in-vivo-leaf-inoculation-an-alternative-method-to-assess-disease

In Vivo Leaf Inoculation: An Alternative Method to Assess the Disease Resistance of Hybrid Clones in Poplar Breeding of Stem Canker Disease

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. &#38; de Not. are the two ...

poplar-adventitious-roots-induced-stem-canker-pathogens-an

Poplar Adventitious Roots Induced by Stem Canker Pathogens: An Experimental System for Studying Roots Biology and Light Response-Related Processes

Valsa sordida and Botryosphaeria dothidea are two crucial necrotrophic fungal pathogens that damage many plant hosts, particularly species in the genus ...

inducing-entry-third-stage-dispersal-juveniles-bursaphelenchus

Inducing the Entry of Third Stage Dispersal Juveniles of  <em>Bursaphelenchus xylophilus</em> into Cryptobiosis Through Osmotic Regulation

Cryptobiosis is a state where organisms lose nearly all their internal water and enter anhydrobiosis under extreme environmental stress. The dispersal ...

chinese academy of forestry

Comprehensive Experimental Center in Yellow River Delta of Chinese Academy of Forestry

The microRNAs (miRNAs) miR482 and miR1448 are disease resistance-related miRNAs; the former is ubiquitously distributed in seed plants whereas the latter has only been reported in Populus trichocarpa. The precursor and mature sequences of poplar miR1448 are highly homologous to those of poplar miR482, and these two miRNAs are located in one transcript as a polycistron. Therefore, we hypothesized that the MIR1448 gene may have evolved from the MIR482 gene in poplar. However, the molecular evolution patterns of this process remain unclear. In this study, utilizing cloning and Blast analysis in NCBI ESTs and whole-genome shotgun contigs (WGS) dataset, we determined that the MIR482-MIR1448 polycistron is a family-specific clustered miRNA in Salicaceae. Moreover, phylogenetic analysis illustrated that MIR1448 is the product of a tandem duplication event from MIR482. Nucleotide substitution analysis revealed that both MIR482 and MIR1448 have more rapid evolution ratios than ribosomal DNA (rDNA) genes, and that compensatory mutations that occurred in the stem region of the secondary structure were the main mechanisms that drove the evolution of these MIRNA genes. Furthermore, by comparing the substitution patterns in the miRNA-target complexes of miR482 and miR1448, we inferred that co-evolution between miRNAs and their targets was the major force that drove the "duplicated MIR482" evolve to MIR1448. We propose a novel miRNA-target pairing pattern called the "frameshift targeted mechanism" to explain the gain of target genes by miR1448. The results also imply that the major role of miR482 was in resistance to disease or other stresses via NBS-LRR proteins, whereas the biological functions of miR1448 are more diverse.

Phylogenetic analysis and molecular evolution patterns in the MIR482-MIR1448 polycistron of Populus L.

A number of transcriptome datasets for differential expression (DE) genes have been widely used for understanding organismal biology, but these datasets also contain untapped information that can be used to develop more precise analytical tools. With the use of transcriptome data generated from poplar/canker disease interaction system, we describe a methodology to identify candidate reference genes from high-throughput sequencing data. This methodology will improve the accuracy of RT-qPCR and will lead to better standards for the normalization of expression data. Expression stability analysis from xylem and phloem of  inoculated with the fungal canker pathogen  revealed that 729 poplar transcripts (1.11%) were stably expressed, at a threshold level of coefficient of variance (CV) of FPKM < 20% and maximum fold change (MFC) of FPKM < 2.0. Expression stability and bioinformatics analysis suggested that commonly used house-keeping (HK) genes were not the most appropriate internal controls: 70 of the 72 commonly used HK genes were not stably expressed, 45 of the 72 produced multiple isoform transcripts, and some of their reported primers produced unspecific amplicons in PCR amplification. RT-qPCR analysis to compare and evaluate the expression stability of 10 commonly used poplar HK genes and 20 of the 729 newly-identified stably expressed transcripts showed that some of the newly-identified genes (such as SSU_S8e, LSU_L5e, and 20S_PSU) had higher stability ranking than most of commonly used HK genes. Based on these results, we recommend a pipeline for deriving reference genes from transcriptome data. An appropriate candidate gene should have a unique transcript, constitutive expression, CV value of expression < 20% (or possibly 30%) and MFC value of expression <2, and an expression level of 50-1,000 units. Lastly, when four of the newly identified HK genes were used in the normalization of expression data for 20 differential expressed genes, expression analysis gave similar values to Cufflinks output. The methods described here provide an alternative pathway for the normalization of transcriptome data, a process that is essential for integrating analyses of transcriptome data across environments, laboratories, sequencing platforms, and species.

Genome-Wide Constitutively Expressed Gene Analysis and New Reference Gene Selection Based on Transcriptome Data: A Case Study from Poplar/Canker Disease Interaction.

Canker pathogens cause necrosis of the phloem, but in many host/pathogen systems, they also cause canopy dieback, which implicates xylem, not phloem dysfunction. We hypothesize that this dieback distal to the canker is caused by water stress resulting from the lack of a phloem-to-xylem connection, which in a healthy plant would allow delivery of nonstructural carbohydrates (NSCs) and water inward to aid in xylem embolism refilling. We tested several components of this hypothesis in the host/pathogen system Corylus avellana L./Anisogramma anomala (Peck) E. Müll (Eastern filbert blight). Cankers were non-girdling and usually ≥0.1 m long. As expected, healthy controls had higher specific conductivity (Ks) than diseased stems, but unexpectedly, had similar moisture content (m.c.), showing that the lower Ks did not result from more embolisms in the diseased stems. Moreover, manipulations that removed cambium and phloem to simulate a canker, or that shaded stems to lower NSCs, did not result in lower Ks or m.c. than controls. The outer millimeter of xylem adjacent to a canker had infrequent tyloses and/or fungal hyphae in many but not all samples, and dye studies showed little xylem water transport in that region, but the incidence of these blockages was insufficient to cause the observed 19% decrease in Ks. Healthy stems had higher m.c. than diseased stems above the canker (or analogous) location and were longer for the same leaf weight, suggestive of water stress in the upper portion of diseased stems. These results suggest that dieback distal to cankers in this system results from the bottleneck in water transport in the region adjacent to a canker, but did not find evidence to support the requirement of a phloem-to-xylem connection for continued water transport.

Effects of phloem on canopy dieback, tested with manipulations and a canker pathogen in the Corylus avellana/Anisogramma anomala host/pathogen system.

Carbon starvation is the current leading hypothesis of plant mortality mechanisms under drought stress; recently, it is also used to explain tree die-off in plant diseases. However, the molecular biology of the carbon starvation pathway is unclear. Here, using a punch inoculation system, we conducted transcriptome and physiological assays to investigate pathogen response in poplar stems at the early stages of Botryosphaeria and Valsa canker diseases. Transcriptome assays showed that the majority of differentially expressed genes (DEGs) in stem phloem and xylem, such as genes involved in carbon metabolism and transportation, aquaporin genes (in xylem) and genes related to the biosynthesis of secondary metabolites and the phenylpropanoid pathway (related to lignin synthesis), were downregulated at 7 days after inoculation (DAI). Results also showed that the expression of the majority of disease-resistance genes upregulated in poplar stems, which may be connected with the downregulation expression of the majority of WRKY family genes. Physiological assays showed that transpiration rate decreased but WUE (water use efficiency) increased the 3 and 7 DAI, while the net photosynthetic rate decreased at 11 DAI in Botryosphaeria infected poplars (ANOVA, P < 0.05). The NSC (non-structural carbohydrates) content assays showed that the soluble sugar content of stem phloem samples increased at 3, 7, and 11 DAI that might due to the impede of pathogen infection. However, soluble sugar content of stem xylem and root samples decreased at 11 DAI; in contrast, the starch content unchanged. Therefore, results revealed a chronological order of carbon related molecular and physiological performance: declination of genes involved in carbon and starch metabolism first (at least at 7 DAI), declination of assimilation and carbon reserve (at 11 DAI) second. Results implied a potential mechanism that affects the host carbon reserve, by directly inhibiting the expression of genes involved in carbon metabolism and transport.

Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems.

It is of great importance to explore the selective hydrogenolysis of β--4 linkages, which account for 45-60% of all linkages in native lignin, to produce valued-added chemicals and fuels from biomass employing UV light as catalyst. TiO exhibited satisfactory catalytic performances in various photochemical reactions, due to its versatile advantages involving high catalytic activity, low cost and non-toxicity. In this work, 20 wt.% Ni/TiO and oxidant PCC (Pyridinium chlorochromate) were employed to promote the cleavage of β--4 alcohol to obtain high value chemicals under UV irradiation at room temperature. The Ni/TiO photocatalyst can be magnetically recovered and efficiently reused in the following four consecutive recycling tests in the cleavage of β--4 ether bond in lignin. Mechanism studies suggested that the oxidation of β--4 alcohol to β--4 ketone by oxidant PCC first occurred during the reaction, and was followed by the photocatalysis of the obtained β--4 ketone to corresponding acetophenone and phenol derivates. Furthermore, the system was tested on a variety of lignin model substrates containing β--4 linkage for the generation of fragmentation products in good to excellent results.

Photocatalytic Cleavage of β--4 Ether Bonds in Lignin over Ni/TiO.

 is a latent and important fungal pathogen on a wide range of woody plants. Fruit ring rot caused by  is a major disease in China on apple. This study establishes a high-quality, nearly complete, and well-annotated genome sequence of  strain sdau11-99. The findings of this research provide a reference genome resource for further research on the apple fruit ring rot pathogen on apple and other hosts.

Genome Assembly and Annotation of  sdau11-99, a Latent Pathogen of Apple Fruit Ring Rot in China.

, as a major family of the largest class of kingdom fungi , encompasses phytopathogens, saprobes, and endophytes. Many members of this family are opportunistic phytopathogens with a wide host range and worldwide geographical distribution, and can infect many economically important plants, including food crops and raw material plants for biofuel production. To date, however, little is known about the family evolutionary characterization, mating strategies, and pathogenicity-related genes variation from a comparative genome perspective. Here, we conducted a large-scale whole-genome comparison of 271 , including 19 species in . The comparative genome analysis provided a clear classification of  in  and indicated that the evolution of lifestyle within  underwent four major transitions from non-phytopathogenic to phytopathogenic. Mating strategies analysis demonstrated that at least 3 transitions were found within  from heterothallism to homothallism. Additionally, pathogenicity-related genes contents in different genera varied greatly, indicative of genus-lineage expansion within . These findings shed new light on evolutionary traits, mating strategies and pathogenicity-related genes variation of .

Comparative Genomics Reveals Evolutionary Traits, Mating Strategies, and Pathogenicity-Related Genes Variation of .

Ophiostomatoid fungi exhibit a complex relationship with bark beetles; exhausting of host tree defenses is traditionally regarded as one of the key benefits provided to beetle vectors.  is one of the dominant species of the mycobiota associated with  genus bark beetles which infect the spruce trees across the Eurasian continent. Host spruce trees resist fungal invasion through structural and inducible defenses, but the underlying mechanisms at the molecular level, particularly with respect to the interaction between bark beetle-associated fungi and host trees, remain unclear. The aim of this study was to observe the pathological physiology and molecular changes in  seedlings after artificial inoculation with  strains (TS, BH, QH, MX, and LWQ). This study showed that  was a weakly virulent pathogen of spruce, and that the virulent of the five  strains showed differentiation. All  strains could induce monoterpenoid release. A positive correlation between fungal virulence and release of monoterpenoids was observed. Furthermore, the release rate of monoterpenoids peaked at 4 days post-inoculation (dpi) and then decreased from 4 to 90 dpi. Transcriptomic analysis at 4 dpi showed that many plant-pathogen interaction processes and mitogen-activated protein kinase (MAPK) metabolic processes were activated. The expression of monoterpenoid precursor synthesis genes and diterpenoid synthesis genes was upregulated, indicating that gene expression regulated the release rate of monoterpenoids at 4 dpi. The enriched pathways may reveal the immune response mechanism of spruce to ophiostomatoid fungi. The dominant  possibly induces the host defense rather than defense depletion, which is likely the pattern conducted by the pioneers of beetle-associated mycobiota, such as  spp.. Overall, these results facilitate a better understanding of the interaction mechanism between the dominant association of beetles and the host at the molecular level.

Pathophysiology and transcriptomic analysis of  inoculated by bark beetle-vectored fungus .

Fungal pathogens can induce canker lesions, wilting, and even dieback in many species. Trees can suffer serious physiological effects from stem cankers. In this study, we investigated the effects of  (. ) on  () leaves photosynthesis and stomatal responses, when stems were inoculated with the pathogen. To provide experimental and theoretical basis for preventing poplar canker early. One-year-old poplar stems were inoculated with .  using an epidermal scraping method. In the early stage of .  inoculation (2-14 days post inoculation, dpi), the gas exchange, stomatal dynamics, hormone content, photosynthetic pigments content, chlorophyll fluorescence parameters, and non-structural carbohydrate (NSC) were evaluated to elucidate the pathophysiological mechanism of .  inhibiting photosynthesis. Compared with the control groups, .  noteworthily inhibited the net photosynthetic rate ( ), stomatal conductance ( ), intercellular CO concentration ( ), transpiration rate ( ), and other photosynthetic parameters of poplar leaves, but stomatal limit value ( ) increased. Consistent with the above results, .  also reduced stomatal aperture and stomatal opening rate. In addition, .  not only remarkably reduced the content of photosynthetic pigments, but also decreased the maximum photochemical efficiency ( / ), actual photochemical efficiency ( ), electron transfer efficiency (ETR), and photochemical quenching coefficient ( ). Furthermore, both chlorophyll and   were positively correlated with  . In summary, the main reason for the abated   under stem canker pathogen was that .  not merely inhibited the stomatal opening, but hindered the conversion of light energy, electron transfer and light energy utilization of poplar leaves. In general, the lessened CO and   would reduce the synthesis of photosynthetic products. Whereas, sucrose and starch accumulated in poplar leaves, which may be due to the local damage caused by .  inoculation in phloem, hindering downward transport of these products.

Stem canker pathogen  inhibits poplar leaf photosynthesis in the early stage of inoculation.

Recently, a novel poplar mosaic disease caused by bean common mosaic virus (BCMV) was investigated in  var.  in China. Symptom characteristics, physiological performance of the host, histopathology, genome sequences and vectors, and gene regulation at the transcriptional and posttranscriptional levels were analyzed and RT-qPCR (quantitative reverse transcription PCR) validation of expression was performed in our experiments. In this work, the mechanisms by which the BCMV pathogen impacts physiological performance and the molecular mechanisms of the poplar response to viral infection were reported. The results showed that BCMV infection decreased the chlorophyll content, inhibited the net photosynthesis rate (Pn) and stomatal conductance (Gs), and significantly changed chlorophyll fluorescence parameters in diseased leaves. Transcriptome analysis revealed that the expression of the majority of DEGs (differentially expressed genes) involved in the flavonoid biosynthesis pathway was promoted, but the expression of all or almost all DEGs associated with photosynthesis-antenna proteins and the photosynthesis pathway was inhibited in poplar leaves, suggesting that BCMV infection increased the accumulation of flavonoids but decreased photosynthesis in hosts. Gene set enrichment analysis (GSEA) illustrated that viral infection promoted the expression of genes involved in the defense response or plant-pathogen interaction. MicroRNA-seq analysis illustrated that 10 miRNA families were upregulated while 6 families were downregulated in diseased poplar leaves; moreover, miR156, the largest family with the most miRNA members and target genes, was only differentially upregulated in long-period disease (LD) poplar leaves. Integrated transcriptome and miRNA-seq analyses revealed 29 and 145 candidate miRNA-target gene pairs; however, only 17 and 76 pairs, accounting for 2.2% and 3.2% of all DEGs, were authentically negatively regulated in short-period disease (SD) and LD leaves, respectively. Interestingly, 4 miR156/SPL (squamosa promoter-binding-like protein) miRNA-target gene pairs were identified in LD leaves: the miR156 molecules were upregulated, but SPL genes were downregulated. In conclusion, BCMV infection significantly changed transcriptional and posttranscriptional gene expression in poplar leaves, inhibited photosynthesis, increased the accumulation of flavonoids, induced systematic mosaic symptoms, and decreased physiological performance in diseased poplar leaves. This study elucidated the fine-tuned regulation of poplar gene expression by BCMV; moreover, the results also suggested that miR156/SPL modules played important roles in the virus response and development of viral systematic symptoms in plant virus disease.

Integrated transcriptome and microRNA sequencing analyses reveal gene responses in poplar leaves infected by the novel pathogen bean common mosaic virus (BCMV).

A key step in the study of tree pathology is the identification of an appropriate method for inoculating pathogens of diseases in branches and trunks. Pathogens of diseases in branches and trunks are commonly inoculated through punching, burning, and toothpick inoculation. However, there is a lack of comparative analyses of the inoculation outcomes of these three methods. In this work, six-year-old  var.  were inoculated with  using punching, burning, and toothpick techniques to investigate the differences in the effectiveness of these inoculation methods. Results reveal that the incidence rate was 93.55% in the toothpick inoculation group, significantly higher than the 80.65% in the burning inoculation group (chi-square,  = 90,  = 0.007), while punching inoculation exhibited significant pathological responses in the early stages, with spontaneous healing in the later stage. Additionally, toothpick inoculation was more efficient in inducing  canker when inoculating the pathogen at the bottom of the tree, with lower intra- and inter-row spacing (stand density) providing better outcomes than higher intra- and inter-row spacing. The results of this study demonstrate that toothpick inoculation is an optimal option for studying the artificial inoculation of  in six-year-old  var. , providing technical support for research on poplar diseases and offering a theoretical basis for the inoculation of other diseases in the branch and trunk.

Comparative Study on the Effectiveness of Three Inoculation Methods for  in  var. .

This study revealed a substrate-level synthesis of pigment cyanidin-3-O-glucoside and the redirection of metabolomic flux in the flavonoid/anthocyanin biosynthesis pathway in poplar adventitious roots (ARs) induced by stem canker pathogens. Recently, we observed a novel allometry on poplar stems, with copious colorful adventitious roots (ARs) induced by fungal canker pathogens. Here, we reveal chemical, physiological, and molecular mechanisms of AR coloration in poplar-pathogens (Valsa sordida/Botrosphaeria dothidea) interaction system using our phloem girdling-inoculation system. Light-induced coloration in ARs: red/rosy under sunlight, and milky white under shading. Chemical and metabolomic analyses indicated that numerous (93 in all 110) and high relative intensities/contents of flavonoids metabolites (mainly including flavonols, flavones, and anthocyanins class) accumulate in red ARs, some flavones and anthocyanins metabolites all contribute to the color of poplar ARs, and cyanidin-3-O-glucoside is the most abundant colorant. Integrated analysis of metabolomic and transcriptomic analysis suggested that sunlight exposure redirected the metabolomic flux from the flavonoid biosynthesis pathway to the flavonols and flavones branch pathways, induced by the upregulation of FLS (flavonol synthase/flavanone 3-hydroxylase) and other structural genes. The anthocyanins metabolomic analysis and the downregulation of the ANS (anthocyanin synthase) gene illustrated a retard of metabolomic flux from leucoanthocyanidins to anthocyanidins. Metabolomic results and the upregulation of the gene BZ1 (Bronze 1, anthocyanin 3-O-glucosyltransferase) illustrated that sunlight triggered a rapid biosynthesis of anthocyanin metabolites in poplar ARs, which based on the substrate level of anthocyanidins. Transcriptomic and RT-qPCR analyses showed that transcriptional factor MYB113, HY5 (Elongated hypocotyl 5), and COP1 (Ring-finger protein CONSTITUTIVE PHOTOMORPHOGENIC1) genes positively regulated the expression of the flavonoid/anthocyanin biosynthesis structural genes (such as the BZ1, FLS and LAR gene) in both sunlight-exposed red ARs and white ARs after light-exposure, suggesting sunlight induces anthocyanins biosynthesis through the interaction between "MBW" complex and COP1-HY5 module. Moreover, one SPL gene (squamosa promoter-binding-like protein gene, target of miR156, and one component of miR156-SPL module) was down-regulated in sunlight-exposed poplar ARs, implying the biosynthesis flavonoid/anthocyanin be regulated at the posttranscriptional level. This study provides a potential AR experimental system for research on flavonoid/anthocyanin biosynthesis in tree species.

Why the adventitious roots of poplar are so colorful: RNAseq and metabolomic analysis reveal anthocyanin accumulation in canker pathogens-induced adventitious roots in poplar.

ArticleTotal : 3	Year
Publication title Cited by 1	2024
Publication title	2024
Publication title	2024

Article	Year
Phylogenetic analysis and molecular evolution patterns in the MIR482-MIR1448 polycistron of Populus L. PloS one\| PubMed ID: 23094096	2012
Genome-Wide Constitutively Expressed Gene Analysis and New Reference Gene Selection Based on Transcriptome Data: A Case Study from Poplar/Canker Disease Interaction. Frontiers in plant science\| PubMed ID: 29163601	2017
Effects of phloem on canopy dieback, tested with manipulations and a canker pathogen in the Corylus avellana/Anisogramma anomala host/pathogen system. Tree physiology\| PubMed ID: 30938425	2019
Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems. Scientific reports\| PubMed ID: 31300723	2019
Photocatalytic Cleavage of β--4 Ether Bonds in Lignin over Ni/TiO. Molecules (Basel, Switzerland)\| PubMed ID: 32365962	2020
Genome Assembly and Annotation of sdau11-99, a Latent Pathogen of Apple Fruit Ring Rot in China. Plant disease\| PubMed ID: 33258431	2021
Comparative Genomics Reveals Evolutionary Traits, Mating Strategies, and Pathogenicity-Related Genes Variation of . Frontiers in microbiology\| PubMed ID: 35283828	2022
Pathophysiology and transcriptomic analysis of inoculated by bark beetle-vectored fungus . Frontiers in plant science\| PubMed ID: 35928703	2022
Stem canker pathogen inhibits poplar leaf photosynthesis in the early stage of inoculation. Frontiers in plant science\| PubMed ID: 36204063	2022
Integrated transcriptome and microRNA sequencing analyses reveal gene responses in poplar leaves infected by the novel pathogen bean common mosaic virus (BCMV). Frontiers in plant science\| PubMed ID: 37396641	2023
Comparative Study on the Effectiveness of Three Inoculation Methods for in var. . Biology\| PubMed ID: 38666863	2024
Why the adventitious roots of poplar are so colorful: RNAseq and metabolomic analysis reveal anthocyanin accumulation in canker pathogens-induced adventitious roots in poplar. Planta\| PubMed ID: 39694940	2024

Jiaping Zhao

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