Host-Specific and Homologous Pairs of Melampsora larici-populina Effectors Unveil Novel Nicotiana benthamiana Stromule Induction Factors.

The poplar rust fungus Melampsora larici-populina is part of one of the most devastating group of fungi (Pucciniales) and causes important economic losses to the poplar industry. Because M. larici-populina is a heteroecious obligate biotroph, its spread depends on its ability to carry out its reproductive cycle through larch and then poplar parasitism. Genomic approaches have identified more than 1,000 candidate secreted effector proteins (CSEPs) from the predicted secretome of M. larici-populina that are potentially implicated in the infection process. In this study, we selected CSEP pairs (and one triplet) among CSEP gene families that share high sequence homology but display specific gene expression profiles among the two distinct hosts. We determined their subcellular localization by confocal microscopy through expression in the heterologous plant system Nicotiana benthamiana. Five out of nine showed partial or complete chloroplastic localization. We also screened for potential protein interactors from larch and poplar by yeast two-hybrid assays. One pair of CSEPs and the triplet shared common interactors, whereas the members of the two other pairs did not have common targets from either host. Finally, stromule induction quantification revealed that two pairs and the triplet of CSEPs induced stromules when transiently expressed in N. benthamiana. The use of N. benthamiana eds1 and nrg1 knockout lines showed that CSEPs can induce stromules through an eds1-independent mechanism. However, CSEP homologs shared the same impact on stromule induction and contributed to discovering a new stromule induction cascade that can be partially and/or fully independent of eds1. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Integrative omics studies revealed synergistic link between sucrose metabolic isogenes and carbohydrates in poplar roots infected by Fusarium wilt.

Advances in carbohydrate metabolism prompted its essential role in defense priming and sweet immunity during plant-pathogen interactions. Nevertheless, upstream responding enzymes in the sucrose metabolic pathway and associated carbohydrate derivatives underlying fungal pathogen challenges remain to be deciphered in Populus, a model tree species. In silico deduction of genomic features, including phylogenies, exon/intron distributions, cis-regulatory elements, and chromosomal localization, identified 59 enzyme genes (11 families) in the Populus genome. Spatiotemporal expression of the transcriptome and the quantitative real-time PCR revealed a minuscule number of isogenes that were predominantly expressed in roots. Upon the pathogenic Fusarium solani (Fs) exposure, dynamic changes in the transcriptomics atlas and experimental evaluation verified Susy (PtSusy2 and 3), CWI (PtCWI3), VI (PtVI2), HK (PtHK6), FK (PtFK6), and UGPase (PtUGP2) families, displaying promotions in their expressions at 48 and 72 h of post-inoculation (hpi). Using the gas chromatography-mass spectrometry (GC-MS)-based non-targeted metabolomics combined with a high-performance ion chromatography system (HPICS), approximately 307 metabolites (13 categories) were annotated that led to the quantification of 46 carbohydrates, showing marked changes between three compared groups. By contrast, some sugars (e.g., sorbitol, L-arabitol, trehalose, and galacturonic acid) exhibited a higher accumulation at 72 hpi than 0 hpi, while levels of α-lactose and glucose decreased, facilitating them as potential signaling molecules. The systematic overview of multi-omics approaches to dissect the effects of Fs infection provides theoretical cues for understanding defense immunity depending on fine-tuned Suc metabolic gene clusters and synergistically linked carbohydrate pools in trees.

Genome-wide analysis of R2R3-MYB transcription factors in poplar and functional validation of PagMYB147 in defense against Melampsora magnusiana.

MAIN CONCLUSION: Transcription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease. Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway.

The antagonistic activity of Streptomyces spiroverticillatus (No. HS1) against of poplar canker pathogen Botryosphaeria dothidea.

BACKGROUND: Poplar canker caused by Botryosphaeria dothidea is one of the most severe plant disease of poplars worldwide. In our study, we aimed to investigate the modes of antagonism by fermentation broth supernatant (FBS) of Streptomyces spiroverticillatus HS1 against B. dothidea. RESULTS: In vitro, the strain and FBS of S. spiroverticillatus HS1 significantly inhibited mycelial growth and biomass accumulation, and also disrupted the mycelium morphology of B. dothidea. On the 3rd day after treatment, the inhibition rates of colony growth and dry weight were 80.72% and 52.53%, respectively. In addition, FBS treatment damaged the plasma membrane of B. dothidea based on increased electrical conductivity in the culture medium, and malondialdehyde content of B. dothidea mycelia. Notably, the analysis of key enzymes in glycolysis pathway showed that the activity of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), Ca2+Mg2+-ATPase were significantly increased after FBS treatment. But the glucose contents were significantly reduced, and pyruvate contents were significantly increased in B. dothidea after treatment with FBS. CONCLUSIONS: The inhibitory mechanism of S. spiroverticillatus HS1 against B. dothidea was a complex process, which was associated with multiple levels of mycelial growth, cell membrane structure, material and energy metabolism. The FBS of S. spiroverticillatus HS1 could provide an alternative approach to biological control strategies against B. dothidea.

The transcriptional landscape of Populus pattern/effector-triggered immunity and how PagWRKY18 involved in it.

Plants trigger a robust immune response by activating massive transcriptome reprogramming through crosstalk between PTI and ETI. However, how PTI and ETI contribute to the quantitative or/and qualitative output of immunity and how they work together when both are being activated were unclear. In this study, we performed a comprehensive overview of pathogen-triggered transcriptomic reprogramming by analyzing temporal changes in the transcriptome up to 144 h after Colletotrichum gloeosporioides inoculated in Populus. Moreover, we constructed a hierarchical gene regulatory network of PagWRKY18 and its potential target genes to explore the underlying regulatory mechanisms of PagWRKY18 that are not yet clear. Interestingly, we confirmed that PagWRKY18 protein can directly bind the W-box elements in the promoter of a transmembrane leucine-rich repeat receptor-like kinase, PagSOBIR1 gene, to trigger PTI. At the same time, PagWRKY18 functions in disease tolerance by modulation of ROS homeostasis and induction of cell death via directly targeting PagGSTU7 and PagPR4 respectively. Furthermore, PagPR4 can interact with PagWRKY18 to inhibit the expression of PagPR4 genes, forming a negative feedback loop. Taken together, these results suggest that PagWRKY18 may be involved in regulating crosstalk between PTI and ETI to activate a robust immune response and maintain intracellular homeostasis.

MiRNAs profiles among three poplar varieties provide insights into different molecular responses in resistance to newly emerging bacterial pathogen.

Canker caused by Lonsdalea populi has seriously reduced the economic and ecological benefits of poplar. MicroRNAs play vital roles in the response of plants to biotic stress. However, there is little research about the regulatory mechanism of miRNAs among different tree varieties upon pathogen infection. To dissect miRNAs involved in L. populi resistance, three poplar varieties, 2025 (susceptible), 107 (moderately resistant) and Populus. tomentosa cv 'henan' (resistant) were selected to elucidate the expression profiles of miRNAs using small RNA-seq. A total of 227 miRNAs were identified from all varieties. Intriguingly, miR160, miR169, miR171 and miR482b-5p were only identified in the resistant variety P. tomentosa upon pathogen infection, and these miRNAs might be important candidates for future investigation to improve the tolerance of poplar to L. populi. Among all identified miRNAs, 174 were differentially expressed in all varieties. Functional annotation analysis indicated that an array of miRNAs, including miR482, miR472, miR169, miR481, and miR172, should be involved in disease resistance and phytohormone signal transduction. Furthermore, correlation analysis of small RNA-seq and RNA-seq identified a handful of L. populi-responsive miRNAs and target genes, which exhibited that miR159 and miR172 played key roles in resistant variety P. tomentosa by targeting MYB and ERF, while miR6462c-5p and miR828 were related to the susceptibility of 2025 by targeting MYB. The comprehensive integration analysis in this research provides new insights into the regulatory pathways involved in the defence response of poplar to L. populi and offers crucial candidate miRNAs-target genes modules for poplar resistance improvement.

Biocontrol mechanisms of poplar leaf blight due to Nigrospora oryzae.

Nigrospora oryzae, a newly identified pathogen, is responsible for poplar leaf blight, causing significant harm to poplar growth. Here, we describe, for the first time, a biological control method for the control of poplar leaf blight via the applications of 3 dominant Trichoderma strains/species. In this study, dominant Trichoderma species/strains with the potential for biocontrol were identified and then further characterised via dual culture assays, volatile organic compounds (VOCs), and culture filtrates. The biocontrol efficacy of these strains against N. oryzae was found to exceed 60%. Furthermore, the reactive oxygen species (ROS) content in Populus davidiana × P. alba var. pyramidalis (PdPap) leaves pretreated with these Trichoderma strains significantly decreased. Furthermore, pretreatment of PdPap with a combination of these Trichoderma (Tcom) resulted in 9.71-fold and 1.95-fold increases in peroxidase (POD) and superoxide dismutase (SOD) activity, respectively, and 3.87-fold decrease in the MDA content compared to controls. Moreover, Tcom pretreatment activated the salicylic acid (SA) and jasmonic acid (JA) pathway-dependent defence responses of poplar, upregulating pathogenesis-related protein (PR) and MYC proto-oncogene (MYC-R) by more than 12-fold and 17.32-fold, respectively. In addition, Trichoderma treatments significantly increased the number of lateral roots, aboveground biomass, and stomata number and density of PdPap, and Tcom was superior to the single pretreatments. The soil pH also became weakly acidic in these pretreatments, which is beneficial for the growth of PdPap seedlings. These findings indicate that these dominant Trichoderma strains can effectively increase biocontrol and poplar growth promotion.

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.

Variations in soil microbial communities in different saline soils under typical Populus spp. vegetation in alpine region of the Qaidam Basin, NW China.

Salinization is a severe threat to agriculture and the environment in many areas, and the same in Qaidam Basin, Qinghai Province, Northwestern China. Microorganisms have an important influence on regulating the biochemical cycles of ecosystems; however, systematic research exploring microbial diversity and interactions with saline-soil ecosystems' environmental variables remains scarce. Thus, 16 S rRNA high-throughput sequencing was performed in this paper to characterize microbial diversity under different levels of salinized soils: non-salinized (NS, 2.25 g/L), moderately salinized (MS, 6.14 g/L) and highly salinized (HS, 9.82 g/L). The alpha diversity results showed that the HS soil was significantly different from the NS and MS soils. An analysis of similarity (ANOSIM) and a principal co-ordinates analysis (PCoA) indicated that NS and MS clustered closely while HS separated from the other two. Significant differences in microbial composition were observed at the taxonomic level. Proteobacteria (42.29-79.23 %) were the most abundant phyla in the studied soils. Gammaproteobacteria (52.49 and 66.61 %) had higher abundance in the MS and HS soils at the class level; Methylophaga and Pseudomonas were the predominant bacteria in the HS soil; and Azotobacter and Methylobacillus were abundant in the MS soil. Most genera belonging to Proteobacteria and Actinobacteria were detected via a linear discriminate analysis (LDA) effect size (LEfSe) analysis, which indicated that microbes with the ability to degrade organic matter and accomplish nutrient cycling can be well-adapted to salt conditions. Further analyses (redundancy analysis and Mantel test) showed that the microbial communities were mainly related to the soil salinity, electrical conductivity (EC1:5), total phosphorus (TP) and ammonia nitrogen (NH4+-N). Overall, the findings of the study can provide insights for better understanding the dominant indigenous microbes and their roles in biochemical cycles in different saline soils in the Qaidam Basin, Qinghai Province, China. The researches related to microbial community under typical poplar species should further clarify the mechanism of plant-microbial interaction and benefit for microbial utilization in salt soil remediation.

Combined effects of cadmium and simulated acid rain on soil microbial communities in the early cultivation of Populus beijingensis seedlings.

The combined cadmium (Cd) and acid rain pollution poses a significant threat to the global ecological environment. Previous studies on the combined adverse effects have predominantly focused on the aboveground plant physiological responses, with limited reports on the microbial response in the rhizosphere soil. This study employed Populus beijingensis seedlings and potting experiments to simulate the impacts of combined mild acid rain (pH=4.5, MA) or highly strong acid rain (pH=3.0, HA), and soil Cd pollution on the composition and diversity of microbial communities, as well as the physiochemical properties in the rhizosphere soil. The results showed that Cd decreased the content of inorganic nitrogen, resulting in an overall decrease of 49.10 % and 46.67 % in ammonium nitrogen and nitrate nitrogen, respectively. Conversely, acid rain was found to elevate the content of total potassium and soil organic carbon by 4.68 %-6.18 % and 8.64-19.16 %, respectively. Additionally, simulated acid rain was observed to decrease the pH level by 0.29-0.35, while Cd increased the pH level by 0.11. Moreover, Cd alone reduced the rhizosphere bacterial diversity, however, when combined with acid rain, regardless of its intensity, Cd was observed to increase the diversity. Fungal diversity was not influenced by the acid rain, but Cd increased fungal diversity to some extend under HA as observed in bacterial diversity. In addition, composition of the rhizosphere bacterial community was primarily influenced by the inorganic nitrogen components, while the fungal community was driven mainly by soil pH. Furthermore, "Metabolism" was emerged as the most significant bacterial function, which was markedly affected by the combined pollution, while Cd pollution led to a shift from symbiotroph to other trophic types for fungi. These findings suggest that simulated acid rain has a mitigating effect on the diversity of rhizosphere bacteria affected by Cd pollution, and also alters the trophic type of these microorganisms. This can be attributed to the acid rain-induced direct acidic environment, as well as the indirect changes in the availability or sources of carbon, nitrogen, or potassium.

Secreted Effector Proteins of Poplar Leaf Spot and Stem Canker Pathogen Sphaerulina musiva Manipulate Plant Immunity and Contribute to Virulence in Diverse Ways.

Fungal effectors play critical roles in manipulating plant immune responses and promoting colonization. Sphaerulina musiva is a heterothallic ascomycete fungus that causes Septoria leaf spot and stem canker disease in poplar (Populus spp.) plantations. This disease can result in premature defoliation, branch and stem breakage, increased mortality, and plantation failure. However, little is known about the interaction between S. musiva and poplar. Previous work predicted 142 candidate secreted effector proteins in S. musiva (SmCSEPs), 19 of which were selected for further functional characterization in this study. SmCSEP3 induced plant cell death in Nicotiana benthamiana, while 8 out of 19 tested SmCSEPs suppressed cell death. The signal peptides of these eight SmCSEPs exhibited secretory activity in a yeast signal sequence trap assay. Confocal microscopy revealed that four of these eight SmCSEPs target both the cytoplasm and the nucleus, whereas four predominantly localize to discrete punctate structures. Pathogen challenge assays in N. benthamiana demonstrated that the transient expression of six SmCSEPs promoted Fusarium proliferatum infection. The expression of these six SmCSEP genes were induced during infection. SmCSEP2, SmCSEP13, and SmCSEP25 suppressed chitin-triggered reactive oxygen species burst and callose deposition in N. benthamiana. The candidate secreted effector proteins of S. musiva target multiple compartments in the plant cell and modulate different pattern-triggered immunity pathways. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Elevated O3 concentrations alter the compartment-specific microbial communities inhabiting rust-infected poplars.

Elevated ozone (O3 ) can affect the susceptivity of plants to rust pathogens. However, the collective role of microbiomes involved in such interaction remains largely elusive. We exposed two cultivated poplar clones exhibiting differential O3 sensitivities, to non-filtered ambient air (NF), NF + 40 ppb or NF + 60 ppb O3 -enriched air in field open-top chambers and then inoculated Melampsora larici-populina urediniospores to study their response to rust infection and to investigate how microbiomes inhabiting four compartments (phyllosphere, rhizosphere, root endosphere, bulk soil) are involved in this response. We found that hosts with higher O3 sensitivity had significantly lower rust severity than hosts with lower sensitivity. Furthermore, the effect of increased O3 on the diversity and composition of microbial communities was highly dependent on poplar compartments, with the microbial network complexity patterns being completely opposite between the two clones. Notably, microbial source analysis estimated that phyllosphere fungal communities predominately derived from root endosphere and vice versa, suggesting a potential transmission mechanism between plant above- and below-ground systems. These promising results suggest that further investigations are needed to better understand the interactions of abiotic and biotic stresses on plant performance and the role of the microbiome in driving these changes.

The Transcription Factor CcRlm1 Regulates Cell Wall Maintenance and Poplar Defense Response by Directly Targeting CcChs6 and CcGna1 in Cytospora chrysosperma.

Maintenance of cell wall integrity is important for fungal cell morphology against external stresses and even virulence. Although the transcription factor Rlm1 is known to play major regulatory roles in the maintenance of cell integrity, the underlying mechanism of how Rlm1 contributes to cell wall integrity and virulence in phytopathogenic fungi remains unclear. Here, we demonstrated that CcRlm1 plays important roles in cell wall maintenance and virulence in the poplar canker fungus Cytospora chrysosperma. Among putative downstream targets, CcChs6 (chitin synthase) and CcGna1 (glucosamine 6-phosphate N-acetyltransferase) were found to be direct targets of CcRlm1 and shown to function in chitin synthesis and virulence. Furthermore, we found stronger induction of poplar defense responses when challenged with these gene deletion mutants. Collectively, these results suggest that CcRlm1 plays a critical role in the regulation of cell wall maintenance, stress response, and virulence by directly regulating CcChs6 and CcGna1 in C. chrysosperma. IMPORTANCE Cytospora chrysosperma causes canker diseases on woody plants, and the molecular basis of its infection is not well understood. This study shows that CcRlm1 is the major regulator of chitin synthesis and virulence of the poplar canker fungus. Our research contributes to further understanding the molecular basis of the interaction between C. chrysosperma and poplar.

Isolation and characterization of phosphate-solubilizing bacteria from rhizosphere of poplar on road verge and their antagonistic potential against various phytopathogens.

BACKGROUND: Phosphate-solubilizing bacteria (PSB) can solubilize insoluble phosphate compounds and improve phosphate availability in soil. Road verges are important in urban landscaping, but the population structure of PSB and their ecological functions in the road verge soil is still unclear. RESULTS: Twenty-one mineral PSB strains and 14 organic PSB strains were isolated from the rhizosphere of poplar on urban road verge. All the mineral PSB strains showed better solubilization to Ca3(PO4)2 than FePO4 or AlPO4. Among them, 7 strains showed high phosphate-solubilizing (PS) activities to Ca3(PO4)2 (150-453 mg/L). All the organic PSB strains displayed weak solubilization to lecithin. 16S rRNA gene-based phylogenetic analysis showed good species diversity of the PSB strains, which belongs to 12 genera: Bacillus, Cedecea, Cellulosimicrobium, Delftia, Ensifer, Paenibacillus, Pantoea, Phyllobacterium, Pseudomonas, Rhizobium, Sinorhizobium and Staphylococcus. Moreover, 8 PSB strains showed various degrees of growth inhibition against 4 plant pathogenic fungi, Fusarium oxysporum S1, F. oxysporum S2, Pythium deliense Meurs Z4, Phomopsis sp. AC1 and a plant pathogenic bacterium, Pectobacterium carotovorum TP1. CONCLUSIONS: The results indicated that these PSB strains could perform multiple ecological functions on road verge. The development and application of bio-agents based on the strains would provide a new strategy for maintaining and improving the ecosystem stability of road verges.

Roseomonas populi sp. nov., an acetate-degrading bacteria isolated from the stem of Populus tomentosa.

Strain CN29T, isolated from the stem of 5- to 6-year-old Populus tomentosa in Shandong, China, was characterized using a polyphasic taxonomic approach. Cells of CN29T were Gram-stain negative, aerobic, nonspore-forming, and nonmotile coccoid. Growth occurred at 20-37 °C, pH 4.0-9.0 (optimum, pH 6.0), and with 0-1% NaCl (optimum, 1%). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain CN29T was closely related to members of the genus Roseomonas and closest to Roseomonas pecuniae N75T (96.6%). This classification was further supported by phylogenetic analysis using additional core genes. The average nucleotide identity and digital DNA‒DNA hybridization values between strain CN29T and Roseomonas populi CN29T were 82.7% and 27.8%, respectively. The genome size of strain CN29T was 5.87 Mb, with a G + C content of 70.9%. The major cellular fatty acids included summed feature 8 (C18:1 ω7c/C18:1 ω6c), C19:0 cyclo ω8c and C16:0. The major respiratory quinone was Q-10. The polar lipids were phosphatidylcholine, aminolipid, phosphatidylglycerol, and diphosphatidylglycerol. Strain CN29T can utilize acetate as a carbon source for growth and metabolism. Additionally, it contains acid phosphatase (2-naphthyl phosphate), which catalyzes the hydrolysis of phosphoric monoesters. The CN29T strain contains several genes, including maeB, gdhB, and cysJ, involved in carbon, nitrogen, and sulfur cycling. These findings suggest that the strain may actively participate in ecosystem cycling, leading to soil improvement and promoting the growth of poplar trees. Based on the phylogenetic, phenotypic, and genotypic characteristics, strain CN29T is concluded to represent a novel species of the genus Roseomonas, for which the name Roseomonas populi sp. nov. is proposed. The type strain is CN29T (= JCM 35579T = GDMCC 1.3267T).

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.

Annotation survey and life cycle transcriptomics of transcription factors in rust fungi (Pucciniales) identify a possible role for cold shock proteins in dormancy exit.

Fungi of the order Pucciniales are obligate plant biotrophs causing rust diseases. They exhibit a complex life cycle with the production of up to five spore types, infection of two unrelated hosts and an overwintering stage. Transcription factors (TFs) are key regulators of gene expression in eukaryote cells. In order to better understand genetic programs expressed during major transitions of the rust life cycle, we surveyed the complement of TFs in fungal genomes with an emphasis on Pucciniales. We found that despite their large gene numbers, rust genomes have a reduced repertoire of TFs compared to other fungi. The proportions of C2H2 and Zinc cluster - two of the most represented TF families in fungi - indicate differences in their evolutionary relationships in Pucciniales and other fungal taxa. The regulatory gene family encoding cold shock protein (CSP) showed a striking expansion in Pucciniomycotina with specific duplications in the order Pucciniales. The survey of expression profiles collected by transcriptomics along the life cycle of the poplar rust fungus revealed TF genes related to major biological transitions, e.g. response to environmental cues and host infection. Particularly, poplar rust CSPs were strongly expressed in basidia produced after the overwintering stage suggesting a possible role in dormancy exit. Expression during transition from dormant telia to basidia confirmed the specific expression of the three poplar rust CSP genes. Their heterologous expression in yeast improved cell growth after cold stress exposure, suggesting a probable regulatory function when the poplar rust fungus exits dormancy. This study addresses for the first time TF and regulatory genes involved in developmental transition in the rust life cycle opening perspectives to further explore molecular regulation in the biology of the Pucciniales.

Culturable bacteria diversity in stem liquid and resina from Populus euphratica and screening of plant growth-promoting bacteria.

BACKGROUND: Populus euphratica Olivier is a kind of tree capable of growing in extremely arid desert and semi-desert environments. In this study, a culture-dependent method was used to analyze the bacterial diversity of stem liquid of P. euphratica and resina of P. euphratica, and to further evaluate plant growth promoting (PGP) activity. RESULTS: A total of 434 bacteria were isolated from stem fluid and resina of P. euphratica in Ebinur Lake Wetland Nature Reserve and Mulei Primitive forest. The results of taxonomic composition analysis shows that Gammaproteobacteria, Firmicutes, and Actinobacteria_c are the three dominant groups in all the communities, and the representative genera are Bacillus, Nesterenkonia and Halomonas. The diversity analysis shows that the culturable bacterial community diversity of P. euphratica in Ebinur Lake Wetland Nature Reserve is higher than that in Mulei Primitive forest, and the bacterial community diversity of P. euphratica stem fluid is higher than that of resina. According to PGP activity evaluation, 158 functional bacteria with plant growth promoting potential were screened. Among them, 61 strains havephosphorus solubilizing abilities, 80 strains have potassium solubilizing abilities, 32 strains have nitrogen fixation abilities, and 151 strains have iron ammonia salt utilization abilities. The germination rate, plant height, and dry weight of the maize seedlings treated with strains BB33-1, TC10 and RC6 are significantly higher than those of the control group. CONCLUSION: In this study, a large number of culturable bacteria were isolated from P. euphratica, which provides new functional bacteria sources for promoting plant growth.

Ontogenetic Variation in Macrocyclic and Hemicyclic Poplar Rust Fungi.

Melampsora larici-populina (Mlp), M. medusae (Mmed), M. magnusiana (Mmag), and M. pruinosae (Mpr) are epidemic rust fungi in China. The first two are macrocyclic rust fungi distributed in temperate humid environments. The latter two are hemicyclic rusts, mainly distributed in arid and semi-arid areas. Ontogenetic variation that comes with this arid-resistance is of great interest-and may help us predict the influence of a warmer, drier, climate on fungal phylogeny. To compare the differences in the life history and ontogeny between the two types of rust, we cloned mating type genes, STE3.4 and STE3.3 using RACE-smart technology. Protein structures, functions, and mutant loci were compared across each species. We also used microscopy to compare visible cytological differences at each life stage for the fungal species, looking for variation in structure and developmental timing. Quantitative PCR technology was used to check the expression of nuclear fusion and division genes downstream of STE3.3 and STE3.4. Encoding amino acids of STE3.3 and STE3.4 in hemicyclic rusts are shorter than these in the macrocyclic rusts. Both STE3.3 and STE3.4 interact with a protein kinase superfamily member EGG12818 and an E3 ubiquitin protein ligase EGG09709 directly, and activating G-beta conformational changes. The mutation at site 74th amino acid in the conserved transmembrane domain of STE3.3 ascribes to a positive selection, in which alanine (Ala) is changed to phenylalanine (Phe) in hemicyclic rusts, and a mutation with Tyr lost at site 387th in STE3.4, where it is the binding site for ß-D-Glucan. These mutants are speculated corresponding to the insensitivity of hemicyclic rust pheromone receptors to interact with MFa pheromones, and lead to Mnd1 unexpressed in teliospora, and they result in the diploid nuclei division failure and the sexual stage missing in the life cycle. A Phylogenic tree based on STE3.4 gene suggests these two rust types diverged about 14.36 million years ago. Although these rusts share a similar uredia and telia stage, they show markedly different wintering strategies. Hemicyclic rusts overwinter in the poplar buds endophytically, their urediniospores developing thicker cell walls. They form haustoria with a collar-like extrahaustorial membrane neck and induce host thickened callose cell walls, all ontogenetic adaptations to arid environments.

Jasmonic Acid- and Ethylene-Induced Mitochondrial Alternative Oxidase Stimulates Marssonina brunnea Defense in Poplar.

Mitochondrial processes are implicated in plant response to biotic stress caused by viruses, actinomyces, bacteria and pests, but their function in defense against fungal invasion remains unclear. Here, we investigated the role and regulation of mitochondrial alternative oxidase (AOX) in response to black spot disease caused by the hemibiotrophic fungus Marssonina brunnea in poplar. M. brunnea inoculation induced the transcription of the AOX1a gene in the mitochondrial electron transport chain and of jasmonic acid (JA) and ethylene (ET) biosynthetic genes, with the accumulation of these phytohormones in poplar leaf, while inhibiting the transcript amount of the mitochondrial cytochrome c oxidase gene (COX6b) and genes related to salicylic acid (SA). Enhanced AOX reduced poplar susceptibility to M. brunnea with a higher ATP/ADP ratio while the repressed AOX caused the reverse effect. Exogenous JA and 1-aminocyclopropane-1-carboxylic acid (ACC, a biosynthetic precursor of ET) inhibited the transcript amount of COX6b and consequently increased the ratio of AOX pathway to total respiration. Furthermore, the transcription of CYS C1 and CYS D1 genes catalyzing cyanide metabolism was induced, while the cysteine (CYS) substrate levels reduced upon M. brunnea inoculation; exogenous JA and ACC mimicked the effect of M. brunnea infection on cysteine. Exogenous SA enhanced, while JA and ACC reduced, poplar susceptibility to M. brunnea. Moreover, inhibiting AOX completely prohibited JA- and ET-increased tolerance to M. brunnea in poplar. These observations indicate that the JA- and ET-induced mitochondrial AOX pathway triggers defense against M. brunnea in poplar. This effect probably involves cyanide. These findings deepen our understanding of plant-pathogenic fungi interactions.