Transcriptome Analysis and Reactive Oxygen Species Detection Suggest Contrasting Molecular Mechanisms in Populus canadensis' Response to Different Formae Speciales of Marssonina brunnea.

Revealing plant-pathogen interactions is important for resistance breeding, but it remains a complex process that presents many challenges. Marssonina leaf spot of poplars (MLSP) is the main disease in poplars; in China, its pathogens consist of two formae speciales, namely, Marssonina brunnea f. sp. Monogermtubi (MO) and M. brunnea f. sp. Multigermtubi (MU). However, the mechanism of the molecular interaction between poplars and the two formae speciales, especially for an incompatible system, remains unclear. In this study, we conducted transcriptome sequencing and reactive oxygen species (ROS) staining based on the interactions between Populus canadensis and the two formae speciales. The results show that the gene expression patterns of P. canadensis induced by MO and MU were significantly different, especially for the genes associated with biotic stress. Furthermore, MO and MU also triggered distinct ROS reactions of P. canadensis, and ROS (mainly H2O2) burst was only observed around the cells penetrated by MU. In conclusion, this study suggested that P. canadensis experienced different resistance reactions in response to the two formae speciales of M. brunnea, providing valuable insights for further understanding the host-pathogen interactions of MLSP.

Metabolomics Analysis of Sporulation-Associated Metabolites of Metarhizium anisopliae Based on Gas Chromatography-Mass Spectrometry.

Metarhizium anisopliae, an entomopathogenic fungus, has been widely used for the control of agricultural and forestry pests. However, sporulation degeneration occurs frequently during the process of successive culture, and we currently lack a clear understanding of the underlying mechanisms. In this study, the metabolic profiles of M. anisopliae were comparatively analyzed based on the metabolomics approach of gas chromatography-mass spectrometry (GC-MS). A total of 74 metabolites were detected in both normal and degenerate strains, with 40 differential metabolites contributing significantly to the model. Principal component analysis (PCA) and potential structure discriminant analysis (PLS-DA) showed a clear distinction between the sporulation of normal strains and degenerate strains. Specifically, 23 metabolites were down-regulated and 17 metabolites were up-regulated in degenerate strains compared to normal strains. The KEGG enrichment analysis identified 47 significant pathways. Among them, the alanine, aspartate and glutamate metabolic pathways and the glycine, serine and threonine metabolism had the most significant effects on sporulation, which revealed that significant changes occur in the metabolic phenotypes of strains during sporulation and degeneration processes. Furthermore, our subsequent experiments have substantiated that the addition of amino acids could improve M. anisopliae's spore production. Our study shows that metabolites, especially amino acids, which are significantly up-regulated or down-regulated during the sporulation and degeneration of M. anisopliae, may be involved in the sporulation process of M. anisopliae, and amino acid metabolism (especially glutamate, aspartate, serine, glycine, arginine and leucine) may be an important part of the sporulation mechanism of M. anisopliae. This study provides a foundation and technical support for rejuvenation and production improvement strategies for M. anisopliae.

First record of Fusarium concentricum (Hypocreales: Hypocreaceae) isolated from the moth Polychrosis cunninhamiacola (Lepidoptera: Tortricidae) as an entomopathogenic fungus.

Fusarium concentricum Nirenberg & O' Donnell (Ascomycota: Hypocreales) is a fungal species known to infect plants, but never reported as entomopathogenic. Polychrosis cunninhamiacola Liu et Pei (Lepidoptera: Tortricidae: Olethreutinae) is a major and widespread insect pest causing economic losses to cultivated Chinese fir Cunninghamia lanceolata (Lamb.) Hook. It is routinely controlled by extensive use of chemical insecticides, which is perceived as environmentally unsustainable. During March and April of 2019-2020, muscardine cadavers of larvae and pupae of P. cunninhamiacola infected with growing fungus were collected in a fir forest in northern Guangdong Province, China. Conidia were isolated and cultured on PDA medium, from which the fungal strain was identified as F. concentricum FCPC-L01 by morphology and by sequence alignment match with Tef-1α gene. Pathogenicity bioassays at the conidial concentration 1 × 107 revealed P. cunninhamiacola adults and Danaus chrysippus (L.) (Lepidoptera: Nymphalidae) larvae are sensitive to the fungal infection, but not the fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae). We believe results indicate this fungal strain might be applicable against specific target insect pests. As this is the first record of a natural infection caused by F. concentricum in insects, we propose host specificity tests should be done to evaluate its potential as a biocontrol agent.

Metarhizium Entomopathogenic Fungi Against the Beetle Brontispa longissima (Coleoptera: Hispidae): Isolation and Species Identification.

Seven entomopathogenic fungi strains (M1-7) were isolated from field-obtained dead coconut hispine beetles Brontispa longissima (Gestro), identified to species, and bioassayed for their pathogenicity. According to ITS sequences, all isolates belong in the genus Metarhizium, mainly M. flavoviride and M. anisopliae. Measured median lethal times (LT50) of 1×107 conidia/ml of M1-7 against fourth-instar B. longissima larvae within 15 d following exposure were, respectively: 5.43, 10.64, 11.26, 10.93, 6.62, 4.73, and 5.95 d. The isolate M6 yielded the highest mortality to fourth-instar larvae, and was thus selected to be tested against other larval instars and adults of B. longissima, after Time-Dose-Mortality (TDM) models. M6 proved more pathogenic against larvae than adults. The obtained bioassays data produced a good fit to the TDM models, yielding estimated LC50 and LT50 for each of the tested developmental stages of B. longissima. Both the obtained dose (ß) and time effect (ri) parameters from TDM models suggest that first-instar larvae are the most susceptible life stage of the pest insect, while adults are more resistant to M6 infection. Calculated LC50 values were, respectively, 1.23×103 and 1.15×106 conidia/ml for first-instar larvae and adults, on the 15th day following M6 inoculation. Estimated LT50 were 3.3 and 5.9 d for first-instar larvae and adults, respectively, at 1×108 conidia/ml. Taken together, these results would suggest Metarhizium M6 as an option for the biological control of B. longissima in the field.

Two Verticillium dahliae MAPKKKs, VdSsk2 and VdSte11, Have Distinct Roles in Pathogenicity, Microsclerotial Formation, and Stress Adaptation.

Verticillium dahliae causes destructive vascular wilt diseases on more than 200 plant species, including economically important crops and ornamental trees worldwide. The melanized microsclerotia enable the fungus to survive for years in soil and are crucial for its disease cycle. Previously, we found that the VdPbs2-VdHog1 (V. dahliae Pbs2-V. dahliae Hog1) module plays key roles in microsclerotial formation, stress responses, and virulence in V. dahliae In this study, two mitogen-activated protein kinase kinase kinases (MAPKKKs) homologous to Ssk2p and Ste11p, which activate the Pbs2p-Hog1p module by phosphorylation in budding yeast, were identified in the genome of V. dahliae Both ΔVdSsk2 (V. dahliaeSsk2) and ΔVdSte11 strains showed severe defects in microsclerotial formation and melanin biosynthesis, but the relative importance of these two genes in microsclerotial development was different. Deletion of VdSsk2, but not VdSte11, affected responses to osmotic stress, fungicidal response, and cell wall stressors. The ΔVdSsk2 strain exhibited a significant reduction in virulence, while the ΔVdSte11 strain was nonpathogenic due to failure to penetrate and form hyphopodia. Phosphorylation assays demonstrated that VdSsk2, but not VdSte11, can phosphorylate VdHog1 in V. dahliae Moreover, VdCrz1, encoding a calcineurin-responsive zinc finger transcription factor and a key regulator of calcium signaling in fungi, was misregulated in the ΔVdSsk2, ΔVdPbs2, and ΔVdHog1 mutants.IMPORTANCE These data provide insights into the distinctive functions of VdSsk2 and VdSte11 in pathogenicity, stress adaptation, and microsclerotial formation in V. dahliae.

The Transcription Factor VdHapX Controls Iron Homeostasis and Is Crucial for Virulence in the Vascular Pathogen Verticillium dahliae.

Iron homeostasis is essential for full virulence and viability in many pathogenic fungi. Here, we showed that the bZip transcription factor VdHapX functions as a key regulator of iron homeostasis for adaptation to iron-depleted and iron-excess conditions and is required for full virulence in the vascular wilt fungus, Verticillium dahliae Deletion of VdHapX impaired mycelial growth and conidiation under both iron starvation and iron sufficiency. Furthermore, disruption of VdHapX led to decreased formation of the long-lived survival structures of V. dahliae, known as microsclerotia. Expression of genes involved in iron utilization pathways and siderophore biosynthesis was misregulated in the ΔVdHapX strain under the iron-depleted condition. Additionally, the ΔVdHapX strain exhibited increased sensitivity to high iron concentrations and H2O2, indicating that VdHapX also contributes to iron or H2O2 detoxification. The ΔVdHapX strain showed a strong reduction in virulence on smoke tree seedlings (Cotinus coggygria) and was delayed in its ability to penetrate plant epidermal tissue.IMPORTANCE This study demonstrated that VdHapX is a conserved protein that mediates adaptation to iron starvation and excesses, affects microsclerotium formation, and is crucial for virulence of V. dahliae.

Genome-Wide Transcriptome Analysis Reveals the Comprehensive Response of Two Susceptible Poplar Sections to Marssonina brunnea Infection.

Marssonina leaf spot disease of poplar (MLDP), caused by the hemibiotrophic pathogen Marssonina brunnea, frequently results in damage to many poplar species. In nature, two formae speciales of M. brunnea exist that are susceptible to different poplar subgenera. Marssonina brunnea f. sp. monogermtubi infects poplar hosts from Populus sect. Aigeiros (Aig), while M. brunnea f. sp. multigermtubi always infects poplar hosts from Populus sect. Leuce Duby (Leu). Based on the fungal penetration structures, a comprehensive transcriptomic approach was used to investigate the gene expression patterns of these two poplar subgenera at three crucial infection stages. MLDP significantly altered the expression patterns of many genes involved in mitogen activated protein kinase (MAPKs) and calcium signaling, transcription factors, primary and secondary metabolism, and other processes in both poplar subgenera. However, major differences in gene expression were also observed between the two poplar subgenera. Aig was most responsive at the initial infection stage, while Leu largely interacted with M. brunnea at the necrotrophic phase. Furthermore, the differentially expressed genes (DEGs) involved in pathways related to biotic stress also differed substantially between the two poplar subgenera. Further analysis indicated that the genes involved in cell wall metabolism and phenylpropanoid metabolism were differentially expressed in the progression of the disease. By examining the expression patterns of genes related to the defense against disease, we found that several genes annotated with causing hypersensitive cell death were upregulated at the necrotrophic phase of MLDP, inferring that plant immune response potentially happened at this infection stage. The present research elucidated the potential molecular differences between the two susceptible interaction systems in MLDP and provided novel insight into the temporal regulation of genes during the susceptible response. To the best of our knowledge, this study also constitutes the first to reveal the molecular mechanisms of poplar in response to the transition of hemibiotrophic fungal pathogens from the biotrophic phase to the necrotrophic phase.

The C2H2 transcription factor VdMsn2 controls hyphal growth, microsclerotia formation, and virulence of Verticillium dahliae.

Verticillium dahliae is a notorious pathogen that causes vascular wilt disease in numerous plant species worldwide. The fungus produces melanized microsclerotia, which helps it survive adverse environmental conditions that it may encounter within its hosts and in the soil. Previously, we determined that the high osmolarity glycerol (HOG) pathway is involved in the environmental stress response of V. dahliae. In this study, we investigated the function of VdMsn2, a homologue of the yeast C2H2 transcription factor Msn2, which is predicted to function as a downstream player in the HOG pathway. Disruption of VdMsn2 has a discernible effect on hyphal growth and septation, but not on diverse stresses including hyperosmotic stresses and cell wall inhibitory agents. Furthermore, we show that VdMsn2 deletion mutants produce significantly more microsclerotia than the wild-type and exhibit attenuated virulence to smoke trees because of poor penetration. Taken together, our findings suggest that VdMsn2 controls hyphal growth, microsclerotia formation, and virulence but does not significantly contribute to stress responses in V. dahliae.

Functional characterization of two bZIP transcription factors in Verticillium dahliae.

bZIP transcription factors play various biological roles in stress responses, conidiation, and pathogenicity in pathogenic fungi. Here, we report two bZIP transcription factors (VDAG_08640 and VDAG_08676) of Verticillium dahliae, which were differentially expressed during microsclerotia development and induced by hydrogen peroxide as well. We find that deletion of either gene does not affect microsclerotia formation and the sensitivity to hydrogen peroxide; however, the mutants manifest decreased activity of extracellular peroxidase and laccase. Other phenotypic characterization reveals that VDAG_08676 disruption results in significant reduction of conidial production and virulence, while VDAG_08640 disruption does not lead to observable phenotypic variances compared with the wild-type strain. To elucidate whether they exhibit functional redundancy, double deletion mutants were generated. The double deletion mutants show remarkably increased sensitivity to hydrogen peroxide stress, whereas the two genes are not involved in microsclerotia formation. Taken together, our data demonstrate that a bZIP transcription factor gene VDAG_08676 is involved in the conidial production, oxidative stress response and virulence which may lay a foundation for further analysis of other bZIP transcription factors in V. dahliae.

The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection.

Verticillium dahliae, a ubiquitous phytopathogenic fungus, forms resting structures, known as microsclerotia that play crucial roles in Verticillium wilt diseases. VdHog1, a mitogen-activated protein kinase (MAPK), controls microsclerotia formation, virulence, and stress response in V. dahliae. In this study, we present detailed evidence that the conserved upstream component of VdHog1, VdPbs2, is a key regulator of microsclerotia formation, oxidative stress and fungicide response and plant virulence in V. dahliae. We identified VdPbs2, homologous to the yeast MAPK kinase Pbs2. Similar to the VdHog1 deletion mutant, VdPbs2 deletion strains exhibited delayed melanin synthesis and reduced formation of microsclerotia. When exposed to stresses, VdPbs2 mutants were more sensitive than the wild type to osmotic agents and peroxide, but more resistant to inhibitors of cell wall synthesis and some fungicides. Finally, VdPbs2 deletion mutants exhibited reduced virulence on smoke tree and tobacco seedlings. When taken together, we implicate that VdPbs2 and VdHog1 function in a cascade that regulates microsclerotia formation and virulence, but not all VdHog1 dependent functions are VdPbs2 regulated. This study thus provides novel insights into the signal transduction mechanisms that regulate microsclerotia formation and pathogenesis in this fungus.

MADS-Box Transcription Factor VdMcm1 Regulates Conidiation, Microsclerotia Formation, Pathogenicity, and Secondary Metabolism of Verticillium dahliae.

Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species resulting in devastating yield losses worldwide. Due to its ability to colonize plant xylem and form microsclerotia, V. dahliae is highly persistent and difficult to control. In this study, we show that the MADS-box transcription factor VdMcm1 is a key regulator of conidiation, microsclerotia formation, virulence, and secondary metabolism of V. dahliae. In addition, our findings suggest that VdMcm1 is involved in cell wall integrity. Finally, comparative RNA-Seq analysis reveals 823 significantly downregulated genes in the VdMcm1 deletion mutant, with diverse biological functions in transcriptional regulation, plant infection, cell adhesion, secondary metabolism, transmembrane transport activity, and cell secretion. When taken together, these data suggest that VdMcm1 performs pleiotropic functions in V. dahliae.

The mitogen-activated protein kinase gene, VdHog1, regulates osmotic stress response, microsclerotia formation and virulence in Verticillium dahliae.

The fungus Verticillium dahliae has gained worldwide notoriety as a destructive plant pathogen, causing vascular wilt diseases on diverse plant species. V. dahliae produces melanized resting bodies, known as microsclerotia, which can survive for 15 years in the soil, and are thus critically important in its disease cycle. However, the molecular mechanisms that underpin microsclerotia formation, survival, and germination remain poorly understood. In this study, we observed that deletion of VdHog1 (ΔVdHog1), encoding a homolog of a high-osmolarity glycerol (HOG) response mitogen-activated protein kinase, displayed decreased numbers of melanized microsclerotia in culture, heightened sensitivity to hyperosmotic stress, and increased resistance to the fungicide fludioxonil. Through RNA-Seq analysis, we identified 221 genes differentially expressed in the ΔVdHog1 strain. Interestingly, the expression levels of genes involved in melanin biosynthesis, as well as the hydrophobin gene VDH1, involved in the early stage of microsclerotia formation, were significantly decreased in the ΔVdHog1 strains relative to the wild-type expression levels. The ΔVdHog1 strains exhibited decreased virulence relative to the wild type strain on smoke tree seedlings. These results indicate that VdHog1 regulates hyperosmotic stress responses in V. dahliae, and establishes the Hog1-mediated pathway as a target to further probe the up- and downstream processes that regulate asexual development in this fungus.