Transcription factor CgSte12 regulates pathogenicity by affecting appressorium structural development in the anthracnose-causing fungus Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is the causal agent of poplar anthracnose, which induces major economic losses and adversely affects the ecosystem services of poplar forests. The appressorium serves as a penetration structure for many pathogenic fungi, including C. gloeosporioides. The production of mucilage and the formation of penetration pegs are critically important for the appressorium-mediated penetration of host tissues. We previously found that CgPmk1 is a key protein involved in appressorium formation, penetration, and pathogenicity. Although CgSte12, which is a transcription factor that functions downstream of CgPmk1, regulates the formation of penetration pegs, its role in C. gloeosporioides appressorium development and pathogenicity has not been elucidated. Here, we developed C. gloeosporioides CgSTE12 mutants and characterized the molecular and cellular functions of CgSTE12. The results showed that mycelial growth and morphology were not affected in the CgSTE12 knockout mutants, which produced normal melanized appressoria. However, these mutants had less mucilage secreted around the appressoria, impaired appressorial cone formation, and the inability to form penetration pores and pegs, which ultimately led to a significant loss of pathogenicity. Our comparative transcriptome analysis revealed that CgSte12 controls the expression of genes involved in appressorium development and function, including genes encoding cutinases, NADPH oxidase, spermine biosynthesis-related proteins, ceramide biosynthesis-related proteins, fatty acid metabolism-related proteins, and glycerophospholipid metabolism-related proteins. Overall, our findings indicate that CgSte12 is a critical regulator of appressorium development and affects C. gloeosporioides pathogenicity by modulating the structural integrity of appressoria.

Comparative transcriptome analysis reveals the importance of phenylpropanoid biosynthesis for the induced resistance of 84K poplar to anthracnose.

BACKGROUND: Poplar anthracnose, which is one of the most important tree diseases, is primarily caused by Colletotrichum gloeosporioides, which has been detected in poplar plantations in China and is responsible for serious economic losses. The characteristics of 84K poplar that have made it one of the typical woody model plants used for investigating stress resistance include its rapid growth, simple reproduction, and adaptability. RESULTS: In this study, we found that the resistance of 84K poplar to anthracnose varied considerably depending on how the samples were inoculated of the two seedlings in each tissue culture bottle, one (84K-Cg) was inoculated for 6 days, whereas the 84K-DCg samples were another seedling inoculated at the 6th day and incubated for another 6 days under the same conditions. It was showed that the average anthracnose spot diameter on 84K-Cg and 84K-DCg leaves was 1.23 ± 0.0577 cm and 0.67 ± 0.1154 cm, respectively. Based on the transcriptome sequencing analysis, it was indicated that the upregulated phenylpropanoid biosynthesis-related genes in 84K poplar infected with C. gloeosporioides, including genes encoding PAL, C4H, 4CL, HCT, CCR, COMT, F5H, and CAD, are also involved in other KEGG pathways (i.e., flavonoid biosynthesis and phenylalanine metabolism). The expression levels of these genes were lowest in 84K-Cg and highest in 84K-DCg. CONCLUSIONS: It was found that PAL-related genes may be crucial for the induced resistance of 84K poplar to anthracnose, which enriched in the phenylpropanoid biosynthesis. These results will provide the basis for future research conducted to verify the contribution of phenylpropanoid biosynthesis to induced resistance and explore plant immune resistance-related signals that may regulate plant defense capabilities, which may provide valuable insights relevant to the development of effective and environmentally friendly methods for controlling poplar anthracnose.

The roles of Gti1/Pac2 family proteins in fungal growth, morphogenesis, stress response and pathogenicity.

Gti1/Pac2 is a fungal specific transcription factor family with a stable and conserved N-terminal domains. Generally, there are two members in this family named as Gti1/Wor1/Rpy1/Mit1/Reg1/Ros1/Sge1 and Pac2, which are involving in fungal growth, development, stress response, spore production, pathogenicity and so on. The Gti1/Pac2 family proteins shared some conserved and distinct functions. For example, in Schizosaccharomyces pombe, Gti1 promotes the initiation of gluconate uptake during glucose starvation, while Pac2 controls the onset of sexual development in a pathway independent of the cAMP cascade. In recent two decades, more attention was focused on the Gti1 and its orthologs due to their significant effect on morphology switch and fungal virulence. By contrast, there are limited works on the functions of Pac2 which is required for stress responses and conidiation, but play minor roles in fungal virulence. In this review, we present an overview of our current understanding of the Gti1/Pac2 proteins that contribute to fungal development and/or pathogenicity and of the regulation mechanisms during infection related development. Understanding the working networks of the conserved Gti1/Pac2 transcription factors in fungal pathogenicity not only advances our knowledge of the highly elaborate infection process but may also lead to the development of novel strategies for the control of plant disease.

The mitogen-activated protein kinase module CcSte11-CcSte7-CcPmk1 regulates pathogenicity via the transcription factor CcSte12 in Cytospora chrysosperma.

The pathogen Cytospora chrysosperma is the causal agent of poplar canker disease and causes considerable economic losses in China. Mitogen-activated protein kinase (MAPK) cascades play a crucial role in mediating cellular responses and Pmk1-MAPKs are indispensable for pathogenic related processes in plant pathogenic fungi. In previous studies, we demonstrated that the CcPmk1 acts as a core regulator of fungal pathogenicity by modulating a small number of master downstream targets, such as CcSte12. In this study, we identified and characterized two upstream components of CcPmk1: MAPKKK CcSte11 and MAPKK CcSte7. Deletion of CcSte11 and CcSte7, resulted in slowed growth, loss of sporulation and virulence, similar to the defects observed in the CcPmk1 deletion mutant. In addition, CcSte11, CcSte7 and CcPmk1 interact with each other, and the upstream adaptor protein CcSte50 interact with CcSte11 and CcSte7. Moreover, we explored the global regulation network of CcSte12 by transcriptional analysis between CcSte12 deletion mutants and wild-type during the simulated infection process. Two hydrolase activity GO terms (GO:0004553 and GO:0016798) and starch and sucrose metabolism (mgr00500) KEGG pathway were significantly enriched in the down-regulated genes of CcSte12 deletion mutants. In addition, a subset of glycosyl hydrolase genes and putative effector genes were significantly down-regulated in the CcSte12 deletion mutant, which might be important for fungal pathogenicity. Especially, CcSte12 bound to the CcSp84 promoter region containing the TGAAACA motif. Moreover, comparison of CcSte12-regulated genes with CcPmk1-regulated genes revealed 116 overlapping regulated genes in both CcSte12 and CcPmk1, including some virulence-associated genes. Taken together, the protein complexes CcSte11-CcSte7-CcPmk1 receive signals transmitted by upstream CcSte50 and transmit signals to downstream CcSte12, which regulates hydrolase, effectors and other genes to promote virulence. Overall, these results indicate that the CcPmk1-MAPK signaling pathway of C. chrysosperma plays a key role in the pathogenicity.

Gene Family Expansion during the Adaptation of Colletotrichum gloeosporioides to Woody Plants.

Gene gains/losses during evolution are critical for the adaptation of organisms to new environments or hosts. However, it remains unknown whether gene family expansions facilitated the adaptation of phytopathogenic fungi to woody plants. In this study, we compared the newly sequenced genome of the Colletotrichum gloeosporioides strain CFCC80308 with the genomes of two other C. gloeosporioides strains, Cg-14 and Lc-1, isolated from Persea americana and Liriodendron leaves, respectively. The genes in the expanded families, which were associated with plant surface signal recognition, encoded various proteins, including glycosyde hydrolases (GHs) and cytochrome P450. Interestingly, there was a substantial increase in the number of GH family genes in CFCC80308. Specifically, there were 368 enriched genes in the GH families (e.g., GH1, GH3, GH10, GH12, GH15, GH16, GH17, GH18, GH25, GH32, GH53, GH61, GH76, and GH81); the expression levels of these genes were highly up-regulated during the infection of poplar trees. Additionally, the GH17 family was larger in CFCC80308 than in C. gloeosporioides strains Cg-14 and Lc-1. Furthermore, the expansion of the MP65-encoding gene family during the adaptation of Colletotrichum species to woody plants was consistent with the importance of gene gains/losses for the adaptation of organisms to their environments. This study has clarified how C. gloeosporioides adapted to woody plants during evolution.

Morphological and phylogenetic analyses reveal three new species of Apiospora in China.

Species of Apiospora are distributed worldwide as endophytes, pathogens and saprobes. In this study, we analysed Apiospora strains isolated from diseased leaves in Yunnan Province and dead culms in Shaanxi Province, China and we identified fungal species based on multi-locus phylogeny of ITS, LSU, tef1 and tub2 genes, along with the morphological characters, host and ecological distribution. Analyses revealed three new species, namely A.corylisp. nov., A.lophatherisp. nov. and A.oenotheraesp. nov. and one known species A.arundinis. Illustrations and descriptions of the four taxa are provided, along with comparisons with closely-related taxa in the genus.

Effects of Colletotrichum gloeosporioides and Poplar Secondary Metabolites on the Composition of Poplar Phyllosphere Microbial Communities.

Poplar anthracnose caused by Colletotrichum gloeosporioides is a common disease affecting poplars globally that causes the destruction and alteration of poplar phyllosphere microbial communities; however, few studies have investigated these communities. Therefore, in this study, three species of poplar with different resistances were investigated to explore the effects of Colletotrichum gloeosporioides and poplar secondary metabolites on the composition of poplar phyllosphere microbial communities. Evaluation of the phyllosphere microbial communities before and after inoculation of the poplars with C. gloeosporioides revealed that both bacterial and fungal OTUs decreased after inoculation. Among bacteria, the most abundant genera were Bacillus, Plesiomonas, Pseudomonas, Rhizobium, Cetobacterium, Streptococcus, Massilia, and Shigella for all poplar species. Among fungi, the most abundant genera before inoculation were Cladosporium, Aspergillus, Fusarium, Mortierella, and Colletotrichum, while Colletotrichum was the main genus after inoculation. The inoculation of pathogens may regulate the phyllosphere microorganisms by affecting the secondary metabolites of plants. We investigated metabolite contents in the phyllosphere before and after the inoculation of the three poplar species, as well as the effects of flavonoids, organic acids, coumarins, and indoles on poplar phyllosphere microbial communities. We speculated that coumarin had the greatest recruitment effect on phyllosphere microorganisms, followed by organic acids through regression analysis. Overall, our results provide a foundation for subsequent screening of antagonistic bacteria and fungi against poplar anthracnose and investigations of the mechanism by which poplar phyllosphere microorganisms are recruited. IMPORTANCE Our findings revealed that the inoculation of Colletotrichum gloeosporioides has a greater effect on the fungal community than the bacterial community. In addition, coumarins, organic acids, and flavonoids may have recruitment effects on phyllosphere microorganisms, while indoles may have inhibitory effects on these organisms. These findings may provide the theoretical basis for the prevention and control of poplar anthracnose.

Morphology and molecular analyses reveal three new species of Botryosphaeriales isolated from diseased plant branches in China.

The Botryosphaeriales represents an ecologically diverse group of fungi, comprising endophytes, saprobes, and plant pathogens. In this study, taxonomic analyses were conducted based on morphological characteristics and phylogenetic analyses of multi-gene sequence data from four loci (ITS, LSU, tef1-α, and tub2). Thirteen isolates obtained from Beijing and Yunnan Province were identified as seven species of Botryosphaeriales, including Aplosporellajaveedii, Dothiorellaalpina, Phaeobotryonaplosporum and Ph.rhois, and three previously undescribed species, namely Aplosporellayanqingensis, Dothiorellabaihuashanensis, and Phaeobotryonplatycladi. Additionally, the new records of Dothiorellaalpina from the host species Populusszechuanica, Phaeobotryonaplosporum from Juglansmandshurica, and Phaeobotryonrhois from Populusalbavar.pyramidalis are included.

Comparative Transcriptome Analysis Reveals the Effect of the DHN Melanin Biosynthesis Pathway on the Appressorium Turgor Pressure of the Poplar Anthracnose-Causing Fungus Colletotrichum gloeosporioides.

Anthracnose of poplar caused by Colletotrichum gloeosporioides is a leaf disease that seriously affects poplar growth. The pathogen invades the host in the form of adherent cells, which generate turgor pressure through the metabolism of intracellular substances prior to penetrating the epidermis of poplar leaves. In this study, the expansion-related pressure of the mature appressorium of the wild-type C. gloeosporioides was approximately 13.02 ± 1.54 MPa at 12 h, whereas it was 7.34 ± 1.23 MPa and 9.34 ± 2.22 MPa in the melanin synthesis-related gene knockout mutants ΔCgCmr1 and ΔCgPks1, respectively. The CgCmr1 and CgPks1 genes were highly expressed at 12 h in the wild-type control, implying that the DHN melanin biosynthesis pathway may play an important role in the mature appressorium stage. The transcriptome sequencing analysis indicated that the upregulated melanin biosynthesis genes in C. gloeosporioides, such as CgScd1, CgAyg1, CgThr1, CgThr2, and CgLac1, are involved in specific KEGG pathways (i.e., fatty acid biosynthesis, fatty acid metabolism, and biotin metabolism). Therefore, we speculate that the melanin synthesis-related genes and fatty acid metabolism pathway genes contribute to the regulation of the turgor pressure in the mature C. gloeosporioides appressorium, ultimately leading to the formation of infection pegs that enter plant tissues. These observations may reflect the co-evolution of C. gloeosporioides and its host.

Identification and Characterization of Neocosmospora silvicola Causing Canker Disease on Pinus armandii in China.

Chinese white pine, Pinus armandii, is a source of high-quality timber and an afforestation tree in China, which plays an important ecological and social role in water and soil conservation. Recently, a new canker disease has been reported in Longnan City, Gansu Province, where P. armandii is mainly distributed. In this study, the causal agent was isolated from diseased samples and identified as a fungal pathogen, Neocosmospora silvicola, based on morphological characteristics and molecular analyses (internal transcribed spacer, large subunit, RNA polymerase II, and translation elongation factor-1α). Pathogenicity tests on P. armandii revealed that N. silvicola isolates caused a 60% average mortality rate in artificially inoculated 2-year-old seedlings. The pathogenicity of these isolates was also observed on the branches of 10-year-old P. armandii trees with a 100% mortality rate. These results agree with the isolation of N. silvicola from diseased plants, suggesting the possible role of this fungus in the decline of P. armandii plants. Mycelial growth of N. silvicola was fastest on potato dextrose agar medium, and growth occurred at pH values ranging from 4.0 to 11.0 with temperatures between 5 and 40°C. The fungus also grew rapidly in complete darkness compared with other light conditions. Of the eight carbon and seven nitrogen sources tested, starch and sodium nitrate, respectively, were highly efficient in supporting the mycelial growth of N. silvicola. The ability of N. silvicola to grow at low temperatures (5°C) may explain its occurrence in the Longnan area of Gansu Province. This article is the first report of N. silvicola as an important fungal pathogen causing branch and stem cankers on Pinus tree species, which remains a threat to the forests.

The function of plant PR1 and other members of the CAP protein superfamily in plant-pathogen interactions.

The pathogenesis-related (PR) proteins of plants have originally been identified as proteins that are strongly induced upon biotic and abiotic stress. These proteins fall into 17 distinct classes (PR1-PR17). The mode of action of most of these PR proteins has been well characterized, except for PR1, which belongs to a widespread superfamily of proteins that share a common CAP domain. Proteins of this family are not only expressed in plants but also in humans and in many different pathogens, including phytopathogenic nematodes and fungi. These proteins are associated with a diverse range of physiological functions. However, their precise mode of action has remained elusive. The importance of these proteins in immune defence is illustrated by the fact that PR1 overexpression in plants results in increased resistance against pathogens. However, PR1-like CAP proteins are also produced by pathogens and deletion of these genes results in reduced virulence, suggesting that CAP proteins can exert both defensive and offensive functions. Recent progress has revealed that plant PR1 is proteolytically cleaved to release a C-terminal CAPE1 peptide, which is sufficient to activate an immune response. The release of this signalling peptide is blocked by pathogenic effectors to evade immune defence. Moreover, plant PR1 forms complexes with other PR family members, including PR5, also known as thaumatin, and PR14, a lipid transfer protein, to enhance the host's immune response. Here, we discuss possible functions of PR1 proteins and their interactors, particularly in light of the fact that these proteins can bind lipids, which have important immune signalling functions.

A putative terpene cyclase gene (CcPtc1) is required for fungal development and virulence in Cytospora chrysosperma.

Cytospora chrysosperma is a destructive plant pathogenic fungus, which causes canker disease on numerous woody plants. However, knowledge concerning the interaction between C. chrysosperma and its host remains limited. Secondary metabolites produced by phytopathogens often play important roles in their virulence. Terpene cyclases (TC), polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) are the key components for the synthesis of secondary metabolites. Here, we characterized the functions of a putative terpene type secondary metabolite biosynthetic core gene CcPtc1 in C. chrysosperma, which was significantly up-regulated in the early stages of infection. Importantly, deletion of CcPtc1 greatly reduced fungal virulence to the poplar twigs and they also showed significantly reduced fungal growth and conidiation compared with the wild-type (WT) strain. Furthermore, toxicity test of the crude extraction from each strain showed that the toxicity of crude extraction secreted by ΔCcPtc1 were strongly compromised in comparison with the WT strain. Subsequently, the untargeted metabolomics analyses between ΔCcPtc1 mutant and WT strain were conducted, which revealed 193 significantly different abundant metabolites (DAMs) inΔCcPtc1 mutant compared to the WT strain, including 90 significantly downregulated metabolites and 103 significantly up-regulated metabolites, respectively. Among them, four key metabolic pathways that reported to be important for fungal virulence were enriched, including pantothenate and coenzyme A (CoA) biosynthesis. Moreover, we also detected significant alterations in a series of terpenoids, among which (+)-ar-turmerone, pulegone, ethyl chrysanthemumate, and genipin were significantly down-regulated, while cuminaldehyde and (±)-abscisic acid were significantly up-regulated. In conclusion, our results demonstrated that CcPtc1 acts as a virulence-related secondary metabolism factor and provides new insights into the pathogenesis of C. chrysosperma.

The Potential Fungal Pathogens of Euonymus japonicus in Beijing, China.

Euonymus japonicus tolerates the dry and frigid climate of Beijing, China, and effectively filters out particles during the winter. However, fungal infestation frequently causes extreme illness and can even lead to shrub death. In this study, 104 diseased E. japonicus specimens were collected from seven districts in Beijing. Seventy-nine isolates were identified as 22 fungal species in seven genera. The species were Aplosporella hesperidica, A. javeedii, A. prunicola, Botryosphaeria dothidea, Colletotrichum aenigma, Co. euonymi, Co. euonymicola, Co. gloeosporioides, Cytospora ailanthicola, C. albodisca, C. diopuiensis, C. discotoma, C. elaeagni, C. euonymicola, C. euonymina, C. haidianensis, C. leucostoma, C. sophorae, C. zhaitangensis, Diaporthe eres, Dothiorella acericola, and Pestalotiopsis chaoyangensis. On the basis of morphological and phylogenetic analyses, Colletotrichum euonymi, Co. euonymicola, Cytospora zhaitangensis, and Pestalotiopsis chaoyangensis were introduced as novel species. Colletotrichum euonymi, Co. euonymicola, and Pestalotiopsis chaoyangensis were subsequently confirmed as pathogens of E. japonicus leaves by pathogenicity testing. This study provides an important assessment of the fungi associated with diseases of E. japonicus in Beijing, China.

Identification and pathogenicity of six fungal species causing canker and dieback disease on golden rain tree in Beijing, China.

Golden rain trees (Koelreuteria paniculata) are largely cultivated because of their important ornamental, medicinal, and economic value. However, they are affected by canker and dieback disease to a large extent. To determine the fungi associated with canker and dieback disease of golden rain trees, isolations were obtained from diseased branches and twigs during 2019 and 2020 in greenbelts and nurseries in Beijing, China. Isolates were identified as six species (Allocryptovalsa castaneicola, Botryosphaeria dothidea, Cytospora koelreutericola sp. nov., Dothiorella acericola, Eutypella citricola, and Peroneutypa scoparia) based on morphological features and phylogenetic analyses of ITS, act, rpb2, tef1-α, and tub2. The results of pathogenicity tests indicated that all fungi produced discoloration and Botryosphaeria dothidea was highly aggressive to golden rain tree. In conclusion, this study explored the taxonomy, phylogeny, and pathogenicity of different fungal species associated with canker and dieback disease on golden rain tree and provided fundamental knowledge to improve disease management.

Re-evaluation of the Fungal Diversity and Pathogenicity of Cytospora Species from Populus in China.

Poplar is widely cultivated in China because of its strong ecological adaptability, fast growth, easy reproduction, and short rotation period. However, it suffers from severe threat from canker disease caused by Cytospora species. The present study revealed the presence of Cytospora species from Populus in China. A total of six species of Cytospora were isolated from Populus in six provinces in China, including five known species (C. ailanthicola, C. chrysosperma, C. donglingensis, C. paratranslucens, and C. sophoriopsis) and one novel species (C. populi) based on morphological and phylogenetic analyses of ITS, act, rpb2, tef1-α, and tub2 gene sequences. Cytospora ailanthicola, C. chrysosperma, C. paratranslucens, and C. sophoriopsis are confirmed as pathogens by pathogenicity tests of which C. paratranslucens showed the strongest virulence, followed by C. ailanthicola, C. chrysosperma, and C. sophoriopsis. The mycelial growth rates of isolates from the six species had 22.5 to 27°C as the optimum temperatures, and the optimum pH values were 5.9 to 7.1. The effectiveness of six carbon sources on the mycelial growth showed that colonies grew the fastest in the presence of fructose and grew the slowest using xylose. This study represents a significant evaluation of Cytospora causing poplar canker disease in China.

Comparative Transcriptomic Analysis of MAPK-Mediated Regulation of Pathogenicity, Stress Responses, and Development in Cytospora chrysosperma.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that mediate cellular responses to various biotic and abiotic signals in plant-pathogenic fungi. Generally, there are three MAPKs in filamentous pathogenic fungi: Pmk1/Fus3/Kss1, Hog1, and Stl2. Our previous studies have shown that CcPmk1 is a core regulator of fungal pathogenicity in Cytospora chrysosperma, the causal agent of canker disease in a wide range of woody plants. Here, we identified and functionally characterized the other two MAPK genes (CcHog1 and CcSlt2) and then compared the transcriptional differences among these three MAPKs in C. chrysosperma. We found that the MAPKs shared convergent and distinct roles in fungal development, stress responses, and virulence. For example, CcHog1, CcSlt2, and CcPmk1 were all involved in conidiation and response to stresses, including hyperosmotic pressure, cell wall inhibition agents, and H2O2, but only CcPmk1 and CcSlt2 were required for hyphal growth and fungal pathogenicity. Transcriptomic analysis showed that numerous hyperosmosis- and cell wall-related genes significantly reduced their expression levels in ΔCcHog1 and ΔCcSlt2, respectively. Interestingly, RNA- and ribosome-related processes were significantly enriched in the upregulated genes of ΔCcSlt2, whereas they were significantly enriched in the downregulated genes of ΔCcPmk1. Moreover, two secondary metabolite gene clusters were significantly downregulated in ΔCcPmk1, ΔCcSlt2, and/or ΔCcHog1. Importantly, some virulence-associated genes were significantly downregulated in ΔCcPmk1 and/or ΔCcSlt2, such as candidate effector genes. Collectively, these results suggest that the similar and distinct phenotypes of each MAPK deletion mutant may result from the transcriptional regulation of a series of common or specific downstream genes, which provides a better understanding of the regulation network of MAPKs in C. chrysosperma.

Phosphoproteomic and Metabolomic Profiling Uncovers the Roles of CcPmk1 in the Pathogenicity of Cytospora chrysosperma.

Pmk1, a highly conserved pathogenicity-related mitogen-activated protein kinase (MAPK) in pathogenic fungi, is phosphorylated and activated by MAP2K and acts as a global regulator of fungal infection and invasive growth by modulating downstream targets. However, the hierarchical CcPmk1 regulatory network in Cytospora chrysosperma, the main causal agent of canker disease in many woody plant species, is still unclear. In this study, we analyzed and compared the phosphoproteomes and metabolomes of ΔCcPmk1 and wild-type strains and identified pathogenicity-related downstream targets of CcPmk1. We found that CcPmk1 could interact with the downstream homeobox transcription factor CcSte12 and affect its phosphorylation. In addition, the ΔCcSte12 displayed defective phenotypes that were similar to yet not identical to that of the ΔCcPmk1 and included significantly reduced fungal growth, conidiation, and virulence. Remarkably, CcPmk1 could phosphorylate proteins translated from a putative secondary metabolism-related gene cluster, which is specific to C. chrysosperma, and the phosphorylation of several peptides was completely abolished in the ΔCcPmk1. Functional analysis of the core gene (CcPpns1) in this gene cluster revealed its essential roles in fungal growth and virulence. Metabolomic analysis showed that amino acid metabolism and biosynthesis of secondary metabolites, lipids, and lipid-like molecules significantly differed between wild type and ΔCcPmk1. Importantly, most of the annotated lipids and lipid-like molecules were significantly downregulated in the ΔCcPmk1 compared to the wild type. Collectively, these findings suggest that CcPmk1 may regulate a small number of downstream master regulators to control fungal growth, conidiation, and virulence in C. chrysosperma. IMPORTANCE Understanding the pathogenic mechanisms of plant pathogens is a prerequisite to developing effective disease-control methods. The Pmk1 MAPK is highly conserved among phytopathogenic fungi and acts as a global regulator of fungal pathogenicity by modulating downstream transcription factors or other components. However, the regulatory network of CcPmk1 from C. chrysosperma remains enigmatic. The present data provide evidence that the core pathogenicity regulator CcPmk1 modulates a few downstream master regulators to control fungal virulence in C. chrysosperma through transcription or phosphorylation and that CcPmk1 may be a potential target for disease control.

Elevated Ozone Concentration and Nitrogen Addition Increase Poplar Rust Severity by Shifting the Phyllosphere Microbial Community.

Tropospheric ozone and nitrogen deposition are two major environmental pollutants. A great deal of research has focused on the negative impacts of elevated O3 and the complementary effect of soil N addition on the physiological properties of trees. However, it has been overlooked how elevated O3 and N addition affect tree immunity in face of pathogen infection, as well as of the important roles of phyllosphere microbiome community in host-pathogen-environment interplay. Here, we examined the effects of elevated O3 and soil N addition on poplar leaf rust [Melampsora larici-populina] severity of two susceptible hybrid poplars [clone '107': Populus euramericana cv. '74/76'; clone '546': P. deltoides Í P. cathayana] in Free-Air-Controlled-Environment plots, in addition, the link between Mlp-susceptibility and changes in microbial community was determined using Miseq amplicon sequencing. Rust severity of clone '107' significantly increased under elevated O3 or N addition only; however, the negative impact of elevated O3 could be significantly mitigated when accompanied by N addition, likewise, this trade-off was reflected in its phyllosphere microbial α-diversity responding to elevated O3 and N addition. However, rust severity of clone '546' did not differ significantly in the cases of elevated O3 and N addition. Mlp infection altered microbial community composition and increased its sensitivity to elevated O3, as determined by the markedly different abundance of taxa. Elevated O3 and N addition reduced the complexity of microbial community, which may explain the increased severity of poplar rust. These findings suggest that poplars require a changing phyllosphere microbial associations to optimize plant immunity in response to environmental changes.

Verification of the Protective Effects of Poplar Phenolic Compounds Against Poplar Anthracnose.

Poplar anthracnose caused by Colletotrichum gloeosporioides is one of the most important diseases widely distributed in poplar-growing areas in China, causing serious economic and ecological losses. In this study, three poplar species showed different resistance to poplar anthracnose: Populus × canadensis was resistant, Populus tomentosa was susceptible, and P. × beijingensis showed intermediate resistance. However, it remains uncertain whether phenolic compounds in poplar are involved in this resistance. Therefore, we determined the concentrations of phenolic compounds and their antifungal activity. Before and after the C. gloeosporioides inoculation, 20 phenolic compounds were detected in P. × canadensis and the number increased from 12 to 14 in P. × beijingensis but decreased from seven to four in P. tomentosa. Thus, phenolic compounds may be positively correlated with the degree of disease resistance. We selected seven phenolic compounds for further analysis, which varied considerably in content after inoculation with C. gloeosporioides. These seven compounds were salicin, arbutin, benzoic acid, salicylic acid, chlorogenic acid, ferulic acid, and naringenin, which helped poplar trees to limit the growth of C. gloeosporioides and differed in their antifungal effects, with phenolic acids having the strongest inhibitory effect. In addition, the optimal concentrations of different substances varied. We demonstrate that these phenolic compounds produced by poplar do play a certain role in the process of poplar resistance to anthracnose. These findings lay a foundation for future research into the antifungal mechanism of poplar trees and may be useful for enhancing the prevention and control of poplar anthracnose.

Fungal Richness of Cytospora Species Associated with Willow Canker Disease in China.

Species of Cytospora are considered important plant pathogens of a wide range of plant hosts, especially Salicaceae plants. Salix (Salicaceae, Malpighiales) has been widely cultivated in China because of its strong ecological adaptability, fast growth, and easy reproduction. In this study, a total of eight species of Cytospora were discovered on Salix in China, including C. ailanthicola, C. alba, C. chrysosperma, C. gigaspora, C. nivea, C. paracinnamomea, C. rostrata, and C. sophoriopsis. Among them, C. alba and C. paracinnamomea were identified as novel species based on morphology and phylogenetic analyses of ITS, act, rpb2, tef1-α, and tub2 gene sequences and were confirmed as pathogens of willow canker disease by pathogenicity tests. The mycelial growth rates of strains from these two novel species (C. alba and C. paracinnamomea) had optimum temperatures of 21 to 22 °C and an optimum pH value of 5 to 6. The effectiveness of six carbon sources on the mycelial growth showed that fructose and maltose had the highest influence. Cytospora species richness was significantly positively correlated with dry and wet areas. This study represents a significant evaluation of Cytospora associated with willow canker disease in China and provides a theoretical basis for predicting the potential risk of willow canker disease.