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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.

Valsa canker is a destructive fungal disease that results in a serious loss of production. The pathogen Valsa pyri (Vp) usually infiltrates the bark and xylem via the wound. ‘Duli’ (Pyrus betulifolia), a rootstock with high tolerance to this disease and multiple other stresses, is widely planted in northern China. We found that wound healing plays a crucial role in the resistance of ‘Duli’ against Vp infection. Nevertheless, elucidation of the molecular mechanism of this process has been largely limited. Using RNA sequencing (RNA-seq), we investigated dynamic gene expression profiles of ‘Duli’ and ‘Zaosu’ (Pyrus bretschneideri, susceptible) phloem tissues which 0–3 mm around the wounded site that were cultured for 1, 3, and 7 days after wounding. Quantitative real-time PCR (qRT-PCR) was used to confirm data veracity. After wounding at 1, 3, and 7 days, 4930, 4652, and 6279 differentially expressed genes (DEGs) were found from ‘Duli’, compared to 1641, 1968, and 3347 in ‘Zaosu’, respectively. Functional enrichment analysis revealed that the DEGs associated with “cell wall organization and biogenesis”, “biosynthesis of secondary metabolites” and “cell wall organization. pectin” were enriched both in ‘Duli’ and ‘Zaosu’. Additionally, expressions of DEGs encoding receptor-like kinase (RLK) subfamily Extensin, LRR-III, LRR-IV, LRR-IX, LRR-XI-1, LRR-XII-1, RLCK-IXa and RLCK-V, and transcription factor CRE1, TF, ABF, TGA, and MYC2 were rapidly activated in ‘Duli’, but only a subtle induction in ‘Zaosu’. In conclusion, DEGs related to cell wall thickening are rapidly and continuously induced in ‘Duli’, which plays a positive role in the formation of protective tissues. Notably, JA-, SA-, LRR-, RLCK-, and Extensin-related DEGs probably participated in the regulation of multiple molecular networks and contributed to the quick wound healing of ‘Duli’.

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 CHEIA DE GOSTO.Compositor: Roniere Leite Soares (2017). Arranjo para banda de música: Willian Aparecido Ciriaco da Silva (Nov/2019). Peça dedicada à Professora Dra. Maria Conceição de Araújo Pereira. Histórico: No auditório do Instituto Euvaldo Lodi (Campina Grande-Paraíba), a valsa intitulada "Cheia de Gosto", INÉDITA, composta em 27/08/2017 para agraciar a professora Maria Conceição Araújo, no lançamento do seu livro MEMÓRIAS POSSÍVEIS DE UMA MENINA CHEIA DE GOSTO (28/08/2017), foi executada ao vivo, em flauta transversa C, pelo próprio autor. O evento ocorreu durante a reunião do PEN Clube de Campina Grande-PB, presidida pelo professor de literatura Dr. José Mario da Silva Branco, da Unidade Acadêmica de Letras (UFCG). Autor da melodia: RONIERE LEITE SOARES. Vídeo: https://youtu.be/pphAZH0-2BU - Execução da valsa CHEIO DE GOSTO em plenário da Câmara de Vereadores de Boa Vista-Estado da Paraíba, em sessão solene presidida por Fernando Aires, no dia 16 de Setembro de 2017, em homenagem ao lançamento do livro "Memória Possíveis de uma Menina Cheia de Gosto", da autora Maria da Conceição Gonçalves Pereira de Araújo. Obra musical registrada no Escritório dos Direitos Autorais da Fundação Biblioteca Nacional, Rio de Janeiro-RJ, em 20/03/2018. Requerimento 2016RJ19042; Nº de Registro: 764.167; Livro 1.482; Folha 209. Copyright©2017: Todos os direitos autorais reservados: Nº de depósito #DEP636399660419515982. MD5: FNxOW5YCTOlXTuez8xYt6w==

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.

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.

These case reports describe locally invasive black pigmented mycotic infections of the skin and gills of saffron cod Eleginus gracilis associated with 8 different opportunistic ascomycete fungi: Alternaria sp., Cladosporium herbarum, Chaetomium globosum, Cadophora luteo-olivacea, Penicillium sp., Phoma herbarum, Pseudophacidium ledi and Valsa sordida. These fungi were isolated on conventional media, identified according to morphological structures and confirmed by genetic sequencing. Several of these fungi are primary plant pathogens as well as opportunistic human pathogens in immunocompromised individuals. Several have also been described as causing opportunistic infections of fish. This case material represents the first report of C. luteo-olivacea, C. globosum, P. ledi and V. sordida as likely opportunistic fish pathogens in Alaskan watersheds of Norton Sound and south in Toksook Bay and possibly elsewhere.

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.

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.

Apple Valsa canker, caused by the necrotrophic pathogen Valsa mali, is a devastating disease of apples and causes great financial loss in East Asia. Improving the understanding of apple - V. mali interactions will contribute to disease management. In this study, three predicted secreted peroxidases (VmPOD1, VmPOD2 and VmPOD3) were uncovered based on the secretome and genome information of V. mali. Phylogenetic analysis showed that VmPOD1 is a catalase peroxidase, VmPOD2 is a chloroperoxidase, and VmPOD3 is a plant peroxidase-like peroxidase. The secretion function of the corresponding genes was confirmed using the yeast invertase secretion system. The deletion of VmPODs did not affect the vegetative growth when the mutants (ΔVmPOD1, ΔVmPOD2 and ΔVmPOD3) and the wild-type strain 03-8 were grown on PDA medium at 25 °C in the dark. However, the respective mutants showed impaired conidiation ability with fewer pycnidia, and all gene deletion mutants grew more slowly than 03–8 on PDA supplemented with H2O2 (Final concentration: 0.06 mol/L H2O2). In addition, VmPOD1 and VmPOD2 were found to be significantly up-regulated at an early infection stage, and VmPOD3 showed sustained high expression during the whole infection progress of V. mali. In addition, the virulence of ΔVmPOD3 was significantly reduced, implying that VmPOD3 plays a critical role during the interaction between V. mali and apple. All of the defective phenotypes could be nearly restored by re-introducing the wild-type VmPOD1, VmPOD2 or VmPOD3 allele. The results enhanced our understanding of the secreted peroxidase, which could also act as a type of virulence factor from the necrotrophic pathogen V. mali and provided new insight into the role of the pathogen-secreted peroxidase.

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.

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.

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.

Four fungi isolated from trunks and branches of European hazelnut (Corylus avellana L.) from commercial orchards in the Willamette Valley, Oregon were characterized and pathogenicity was tested on potted hazelnut trees. The acreage of hazelnuts in Oregon has expanded greatly in recent years in response to the availability of Eastern filbert blight resistant cultivars. Fungi were characterized using the BLASTn algorithm and the GenBank database with multiple partial gene sequence(s). If BLASTn and GenBank were not sufficient for species-level identification, then a multilocus sequence analysis (MLSA) was performed. The four pathogens were identified as Diplodia mutilla (Fr.) Mont., Dothiorella omnivora B.T. Linaldeddu, A. Deidda & B. Scanu, Valsa cf. eucalypti Cooke & Harkn., and Diaporthe eres Nitschke. All pathogens but D. omnivora have not been previously reported from European hazelnut in the literature. All four pathogens caused lesions on trunks bare root hazelnut trees cv. ‘Jefferson’ planted in pots in the greenhouse and fungi were re-isolated from inoculated trees. D. mutilla appeared particularly aggressive in repeated inoculation experiments.

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.

F. excelsior is affected by dieback in the major part of its natural geographical range in Europe, which results in economic and ecological losses. The disease is caused by the ascomycetous fungus Hymenoscyphus fraxineus, a pathogen introduced to Europe most probably from East Asia. This paper presents data on fungi identified on F. excelsior trees representing two different stages of ash dieback in Poland. Fungal communities were identified in initial necrotic lesions on living stems and twigs using the classical method of isolation on malt extract agar and morphological and molecular analyses. In dead apical parts of stems and twigs, fungi were identified by microscopic analyses of fruit bodies formed in situ. Seventy-one fungal taxa were found in 720 samples with symptoms of initial or advanced necrosis. The most common fungus detected in initial necrotic lesions in each forest site was Hymenoscyphus fraxineus (59.2 % of analysed samples). Other frequently isolated fungi included Alternaria alternata, Diaporthe eres, Diplodia mutila, Fusarium avenaceum, F. lateritium and Phomopsis spp. Fruit bodies on dead apical parts of stems and twigs were produced mostly by Diaporthe eres, Diplodia mutila, Lophiostoma corticola, Phomopsis spp., Sirodothis sp. and Valsa cypri. Fungal communities from different sites were similar, as shown by high Sorensen similarity index values. Greatest variation in fungal community structure at the initial necrotic stage was realized by D. mutila and F. avenaceum, and at the advanced necrotic stage by D. eres, D. mutila and Phomopsis spp. Data show a close affinity of certain fungi to site, suggesting geographical relatedness. The ecological effects of distribution of the invasive H. fraxineus and of other fungi sporulating on diseased ash trees in Europe are discussed.

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.

Apple Valsa canker (AVC), caused by the fungus Valsa mali (Vm), is a serious and persistent disease affecting apple production in China that is difficult to control chemically and agriculturally. In order to develop biological control agents (BCAs) for this disease, an antifungal activity-deficient Bacillus amyloliquefaciens mutant, ED-2544, was characterized using PCR amplification, Southern blotting, and SiteFinding-PCR. The B. amyloliquefaciens ChbB (chitin-binding proteins from B. amyloliquefaciens) and chitinase genes were cloned and heterologously expressed in Escherichia coli. The purified recombinant ChbB and chitinase were used to study antifungal, substrate binding, and synergistic activities. The results show that combining ChbB with chitinase increased the activity of chitinase and its antifungal activity. ChbB and chitinase show a synergistic effect on B. amyloliquefaciens EDR2 activity against Vm. These results provide a theoretical basis for the use of B. amyloliquefaciens EDR2 to control AVC.

ClpXP protease is widely distributed in the bacterial kingdom and plays a vital role in maintaining protein quality, physiology, metabolism, and virulence in pathogenic bacteria; however, its functionality in crop-protecting bacteria is rarely investigated. Here, we uncover the role of ClpXP in Lysobacter enzymogenes, an environmental bacterial predator of crop fungal pathogens. We showed that ClpXP, as represented by ClpP, was involved in the antagonistic interaction between L. enzymogenes and a pear fungal pathogen, Valsa pyri. We found that ClpP is required for the full antifungal activity against V. pyri by affecting the secretion of a secondary antifungal metabolite (heat-stable antifungal factor, HSAF), the formation of twitching motility to help its infection to fungal mycelium, and the production of extracellular protease to degrade fungal cells. Together, our findings uncover key unidentified roles of the widespread bacterial ClpXP protease in a crop-protecting bacterium, expanding the diversified functionality of ClpXP from crop-pathogenic to protecting bacteria, particularly in crop bacterial-fungal antagonistic interaction.

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.

A book review of Laura Carvalho's Valsa Brasileira, focusing on the Brazilian economy of the past Workers' Party administrations. The book was published in Brazil in Portuguese.

The Valsa canker, caused by Valsa mali (V. mali), is a destructive disease of apple in Eastern Asia. Effector proteins are important for fungal pathogenicity. We studied a candidate effector VmPxE1 isolated based on the genome information of V. mali. By using the yeast invertase secretion assay system, VmPxE1 was shown to contain a signal peptide with secretory functions. VmPxE1 can suppress BCL-2-associated X protein (BAX)-induced cell death with a high efficacy of 92% in Nicotiana benthamiana. The expression of VmPxE1 was upregulated during the early infection stage and deletion of VmPxE1 led to significant reductions in virulence on both apple twigs and leaves. VmPxE1 was also shown to target an apple ascorbate peroxidase (MdAPX1) by the yeast two-hybrid screening, bimolecular fluorescence complementation and in vivo co-immunoprecipitation. Sequence phylogenetic analysis suggested that MdAPX1 was an ascorbate peroxidase belonging to a subgroup of heme-dependent peroxidases of the plant superfamily. The ectopic expression of MdAPX1 in the mutant of VmPxE1 significantly enhanced resistance to H2O2, while the presence of VmPxE1 seems to disturb MdAPX1 function. The present results provide insights into the functions of VmPxE1 as a candidate effector of V. mali in causing apple canker.

Valsa pyri, an ascomycete pathogen that is a member of the Valsaceae family (Sordariomycetes, Diaporthales), which causes pear or apple canker and leads to tree death and massive yield losses. Here, we selected two V. pyri isolates (Vp14 and Vp297) that exhibited different invasion abilities for transcriptomics analyses. Compared toVp297, Vp14 had stronger virulence and spread faster on host-like nutrients. Four samples, including mycelium or infectious mycelium, of the two isolates were sequenced. Clean reads were mapped to the V. pyri genome, and 12490 transcripts and 178 new genes were identified. There were dramatically fewer differentially expressed genes (DEGs) in Vp14 than in Vp297. According to GO and COG annotations, there were both more up- and down-regulated genes in Vp297 than in Vp14 except for genes involved in amino acid transport and metabolism, carbohydrate transport and metabolism, peroxidases and so on. Specific up-regulated DEGs, including genes encoding cell wall degrading enzymes and genes involved in nitrogen metabolism and peroxidases which play crucial roles in virulence and infectious growth, were especially enriched inVp14. These results indicate that the Vp14 isolate may infect its host and take up nutrition more efficiently, reflecting a stronger ability for invasion or infectious growth. Our analysesindicate that a successful V. pyri infection involves multiple instances of transcriptome remodeling to regulate gene functions. Comparative transcriptomics between isolates of V. pyri may aid in our understanding of the virulence mechanism of this pathogen.

miRNAs are important regulators involving in plant-pathogen interactions. However, their roles in apple tree response to Valsa canker pathogen (Valsa mali, Vm) infection were poorly understood. In this study, we constructed two miRNA libraries using the twig bark tissues of apple tree (Malus domestica Borkh. cv. “Fuji”) inoculated with Vm (IVm) and PDA medium (control, BMd). Among all detected miRNAs, 23 miRNAs were specifically isolated from BMd and 39 miRNAs were specifically isolated from IVm. Meanwhile, the expression of 294 miRNAs decreased; and another 172 miRNAs showed an increased expression trend in IVm compared with that in BMd. Furthermore, two degradome sequencing libraries were also constructed to identify the target genes of these miRNAs. In total, 353 differentially expressed miRNAs between IVm and BMd were detected to be able to target 1,077 unigenes with 2,251 cleavage sites. Based on GO and KEGG analysis, these genes were found to be mainly related to transcription regulation and signal transduction. In addition, we selected 17 miRNAs and 22 corresponding target genes to screen the expression profiles when apple twigs were infected by Vm. The expression trends of most miRNAs/target genes were consist with the results of deep sequencing. Many of them may involve in the apple twig-Vm interaction by inducing/reducing their expression. What's more, miRNAs and their target genes regulate the apple twig-Vm interaction by forming many complicated regulation networks rather than one to one model. It is worth that a conserved miRNAs mdm-miR482b, which was down regulated in IVm compared with BMd, has 14 potential target genes, most of which are disease resistance related genes. This indicates that mdm-miR482b may play important roles in apple twig response to Vm. More important, the feedback regulation of sRNA pathway in apple twig is also very complex, and play critical role in the interaction between apple twig and Vm based on the results of expression analysis. In all, the results will provide insights into the crucial functions of miRNAs in the woody plant, apple tree-Vm interaction.

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.

Poplar and willow plantations have become widespread in China, in order to meet national economic and environmental needs. The emergence of several pathogens is enhanced by climatic change and associated human factors. Species of Cytospora are well-known pathogens on poplar and willow, and cause stem cankers and diebacks. In the present study, we conducted a survey of Cytospora species occurring on Populus spp. and Salix spp. in China. We used morphological examination and phylogenetic inferences, based on the DNA sequence data from the internal transcribed spacer regions (ITS1, 5.8S rDNA, and ITS2) and partial β-tubulin gene, to identify six Cytospora species occurring on poplar and willow. Five of these species belonged to known taxa, viz. Cytospora chrysosperma (asexual state of Valsa sordida), Cytospora translucens (asexual state of Leucostoma translucens), Cytospora fugax (asexual state of Valsa salicina), Cytospora atrocirrhata, and Cytospora kantschavelii. Our study yielded a new species, Cytospora davidiana sp. nov., on poplar. The new species is characterized by typical torsellioid conidiomata. An additional Cytospora sp. 1, which formed a distinct clade in the phylogenetic inferences, remains unnamed; the paucity of available materials prevented phenotypical characterization.

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.

Valsa mali (V. mali) is the causative agent of apple tree Valsa canker, which heavily damages the production of apples in China. However, the biological roles of the RNA interfering (RNAi) pathway in the pathogenicity of V. mali remain unknown. Dicer-like proteins (DCLs) are important components that control the initiation of the RNAi pathway. In this study, VmDCL1 and VmDCL2 were isolated and functionally characterized in V. mali. VmDCL1 and VmDCL2 are orthologous in evolution to the DCLs in Cryphonectria parasitica. The deletion of VmDCL1 and VmDCL2 did not affect vegetative growth when the mutants (△VmDCL1, △VmDCL2 and △VmDCL1DCL2) and wild type strain 03-8 were grown on a PDA medium at 25°C in the dark. However, the colony of △VmDCL1 increased by 37.1% compared to the 03-8 colony in a medium containing 0.05% H2O2 3 days after inoculation, and the growth of △VmDCL1 was significantly inhibited in a medium containing 0.5 M KCl at a ratio of 25.7%. Meanwhile, in the presence of 0.05% H2O2, the growth of △VmDCL2 decreased by 34.5% compared with the growth of 03-8, but △VmDCL2 grew normally in the presence of 0.5 M KCl. More importantly, the expression of VmDCL2 was up-regulated 125-fold during the pathogen infection. In the infection assays using apple twigs, the pathogenicity of △VmDCL2 and △VmDCL1DCL2 was significantly reduced compared with that of 03-8 at a ratio of 24.7% and 41.3%, respectively. All defective phenotypes could be nearly rescued by re-introducing the wild type VmDCL1 and VmDCL2 alleles. Furthermore, the number and length distribution of unique small RNAs (unisRNAs) in the mutants and 03-8 were analyzed using deep sequencing. The number of unisRNAs was obviously lower in △VmDCL1, △VmDCL2 and △VmDCL1DCL2 than that in 03-8, and the length distribution of the sRNAs also markedly changed after the VmDCLs were deleted. These results indicated that VmDCLs function in the H2O2 and KCl stress response, pathogenicity and generation of sRNAs.

Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.

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.

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.

O presente artigo procura desmistificar a tese da ameaça chinesa na ordem mundial, particularmente no que respeita à hegemonia dos Estados Unidos neste novo século, traçando, em simultâneo, algumas pistas de reflexão sobre o lugar da China na conjuntura internacional. Gigante frágil, enquanto a China não exorcizar primeiro os seus ‘demónios internos’, freios ao desenvolvimento, o país dificilmente poderá ser entendido como uma ameaça, ou, mesmo, como a próxima superpotência.

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.

Forty-three endophytic fungal isolates were separated from the healthy twigs and barks of the hybrid poplar 'Neva' of Populus deltoides Marsh × Populus nigra L. Eleven distinct isolates were selected for further taxonomical identification by morphological traits and internal transcribed spacer (ITS) rRNA gene sequence analysis. Eight genera namely Alternaria, Aureobasidium, Botryosphaeria, Hyalodendriella, Mycosphaerella, Peyronellaea, Phoma and Valsa were identified on the basis of their morphological characterizations. Of them, the most frequent genus was Alternaria (that is, Ponipodef01, Ponipodef04 and Ponipodef07). Their ITS-rDNA sequences were compared with those available in the GenBank database to obtain the closest related species by BLAST analysis. A modified thin layer chromatography-bioautography assay was used to detect the antimicrobial activity of the n-butanol extracts of mycelia and culture filtrates of the isolates. Most of the fungal isolates were observed to have antibacterial activity, and antibacterial components mainly existed in mycelia. Two isolates Ponipodef05 and Ponipodef12 were also observed to have antifungal activity. The endophytic fungi isolated from the hybrid poplar 'Neva' of P. deltoides Marsh × P. nigra L. may be used as potential producers of antimicrobial natural products. Key words: Poplar hybrid 'Neva', endophytic fungi, extract, antimicrobial activity.

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.