First report of leaf spot disease caused by Alternaria alternata on Polygonatum cyrtonema Hua in Hunan Province of China.

Polygonatum cyrtonema Hua, a perennial plant of the Asparagaceae family, is an important herb in Chinese medicine and is mainly grown in the Chinese provinces of Guizhou, Hunan, Yunnan, Anhui, and Zhejiang (Chen et al. 2021). In June 2021, a new case of leaf spot disease was detected in an 80 m2 plantation of P. cyrtonema on Xuefeng Mountain, Huaihua City, Hunan Province (27°17'30″N, 110°24'20″E). It infected almost 40% of the total planted area. Initially, irregular light brown spots appeared on the leaves, gradually turning dark brown and coalescing to form large necrotic areas, after which the affected plant turned yellow and eventually died. Ten disease samples were collected from ten plants in the plantation area. The leading edge of necrotic tissues were rinsed with sterile water and then disinfected with 3% hydrogen peroxide for 30 s, followed by 75% ethanol for 90 s, and rinsed three times with sterile water. Samples were then placed on water agar plates and incubated in the dark in a constant temperature incubator at 28 ℃ for 3-5 days. After mycelial growth was observed in the media, the hyphae were transferred to potato dextrose agar plates and incubated for 3-5 days at 28 ℃ in the dark. Ultimately, 12 purified fungal isolates were obtained, some of which were morphologically similar, including 10 that were Alternaria (83.3% isolation rate). Three representative isolates (HJYB1, HJYB2, and HJYB3) were selected for further study. The initial colonies were grayish green with white fluffy mycelia on the surface and a prominent white rim, which became brown with dense, cottony aerial mycelia as the colonies matured. The conidia were obpyriform or ellipsoidal, pale to dark brown, with 0-4 transverse and 0-3 longitudinal septa, some with a short cylindrical beak at the tip. They measured 11.826-28.873 × 6.231-26.018 µm (n = 100). To further confirm the identity of the isolates, their rDNA internal transcribed spacer region (ITS), ß-microtubulin (TUB2) and translation elongation factor-1 (TEF-1) genes were amplified and sequenced using the ITS4/ITS5, TUB2F/R and EF-526F/1567R primers, respectively (Hong et al. 2006). The sequences were submitted to GenBank (ITS: OR513924, OR513964, OR519874; TUB2: OR526928, OR533421, OR526929; TEF: OR526926, OR533420, OR526927). A concatenated phylogenetic tree of the three genes showed that the isolate clustered significantly with Alternaria alternata. Based on morphological identification and phylogenetic tree analysis, the isolate was identified as A. alternata. We carried out pathogenicity tests on four uniformly growing P. cyrtonema plants. Three of these plants were used as experimental plants and one as a control. For each plant, three young leaves were selected and inoculated with 6 × 6 mm PDA blocks, while sterile PDA blocks were used as controls. The treated plants were subjected to 10 days of stable temperature in a climatic chamber set at 28°C, 80% constant relative humidity and 12 hours of light per day. The pathogenic lesions appeared and the pathogens re-isolated from the diseased leaves showed similar morphological characteristics to representative isolates and were confirmed as A. alternata by DNA sequencing, thus fulfilling Koch's postulates. A. alternata is the major causal agent of leaf spot on P. sibiricum (Zou et al. 2023) and Agrimonia pilosa (Jiang et al. 2023). As far as we know, leaf necrosis caused by A. tenuissima has been found on P. cyrtonema (Li et al. 2020). To our knowledge, this is the first report of A. alternata causing leaf spot disease in P. cyrtonema. These findings form the basis for the management of this leaf spot disease.

[Diagnosis and treatment of traumatic optic neuropathy with internal carotid artery trauma].

Objective: To summarize our experience in the diagnosis of internal carotid artery trauma in patients with traumatic optic neuropathy, and to make recommendations for the treatment. Methods: The clinic data of 6 cases who had traumatic optic neuropathy with internal carotid artery trauma and who were admited in Department of Otorhinolaryngology, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University from Jan. 2013 to Dec. 2015 were analyzed retrospectively. Results: All 6 cases were monocular blindness. Four cases did not undergo nasal endoscopic optic nerve decompression because of the diagnoses of internal carotid artery trauma. One case was diagnosed after nasal endoscopic optic nerve decompression because of fatal bleeding during the operation. One case was diagnosed because of late-onset recurrent epistaxis. Among the 6 cases with internal carotid artery trauma, 3 cases were successfully treated with endovascular interventional treatment (stent embolization was used in one case, Coil embolization was used in two cases), and 3 patients refused treatment. Conclusions: The patients with traumatic optic neuropathy have the possibility of severe carotid artery trauma. Endoscopic optic nerve decompression is not suitable for these cases. It should pay more attention to patients with traumatic optic neuropathy. For suspected cases, vascular-enhanced computed tomography screening and digital subtraction angiography should be recommended and patients should be treated by endovascular intervention in a timely manner.

Identification and characterization of low temperature stress responsive genes in Poncirus trifoliata by suppression subtractive hybridization.

Trifoliate orange (Poncirus trifoliata (L.) Raf.) is extremely cold hardy when fully acclimated, but knowledge relevant to the molecular events underlying the acclimation is still limited so far. In this study, forward (4 °C over 25 °C) and reverse (25 °C over 4 °C) suppression subtractive hybridization (SSH) libraries were constructed in order to identify the genes involved in cold acclimation in trifoliate orange. After reverse northern blotting analysis and sequencing, a total of 105 and 117 non-redundant differentially expressed sequence tags (ESTs) were obtained from the forward and reverse libraries, respectively. Blast2go analysis revealed that 91 ESTs, 31 from the forward library and 60 from the reverse library, displayed significant sequence homology to the genes with known or putative functions. They were categorized into various functional groups, including catalytic activity, binding protein, structural molecule, enzyme regulator, molecular transducer, electron carrier, and transport activity/transcription regulation. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction was consistent with the results of differential screening. In addition, time-course expression patterns of the genes further confirmed that they were responsive to low temperature treatment. Among the genes of known functions, many are related to maintenance of cell wall integrity, adjustment of osmotic potential and maintenance of reactive oxygen species homeostasis, implying that these physiological processes might be of paramount significance in rendering protective mechanisms against the low temperature stress. The data presented here gain an insight into the molecular changes underlying the cold acclimation of trifoliate orange, and the results can be of reference for unraveling candidate genes that hold great potential for genetic engineering in an effort to create novel germplasms with enhanced cold stress tolerance.