First Report of Neopestalotiopsis clavispora Causing Fruit Brown Spot of Pomegranate in China.

Pomegranate (Punica granatum) is an important fruit crop for therapeutic and food applications. In June 2022, brown spots were observed on the fruit surface of pomegranate cultivar named Guangyan in Mengzi (23°20'6''N,103°25'5''E), Yunnan, China. The early spots appeared as circular or irregular lesions, measuring 1~1.5 mm in diameter. They were light brown with a clear boundary between disease and healthy tissues. Over time, these spots developed into polygonal lesions covering the entire fruit surface. Eventually, the diseased fruits decayed, and more than 50% of fruits were infected in pomegranate orchards. The tissues from the interface between health and disease were cut down, immersed in 75% ethanol for 15 s, then 5% NaOCl disinfecting for 2 min, washed three times with sterile water, and the PDA cultured at 26 °C in an incubator under dark conditions. Twenty-five samples were collected for pathogen isolation, ten fungal isolates were obtained by single spore germination, and these isolates had similar morphological characters. The colonies were white with 81 mm diameter at 7 days of incubation, containing undulate edges with dense aerial mycelium. After 14 days, the black conidiomata formed superficially, gathering into black droplets. Conidiogenous cells were hyaline, short, and filiform. Conidia were fusiform, straight or slightly curved, and comprised five cells, 24.12 to 34.53 (x̄=29.78) µm × 4.21 to 12.15 (x̄=8.68) µm (n=50). The three median cells were 13.13 to 25.22 µm (x̄=18.54), dark brown, whose septa and periclinal walls were darker than the other two cells. The apical cells showed two to four appendages, 12.31 to 29.15 (x̄=21.56) µm. Only a single appendage was found on the basal cell, 2.34 to 7.16 µm. Based on morphological features, these isolates were identified as Neopestalotiopsis clavispora (Maharachchikumbura et al., 2012, 2014). Molecular identification of isolate YNSL-3 was performed by amplification and sequencing of ITS4/ITS5, BT2A/ BT2B and EF1-728F/EF-2, respectively (White et al. 1990, Glass et al.1995, Carbone et al. 1999, O'Donnell et al. 1998). These base sequences were deposited in GenBank with accession numbers OQ891378 (ITS), OR088917 (Tef) and OR513439(Tub), respectively. BLAST searches of the sequences revealed 100% (478/478 bp), 100% (484/484 bp), and 94.67% (426/450 bp) homology with those of N. clavispora NM16311a from GenBank (LC209216, LC209220, and LC209221), respectively. Phylogenetic analysis (IQ-TREE) by maximum likelihood method showed that the isolate YNSL-3 was clustered with N. clavispora. The pathogenicity was tested with the isolate of YNSL-3, YNSL-5 and YNSL-8 by detached assay. The fruit surface of pomegranate cultivar Guangyan was wounded with a sterilized needle. The mycelial blocks (5mm2) of isolates cultured on PDA for 7 days were attached to the points of inoculation. Controls were inoculated with sterile PDA agar. All inoculated fruits were maintained in a growth chamber at 26°C with 75% relative humidity. The test was performed thrice. The brown lesions were observed after 7 days, whereas the controls showed no symptoms. The same pathogens reisolated were identical to the original isolates based on morphological characterization and molecular analyses. N. clavispora could cause different diseases in many plants (Rajashekara et al. 2023, Loredana et al. 2020). To our knowledge, this is the first report of fruit brown spot on Punica granatum caused by N. clavispora in China. This finding will help improve management strategies against the fruit brown spots on P. granatum in China.

First Report of Lasiodiplodia theobromae Causing Stem Brown Rot of Loquat in China.

Loquat (Rhaphiolepis biabas, heterotypic synonym: Eriobotrya japonica) is an important edible and medicinal plant that is widely cultivated on 133 thousand hectares (recorded in 2022) in China. A stem brown rot was observed on young and old trees in Mengzi city (23°23' N; 103°23' E), Yunnan Province, southwest China, during October 2014 and September 2021. Incidence ranged from 20% of trees in surrounding plantations to 50% incidence of a 160 tree orchard that was the focal point of the disease survey. Circular brown lesions occurred initially on the stems and gradually covered all the epidermis of the stem, leading to irregular dents within the bark that developed a dark brown powdery appearance (Fig.1A). Larger lesions affected vascular tissues, causing diseased trees to wither and die. Diseased tissues were surface-disinfected in a 5% sodium hypochlorite solution for 3 min, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), and incubated in the dark at 28°C. Twenty samples were collected for tissue isolation, and 11 isolates were single-spored on water agar. In culture, the colonies on PDA were white to dark-gray, velvet, with dense hyphae, diameter 7.64 cm after 5 days. After 18 days, spherical or subglobose pycnidia were developed and semi-buried in medium, their walls were thicker and dark-brown, which were black particles surrounded by gray-black hyphae. Conidiogenous cells were hyaline, cylindrical, holoblastic, slightly swollen at the base, with rounded apex. Conidia were initially hyaline and aseptate with elliptic or ovate shape, becoming dark brown with a single septate and developing longitudinal striations along thick walls at maturity. Conidia dimensions varied from 8.0 to 12.2 × 3.8 to 6.1µm (n=50) (Fig.1D). The morphological characteristics of eleven isolates were consistent with the description of Lasiodiplodia theobromae (Alves et al. 2008). Further confirmation was also determined by sequencing the internal transcribed spacer (ITS), ß-tubulin genes, partial translation elongation factor-1α (TEF-1α) (White et al. 1990, Carbone et al. 1999, Glass et al.1995). The isolate LSB-1 was selected for DNA sequence analysis. Based on BLASTn analysis, ITS sequences (OM617921) had 98.3% similarity with L. theobromae CBS164.96 (accession AY640255), CBS124.13(accession DQ458890), CAA006 (accession DQ458891) and CBS111530 (accession EF622074), ß-tubulin sequences (OM643838) showed 99.1% similarity with L. theobromae accessions EU673110. The TEF-1α (OM643839) had 99.0% identity with L. theobromae accession EF633054. The isolate LSB-1 clustered on the same clade with other L. theobromae. Pathogenicity testing of isolate LSB-1, LSB-2, LSB-3 was conducted by inoculating the stems of l-year-old seedlings growing in pots. The epidermis at the inoculation site, 15-20 cm below the crown, was wiped with 75% alcohol cotton ball, washed three times with sterile water, and then punctured (5mm diameter) with sterile inoculation needle. A 5mm block of each isolate cultured on PDA for seven days was attached to the inoculation site. Controls were inoculated with sterile PDA blocks. The inoculation area was covered with polyethylene cling film. All inoculated seedlings were kept in controlled greenhouse at 27°C with 80% relative humidity under natural daylight conditions, and watered weekly. Each treatment was repeated three times. Eight days after inoculation, all diseased plants showed dark brown discoloration at the point of inoculation (Fig. 1G) with the bark at the inoculation site gradually raising as the disease progressed. Thirty days after inoculation, all inoculated seedlings produced typical symptoms, whereas the control seedlings remained healthy. Fungal isolates were only recovered from symptomatic stems and were morphologically identical to L. theobromae, completing Koch's postulates. According to the relevant literature, Lasiodiplodia theobromae has a broad host range, causing numerous diseases, including canker and dieback of branch (Aguilera-Cogley et al., 2021), panicle blight (Mahadevakumar et al, 2022), root rot (Abd-El Ghani and Fatouh, 2005), fruit rot(Freire et al., 2011) in diverse geographical regions. To our knowledge, this is the first report of L. theobromae causing stem brown rot of loquat in China and provides a foundation for further study of the epidemiology and integrated management of this disease.

Regulation of Hippo signaling and triple negative breast cancer progression by an ubiquitin ligase RNF187.

Breast cancer is the most common malignancy for women worldwide, while Triple Negative Breast Cancer (TNBC) accounts for 20% in all patients. Compared with estrogen receptor positive breast cancer, which could be effectively controlled via endocrine therapy, TNBC is more aggressive and worse in prognosis. It is therefore urgent and necessary to develop a novel therapeutic strategy for TNBC treatment. Recent studies identified Hippo signaling is highly activated in TNBC, which could be a driving pathway for TNBC progression. In our study, we determine RNF187 as a negative regulator for Hippo signaling activation. RNF187 depletion significantly decreases cell migration and invasion capacity in TNBC. These effects could be rescued by further YAP depletion. Depletion of RNF187 increases the YAP protein level and Hippo signaling target genes, such as CTGF and CYR61 in TNBC. Immuno-precipitation assay shows that RNF187 associates with YAP, promoting its degradation possibly via inducing YAP K48-dependent poly-ubiquitination. Interestingly, Our clinical data reveals that RNF187 reversely correlates with YAP protein level and Hippo target genes. RNF187 tends to correlate with good prognosis in TNBC patients. Our study provides evidence to establish a proteolytic mechanism in regulation Hippo signaling activation in TNBC.

Prognostic value of mutant p53, Ki-67, and TTF-1 and their correlation with EGFR mutation in patients with non-small cell lung cancer.

INTRODUCTION: The clinical characteristics of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutation have been well studied. However, the correlation of EGFR mutation with mutant p53, Ki-67, and thyroid transcription factor 1 (TTF-1) and their prognostic value remain indistinct. MATERIAL AND METHODS: Clinical and pathological characteristics and overall survival were analysed retrospectively in 523 surgically resected NSCLC patients. The expression levels of p53, Ki-67, and TTF-1 protein were detected by immunohistochemistry, and an amplification refractory mutation system was used to access the status of EGFR mutations. RESULTS: Of 523 patients with surgically resected NSCLC, 210 patients (38.4%) harboured EGFR mutations. Compared to the EGFR wild-type lung cancer, mutated EGFR harboured significantly increased mutant p53-positive or TTF-1-positive tumors (P<0.001 and<0.001, respectively). Former or current smokers, pathological stage and mutant p53-or TTF-1-positive status were independent predictors of EGFR mutation (P=0.001, 0.014, 0.014 and <0.001, respectively). Patients with p53 under expression had significantly better overall survival in the whole cohort and wild-type EGFR cohort (P=0.0010 and 0.0020, respectively) as well as in Ki-67-negative and TTF-1-positive patients (P<0.0001 and 0.0009, and P<0.0001 and 0.0004, respectively). Interestingly, in patients harbouring EGFR mutations, p53-under expression and Ki-67-negative cases still had better survival than positive cases, whereas there was no obvious difference between TTF-1-negative and TTF-1-positive cases (P=0.0198, 0.0068 and 0.3684, respectively). Finally, in NSCLC patients with wild-type EGFR, positive Ki-67 expression was the independent predictor for the worst survival (P=0.022). CONCLUSION: The expression levels of mutant p53, Ki-67, and TTF-1 were correlated with EGFR mutation. High expression of mutant p53 and Ki-67 correlated with poor survival in the entire cohort, EGFR mutation or wild-type cohort. In addition, Ki-67 might have an impact on the prognosis for patients with NSCLC with wild-type EGFR.

Regulation of estrogen signaling and breast cancer proliferation by an ubiquitin ligase TRIM56.

Breast cancer ranks no. 1 in women cancer worldwide, while 60-70% are estrogen receptor alpha positive. The estrogen selective modulators, such as tamoxifen, become the effective drugs for controlling ER alpha breast cancer progression. However, tamoxifen resistance will develop during long-time treatment and cancer progression. Thus, further understanding of ER alpha signaling becomes necessary for the improvement of breast cancer therapy. Here, we identify TRIM56 as a novel regulatory factor in ER alpha signaling. TRIM56 expression is positively correlated with ER alpha and PR in breast cancer samples and is related to poor prognosis in endocrine therapy patients. TRIM56 depletion significantly decreases ER alpha signaling activity and ER-alpha-positive breast cancer proliferation in vitro and in vivo. TRIM56 associates with AF1 domain of ER alpha via its WD40 domain in the cytoplasm. TRIM56 prolongs ER alpha protein stability, possibly through targeting ER alpha K63-linked ubiquitination. In conclusion, our study reveals an interesting posttranslational mechanism between TRIM56 and ER alpha in breast cancer progression. Targeting TRIM56 could be a promising approach for ER-alpha-positive breast cancer.

The Expression and Related Clinical Significance of SIRT3 in Non-Small-Cell Lung Cancer.

OBJECTIVE: To examine the relationship between the Sirtuin-3 (SIRT3) expression and the clinical indicators/prognosis of patients with non-small-cell lung cancer (NSCLC). METHODS: The mRNA level of SIRT3 was detected by real-time PCR, while the protein level was detected by Western blot and immunohistochemical staining. SPSS 16.0 software was used for statistical analysis. RESULTS: The expression of SIRT3 was significantly higher in NSCLC tissue than in adjacent tissue. The SIRT3 level was correlated significantly with lymph node metastasis and clinical stage of NSCLC patients. Moreover, univariate analysis showed that the expression of SIRT3, tumor size, lymph node metastasis, degree of differentiation, and clinical stage were correlated with the prognosis of NSCLC patients. Multivariate analysis demonstrated that lymph node metastasis, the tumor size, and SIRT3 expression were independent prognostic factors for NSCLC patients. CONCLUSIONS: SIRT3 is associated with the development and progression of NSCLC. The SIRT3 expression can be used as an independent prognostic factor for NSCLC patients and help identify prognosis of NSCLC. Therefore, SIRT3 has the potential to become a new factor for prognosis prediction and personalized treatment of NSCLC.