpression of MdCN11 induced expression of hypersensitive response (HR)-related genes. In conclusion, tandem duplication resulted in a drastic expansion of CNGC Group I members in Rosaceae. Among these, MdCN11 and MdCN19 negatively regulate the Valsa canker resistance via inducting HR.

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.

Members of the genus Cytospora are often reported as endophytes, saprobes or phytopathogens, primarily causing canker diseases of woody host plants. They occur on a wide range of hosts and have a worldwide distribution. Although several species have in the past been reported from China, the vast majority are not known from culture or DNA phylogeny. The primary aim of the present study was thus to clarify the taxonomy and phylogeny of a large collection of Cytospora species associated with diverse hosts in China. Cytospora spp. were collected in northeast, northwest, north and southwest China, indicating that the cold and dry environments favour these fungi. In this paper, we provide an assessment of 52 Cytospora spp. in China, focussing on 40 species represented by 88 isolates from 28 host genera. Based on a combination of morphology and a six-locus phylogeny (ITS, LSU, act1, rpb2, tef1-α and tub2), 13 new species and one new combination are introduced. The majority of the species investigated here appear to be host-specific, although further collections and pathogenicity studies will be required to confirm this conclusion.

Since its discovery more than 110 years ago, Valsa canker has emerged as a devastating disease of apple in East Asia. However, our understanding of this disease, particularly the identity of the causative agents, has been in a state of confusion. Here we provide a synopsis for the current understanding of Valsa canker and the taxonomy of its causal agents. We highlight the major changes concerning the identity of pathogens and the conflicting viewpoints in moving to “One Fungus = One Name” system for this group of fungal species. We compiled a list of 21Cytosporaspecies associated withMalushosts worldwide and curated 12 of them with rDNA-ITS sequences. The inadequacy of rDNA-ITS in discriminatingCytosporaspecies suggests that additional molecular markers, more intraspecific samples and robust methods are required to achieve reliable species recognition.

Pyraclostrobin, a quinone outside inhibitor (QoI) fungicide, has been registered to control apple tree Valsa canker (AVC) caused by Valsa mali in China. However, there is no data available regarding the resistance risk of V. mali to pyraclostrobin. In this study, the sensitivities of 120 V. mali isolates to pyraclostrobin were detected. The isolates were collected from apple orchards with no application of pyraclostrob at six provinces in China during 2013–2015, and showed similar sensitivity to pyraclostrobin. The EC50 values of these 120 V. mali isolates to pyraclostrobin ranged from 0.0014 to 0.0240 μg/mL, indicating an excellent inhibitory efficacy of pyraclostrobin to the pathogen. The EC50 values were distributed as a unimodal curve with a mean value of 0.0091 μg/mL, and the mean EC50 displayed correlation with geographic location. Meanwhile, three pyraclostrobin-resistant mutants (PR mutants) of V. mali were obtained using fungicide adaption method, with a resistance factor (RF) of 41.0, 56.8 and 22.0, respectively. The mutants showed a stable resistance to pyraclostrobin after 10 transfers on pyraclostrobin-free medium. Comparing with the corresponding parental isolates, the hyphal growth, mycelial dry weight and pathogenicity of PR mutants were significantly reduced, but the number of propagules showed no significant difference. More importantly, no cross-resistance of PR mutants to pyraclostrobin, tebuconazole, difenoconazole, imazalil and thiophanate-methyl was detected. In conclusion, V. mali showed a moderate risk to pyraclostrobin, and pyraclostrobin could be used as an alternative fungicide to control AVC in the field in China.

Background: Apple Valsa Canker (AVC) with early incubation characteristics is a severe apple tree disease. Therefore, early detection of the infected trees is necessary to prevent the rapid development of the disease. Surface enhanced Raman Scattering (SERS) spectroscopy is a promising technique that simplifies detection procedures and reduces detection time. Meanwhile, SERS enhance signals at low laser powers and suppress biological fluorescence. In this study, the early detection of the AVC disease was carried out by combining SERS spectroscopy with the chemometrics methods and machine learning algorithms, and then chemical distribution imaging was successfully applied to the analysis of disease dynamics.Results: Firstly, the microstructure, UV-Vis spectrum, and Raman spectrum of SERS metallic nano-substrates were proved to investigate the enhancement effects of the synthesized AgNPs. Secondly, the multiple spectral baseline correction (MSBC), the asymmetric least squares (AsLS), and the adaptive iterative reweighted penalized least squares (air-PLS) were adopted to eliminate the disturbances of the baseline offset. The correlation analysis method was employed to identify the best baseline correction algorithm, which was the air-PLS algorithm herein. Meanwhile, principal component analysis (PCA) was used to perform clustering analysis based on the healthy, early disease, and late disease sample datasets, demonstrating obvious clustering effects. After that, optimal spectral variables were selected to build machine learning models to detect AVC disease, incorporating the BP-ANN, ELM, RForest, and LS-SVM algorithms. The accuracy of these models was above 90%, showing excellent discriminant performance. Finally, SERS chemical imaging provided the spatiotemporal dynamic characteristics of changes in the cellulose and lignin of the phloem disease-health junction under AVC stress. The results suggested that cellulose and lignin in the cell walls of infected tissues reduced significantly.Conclusions: SERS spectroscopy combining with chemical imaging analysis for early detection of the AVC disease was considered feasible and promising. This study provided a practical method for the rapid diagnosis of apple orchard diseases.

Valsa mali is the causative agent of apple tree valsa canker, which causes significant losses in apple production. It produces various toxic compounds that kill plant cells, facilitating infection. Among these, protocatechuic acid exhibits the highest phytotoxic activity. However, those genes involved in toxin production have not been studied. In this study we identified four hydroxybenzoate hydroxylase genes (VmHbh1, VmHbh2, VmHbh3, and VmHbh4) from the transcriptome of V. mali. The VmHbh protein had high enzymatic activities of hydroxybenzoate hydroxylase, which could convert 4‐hydroxybenzoate to protocatechuic acid. These four VmHbh genes all had highly elevated transcript levels during the V. mali infection process, especially VmHbh1 and VmHbh4, with 26.0‐ and 53.4‐fold increases, respectively. Mutants of the four genes were generated to study whether VmHbhs are required for V. mali pathogenicity. Of the four genes, the VmHbh1 and VmHbh4 deletion mutants considerably attenuated V. mali virulence in apple leaves and in twigs, coupled with much reduced toxin levels. The VmHbh2 and VmHbh3 deletion mutants promoted the transcript levels of the other VmHbhs, suggesting functional redundancies of VmHbhs in V. mali virulence. The results provide insights into the functions of VmHbhs in the production of protocatechuic acid by V. mali during its infection of apple trees., Hydroxybenzoate hydroxylase genes of Valsa mali contribute to full pathogenicity during pathogen infection apple trees by regulating protocatechuic acid production.

No doubt that studying the secondary metabolites produced by plants can lead to the detection of new pesticides. In this study, we followed the bioassay-guided method to isolate active ingredients from Periploca sepium root barks (PSRB) and verified their antifungal activity against seven phytopathogenic fungi in vitro. A total of six pregnane glycosides were isolated and identified as periplocoside A (PSA), periplocoside D (PSD), periplocoside E (PSE), periplocoside F (PSF), periplocoside N (PSN) and periploside W (PSW). Bioassay results revealed that PSW exhibited highly significant antifungal activity against Valsa mali and Fusarium graminearum with EC50 values of 26.50 mg/L and 93.94 mg/L, respectively. In controlling apple Valsa canker, PSW (1000 mg/L) exhibited significantly higher protective activity with 85.40% than that efficacy with 64.62% obtained by carbendazim (200 mg/L). Whereas there is no significant difference between both of them in the curative efficacy with 71.32% and 69.70%, respectively. In conclusion, our study recommended the pregnane glycosides from PSRB as a new class of fungicides or fungicide lead compounds not only because of their potential antifungal activity, but also because of their safety for human beings, mammals and non-target organisms.

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.

Apple valsa canker (AVC), caused by Valsa mali, is a serious wood disease of apple trees. The pathogen decays the barks and branches of trees and ruins entire orchards under severe conditions. However, studies have rarely focused on the suitable habitat of the pathogen, especially on a relatively large scale. In this study, we applied the maximum entropy model (MaxEnt 3.4.1, Princeton, NJ, USA) to predict the distribution of V. mali using climate factors, topographic factors, and soil factors under current and future climate scenarios. We measured the area of suitable habitat, change ratio of the suitable habitat area, increase and decrease maps under climate change, direction and distance of range shifts from the present to the end of the 21st century, and the contribution of environmental variables. The results showed that the area of suitable habitat is currently 183.46 &times, 104 km2 in China, among which 27.54% is moderately suitable habitat (MSH) and 13.13% is highly suitable habitat (HSH). Compared with current distribution, the area of MSH and HSH increases in future and the change ratio are positive. The Shared Socioeconomic Pathways (SSPs) 3&ndash, 70 is considered the optimum climate scenario for V. mali. The suitability of V. mali increased mainly in Northwest, North, and Northeast China. V. mali will shift to the northwest with climate change. The shift distance optimistically increased from the SSP1&ndash, 26 to the SSP5&ndash, 85, with the biggest shift distance of 758.44 km in the 2090s under the SSP5&ndash, 85 scenario. Minimum temperature of the coldest month (bio6) was the most critical climate factor affecting the distribution of the pathogen, and topographic factors played a more important role than soil factors. This study demonstrates that the potential distribution of V. mali is vitally affected by climate change and provides a method for large&ndash, scale research on the distribution of pathogens.

Valsa pyri is a fatal canker pathogen that causes significant reduction of crop yield in pear orchards. V. pyri invades the trunk phloem, and is difficult to control by chemical treatment. In this work, it was found for the first time that Bacillus subtilis-produced dipicolinic acid (DPA) exhibits antifungal activity against different canker pathogens, including Alteraria alternata, Botryosphaeria dothidea, Rhizoctonia solani, and V. pyri. Growth inhibition of V. pyri was observed at less than 5 mM concentration (pH = 5.6). DPA showed the highest antifungal activity at acidic pH values and in the presence of bivalent metals, such as zinc(II), cobalt(II), and copper(II). Measurement of mRNA expression levels and scanning electron microscope (SEM) observations revealed that DPA causes V. pyri apoptosis via inhibition of chitin biosynthesis and subsequent cell lysis. Interestingly, DPA showed high stability in the pear bark and was able to cross the pear tree bark into the phloem, protecting the internal phases of the pear trunk. In preventive applications, DPA reduced the canker symptoms of V. pyri on Cuigan pear trees by 90%. Taken together, an efficient strategy for the management of V. pyri-caused canker disease was developed using a novel antifungal agent, DPA, with strong antifungal activity and particular diffusion properties.

Pear Valsa canker is a fungal trunk disease caused by Valsa pyri. Phenolic compounds are ubiquitous in plants and usually contribute to plant resistance against biotic stress. To investigate the association between phenolic compounds and level of resistance to V. pyri, we quantified the contents of individual phenolic compounds in the cortex and phloem of stems from 8 cultivars of Pyrus bretschneideri. Significant variation in the levels of all compounds was found among the cultivars. Correlation analysis revealed an inverse correlation between levels of arbutin and gallic acid with the degree of canker resistance. This suggested that these phenolic compounds are beneficial to V. pyri infection. These data could be valuable for breeding cultivars of P. bretschneideri with greater resistance to V. pyri. Keywords: Pyrus bretschneideri, phenolic compound, resistance, Valsa canker, LC–MS

Apple valsa canker (AVC), caused by Valsa mali (Vm), is a serious disease worldwide. In this study, a total of 193 actinomycete cultures were isolated from the rhizosphere of healthy wild apple trees in the Qinling Mountains in China. Six of these isolates had a strong antagonistic activity towards V. mali (inhibition rates >50%). The hyphal growth and conidial germination of V. mali were significantly inhibited by these isolates. The strain SL01 could induce the protoplasm leakage from V. mali hyphae. In a detached twig inoculation experiment, the fermentation broth of the six isolates showed significant inhibiting effects on disease development by V. mali. Among the six candidates, strain SL01 showed the best potential for biocontrol of AVC based on the inhibitory effects on hyphal growth, conidial germination and disease development by V. mali. Strain SL01 was identified as Streptomyces longissimus based on morphological, biochemical, physiological characteristics and 16S rDNA sequence analysis. In field studies, the SL01 strain showed an average of 90.3% inhibition on AVC recurrence in a two-year experiment. This study indicated that the actinomycete strain SL01 has the potential for biological control of AVC.

Apple Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple trees in East Asia. Feruloyl esterases (ferulic acid esterases, FAEs), which belong to a subclass of carboxylic esterases, can cleave ester bonds that crosslink hydroxycinnamic acids and arabinoxylans or certain pectins in plant cell walls. However, a pathogenic role of FAE has not been demonstrated in plant-pathogenic fungi. In this study, the FAE gene family, including one type A, one type B, three type C and two type D FAE genes, was identified in V. mali. Five of the seven FAE genes had highly elevated transcript levels in V. mali-apple tree bark interactions compared with mycelia grown in axenic culture. Signal peptides of the VmFAEs were confirmed using yeast signal sequence trap assays. To examine whether FAEs are required for the pathogenicity of V. mali, seven single- and six double-gene deletion mutants were generated. Compared with the wild-type, three of the seven FAE single-deletion mutants showed significantly reduced pathogenicity and three of the six FAE double-deletion mutants exhibited greater reductions in pathogenicity, suggesting the joint action of FAEs in the V. mali-apple tree interaction. Most of the FAE mutants that exhibited a significant reduction in pathogenicity had significantly lower FAE activity than the wild-type fungus. These results indicate that secreted FAEs are required for the full pathogenicity of the phytopathogenic fungus V. mali.

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.

Valsa canker caused by Valsa pyri is one of the most destructive diseases of commercial pear. For the present analysis, 29 different endophytic fungal strains were isolated from the branches of a healthy pear tree. In dual culture assays, strain ZZ1 exhibited robust antifungal activity against all tested pathogens including Valsa pyri. Microscopic analyses suggested that following co-culture with ZZ1, the hyphae of V. pyri were ragged, thin, and ruptured. ZZ1 also induced significant decreases in lesion length and disease incidence on detached pear branches inoculated with V. pyri. ZZ1 isolate-derived culture filtrates also exhibited antifungal activity against V. pyri, decreasing mycelial growth and conidium germination and inhibiting V. pyri-associated lesion development on pear branches. These results suggest that the ZZ1 isolate has the potential for use as a biological control agent against V. pyri. The strain was further identified as Penicillium&nbsp, citrinum based on its morphological characteristics and molecular analyses. Overall, these data highlight a potentially valuable new biocontrol resource for combating pear Valsa canker.

Valsa canker is a destructive fungal disease for the apple industry, especially in Asian regions. Here, individuals in “Jonathan” × “Golden Delicious” hybrid population were phenotyped by off situ inoculation with Valsa mali isolates 03‐8 and xc56 from 2012 to 2015. Thirty‐one QTLs for the resistance were detected using high‐density genetic maps. Based on year‐stable and isolate‐robust QTLs, resistant loci G13‐1 and G13‐2 were mapped on chromosome 13 of the male parent, meanwhile locus J15‐1 was mapped on chromosome 15 of the female parent. Using the parental whole‐genome resequencing SNP databases, eighteen SNPs that could change predicted cis‐elements of gene or led to non‐synonymous substitution were screened in the three loci. After segregation analysis in hybrids and gene expression verification, three SNPs in an RNA‐binding protein gene, a serine/threonine‐protein kinase gene and a MYB transcription factor gene showed a close relationship to Valsa canker resistance in apple. These results will be useful for the application of marker‐assisted selection for Valsa canker resistance in apple.

Valsa canker caused by Valsa mali is commonly present in eastern Asia and cause large economic losses. Because of limited agricultural measures and chemical residues of commonly used fungicides there is an urgent need of alternative plant protecting agents. On this background the activity of cuminic acid, a plant extract from the seed of Cuminum cyminum L, was assessed. The median effective concentration (EC50) values for inhibition of mycelial growth of seven V. mali strains ranged from 3.046 to 8.342 μg/mL, with an average EC50 value of 4.956 ± 0.281 μg/mL. The antifungal activity was the direct activity of cuminic acid instead of the influence on the pH of media by cuminic acid. After treated with cuminic acid, mycelia dissolved with decreased branches and swelling; cell membrane permeability increased while pectinases activity decreased significantly. Moreover, peroxidase (POD) activity of the apple leaves increased after treated with cuminic acid. Importantly, on detached branches of apple tree, cuminic acid exhibited both protective and curative activity. These results indicated that cuminic acid not only showed the antifungal activity, but also could improve the defense capacity of the plants. Taken together, cuminic acid showed the potential as a natural alternative to commercial fungicides or a lead compound to develop new fungicides for the control of Valsa canker.

Sexual reproduction in filamentous ascomycetes is controlled by the mating type (MAT) locus, including two idiomorphs MAT1-1 and MAT1-2. Understanding the MAT locus can provide clues for unveiling the sexual development and virulence factors for fungal pathogens. The genus Valsa (Sordariomycetes, Diaporthales) contains many tree pathogens responsible for destructive canker diseases. The sexual stage of these ascomycetes is occasionally observed in nature, and no MAT locus has been reported to date. Here, we identified the MAT locus of the apple canker pathogen Valsa mali, which causes extensive damage, and even death, to trees. V. mali is heterothallic in that each isolate carries either the MAT1-1 or MAT1-2 idiomorph. However, the MAT structure is distinct from that of many other heterothallic fungi in the Sordariomycetes. Two flanking genes, COX13 and APN2, were coopted into the MAT locus, possibly by intrachromosomal rearrangement. After the acquisition of foreign genes, unequal recombination occurred between MAT1-1/2 idiomorphs, resulting in a reverse insertion in the MAT1-2 idiomorph. Evolutionary analysis showed that the three complete MAT1-1-2, COX13, and APN2 genes in this region diverged independently due to different selection pressure. Null hypothesis tests of a 1:1 MAT ratio of 86 V. mali isolates from four different provinces showed a relatively balanced distribution of the two idiomorphs in the fields. These results provide insights into the evolution of the mating systems in Sordariomycetes.

Valsa canker, caused by Valsa mali, is a destructive disease of apple in China. The pathogen infects apple branches, mainly through pruning wounds, and causes branch and tree death. To determine the conditions required for V. mali infection through pruning wounds and growth within the xylem, pruning wounds on 1- to 4-year-old apple branches were inoculated with conidia in vitro under artificially controlled conditions and in vivo in the orchard. The effects of temperature, wetness duration, and wound age on conidial infection through pruning wounds as well as hyphal growth in the xylem were examined. The results showed that, after invading through pruning wounds, V. mali hyphae grew along xylem vessels, tracheids, and rays, expanding longitudinally and laterally. The hyphae could enter adjacent xylem vessels and tracheids through micropores to form a dense hyphal network. Wetness duration did not exhibit an essential effect on conidial infection from pruning wounds. Conidia spread to pruning wounds with rainwater could infect the xylem without any other extra moisture. Temperature for V. mali conidia infection through pruning wounds and hyphal extension in the xylem ranged from 5 to 35°C, with the optimum at 20°C. Pruning wounds made in late March were susceptible to V. mali infection in March, April, and May; the susceptibility was markedly deceased by June, and the pathogen could barely infect through the pruning wounds in November. The infected pruning wounds began to show symptoms from the spring of the following year. More than half of the observed Valsa canker lesions emerged in the spring of the second year, and new canker twigs were also developed from the inoculations in the spring of the third year. March, April, and May are the critical periods for protecting pruning wounds against infection by V. mali in China, and coating pruning wounds with protective film immediately after pruning is an easy and effective measure to protect the pruning wounds.

In apple (Malus domestica), the polyphenol profile is dominated by phloridzin, but its physiological role remains largely elusive. Here, we used MdUGT88F1 (a key UDP-glucose:phloretin 2'-O-glucosyltransferase gene) transgenic apple lines and Malus spp. germplasm to gain more insight into the physiological role of phloridzin in apple. Decreasing phloridzin biosynthesis in apple lines by RNA silencing of MdUGT88F1 led to a series of severe phenotypic changes that included severe stunting, reduced internode length, spindly leaf shape, increased stem numbers, and weak adventitious roots. These changes were associated directly with reduced lignin levels and disorders in cell wall polysaccharides. Moreover, compact organization of tissues and thickened bark enhanced resistance to Valsa canker (caused by the fungus Valsa mali), which was associated with lignin- and cell wall polysaccharide-mediated increases of salicylic acid and reactive oxygen species. Phloridzin was also assumed to be utilized directly as a sugar alternative and a toxin accelerator by V. mali in apple. Therefore, after infection with V. mali, a higher level of phloridzin slightly compromised resistance to Valsa canker in MdUGT88F1-overexpressing apple lines. Taken together, our results shed light on the importance of MdUGT88F1-mediated biosynthesis of phloridzin in the interplay between plant development and pathogen resistance in apple trees.

Leucostoma canker, also called perennial canker, Cytospora canker, and Valsa canker, is recognized as an important disease of peach in the northern portions of the region suitable for production of temperate fruits, including Canada, and the northeastern US It is mentioned as part of a complex set of factors associated with peach tree short-life syndrome in the southeastern US There are a great many fungal pathogens of fruit trees that cause twig and stem cankers and that also cause leaf spots and/or fruit rots. There are some fungal pathogens, however, that primarily cause stem cankers and that cause major economic losses in some regions. In 1-year-old peach shoots exhibiting tip dieback from natural infection in the field by either L. cincta or L. persoonii, fungal mycelium was present within the cankered area in the cortical tissue, phloem, xylem vessels, and pith. The mycelium was intracellular in the xylem vessels and pith.

A real-time quantitative PCR assay using a species-specific primer pair was developed to rapidly and accurately quantify Valsa mali, the causative pathogen of apple Valsa canker (AVC), in crabapple seeds, crabapple seedlings, apple twigs and apple seeds. Surveys were conducted in different regions, and crabapple or apple seeds were collected for V. mali detection by qPCR assay. Our results showed that 12.87% to 49.01% of crabapple seeds collected from different regions were positive for V. mali. The exopleura and endopleura were the two major areas of V. mali infection in crabapple seeds. The presence of V. mali infection in crabapple seeds was also confirmed by a high-throughput sequencing approach. With the growth of crabapple seedlings, the concentration of V. mali gDNA in crabapple seedlings gradually increased until eight or more leaf blades emerged. One-year-old twigs from an apple scion nursery were infected with V. mali, and only apple seeds from infected apple trees showing evident Valsa canker symptoms carried V. mali. In conclusion, this study reports that crabapple seeds and apple seeds carried V. mali as latent inoculum sources. V. mali infected not only apple tissues but also crabapple seedlings, which are the rootstocks of apple trees. This study indicated that the inoculum sources for AVC vary. Application of a novel qPCR assay can potentially improve the accuracy of early diagnosis, and is helpful to reveal the epidemic regularity of AVC.

Pear is a popular fruit in the world market, and has been widely cultivated in China. Since 2008, a severe canker disease has consistently been observed on 20-year-old pear trees (Pyrus communis cv. Duchess de' Angouleme) grown in a nursery in Xingcheng, Liaoning Province, China. Observed symptoms include brown elongated ulcerative lesions (more than 20 cm in length in general), with red brown conidia produced on wet lesions. Reductions in tree vigor and yield were observed for infected trees. Tree mortality was observed for severe infections. To diagnose the pathogen, 15 canker samples were collected from five pear trees in April, 2012. Bark pieces (3 to 5 mm) taken from the border of healthy and diseased tissue were surface-disinfected with 0.1% mercury bichloride and 75% ethanol for 45 s, and placed on potato dextrose agar (PDA) medium at 25°C in darkness. Fungal colonies with a common colony morphology were consistently recovered from three samples. These fungal colonies were initially white, becoming olive green in 3 days. Conidia produced on colonies were hyaline, allantoid, and single-celled with average length × width of 6.04 (5.43 to 6.59) × 0.65 (0.51 to 0.73) μm, which were consistent with descriptions of Valsa leucostoma (1). Genomic DNA was extracted from a representative isolate F-LN-32b, and subjected to PCR amplification of the internal transcribed spacer region (ITS), β-tubulin gene, and EF1 gene using the primer pairs ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R (3), respectively. Sequence alignment of the amplified fragments with the deposited data in NCBI showed that sequences of EF1, ITS, and β-tubulin (GenBank Accession No. KF293296 to KF293298, respectively) of isolate F-LN-32b had the highest similarity of 99% to those of V. leucostoma strain 32-2w (JQ900340, JN584644, and JQ900374), and suggested that isolate F-LN-32b is a V. leucostoma strain. Pathogenicity tests was carried out by placing a 5-mm-diameter, 2-day-old mycelium agar plug of isolate F-LN-32b onto a punched bark hole of a detached 1-year-old pear shoot after it was surface disinfested with ethanol. Inoculated shoots were incubated at 25°C in plastic containers covered with plastic film. Pathogenicity assays were conducted on 18 pear varieties (cvs. Qiuyue, Jinshui 2, Hohsui, Huali 1, Cuiguan, Shinseiki, Xuehua, Dangshansu, Zaosu, Hongxiangsu, Yuluxiang, Nanguoli, Xizilv, Bartlett, Huanghua, Huashan, Duchess de' Angouleme, and Packham's) collected from a nursery in Wuhan, Hubei Province, China. Six shoots were inoculated for each variety and the assay was conducted three times. All inoculated shoots developed the typical canker symptoms after 6 days post inoculation (dpi) and sporulated at 25 dpi while the control shoots inoculated with non-colonized PDA plugs remained asymptomatic. Isolates recovered from inoculated samples were of the same morphology and ITS sequence as F-LN-32b. Based on these results, V. leucostoma was determined as the pathogen responsible for the Valsa canker disease on pear. Valsa mali var. pyri was identified as the only pathogen causing Valsa canker disease on pear in China (2). To our knowledge, this is the first report of V. leucostoma causing a canker disease on pear in China. References: (1) G. C. Adams et al. Australas. Plant Pathol. 35:521, 2006. (2) X. L. Wang et al. Mycologia 103:317, 2011. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple in the primary production areas of China and other East Asian countries. Currently, there are no effective control methods for this disease. We investigated the occurrence of Valsa canker in 24 apple orchards in Shaanxi Province in concert with foliar nutrient analysis, and found that there was a significant negative correlation of leaf potassium (K) content with incidence and severity of Valsa canker. Fertilization experiments showed that increasing tree K content enhanced resistance to pathogen colonization and establishment. Apple trees with leaf K content greater than 1.30% exhibited almost complete resistance to Valsa mali. Field trials demonstrated that increasing K fertilization could significantly reduce disease incidence. Improved management of tree nutrition, especially K content, could effectively control the occurrence and development of Valsa canker.

The apple Valsa canker caused by Valsa mali is a devastating branch disease that has seriously threatened the development of the apple industry worldwide. In current study, a total of 115 V. mali strains collected from different apple orchards in Shaanxi Province of China during 2016 and 2017 were tested for their sensitivity to flusilazole. The average EC50 (effective concentrations causing 50% mycelial growth inhibition) value of all tested strains for flusilazole was 0.0892 (±0.0036) μg/mL and the frequency distribution of the EC50 values was unimodal. Flusilazole exhibited both excellent protective and curative activity on detached apple branches, which was significantly better than the commonly used fungicide thiophanate-methyl. After flusilazole treatment, mycelia twisted with offshoot of top increased, the V. mali strains lost the ability of fruiting body production, and cell membrane permeability of the mycelia increased while ergosterol content and pectinase activity decreased. The expression of pectinase genes involved in virulence down-regulated after flusilazole treatment. This study is the first report on the baseline sensitivity of V. mali to flusilazole. These results indicated that flusilazole has a great potential to play an important role in the management of Valsa canker.

Apple canker caused by Valsa mali is a destructive disease that causes serious economic losses in China. In this study, the dynamics of release of conidial masses (cirri) in apple orchards were studied in different seasons, and in laboratory tests the effects of temperature on conidial germination were determined. Using artificial inoculations in the field, the influence of the pruning process on year-round disease development, and the possibility of the pruning saws carrying conidia of V. mali were tested to determine disease transmission in the winter months. Conidial masses released from pycnidia demonstrated strong adaptability to the changing environment. Conidial exudation from pycnidia could last up to a year and take place even in the winter, in the period when the bark was wet. The germination rate of conidia reached 90 % after 6 days incubation at 5 °C and 50 % after 19 days at 0 °C. Infection rates of conidia in different seasons were determined in a 3-year experiment. Fresh pruning wounds were more susceptible to infection than old wounds, and conidia were able to infect apple trees and induce canker lesions at different times of the year, and especially in winter. Pruning in early winter may promote heavy infections of apple by V. mali and could play an important role for the spread of the disease.

Valsa canker caused by the necrotrophic ascomycete Valsa mali is a destructive disease on apple in eastern Asia. To date, the disease still cannot be effectively controlled, mainly because V. mali extensively penetrates into the phloem and xylem and limited information about the resistance mechanism is available. In this study, we analyzed the transcriptome of V. mali-infected apple (Malus x domestica) to identify genes associated with V. mali induced resistance responses. Totally, 2713 apple genes were significantly up-regulated upon V. mali infection. Functional enrichment analysis showed that resistance responses were significantly over-represented in up-regulated genes, including chitin, hormone and cell death responses. Two chitin-elicitor receptors, MDP0000136494 and MDP0000169047, probably were the key chitin receptors responded to V. mali-infection. Jasmonic acid and salicylic acid signaling were revealed to be the primary phytohormone pathways of apple against V. mali. In addition, 11 cytochrome P450 genes likely to be involved in the xenobiotics biodegradation or phytoalexins biosynthesis were identified. This new information provides insights into the resistance responses against V. mali. Functional verification of these genes identified here will facilitate understanding the resistance mechanism of apple and establishing new effective disease arrangement strategies.

Valsa canker caused by the necrotrophic pathogen Valsa mali (Vm) severely affects apple production in Eastern Asia. The molecular basis underlying the apple response to Vm infection is poorly understood. Hence, we performed RNA sequencing (RNA-seq) to investigate the dynamic gene expression profiles of a major apple cultivar, ‘Changfu No.2’, during Vm infection. Compared with the control (C), 104, 313, and 1059 differentially expressed genes (DEGs) were detected from the phloem tissue within the range of 0.9–1.3 cm (T1), 0.5–0.9 cm (T2), and 0.1–0.5 cm (T3) beyond the lesion periphery, respectively. Gene ontology (GO) enrichment analysis revealed that the DEGs associated with plant growth and development were down-regulated, whereas those related to defense responses were up-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that hormonal and Ca2+ signaling and phenylpropanoid biosynthesis were involved in the defense responses. In conclusion, multiple defense responses associated with ABA, JA, ET, Ca2+, and cell wall signals contributed to the defense against Vm infection in ‘Changfu No.2’. In contrast, the DEGs with inhibited expression were involved in plant growth and development; auxin signaling and several resistance genes might weaken the resistance of ‘Changfu No.2’ to pathogens. Our results offer a new insight into plant responses against necrotrophs and could benefit programs aimed at breeding for Vm resistance.

Apple valsa canker (AVC), caused by Valsa mali , is one of the most serious diseases of apple trees in eastern Asia, and the most important factor limiting apple production in China. This disease is difficult to control by chemical and agricultural measures, thus biocontrol may constitute a desirable alternative strategy. A Bacillus amyloliquefaciens strain denoted GB1 isolated from ageing cucumber stems, exhibited a strong antagonistic activity against V . mali , inhibiting significantly the germination of conidia and the growth of hyphae. GB1 conidial suspensions (above 10 6 CFU/ml) applied prior to wound inoculation of apple twigs with V. mali resulted in total inhibition of infection. Strain GB1 colonized xylem and phloem tissues surrounding the wounds made on apple twigs and formed biofilms over them. Results indicate that B. amyloliquefaciens GB1 may be a promising agent for the biocontrol of AVC, and provide new insights into the ability of B . amyloliquefaciens to colonize apple trees.

The pear pyralid moth, Euzophera pyriella Yang, has been associated with the Valsa canker (Valsa ceratosperma), which is responsible for the death of Xinjiang pear trees (Pyrus sinkiangensis). According to a field investigation and an indoor experiment, larval boring and fungal colonization always happen in the same phloem area. The larvae preferred to feed on diseased phloem colonized by V. ceratosperma, and even completed their entire life feeding only on the mycelium. Further, different strains had different effects on larval growth and development. The E. pyriella cohorts developed well on a high pathogenicity isolate but did not complete their lifecycle on a low pathogenicity isolate. V. ceratosperma plays an important role as a mediator in plant–herbivore interactions.

In the current study, baseline sensitivity of Valsa mali to propamidine was determined using 80 strains collected from apple orchards in Shaanxi Province, China. The median effective concentration (EC 50 ) values for propamidine inhibiting mycelial growth ranged from 0.086 to 0.852 μg/mL, with a mean of 0.405 ± 0.137 μg/mL. After treated with propamidine, mycelia were contorted with an increased number of branches, loss of fruiting body production, and decreased cell membrane permeability. Moreover, the enzyme activities of the complexes I, II, IV and ATPase in the mitochondrial respiratory chain were increased significantly, while the enzyme activities of complexes III decreased. Importantly, both on detached leaves and branches of apple trees, propamidine applied at 100 μg/mL exhibited over 75% protective and curative efficacies, which were even better than the efficacies obtained by carbendazim at the same concentration. These results indicated that propamidine could be used as an alternative compound in controlling Valsa canker and mitochondrial respiratory chains might be correlated with the action mode of propamidine. This study encourages further investigation for the action mechanism of propamidine against plant pathogens and the information could be valuable for synthesis of new antifungal drugs with novel modes of action.

Apple Valsa canker, caused by the fungus Valsa mali (Vm), is one of the most destructive diseases of apple in China. A better understanding of this host-pathogen interaction is urgently needed to improve management strategies. In the current study we sequenced the transcriptomes of Vm during infection of apple bark and mycelium grown in axenic culture using Illumina RNA-Seq technology. We identified 437 genes that were differentially expressed during fungal infection compared to fungal mycelium grown in axenic culture. One hundred and thirty nine of these 437 genes showed more than two fold higher transcript abundance during infection. GO and KEGG enrichment analyses of the up-regulated genes suggest prevalence of genes associated with pectin catabolic, hydrolase activity and secondary metabolite biosynthesis during fungal infection. Some of the up-regulated genes associated with loss of pathogenicity and reduced virulence annotated by host-pathogen interaction databases may also be involved in cell wall hydrolysis and secondary metabolite transport, including a glycoside hydrolase family 28 protein, a peptidase and two major facilitator superfamily proteins. This highlights the importance of secondary metabolites and cell wall hydrolases during establishment of apple Valsa canker. Functional verification of the genes involved in pathogenicity of Vm will allow us to better understand how the fungus interferes with the host machinery and assists in apple canker establishment.

Fungal diseases are posing tremendous threats to global economy and food safety. Among them, Valsa canker, caused by fungi of Valsa and their Cytospora anamorphs, has been a serious threat to fruit and forest trees and is one of the most destructive diseases of apple in East Asia, particularly. Accurate and robust delimitation of pathogen species is not only essential for the development of effective disease control programs, but also will advance our understanding of the emergence of plant diseases. However, species delimitation is especially difficult in Valsa because of the high variability of morphological traits and in many cases the lack of the teleomorph. In this study, we delimitated species boundary for pathogens causing apple Valsa canker with a multifaceted approach. Based on three independent loci, the internal transcribed spacer (ITS), β-tubulin (Btu), and translation elongation factor-1 alpha (EF1α), we inferred gene trees with both maximum likelihood and Bayesian methods, estimated species tree with Bayesian multispecies coalescent approaches, and validated species tree with Bayesian species delimitation. Through divergence time estimation and ancestral host reconstruction, we tested the possible underlying mechanisms for fungal speciation and host-range change. Our results proved that two varieties of the former morphological species V. mali represented two distinct species, V. mali and V. pyri, which diverged about 5 million years ago, much later than the divergence of their preferred hosts, excluding a scenario of fungi–host co-speciation. The marked different thermal preferences and contrasting pathogenicity in cross-inoculation suggest ecological divergences between the two species. Apple was the most likely ancestral host for both V. mali and V. pyri. Host-range expansion led to the occurrence of V. pyri on both pear and apple. Our results also represent an example in which ITS data might underestimate species diversity.

Valsa canker (Valsa ceratosperma (Tode ex Fr.) Maire) is one of the most destructive fungal diseases of apple, especially in Eastern Asia. In this study, the first high density genetic linkage map of Malus asiatica × Malus domestica was constructed by 640 simple sequence repeats (SSRs) and 490 single nucleotide polymorphisms (SNPs), which spanned 1497.5 cM with an average marker interval of 1.33 cM per marker. Quantitative trait loci (QTLs) for apple’s resistance to V. ceratosperma isolates 03-8 and xc56 were identified using the linkage map. Lesion lengths were used as the phenotypic data for the QTL analysis, which were measured on 1-year-old shoots inoculated with conidia of the two isolates. One QTL for resistance to isolate 03-8 was mapped on LG 16, and one QTL for resistance to isolate xc56 was detected on LG 9. Our research not only promoted the further understanding of the genetic basis of apple’s resistance to Valsa canker but also provided two molecular markers that might be used in future marker-assisted selection for resistance in apple breeding programs.

A nested polymerase chain reaction (PCR) assay for detecting Valsa mali var. mali, the causal agent of apple tree Valsa canker, was developed. One pair of genus-specific primers was designed based on the ribosomal DNA internal transcribed spacer conservative sequence of the Valsa genus and one pair of species-specific primers was designed based on the specific sequence of V. mali var. mali. The specificity of the genus-specific and species-specific primers was evaluated against 10 V. mali var. mali isolates, 10 V. mali var. pyri isolates, 4 isolates from closely related Valsa spp., and 8 isolates from fungal species that are commonly isolated from naturally infected apple bark tissue. A distinct band of 348 bp in length was detected in all V. mali var. mali isolates but not in other tested species and the V. mali var. pyri variety. The sensitivity of this assay was evaluated by serial dilutions of DNA extracted from V. mali var. mali pure cultures and apple bark tissues with or without visible symptoms. The results showed that the assay was able to detect as little as 100 fg of DNA in mycelial samples and apple bark tissues with visible symptoms, whereas the lowest detectable concentration was 10 pg of DNA in symptomless apple bark tissues. The efficiency of the nested PCR assay was compared with that of fungal isolation assays. All symptomless and symptomatic samples from which the pathogen was successfully isolated yielded a PCR product of the expected size. The detection rate of nested PCR for symptomless samples was 64.7%, which was much higher than the detection rate of 20.6% by fungal isolation. The PCR analysis of different symptomless tissues showed that the incidence of V. mali var. mali was different in different tissues of apple trees. The average incidence of V. mali var. mali was 89% in terminal buds, 71% in internodes, and 48% in bud scale scars. Moreover, the incidence of V. mali var. mali in nonsymptomatic tissues was higher in orchards where more trees were infected. Taken together, the assay developed in this study can be used for rapid and reliable detection of V. mali var. mali in tissues of apple trees with or without symptoms and also for monitoring the presence of the pathogen at an early stage of disease development.

The infection process of Valsa mali var. mali (Vmm), causal agent of canker, in apple (Malus domestica cv. Fuji) tissue was investigated by light and electron microscopy. The germination pattern of conidia was similar on wounded or intact twigs, but there was a time delay on intact bark. On intact bark tissue, conidia swelling could be observed at 16 h post inoculation (hpi) and conidia germinated at 20 hpi, however, no infection was observed. On wounded twig surface, conidia swelled at 6 hpi, and germinated at 16 hpi. Direct penetration of germ tubes was observed by 20 hpi and the pathogen caused canker lesions 4 days post inoculation (dpi) on wounded twigs. Electron microscopy observation of the canker region showed that fungal hyphae mainly spread intercellularly causing severe tissue maceration and cell necrosis. By 15 dpi, hyphae colonized all bark tissues and xylem vessels and generated pycnidia on the canker region. Fungal colonization resulted in marked alterations in host tissues including plasmolysis and degeneration of protoplasts and cell walls. Based on enzyme- and immuno-gold labeling of cell wall components, pectinases were involved in pathogenesis of Vmm of apple tissue.

Valsa melanodiscus (anamorph Cytospora umbrina) has been associated with cankers, dieback, and death of alder (Alnus) stems in western North America. To determine the ability of this fungus to induce these symptoms, the responses of thinleaf alder (Alnus tenuifolia) stems to inoculation with V. melanodiscus were studied in field locations in Alaska and on plants in a greenhouse. In the field, woody stems were wounded to expose both inner bark and sapwood and inoculated in early May 2007 or September 2007 by placing a colonized agar plug over the wound. Sunken, elongated cankers that developed on inoculated stems in the field closely resembled those attributed to natural infection of thinleaf alders by V. melanodiscus. In contrast, wounded control stems exhibited strong callus production and wound closure. In the greenhouse, actively growing lateral shoots were inoculated by placing a colonized agar plug over a fresh leaf scar. Inoculation in the greenhouse resulted in development of cankers, and ...

Valsa canker is a destructive disease on apple that causes serious economic losses in eastern Asia. In the present study fungal isolates from cankered apple and pear bark were examined and compared with morphology and rDNA-ITS sequences. Valsa mali was confirmed to be an independent species and a causal pathogen of Valsa canker on apple and pear in China. It was the predominant species (96.7% of isolates) on apple and was complemented by V. malicola (3.3% of isolates). Significant intraspecific genetic differentiation was detected in V. mali with two varieties recognized, V. mali var. mali occurring exclusively on apple and V. mali var. pyri occurring on both apple and pear. Results from genetic analysis and cross-inoculation tests provided support for the hypothesis that host preference probably catalyzed such genetic changes within the pathogen populations.

Cytospora spp. and associated teleo- morphic species (Ascomycota, Diaporthales, Valsa- ceae) are among the most common and widespread canker- and dieback-causing fungi on trees, shrubs and herbaceous plants worldwide. From specimens collected all over Iran a total of 114 isolates were morphologically identified, representing 20 Cytos- pora, one Leucostoma and five Valsa species from 38 plant species. Nine of the identified taxa were new records for Iran, and many new hosts were identified. The phylogenetic relationships of the Iranian strains, along with sequences of 13 reference strains from GenBank, were inferred from ITS1-5.8S-ITS2 nuclear rDNA sequences. Parsimony analysis established five distinct major clades and 12 subclades, which represented accepted species and genera. Some of these subclades corresponded to morphologically based taxonomic concepts of single Cytospora species, while others contained more than one morphospe- cies. Teleomorphic states were present in six sub- clades, and most clustered with the corresponding anamorphs. This suggests that morphological and phylogenetic species concepts overlap and that in most cases they are meaningful for correct species identification.

Malus sieboldii Rehd. exhibits high levels of resistance to Valsa canker caused by Valsa ceratosperma (Tode ex Fr.) Maire while cultivated apples (Malus domestica Borkh.) are susceptible to the disease. In this study, progenies from 23 full-sib families derived from both inter- and intra-specific hybridization among 16 Malus genotypes as parents were assessed for resistance to V. ceratosperma (Vc) for two seasons using an excised shoot assay to determine the pattern of inheritance of the resistance and to also estimate the variance components, narrow-sense heritability, and breeding values of parental genotypes. Generally, M. sieboldii × M. domestica and its reciprocal crosses had more resistant progenies to Vc than intra-specific crosses of M. domestica. Resistance to Vc expressed as the relative lesion length among progenies showed continuous variation irrespective of cross, suggesting the quantitative nature of the resistance to the three virulent isolates of Vc that were tested. Resistance to Vc using the progeny population was analyzed using a mixed linear model based on restricted maximum likelihood. The parental effect (general combining ability (GCA)) was significant while the interaction effect between parents (specific combining ability (SCA)) was relatively small and non-significant. The ratio of SCA/GCA variance was about 32%, suggesting that additive genetic variance had a major contribution to the total genetic variance for resistance to Vc. There was a positive correlation (r = 0.49, p

An epidemic of cytospora canker [Valsa melanodiscus, anamorph Cytospora umbrina] is associated with dieback and mortality of Alnus incana ssp. tenuifolia in the Southern Rocky Mountains and had begun by the late 1980s. Isolations showed that inoculum was often abundant on bark and bud surfaces even in winter, especially near diseased stems. The pathogen was occasionally isolated from internal tissues of dormant buds and asymptomatic wood and phloem. In infected stems, the pathogen was detected up to 5 cm beyond visible canker margins. The data suggest that the pathogen can cause latent infections, but the infection court remains unclear. Inoculations of healthy stems in the field did not induce canker formation. Fruiting of the anamorph was primarily in late winter and spring, and sexual maturation occurred in late summer and fall. Stem age and growth data support earlier conclusions that the mortality does not represent a steady-state condition with normal attrition of older stems. Canker expansion and k...

Members of Cytospora encompass important plant-associated pathogens, endophytes and saprobes, commonly isolated from a wide range of hosts with a worldwide distribution. Two specimens were collected associated with symptomatic canker and dieback disease of Spiraea salicifolia in Gansu, China. These isolates are characterized by its hyaline, biseriate, aseptate, elongate-allantoid ascospores and allantoid conidia. Cytospora spiraeae sp. nov. is introduced based on its holomorphic morphology plus support from phylogenetic analysis (ITS, LSU, ACT and RPB2), and differs from similar species in its host association.

On the basis of mycelial compatibility, the population structure of Valsa ceratosperma, the causal fungus of Valsa canker on broadleaf trees, was investigated in apple and pear orchards in Japan. Field strains of V. ceratosperma from a single canker on trunks or scaffold limbs belonged to different mycelial compatibility groups. Thus, the population structure of this fungus was complex in most orchards. Because mycelia of strains originating from different conidia from the same pycnidium were compatible, infection by this fungus is thought to be ascospores.

Although Valsa canker caused by Valsa ceratosperma (Tode ex Fr.) Maire is one of the most destructive diseases in apple (Malus x domestica Borkh.) especially in eastern Asia, information available to help with breeding against Valsa canker in apples is limited. In this work, 53 accessions of diverse Malus species and their interspecific hybrids were tested for resistance to V. ceratosperma, using an excised shoot assay. Dormant shoots and succulent growing shoots from each accession were inoculated with a virulent isolate AVC-12 of V. ceratosperma, and the length of necrosis was measured at 10 days post-inoculation for the dormant shoots and at 7 days post-inoculation for the growing shoots. The lesion length relative to the susceptible control 'Fuji' in dormant shoots (RL D ) and to that in growing shoots (RL G ) were simple but useful parameters to differentiate between resistant and susceptible accessions. Fourteen accessions from M. baccata, M. florentina, M. halliana, M. micromalus, M. pratii, M. sieboldii, M. yunnanensis, M. × floribunda and M. x platycarpa gave low RL D and RL G values of less than 0.6 and were evaluated as resistant regardless of the difference in the stage of growth. The highest level of resistance was found in M. sieboldii. This high level of resistance in M. sieboldii was effective against different isolates of V. ceratosperma.

Valsa mali var. mali (Vmm), which is the causative agent of Valsa canker of apple tree, causes heavy damage to apple production in eastern Asia. In this article, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of Vmm and expression of gfp (green fluorescent protein) in this fungus. The transformation system was optimized to a transformation efficiency of approximately 150 transformants/10(6) conidia, and a library containing over 4,000 transformants was generated. The tested transformants were mitotically stable. One hundred percent hph (hygromycin B phosphotransferase) integration into Vmm was identified by PCR and five single-copy integration of T-DNA was detected in the eighteen transformants by Southern blot. To our knowledge, this is the first report of ATMT of Vmm. Furthermore, this library has been used to identify genes involved in the virulence of the pathogen, and the transformation system may also be useful to the transformation of other species of the genus Valsa.