Chemical Constituents from Nicotiana tabacum L. and Their Antifungal Activity

A phytochemical investigation of an aqueous ethyl alcohol (EtOH) extract of the flowers of Nicotiana tabacum L . led to the identification of one new eudesmane-type sesquiterpenoid (1) and 14 known compounds, including 7 flavones (2-8), 6 phenolic acids (9-14), and 1 coumarin (15). The chemical structure of 1 was established on the basis of high-resolution electrospray ionization (HRESIMS) and 1D and 2D NMR analyses, while the other structures were elucidated by 1D NMR and LC–MS data. The antiphytopathogenic activities of all the isolated compounds were tested to determine their antifungal activity against Valsa mali var. mali, Alternaria porri, and Botrytis cinerea at a concentration of 10 µg/mL. Compound 4 exhibited more potent antifungal effects against Valsa mali var. mali with a % growth inhibition of 47.9 ± 1.2%, while the others only showed insignificant activities.

Dopamine enhances the resistance of apple to Valsa mali infection

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 MdTYDC (Malus domestica TYDC) overexpression (OE) enhances the resistance of apple to V. mali (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 overexpression 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.

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

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

Nep1-like Proteins from Valsa mali Differentially Regulate Pathogen Virulence and Response to Abiotic Stresses

Necrosis and ethylene-inducing peptide 1(Nep1)-like protein (NLP) is well known for its cytotoxicity and immunogenicity on dicotyledonous, and it has attracted large attention due to its gene expansion and functional diversification in numerous phytopathogens. Here, two NLP family proteins, VmNLP1 and VmNLP2, were identified in the pathogenic fungus Valsa mali. We showed that VmNLP2 but not VmNLP1 induced cell death when transiently expressed in Nicotiana benthamiana. VmNLP2 was also shown to induce cell death in apple leaves via the treatment of the Escherichia coli-produced recombinant protein. VmNLP1 and VmNLP2 transcripts were drastically induced at the early stage of V. mali infection, whereas only VmNLP2 was shown to be essential for pathogen virulence. We also found that VmNLP1 and VmNLP2 are required for maintaining the integrity of cell membranes, and they differentially contribute to V. mali tolerance to salt- and osmo-stresses. Notably, multiple sequence alignment revealed that the second histidine (H) among the conserved heptapeptide (GHRHDWE) of VmNLP2 is mutated to tyrosine (Y). When this tyrosine (Y) was substituted by histidine (H), the variant displayed enhanced cytotoxicity in N. benthamiana, as well as enhanced virulence on apple leaves, suggesting that the virulence role of VmNLP2 probably correlates to its cytotoxicity activity. We further showed that the peptide among VmNLP2, called nlp25 (VmNLP2), triggered strong immune response in Arabidopsis thaliana. This work demonstrates that NLPs from V. mali involve multiple biological roles, and shed new light on how intricately complex the functions of NLP might be.

A Novel DCL2-Dependent Micro-Like RNA Vm-PC-3p-92107_6 Affects Pathogenicity by Regulating the Expression of Vm-VPS10 in Valsa mali

Dicer proteins are mainly responsible for generating small RNAs (sRNAs), which are involved in gene silencing in most eukaryotes. In previous research, two DCL proteins in Valsa mali, the pathogenic fungus causing apple tree Valsa canker, were found associated with both the pathogenicity and generation of sRNAs. In this study, the differential expression of small interfering RNAs (siRNAs) and miRNA-like RNAs (milRNAs) was analyzed based on the deep sequencing of the wild type and Vm-DCL2 mutant, respectively. Overall, the generation of 40 siRNAs and 18 milRNAs was evidently associated with Vm-DCL2. The target genes of milRNAs were then identified using degradome sequencing; according to the prediction results, most candidate targets are related to pathogenicity. Further, expression of Vm-PC-3p-92107_6 was confirmed in the wild type but not in the Vm-DCL2 mutant. Moreover, the pathogenicity of Vm-PC-3p-92107_6 deletion mutants (ΔVm-PC-3p-92107_6) and the over-expression transformants (Vm-PC-3p-92107_6-OE) was significantly increased and decreased, respectively. Based on those degradome results, vacuolar protein sorting 10 (Vm-VPS10) was identified as the target of Vm-PC-3p-92107_6. Co-expression analysis in tobacco leaves further confirmed that Vm-PC-3p-92107_6 could suppress the expression of Vm-VPS10. Meanwhile, the expression levels of Vm-PC-3p-92107_6 and Vm-VPS10 displayed divergent trends in ΔVm-PC-3p-92107_6 and Vm-PC-3p-92107_6-OE, respectively. Perhaps most importantly, ΔVm-VPS10 featured a significant reduction in pathogenicity. Taken together, our results indicate that a DCL2-dependent milRNA Vm-PC-3p-92107_6 plays roles in pathogenicity by regulating the expression of Vm-VPS10. This study lays a foundation for the comprehensive analysis of pathogenic mechanisms of V. mali and deepens our understanding of the generation and function of fungal sRNA.

Genome-Wide Characterization of HSP90 Gene Family in Malus sieversii and Their Potential Roles in Response to Valsa mali Infection

Heat shock protein 90 (HSP90) is highly conservative molecular chaperon produced by plants in response to adverse environmental stresses including fungal infection. In China, canker disease, caused by Valsa mali, is the main threat for Malus sieversii, an ancestor of the cultivated apple. In this study, a total of eight HSP90 genes were identified from the M. sieversii genome and randomly distributed on eight chromosomes. According to gene structure and phylogenetic analysis, the MsHSP90s can be divided into five categories. The transcriptome analysis of M. sieversii under V. mali infection showed that the plant pathogen interaction pathway was identified as significantly enriched. RNA-seq data and qRT-PCR analysis demonstrated that the MsHSP90-6a gene was significantly repressed by V. mali infection. We further predicted cis-regulatory elements on the promotor region of MsHSP90 genes and identified canonical SHE motifs. Our results improve our understanding of the HSP90 gene family and provide a foundation for further studies of disease prevention in M. sieversii.

β-Glucosidase VmGlu2 Contributes to the Virulence of Valsa mali in Apple Tree

The apple tree canker is caused by Valsa mali, which produces major pathogenic factors involving multiple cell wall-degrading enzymes (CWDEs) and toxins. The β-glucosidases are among the main CWDEs, and thus, they play important roles in the virulence of necrotrophic pathogens. However, the specific roles of β-glucosidases in the virulence of V. mlai remain largely unknown. In this study, we identified a β-glucosidase gene, VmGlu2, which was upregulated during the V. mali infection. We found that VmGlu2 protein had high enzyme activity of β-glucosidase using p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate, while the VmGlu2 could convert phloridzin to phloretin with the release of glucose. The deletion and overexpression of VmGlu2 showed no effect on vegetative growth, but gene deletion mutants of V. mlai showed significantly reduced pycnidia formation. The gene deletion mutants had lower β-glucosidase activities and toxin levels as compared to the wild-type strain. Therefore, these mutants showed a reduced virulence. Moreover, the overexpression of VmGlu2 did not affect toxin levels, but it significantly enhanced β-glucosidase activities, which resulted in an increased pathogenicity. Thus, we conclude that VmGlu2 is required for the full virulence of V. mali. These results provide valuable evidence to the complex role of CWDEs in the fungal pathogenicity.

Inhibitory Effect of Osthole from Cnidium Monnieri (L.) Cusson on Fusarium oxysporum, a Common Fungal Pathogen of Potato

Fusarium wilt of potato is one of the most common diseases of potato in China, and is becoming a serious threat in potato production. It has been reported that osthole from Cnidium monnieri (L.) Cusson can inhibit plant pathogens. Here, we test the anti-fungal activity of C. monnieri osthole against Fusarium oxysporum in potatoes. The results showed that at a concentration of 5 mg/mL, osthole was able to obviously inhibit mycelial growth of F. oxysporum. We found that osthole caused changes of mycelial morphology, notably hyphal swelling and darkening. Osthole significantly reduced the spore germination of Fusarium by 57.40%. In addition, osthole also inhibited the growth of other pathogens such as Fusarium moniliforme J. Sheld, Thanatephorus cucumeris Donk, and Alternaria alternata (Fr.) Keissl, but not Alternaria solani Jonesetgrout and Valsa mali Miyabe and G. Yamada. Our results suggest that osthole has considerable potential as an agent for the prevention and treatment of potato Fusarium wilt.