First report of Fusarium verticillioides causing leaf rot on Artemisia argyi in China.

Artemisia argyi is a perennial herb native to East Asia. It is an important traditional Chinese medicinal plant known for its strong flavor and medicinal effects. It is rich in active ingredients and has a wide range of biological activities, including anti-inflammatory, antioxidant, and immune regulation properties. From May to July in 2023, a serious leaf rot outbreak occurred on A. argyi in several farms (approximately 200 acres) in Tanghe county (32°46'44" N, 112°43'13" E), Henan Province, China. The incidence rate reached 65% (n=200). Pale yellow spots (1-2 cm in diameter) first appeared on the leaves, then expanded to form irregular yellowish-brown lesions, eventually causing the entire leaves to wither. Diseased leaves (30) were collected and cut into 5 x 5 mm2 pieces in the areas between infected and healthy tissues. The excised plant tissues were sterilized in 75% ethanol and 1% sodium hypochlorite solution for 30 seconds and one minute, respectively. The tissues were then rinsed with sterile water and placed on potato dextrose agar (PDA) followed by incubating at 25 °C for 3 days. The isolated strains belonged to the genera Fusarium and Alternaria. After pathogenicity verification, 25 purified Fusarium strains were obtained. Three representative strains (AC-Q, AC-X, AC-Y) from different regions were used for further studies. Each strain formed abundant aerial mycelium that was initially white and later developed into purple pigments. Aerial conidiophores were sparsely branched, terminating with verticillate phialides. Macroconidia were slender, straight, and measured 21.8 to 47.5 × 3.1 to 4.4 µm, with two to four septa. Microconidia were clavate and measured 8.31 to 11.6 × 2.1 to 3.5 µm. Morphological characteristics were consistent with the species description of Fusarium verticillioides (Sacc.) Nirenberg 1976 (Leslie and Summerell, 2006). The rDNA internal transcribed spacer (ITS), ß-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase II largest subunit (rpb1) and RNA polymerase II second largest subunit (rpb2) were amplified for molecular identification (O'Donnell et al., 2022). The sequences were deposited in GenBank with accession Nos. OR960548, OR960552, OR960555 (ITS), OR972413, OR972414, OR972415 (tub2), OR797685, OR797686, OR797687 (tef1), OR972410, OR972411, OR972412 (cmdA), PP035106, PP035107, PP035108 (rpb1), and PP035109, PP035110, PP035111 (rpb2). BLASTn analysis of AC-Q sequences exhibited 99 to 100% similarity with F. verticillioides sequences (strains CBS 576.78) MT010888 of cmdA, MT0109566 of rpb1, and MT010972 of rpb2. A phylogenetic tree was constructed with concatenated sequences (tub2, tef1, cmdA, rpb1, rpb2), alongside the sequences of the type strains using the neighbor-joining method. The three strains formed a clade with the type strain CBS 576.78 of F. verticillioides, and were separated from other Fusarium spp. These morphological and molecular identifications indicated that the pathogen was F. verticillioides. Pathogenicity was tested on 10 healthy 2-month-old potted seedlings by spraying them with a conidial suspension (106 conidia ml-1), and 5 seedlings were sprayed with sterilized water as a control. The plants were placed in a climate incubator at 28°C and a relative humidity of approximately 90%. Ten days after seedling inoculation, typical lesions were observed on the treated plants, except in the control group. The reisolated strains were identified as F. verticillioides by morphological and molecular characterization, fulfilling Koch's postulates. F. verticillioides is known to cause Fusarium ear rot on maize, as well as diseases on other plants in China such as Brassica rapa (Akram et al., 2020) and Schizonepeta tenuifolia (Li et al., 2024). This is the first report of F. verticillioides causing leaf rot on A. argyi worldwide. Identification of the pathogen is crucial for implementing management approaches to reduce yield losses.

Occurrence of Fusarium fujikuroi Causing Fusarium Wilt on Carthusian Pink in China.

Carthusian pink (Dianthus carthusianorum) is native to Europe and is widely grown in China for landscaping. In September 2022, wilting symptoms of carthusian pink were found in Xixia City (33°18'31″ N, 111°29'45″ E), Henan Province, China, with a disease incidence of 65%. Approximately 100 plants were surveyed on the landscaping lawns of the park. Initial symptoms were yellow to brown lesions on the base of stems and leaves. Later, the lesions spread throughout the plants, turning leaves yellow, and leading to root and leaf rot. Eventually, the plants shriveled and died (Figure S1a). Thirty diseased tissues isolated from the roots and leaves were cut into 5×5 mm pieces, which were surface sterilized with 75% ethanol solution for 30 seconds and 1% NaClO solution for 1 minute, rinsed three times in sterilized water, placed on potato dextrose agar (PDA) plates supplemented with 50 µg ml-1 streptomycin, and incubated at 28°C for five days. A total of 25 purified fungal strains with similar phenotypic features were obtained. Three representative strains named OSZ-P1, OSZ-P2, and OSZ-P3 were selected for identification. Fungal colonies developed an abundant aerial mycelium, initially white, which subsequently developed red to purple pigments (Figure S1b). Macroconidia were slender, straight, and measured 12.74 to 49.39 × 2.07 to 4.39 µm (n=50), with two to five septa. Microconidia were clavate and measured 6.31 to 11.61 × 2.15 to 4.02 µm (n=50) (Figure S1c). These morphological characteristics were consistent with Fusarium spp.. The rDNA internal transcribed spacer (ITS), ß-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified with primers ITS1/ITS4, BT-2a/BT-2b, EF1/EF2, CL1/CL2A, Fa/G2R, and 5F2/7Cr, respectively, for further identification (Yilmaz et al. 2021, O'Donnell et al. 2022). ITS (OQ726389, OQ726390, OQ726391), tub2 (OQ730191, OQ789645, OQ789646), tef1 (OR088904, OR088905, OR088906), cmdA (OR133730, OR133731, OR133732), rpb1 (OR088907, OR088908, OR133729), and rpb2 (OR133733, OR133734, OR133735) nucleotide sequences of the strains OSZ-P1, OSZ-P2, and OSZ-P3 were submitted to GenBank. BLASTn analysis of OSZ-P1 sequences exhibited 99 to 100% similarity with Fusarium fujikuroi sequences (strains Augusto2, I1.3, and CSV1) CP023096, CP023108, CP023084 of cmdA, CP023089, CP023077 of rpb1, and CP023093, CP023105, CP023081 of rpb2. A Phylogenetic tree was constructed of combined genes (tub2, tef1, cmdA, rpb1, rpb2) of sequences, alongside the sequences of the type strains by the neighbor-joining method. The three strains formed a clade with the type strains CBS257.52 and Augusto2 of F. fujikuroi in phylogenetic trees, being clearly separated from other Fusarium spp. (Figure S2). The morphological features and molecular analyses supported the strains as members of F. fujikuroi. To verify the pathogenicity, aboveground parts of the plants of five healthy six-month-old potted plants were sprayed with 100 µl of conidial suspension per pot (106 conidia ml-1), and five similar plants were sprayed with sterilized water as a control. All plants were placed in a climate incubator at 28°C and 90% relative humidity. Seven days after inoculation, withered and yellowed lesions were observed, similar to the natural lesions (Figure S1e). No symptoms were observed on the control plants. The whole pathogenicity tests were performed thrice. Reisolation resulted in cultures that were morphologically and molecularly identical to the original isolates, fulfilling Koch's postulates. Fusarium wilt disease has been reported on other plants of the genus Dianthus. Vascular wilt on Dianthus caryophyllus (carnation) caused by Fusarium oxysporum is the most destructive disease of carnation crops worldwide (Ardila et al. 2014). Fusarium acuminatum causing Dianthus chinensis root rot and foliage blight has recently been reported in Nanjing, China (Xu et al. 2022). To our knowledge, this is the first report of F. fujikuroi causing Fusarium wilt on carthusian pink worldwide. The host range of F. fujikuroi still needs to be clarified for accurate disease management in the selection of plant species for landscape.

Principles and applications of green fluorescent protein-based biosensors: a mini-review.

Green fluorescent proteins (GFPs) are useful and essential biomolecules that have revolutionized biosensor research. Researchers have extensively utilized GFPs for designing fluorescence biosensors due to their intrinsic fluorescence, high stability, and ability to undergo permutation or mutation. This review provides a concise summary of recent advancements in developing GFP-based biosensors. The construction approaches for GFP-based biosensors can be categorized into four types: (1) single GFP-based biosensors; (2) fluorescence resonance energy transfer-based biosensors; (3) GFP-based split biosensors; and (4) GFP chromophore analogy-based biosensors. We highlight the applications of these sensors in biomolecule detection and life sciences, while also sharing personal insights into the challenges associated with GFP-based biosensors and proposing future research directions.

Gray Blight Disease on Euonymus japonicas Caused by Pestalotiopsis disseminata in China.

Euonymus japonicas is widely planted as an important landscape species throughout China. In June 2021, a serious gray blight disease was detected on E. japonicas in Henan Province (32°30'58" N, 112°19'44" E), causing severe defoliation of infected trees with a foliar disease incidence of 52 to 70% (n = 100). Gray spots initially appeared on leaves, gradually expanded into irregular white blotches with dark brown borders, eventually leading to wilting and death of the leaves. The junctions between the lesion and healthy tissue of infected leaves were cut into 3 × 3-mm pieces, surface sterilized with 1% NaClO solution for 1 min, rinsed in sterile water, and placed on PDA plates with 50 µg/ml of streptomycin. Three isolates (HY94, HY95, and HY98) were selected for subsequent experiments. The colonies reached 80-85 mm diam after 7 days at 25°C, with undulated margins, white to pale in color, with moderate aerial mycelium on the surface. Conidiomata were globose, solitary, and dark black. Conidia were ellipsoid, straight to slightly curved, 4-septate, 19 to 26.4 × 5 to 7.5 µm (n=100). The apical cell was cylindrical and hyaline, with 2 to 3 tubular apical appendages, unbranched, filiform, 2.5 to 3.5 µm in length. The basal appendage was single, unbranched, centric, 1.5 to 3 µm long. The characteristics were close to those of Pestalotiopsis spp. (Maharachchikumbura et al. 2013). The genomic DNA was extracted, and the rDNA internal transcribed spacer (ITS), the ß-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified by primers ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively (Carbone and Kohn, 1999). Sequences were submitted to GenBank with accession numbers OL840327-OL840329(ITS), OL961454-OL961456(TUB), and OL961448-OL961450 (TEF1). BLASTn analyses of ITS, TUB, and TEF1 sequences exhibited 99.46, 99.05, and 96.53% similarity to the sequences of Pestalotiopsis disseminata strain MEAN1166 (ITS, 548/551 bp; MT374688) (Silva et al. 2020), PSH2000I-066 (TUB, 418/422 bp; DQ333575), and TAP29O082 (TEF1, 250/259 bp; AB453850), respectively in GenBank. The three isolates formed a clade with the type strains, MEAN 1166 and MAFF238347 of P. disseminata in phylogenetic trees, being clearly seperated from other Pestalotiopsis spp. Based on morphological and molecular evidence, the pathogen was identified as P. disseminata (Maharachchikumbura et al. 2011). To fulfill Koch's postulates, pathogenicity was tested with three isolates. Ten healthy leaves of 5-year-old intact plants were used per isolate and inoculated with mycelial plugs on both nonwounded and wounded leaves. Control leaves were inoculated with agar plugs. The inoculated plants were placed at 28°C in a greenhouse (90% relative humidity). Distinct lesions were observed after 10 days. The pathogen reisolated was identical to that of the original cultures according to phenotype and ITS sequences. The control leaves showed no obvious symptoms. P. disseminata is known to cause disease on several important plants in China, such as Camellia japonica (Zhang et al. 2012), Pinus armandii (Hu et al. 2007), and Tripterygium wilfordii (Kumar et al. 2004). This is the first report of gray blight disease caused by P. disseminata on E. japonicas in China and worldwide. The fungal pathogen identification will provide valuable information for prevention and management of gray blight disease associated with E. japonicas.

Comparison of the actual diameter of capsulotomy and predicted diameter of capsulotomy after femtosecond laser-assisted capsulotomy.

PURPOSE: To compare the consistency between the actual diameter of capsulotomy (ADC) and the predicted diameter of capsulotomy (PDC) in femtosecond laser-assisted cataract surgery (FLACS) and analyze the factors that influence the deviation of the diameter of capsulotomy (DDC) between the actual and predicted. SETTING: Aier Eye Hospital of Wuhan University, China. DESIGN: Retrospective observational case series. METHODS: Patients who underwent FLACS from March 2020 to March 2021 were reviewed. The ADC in FLACS was measured and compared with the PDC. The effects of age, sex, and biometrics on DDC were analyzed. RESULTS: 412 eyes of 336 patients were included. The mean age was 53.0 ± 0.91 years (range 3 to 91 years). When the PDC was set to 4.50 mm, the results showed that the ADC was 5.21 ± 0.21 mm with a significant difference between them ( P < .05). However, when the PDC was set to 5.2 mm, the ADC was 5.10 ± 0.38 mm without a significant difference between them ( P > .05). No correlation ( P > .05) was found between the DDC and the axial length, the DDC and the anterior chamber depth (ACD), and the DDC and the mean keratometry (Km), but a negative correlation was found between the DDC and the lens thickness (LT) ( r = -0.21; P < .05) and the DDC with age ( r = -0.70; P < .05). Using curvilinear regression analysis, a development of an age-depending correction formula was predicted: ADC = PDC + 1.23 - 0.30 ln (x) (x = age ≥3) ( R2 = 0.65; F = 752.39; P = .00). CONCLUSIONS: The consistency of the ADC and PDC was influenced by age and LT. For patients aged 40 years or younger, the younger the patient, the wider the DDC; for patients older than 40 years, the DDC was small. The thicker the LT, the smaller the DDC.

[Atmospheric Dry Deposition Fluxes and Sources of Polycyclic Aromatic Hydrocarbons in Lanzhou Valley, Northwest China].

The atmospheric dry deposition flux pollution levels were determined and the sources apportioned for 15 US EPA priority polycyclic aromatic hydrocarbons (PAHs) collected at 13 sampling sites in the Lanzhou valley using passive sampling techniques. The results are as follows. The annual atmospheric dry deposition fluxes ranged from 7.48 to 53.94 µg·(m2·d)-1, with a mean of 18.65 µg·(m2·d)-1. The highest flux levels for both the heating and non-heating seasons, 60.85 µg·(m2·d)-1 and 47.03 µg·(m2·d)-1, respectively, appeared at the traffic site (Donggang Bridge), where the heaviest traffic in the Lanzhou valley occurs. The lowest flux levels of 8.16 µg·(m2·d)-1 and 6.80 µg·(m2·d)-1 for the heating and non-heating seasons, respectively, were found at Baita Mountain, which has a higher percentage of vegetation cover. Meanwhile, the flux levels at the background site (Guantangou Mountain) were significantly lower than those of the other sampling sites. For dry deposition, the PAH components Phe, Flua, Flu, and Pyr were dominant in the heating and non-heating periods, and the sums of the percentages of 3- and 4-ring PAHs were 87.53% for the heating season and 82.73% for the non-heating season. Moreover, the percentage of 5- and 6-ring PAHs for the non-heating period was higher than that for the heating period, which may be because the lighter PAHs were easily volatilized, and thus escaped. Furthermore, the sources of atmospheric dry deposition were identified by principal component analysis (PCA). The results of source apportionment showed that the main atmospheric PAH dry deposition in the Lanzhou valley were from vehicle exhaust emissions, coal combustion, and the coking industry. In further detail, coal combustion and the coking industry contributed most of the PAH dry deposition emissions during the heating period except for the traffic area site of Donggang Bridge, whereas vehicle exhaust was the dominant contributor during the non-heating season. In addition, the annual average dry deposition velocities of atmospheric PAHs were calculated using the dry deposition model for three sites: 0.20 cm·s-1 downtown (Environmental Protection Agency of Lanzhou; JCZ), 0.15 cm·s-1 in an industrial area (Lanyuan Hotel of Xigu; LLH), and 0.17 cm·s-1 in a traffic area (the Staff Hospital of Gansu Province; ZGH), of which the latter two were relatively lower because of comprehensive meteorological factors such as wind speed, temperature, and land use categories. Regarding the dry deposition flux values of the 3- and 4-ring PAHs, the simulated values were a bit larger than the observed values, but all were at the same level of magnitude. However, the simulated flux values were closer to their observed values for 4-ring PAHs than for 3-ring PAHs, which indicated that 3-ring PAHs were lost more easily than 4-ring PAHs were during monitoring.