Identification of Fusarium sacchari Causing Foliar Blight on Begonia semperflorens in China.

Begonia semperflorens Link et Otto (Begoniaceae) is a flowering, ornamental plant widely cultivated in China. In April of 2020, a foliar blight disease on B. semperflorens was observed in plant nurseries (∼0.2 ha), with ~ 20% disease incidence (n = 150) in Nanning, Guangxi Province, China. Initial symptoms included irregular to circular, grayish white spots surrounded by a dark brown halo, mainly scattered on the edges the leaves. In severe infections, the spots frequently coalesced to form large, blighted areas, followed by defoliation. To isolate the pathogen, three representative plants exhibiting symptoms were collected from the nurseries. Leaf tissues (5 × 5 mm) were cut from the margin of necrotic lesions (n = 18), surface disinfected in 1% NaOCl for 2 min, then rinsed three times in sterile H2O. Then the tissues were plated on potato dextrose agar (PDA), and incubated at 28°C (12 h photoperiod) for 3 days. Hyphal tips from recently germinated spores were transferred to PDA to purify fungal isolates. A total of 11 isolates (85% isolation frequency) with similar morphological characteristics were obtained. Colonies on PDA plates were villose, had a dense growth of white aerial mycelia and appeared pale but becoming violet with age. On Spezieller Nährstoffarmer Agar (SNA), the macroconidia were slender, slight falcate, two to three septate, and 23.5 to 48.8 × 2.8 to 4.8 µm (n = 60), and the microconidia were abundant and formed in false heads on monophialides or polyphialides, slim, oval, zero to one septate, and 7.8 to 22.4 × 2.4 to 4.0 µm (n = 60). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, and partial translation elongation factor-1 alpha (TEF-1α), and RNA polymerase second largest subunit (RPB2) genes of the representative isolate HT-2B were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (O'Donnell et al. 1998), and 5f2/11ar (Liu et al. 1999, Reeb et al. 2004), respectively. The obtained sequences were deposited in NCBI GenBank under the accession numbers OQ048268 (TIS), OP994260 (TEF-1α), OP994262 (RPB2) and showed 99.4%, 99.8%, and 99.4% similarity with the corresponding sequences (X94168，AF160278，and JX171580, respectively) of Fusarium sacchari from type material. In addition, a phylogenetic analysis showed that HT-2B was grouped with F. sacchari. Therefore, based on morphological (Leslie et al. 2005) and molecular characteristics, the isolates were identified as F. sacchari. To test pathogenicity, three healthy leaves on each of three B. semperflorens plants were stab-wounded with a sterile syringe and inoculated with a 10-µl droplet of a conidial suspension (106 spores/ml) of the isolate HT-2B. As a control, another three leaves were wound inoculated with sterilized dH2O. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 28°C (12 h photoperiod, ~ 80% relative humidity). Six days post-inoculation, symptoms appeared on the inoculated leaves. No symptoms were detected on control plants. Experiments were replicated three times with similar results. To fulfill Koch's postulates, the F. sacchari isolates were consistently re-isolated from symptomatic tissue and confirmed by morphology and sequencing, whereas no fungus was isolated from the control plants. To our knowledge, this is the first report of F. sacchari causing foliar blight on B. semperflorens in China. This result will help develop management strategies for this disease.

[Comparative Study of PD-L1 Expression in Different Sites of 
Non-small Cell Lung Cancer].

BACKGROUND: The expression of programmed cell death ligand 1 (PD-L1) as a biomarker for immunotherapy in non-small cell lung cancer (NSCLC) is routinely detected in clinical pathology department. However, the spatial heterogeneity of PD-L1 expression in intrapulmonary tumors and extrapulmonary metastases is still a challenge for the clinical testing. This study aims to explore the differences of PD-L1 expression in test samples obtaining from different sites of NSCLC. This study may contribute to the detection strategy of PD-L1 in patients with advanced lung cancer. METHODS: One hundred and thirty-one cases of consecutively detected PD-L1 (22c3 assay, Dako) staining in metastatic NSCLC and 972 cases of non-paired intrapulmonary NSCLC were collected. The discrepancies of tumor proportion score (TPS) of PD-L1 expression in intrapulmonary samples and extrapulmonary metastatic samples of different sites were compared. RESULTS: The positive expression rate of PD-L1 in extrapulmonary metastatic NSCLC (TPS ≥ 1%) was 61.83%, and the TPS was significantly higher than that in intrapulmonary tumors (P=0.03). The PD-L1 scores of the specimens obtained from different sites were significantly different (P=0.007). The positive rates of PD-L1 in liver and adrenal metastases were 85.71% and 77.78% respectively, and their TPS were significantly higher than that of the intrapulmonary samples (P<0.05). The positive rates of PD-L1 in lymph node, bone, brain, soft tissue, and pleural metastases was 40.00%-66.67%, with no significant differences compared to intrapulmonary tumors. The analysis of histological subtype and sample type showed that the PD-L1 score of extrapulmonary samples of adenocarcinoma subtype or surgical specimen was significantly higher than that of intrapulmonary tumors. The analysis of clinicopathological parameters showed that the PD-L1 positive expression or high expression were significantly correlated with male patients, smoking history, and epidermal growth factor receptor (EGFR) wild type. CONCLUSIONS: The expression of PD-L1 in metastatic NSCLC is generally higher than that in intrapulmonary tumor, and the positive rate of PD-L1 expression was discrepant in different sites of specimen. The differences of PD-L1 score between extrapulmonary metastatic samples and intrapulmonary samples may be associated with different metastatic sites, histological subtype, and specimen type.