A tissue injury sensing and repair pathway distinct from host pathogen defense.

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.

The Cytoplasmic DNA Sensor cGAS Promotes Mitotic Cell Death.

Pathogenic and other cytoplasmic DNAs activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce inflammation via transcriptional activation by IRF3 and nuclear factor κB (NF-κB), but the functional consequences of exposing cGAS to chromosomes upon mitotic nuclear envelope breakdown are unknown. Here, we show that nucleosomes competitively inhibit DNA-dependent cGAS activation and that the cGAS-STING pathway is not effectively activated during normal mitosis. However, during mitotic arrest, low level cGAS-dependent IRF3 phosphorylation slowly accumulates without triggering inflammation. Phosphorylated IRF3, independently of its DNA-binding domain, stimulates apoptosis through alleviating Bcl-xL-dependent suppression of mitochondrial outer membrane permeabilization. We propose that slow accumulation of phosphorylated IRF3, normally not sufficient for inducing inflammation, can trigger transcription-independent induction of apoptosis upon mitotic aberrations. Accordingly, expression of cGAS and IRF3 in cancer cells makes mouse xenograft tumors responsive to the anti-mitotic agent Taxol. The Cancer Genome Atlas (TCGA) datasets for non-small cell lung cancer patients also suggest an effect of cGAS expression on taxane response.

Brainstem ADCYAP1+ neurons control multiple aspects of sickness behaviour.

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.

Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Ethylene and jasmonate signaling converge on gibberellin catabolism during thigmomorphogenesis in Arabidopsis.

Touch induces marked morphological changes in plants, including reduced rosette diameters and delayed flowering, a process called thigmomorphogenesis. Previous studies have revealed that thigmomorphogenesis in Arabidopsis (Arabidopsis thaliana) results from touch-induced accumulation of jasmonic acid (JA) and GIBBERELLIN 2-OXIDASE7 (GA2ox7) transcripts, which encode a gibberellin (GA) catabolism enzyme, leading to reduced levels of active GAs. However, the mechanisms underlying thigmomorphogenesis remain uncharacterized. Here, we showed that touch induces ethylene (ET) production in Arabidopsis. After touch treatment, ET biosynthesis and signaling mutants exhibited even greater thigmomorphogenic changes and more decreased GA4 contents than did wild-type (WT) plants. Biochemical analysis indicated that the transcription factor ETHYLENE INSENSITIVE3 (EIN3) of the ET pathway binds to the promoter of GA2ox8 (encoding another GA 2-oxidase performing the same GA modification as GA2ox7) and represses GA2ox8 transcription. Moreover, MYC2, the master regulator of JA signaling, directly promoted GA2ox7 expression by binding the G-box motif on GA2ox7 promoter. Further genetic analysis suggested that the ET and JA pathways independently control the expression of GA2ox8 and GA2ox7, respectively. This study reveals that the ET pathway is a novel repressor of touch-induced thigmomorphogenesis and highlights that the ET and JA pathways converge on GA catabolism but play opposite roles to fine-tune GA4 content during thigmomorphogenesis.

Global Inhibition with Specific Activation: How p53 and MYC Redistribute the Transcriptome in the DNA Double-Strand Break Response.

In response to stresses, cells often halt normal cellular processes, yet stress-specific pathways must bypass such inhibition to generate effective responses. We investigated how cells redistribute global transcriptional activity in response to DNA damage. We show that an oscillatory increase of p53 levels in response to double-strand breaks drives a counter-oscillatory decrease of MYC levels. Using RNA sequencing (RNA-seq) of newly synthesized transcripts, we found that p53-mediated reduction of MYC suppressed general transcription, with the most highly expressed transcripts reduced to a greater extent. In contrast, upregulation of p53 targets was relatively unaffected by MYC suppression. Reducing MYC during the DNA damage response was important for cell-fate regulation, as counteracting MYC repression reduced cell-cycle arrest and elevated apoptosis. Our study shows that global inhibition with specific activation of transcriptional pathways is important for the proper response to DNA damage; this mechanism may be a general principle used in many stress responses.

Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence.

The TGF-ß-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-ß pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

Genetic interrogation of replicative senescence uncovers a dual role for USP28 in coordinating the p53 and GATA4 branches of the senescence program.

Senescence is a terminal differentiation program that halts the growth of damaged cells and must be circumvented for cancer to arise. Here we describe a panel of genetic screens to identify genes required for replicative senescence. We uncover a role in senescence for the potent tumor suppressor and ATM substrate USP28. USP28 controls activation of both the TP53 branch and the GATA4/NFkB branch that controls the senescence-associated secretory phenotype (SASP). These results suggest a role for ubiquitination in senescence and imply a common node downstream from ATM that links the TP53 and GATA4 branches of the senescence response.

Symbol-level fiber-longitudinal power profile estimation.

Symbol-level fiber-longitudinal power profile estimation (PPE) greatly reduces the implementation complexity compared with the waveform-level PPE using oversampled data. However, symbol-rate data cannot account for the inter-sample interaction, which leads to inaccuracy of the absolute power estimation. To realize an accurate symbol-level PPE, we provide an in-depth analysis of the differences between symbol-level and waveform-level perturbation matrices and propose a power calibration method based on the trace of the inverse matrix. Evaluated in the probabilistic constellation shaping (PCS) 64QAM 130 Gbaud 5 × 50 km optical links, the root mean squared error (RMSE) of the symbol-level PPE decreases by 0.98 and 0.62 dB at erbium-doped fiber amplifier (EDFA) positions and all estimated positions with the aid of matrix calibration.

Principles of cancer therapy: oncogene and non-oncogene addiction.

Cancer is a complex collection of distinct genetic diseases united by common hallmarks. Here, we expand upon the classic hallmarks to include the stress phenotypes of tumorigenesis. We describe a conceptual framework of how oncogene and non-oncogene addictions contribute to these hallmarks and how they can be exploited through stress sensitization and stress overload to selectively kill cancer cells. In particular, we present evidence for a large class of non-oncogenes that are essential for cancer cell survival and present attractive drug targets. Finally, we discuss the path ahead to therapeutic discovery and provide theoretical considerations for combining orthogonal cancer therapies.

A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene.

Oncogenic mutations in the small GTPase Ras are highly prevalent in cancer, but an understanding of the vulnerabilities of these cancers is lacking. We undertook a genome-wide RNAi screen to identify synthetic lethal interactions with the KRAS oncogene. We discovered a diverse set of proteins whose depletion selectively impaired the viability of Ras mutant cells. Among these we observed a strong enrichment for genes with mitotic functions. We describe a pathway involving the mitotic kinase PLK1, the anaphase-promoting complex/cyclosome, and the proteasome that, when inhibited, results in prometaphase accumulation and the subsequent death of Ras mutant cells. Gene expression analysis indicates that reduced expression of genes in this pathway correlates with increased survival of patients bearing tumors with a Ras transcriptional signature. Our results suggest a previously underappreciated role for Ras in mitotic progression and demonstrate a pharmacologically tractable pathway for the potential treatment of cancers harboring Ras mutations.

Metabolic supervision by PPIP5K, an inositol pyrophosphate kinase/phosphatase, controls proliferation of the HCT116 tumor cell line.

Identification of common patterns of cancer metabolic reprogramming could assist the development of new therapeutic strategies. Recent attention in this field has focused on identifying and targeting signal transduction pathways that interface directly with major metabolic control processes. In the current study we demonstrate the importance of signaling by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) to the metabolism and proliferation of the HCT116 colonic tumor cell line. We observed reciprocal cross talk between PPIP5K catalytic activity and glucose metabolism, and we show that CRISPR-mediated PPIP5K deletion suppresses HCT116 cell proliferation in glucose-limited culture conditions that mimic the tumor cell microenvironment. We conducted detailed, global metabolomic analyses of wild-type and PPIP5K knockout (KO) cells by measuring both steady-state metabolite levels and by performing isotope tracing experiments. We attribute the growth-impaired phenotype to a specific reduction in the supply of precursor material for de novo nucleotide biosynthesis from the one carbon serine/glycine pathway and the pentose phosphate pathway. We identify two enzymatic control points that are inhibited in the PPIP5K KO cells: serine hydroxymethyltransferase and phosphoribosyl pyrophosphate synthetase, a known downstream target of AMP-regulated protein kinase, which we show is noncanonically activated independently of adenine nucleotide status. Finally, we show the proliferative defect in PPIP5K KO cells can be significantly rescued either by addition of inosine monophosphate or a nucleoside mixture or by stable expression of PPIP5K activity. Overall, our data describe multiple, far-reaching metabolic consequences for metabolic supervision by PPIP5Ks in a tumor cell line.

Decoupling expression and editing preferences of ADAR1 p150 and p110 isoforms.

Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.

Polyhydroxylated Spirostanol Saponins from the Rhizomes of Paris dulongensis.

Three new polyhydroxylated spirostanol steroidal saponins, dulongenosides B-D (2-4), along with 14 known compounds, dulongenoside A (1), padelaoside B (5), parisyunnanoside G (6), polyphyllin D (7), ophiopogonin C' (8), formosanin C (9), dioscin (10), paris saponin VII (11), paris H (12), parisyunnanoside I (13), protodioscin (14), proprotogracillin (15), crustecdysone (16), and stigmasterol-3-O-ß-d-glucopyranoside (17), were isolated from the rhizomes of Paris dulongensis (Melanthiaceae). Their chemical structures were elucidated based on extensive analyses of NMR and MS data and acidic hydrolyses. The isolates were evaluated for their cytotoxicity to five human cancer cell lines (HL-60, SW480, MDA-MB-231, A549, and A549/Taxol) and the normal human bronchial epithelial cell line BEAS-2B by the MTS test. Compounds 7-12 and 14 showed cytotoxic activity, with IC50 values ranging from 0.20 to 4.35 µM. Proprotogracillin selectively inhibited A549 (IC50=0.58 µM) and A549/Taxol (IC50=0.74 µM) cells, with no significant cytotoxic activity against HL-60, SW480, MDA-MB-231, or BEAS-2B cells, with IC50 values greater than 40 µM.

Harmonizing florbetapir and PiB PET measurements of cortical Aß plaque burden using multiple regions-of-interest and machine learning techniques: An alternative to the Centiloid approach.

INTRODUCTION: Machine learning (ML) can optimize amyloid (Aß) comparability among positron emission tomography (PET) radiotracers. Using multi-regional florbetapir (FBP) measures and ML, we report better Pittsburgh compound-B (PiB)/FBP harmonization of mean-cortical Aß (mcAß) than Centiloid. METHODS: PiB-FBP pairs from 92 subjects in www.oasis-brains.org and 46 in www.gaain.org/centiloid-project were used as the training/testing sets. FreeSurfer-extracted FBP multi-regional Aß and actual PiB mcAß in the training set were used to train ML models generating synthetic PiB mcAß. The correlation coefficient (R) between the synthetic/actual PiB mcAß in the testing set was assessed. RESULTS: In the testing set, the synthetic/actual PiB mcAß correlation R = 0.985 (R2  = 0.970) using artificial neural network was significantly higher (p ≤ 6.6e-4) than the FBP/PiB correlation R = 0.927 (R2  = 0.860), improving total variance percentage (R2 ) from 86% to 97%. Other ML models such as partial least square, ensemble, and relevance vector regressions also improved R (p = 9.677e-05 /0.045/0.0017). DISCUSSION: ML improved mcAß comparability. Additional studies are needed for the generalizability to other amyloid tracers, and to tau PET. Highlights Centiloid is a calibration of the amyloid scale, not harmonization. Centiloid unifies the amyloid scale without improving inter-tracer association (R2 ). Machine learning (ML) can harmonize the amyloid scale by improving R2 . ML harmonization maps multi-regional florbetapir SUVRs to PiB mean-cortical SUVR. Artificial neural network ML increases Centiloid R2 from 86% to 97%.

Flortaucipir tau PET findings from former professional and college American football players in the DIAGNOSE CTE research project.

INTRODUCTION: Tau is a key pathology in chronic traumatic encephalopathy (CTE). Here, we report our findings in tau positron emission tomography (PET) measurements from the DIAGNOSE CTE Research Project. METHOD: We compare flortaucipir PET measures from 104 former professional players (PRO), 58 former college football players (COL), and 56 same-age men without exposure to repetitive head impacts (RHI) or traumatic brain injury (unexposed [UE]); characterize their associations with RHI exposure; and compare players who did or did not meet diagnostic criteria for traumatic encephalopathy syndrome (TES). RESULTS: Significantly elevated flortaucipir uptake was observed in former football players (PRO+COL) in prespecified regions (p < 0.05). Association between regional flortaucipir uptake and estimated cumulative head impact exposure was only observed in the superior frontal region in former players over 60 years old. Flortaucipir PET was not able to differentiate TES groups. DISCUSSION: Additional studies are needed to further understand tau pathology in CTE and other individuals with a history of RHI.

[Study on Circulating Tumor Cell Detection System Based on Microfluidic Chip].

Objective: To achieve high throughput and high detection rate of circulating tumor cells (CTCs) in human peripheral blood, and to provide efficient and accurate early screening for cancer patients. Methods: A microfluidic chip with the integration of sorting, enrichment and detection was designed, and CTCs at the single cell level were detected by fluorescence detection system to obtain the number of CTCs in samples. Results: The peripheral blood samples after lysed red blood cells were used for 6 experiments. When the injection rate reached 0.2 mL/h, CTCs could reach the best detection rate of 78.6%, and the correlation coefficient within the group was above 0.8. Conclusion: CTCs detection system can achieve high detection rate and has good reliability, which can provide a reliable reference for clinical research in related fields.

Size distribution analysis of residual host cell DNA fragments in lentivirus by CGE-LIF.

During the production of cell and gene therapy products, residual host cell DNA (HCD) could cause safety risks of the biological products, and the longer the residual HCD fragment, the greater the risk to the human body. For this reason, it was necessary to develop an effective method for the size distribution analysis of residual HCD fragments with high accuracy and sensitivity. In this study, capillary gel electrophoresis with laser-induced fluorescence detector (CGE-LIF) was used to analyze the size distribution of residual HCD fragments in lentivirus products. The results confirmed that lentiviral RNA genome could interfere with the size distribution analysis of residual HCD fragments. By optimizing the amount of RNase I and digestion time in sample pretreatment process, the interfere of RNA genome could be avoided. The specificity, precision, accuracy, linear range, the detection of limit (LOD), and the quantification of limit (LOQ) of CGE-LIF method were also validated. The results showed that the CGE-LIF method had a good performance both in terms of specificity and reproducibility. The intra- and inter-day relative standard deviations of migration time and corrected peak area were all less than 1% and 2%, respectively. The 200 bp DNA marker had a good linearity between 50 and 1000 pg/ml. The LOD and LOQ of 200 bp DNA marker were 2.59 and 8.64 pg/ml, respectively. In addition, this method was successfully used to analyze the size distribution analysis of residual HCD fragments in lentivirus products with different production processes.

Global warming accelerates uptake of atmospheric mercury in regions experiencing glacier retreat.

As global climate continues to warm, melting of glaciers releases a large quantity of mercury (Hg) originally locked in ice into the atmosphere and downstream ecosystems. Here, we show an opposite process that captures atmospheric Hg through glacier-to-vegetation succession. Our study using stable isotope techniques at 3 succession sites on the Tibetan Plateau reveals that evolving vegetation serves as an active "pump" to take up gaseous elemental mercury (Hg0) from the atmosphere. The accelerated uptake enriches the Hg pool size in glacier-retreated areas by a factor of ∼10 compared with the original pool size in the glacier. Through an assessment of Hg source-sink relationship observed in documented glacier-retreated areas in the world (7 sites of tundra/steppe succession and 5 sites of forest succession), we estimate that 400 to 600 Mg of Hg has been accumulated in glacier-retreated areas (5‰ of the global land surface) since the Little Ice Age (∼1850). By 2100, an additional ∼300 Mg of Hg will be sequestered from the atmosphere in glacier-retreated regions globally, which is ∼3 times the total Hg mass loss by meltwater efflux (∼95 Mg) in alpine and subpolar glacier regions. The recapturing of atmospheric Hg by vegetation in glacier-retreated areas is not accounted for in current global Hg models. Similar processes are likely to occur in other regions that experience increased vegetation due to climate or land use changes, which need to be considered in the assessment of global Hg cycling.

First report of Black Spot on Eucalyptus grandis × Eucalyptus urophylla caused by Pseudoplagiostoma eucalypti in Guangxi, China.

Eucalyptus grandis × Eucalyptus urophylla hybrid clone is an economically and ecologically important forest variety and is widely planted in Guangxi, China. Black spot, a newly found disease, occurred nearly 5333.3 hectares in an E. grandis × E. urophylla plantation of Qinlian forest farm (N: 21.866°, E: 108.921°) in Guangxi in October, 2019. Infected plants had lesions of black spots with watery margins on petioles and veins of E. grandis × E. urophylla. The size of spots ranged between 3 to 5 mm in diameter. When lesions expanded to girdle the petioles, wilt and death of leaves was observed, which subsequently affected growth of the trees. To isolate the causal agent, symptomatic plant tissues (leaves and petioles) were collected from two different sites, sampled from five plants each site. In the lab, infected tissues were surface sterilized with 75% ethanol for 10 seconds, then 2% sodium hypochlorite for 120 seconds, and rinsed with sterile distilled water three times. Small segments (5×5 mm) were cut from the margins of the lesions, then placed on potato dextrose agar (PDA) plates. The plates were incubated at 26°C in dark for 7 to 10 days. Fungal isolates YJ1 and YM6 with a similar morphology, which were obtained from 14 of 60 petioles and 19 of 60 veins respectively. These two colonies were initially light orange, then turned to olive brown as time progressed. Conidia were hyaline, smooth, aseptate, ellipsoidal, apex obtuse, and base tapering to flat protruding scar, 16.8 to 26.5µm long, and 6.6 to 10.4 µm wide (n=50). Some conidia had one or two guttules. The morphological characteristics were consistent with the description of Pseudoplagiostoma eucalypti Cheew., M. J. Wingf. & Crous (Cheewangkoon et al. 2010). For molecular identification, the internal transcribed spacer (ITS), ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 and T1/Bt2b, respectively (White et al. 1990; O'Donnell et al.1998; Glass and Donaldson 1995). Sequences of the two strains were deposited in GenBank (ITS: MT801070 and MT801071; BT2: MT829072 and MT829073). Phylogenetic tree was constructed with a maximum likelihood method, revealing that YJ1 and YM6 were on the same branch with P. eucalypti. Pathogenicity tests of the two strains were performed on three-month-old E. grandis × E. urophylla seedlings, by inoculating 6 wounded (by stabbing on petioles or veins) leaves of seedlings with mycelial PDA plugs (5 ×5 mm) from the edge of a 10-day old colony of strain YJ1 or YM6. Another 6 leaves were treated in the same manner but with PDA plugs as controls. All treatments were incubated in humidity chambers at 27°C and 80% relative humidity, under ambient light. All experiments were conducted three times. Lesions were observed at the points of inoculation, the petioles or veins turned black on inoculated leaves after 7 days, wilting of the leaves were also observed after 30 days, however the controls remained asymptomatic. Re-isolation was made and the fungus had same morphological measurements as the inoculated fungus, thus completing Koch's postulates. P. eucalypti had been reported as a pathogen of leaf spot on E. robusta in Taiwan island (Wang et al. 2016), leaf and shoot blight on E. pulverulenta in Japan (Inuma et al. 2015). To our knowledge, this is the first report of P. eucalypti affecting E. grandis × E. urophylla in mainland China. This report provides basis for the rational prevention and control of this new disease in the cultivation process of E. grandis × E. urophylla.