First Report of Pseudopestalotiopsis ixorae Causing Leaf Spot of Photinia × fraseri in China.

Photinia × fraseri is a well-known ornamental shrub in southern China. In December 2021, we observed leaf spots that were circular to irregular, gray with dark red margins and violet brown with brownish violet edges on the leaves of Photinia × fraseri shrubs in the scenic area of Shenlongtan (28°46'10″N, 115°42'93″E), Jiangxi Province, China. Almost 15% of the leaves in the 1300 m2 Photinia × fraseri planting area were symptomatic. Thirty symptomatic leaves were randomly collected from different plants, and sectioned into 5-mm2 pieces, which were surface-sterilized using 1% NaOCl for 30 s. After rinsing thrice in sterile distilled water and drying, the pieces were transferred onto potato dextrose agar (PDA) and incubated at 28 ℃ for 5-7 days. A total of sixteen morphologically similar isolates were obtained. After incubation on PDA for 20 days, the fungi had irregular edges, were white to pale brown, and had spare aerial mycelium on the surface with irregularly distributed black, gregarious conidiomata. Conidia were fusoid, subcylindrical, straight to slightly curved, 4-septated, slightly constricted at the septa, and 23 to 36 × 6 to 10 µm (mean: 27.6 × 7.7 µm). The morphological characteristics were consistent with the features of Pseudopestalotiopsis species (Maharachchikumbura et al. 2014). The genomic DNA of two representative isolates (JFRL032 and JFRL033) was extracted for further identification. The internal transcribed spacer (ITS) region, translation elongation factor 1-ɑ (tef1-ɑ) and ß-tubulin (tub2) genes were amplified and sequenced using primers ITS5/ITS4, EF1-526F/EF1-1567R, and Bt2A/Bt2B, respectively (Maharachchikumbura et al. 2012). The sequences of the two representative isolates were 100% identical to each other. These nucleotide sequences were deposited in GenBank with accession numbers, ON342794 and ON342795 (ITS); ON375851 and ON375852 (tef1-ɑ); ON375853 and ON375854 (tub2). BLASTn searches of the obtained sequences revealed 99%-100% to ITS (MG816316, 478/478 nucleotides), tef1-ɑ (MG816336, 924/926 nucleotides), tub2 (MG816326, 441/442 nucleotides) sequences of the ex-type strain of Pseudopestalotiopsis ixorae (NTUCC17-001.1). Phylogenetic analysis was conducted using the concatenation of multiple sequences (ITS, tef1-ɑ and tub2) with the Maximum likelihood statistics in PhyloSuite v1.2.2 (Zhang et al.2020). The phylogenetic tree showed the two isolates clustered with P. ixorae in a clade with 100% bootstrap support. The isolates were identified as P. ixorae based on morphological and molecular data. To confirm pathogenicity, eight healthy leaves of 3-year-old Photinia × fraseri were surface sterilized, scratched with a pair of sterilized tweezers, and ten µl of conidial suspension (106 conidia/ml) was sprayed on the injured leaves and the control was sprayed with sterile distilled water. Then, All plants were potted in a climate chamber at 25℃ and 85% relative humidity. After 3 days, leaf spot symptoms similar to those described above were observed on inoculated leaves, while the non-inoculated leaves remained symptomless. The pathogen was reisolated from the inoculated leaves to fulfill Koch's postulates and confirmed as P. ixorae by morphological and molecular analysis. It has been reported that P. ixorae can infect the Ixora plant (Tsai et al., 2018). To the best of our knowledge, this is the first report of P. ixorae causing leaf spot on Photinia × fraseri in China. The study provides valuable information for identifying and controlling the leaf spot on Photinia × fraseri.

First Report of Anthracnose Caused by Colletotrichum spaethianum on Zephyranthes candida in China.

Zephyranthes candida, an bulbous perennial plant, are planted in almost every park. In October 2023, anthracnose symptoms were observed on Z. candida leaves in Jiangxi Agricultural University (28.75° N, 115.83°E), Nanchang, Jiangxi Province, China, and the incidence of disease were up to 35% (140 of 400 plants). The lesions extended from the leaf apex to the base, appearing as a dark brown color, and later changed to yellow and became dry. To isolate the pathogen, 20 symptomatic leaves were collected and cut into small pieces (4×4 mm, one pieces per leave), surface-sterilized with 70% ethanol for 10 s and 1% NaClO for 30 s, rinsed thrice with sterile water, placed onto potato dextrose agar (PDA) plates and incubated at 25℃ for 5 days. Fifteen isolates (15 out of 20) with similar morphological characteristics were obtained. The colonies on PDA presented effuse mycelium, initially white and later pale gray. Conidia were hyaline, curved or slightly curved, aseptate, with a truncate base and acute apex, measuring 17 to 23 × 3 to 6 µm (n = 50), and were matched to Colletotrichum species (Damm et al. 2009). To further confirm species, two representative isolates (JFRL 03-2873 and JFRL 03-2874) were selected for molecular identification. The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS), histone 3 (HIS3), actin (ACT) and ß-tubulin 2 (TUB2) regions were amplified and sequenced by using primers sets ITS5/ITS4, Gpd1/Gpd2, CHS-79F/CHS354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R and T1/Bt2b (Tan et al. 2022), respectively. These sequences were deposited into GenBank with accession number PP425890-PP425891 (ITS), PP437551-PP437552 (GAPDH), PP437549-PP437550 (CHS), PP480643-PP480644 (HIS3), PP437547-PP437548 (ACT) and PP437553-PP437554 (TUB2). A BLASTN search revealed high similarity of 99%-100% to ITS (GU227807, 518 nt/519 nt), GAPDH (GU228199, 525 nt/526 nt), CHS (GU228297, 251 nt/251 nt), HIS3 (GU228003, 372 nt/373 nt), ACT (GU227905, 236 nt/237 nt) and TUB2 (GU228101, 490 nt/490 nt) sequences of Colletotrichum spaethianum CBS 167.49. A maximum likelihood phylogenetic tree was constructed by combining ITS, GAPDH, CHS , HIS3, ACT and TUB2 sequences in IQtree web server (Ngugen et al. 2015). The result indicated that the two representative isolates were clustered together with Colletotrichum spaethianum in a clade with 100% bootstrap support. Based on morphological observation and sequence analysis, the isolates were identified as C. spaethianum. To confirm pathogenicity, six surface-sterilized leaves of Z. candida were wounded and inoculated with 1 × 106 conidia/ml conidial suspension of JFRL 03-2873, and control leaves were inoculated with sterile water. They were incubated at 25 ℃ with 12 h photoperiod and 80% humidity, the experiment was repeated twice. After five days, all leaves inoculated with JFRL 03-2873 displayed anthracnose symptom, whereas the control leaves remained unaffected. We re-isolated C. spaethianum from the symptomatic leaves and identified it based on morphological and molecular characteristics. Previous studies reported that C. spaethianum caused anthracnose on various common herbaceous plants in China (Vieira et al. 2014, Guo et al. 2013), but to our knowledge, this is the first report of C. spaethianum causing anthracnose on Z. candida in China. Anthracnose disease caused great economic loss to the cultivation of landscape plant Z. candida. Therefore, it is necessary to pay more attention to the anthracnose disease of herbaceous plants caused by C. spaethianum and develop appropriate control strategies.

First Report of Botryosphaeria dothidea Causing Postharvest Fruit Rot of Plum in China.

The Nai plum (Prunus salicina var. cordata cv. Younai) holds significance as an important deciduous fruit crop in China. In July 2023, symptoms of postharvest fruit rot were observed on Nai plum with a 10% disease incidence of harvested fruits in three supermarkets, located in Nanchang City, Jiangxi Province, China. Infected fruits displayed brown, circular lesions, accompanied by a transition in the surrounding peel color from cyan to red. To investigate the causal agent, small sections (3 to 4 mm2) from the periphery of ten infected fruits were subjected to surface sterilization using 75% ethanol for 30 seconds. Following sterilization, the samples were rinsed three times with sterilized distilled water, air-dried, and aseptically placed on potato dextrose agar (PDA) at 25 ℃ for 3 days. Isolated colonies were subcultured by hyphal tip transfer. Ten of the resulting 12 fungal isolates showed similar morphological characteristics. The colonies exhibited an initial white hue, gradually transitioning to gray, and featured short and thick aerial hyphae with an irregular colony margin. Microscopic examination revealed conidiogenous cells that were hyaline, aseptate, and narrowly fusiform. The conidia were measured 11.0 to 15.6 × 3.2 to 4.9 µm (x̅ = 13.5 ± 1.4 × 4.0 ± 0.4 µm, n = 30), and were hyaline and subcylindrical. The morphological characteristics were in accordance with those of the Botryosphaeria species (Crous et al. 2006). To identify the strain, two representative isolates, JFRL03-1792 and JFRL03-1793, were selected for further characterization. Amplification of nucleotide sequences from three regions (ITS, TEF1-a and TUB2) was conducted using the primer sets ITS5/ITS4, EF1-728F/EF1-986R, and BT2A/BT2B, respectively (Guo et al. 2023). The resulting sequences were deposited in GenBank under the accession numbers: OR418373 and OR418374 for ITS; OR424405 and OR424405 for TEF1-a; OR424411 and OR424412 for TUB2. A BLASTN homology search of the obtained sequences revealed a high similarity of 99%-100% to the ITS (AY236949, 511/513 nucleotides), TEF1-a (AY236898, 282/282 nucleotides), and TUB2 (AY236927, 431/431 nucleotides) sequences of Botryosphaeria dothidea CWM8000 (ex-type). Maximum likelihood analyses were performed for the combined ITS, TEF1-a, and TUB2 dataset using Phylosuite V1.2.2 (Zhang et al. 2020). The resulting phylogenetic tree indicated that the two representative isolates were clustered together with Botryosphaeria dothidea in a clade with 95% bootstrap support. Based on the comprehensive assessment of morphological and molecular data, the two isolates were conclusively identified as B. dothidea. To confirm pathogenicity, six healthy Nai plum fruits were surface sterilized with 75% ethanol and were subsequently wounded with a sterile needle. A 5-mm-diameter mycelial plug of the isolate JFRL03-1792, cultured on PDA at 25 ℃ for three days, was applied to the wound. Another set of six fruits was inoculated with sterile agar plugs as control. Following incubation in a climatic chamber at 25 ℃ and 80% relative humidity, the fruits were examined after 5 days. The experiment was repeated twice. The fruits inoculated with B. dothidea displayed typical rot symptoms, while the control fruits remained asymptomatic. In adherence to Koch's postulates, the fungus was successfully re-isolated from the inoculated fruits and confirmed as B. dothidea through morphological and molecular analysis. B. dothidea has previously been reported causing fruit rot on kiwifruit, winter jujube, and apple (Tang et al. 2012; Zhou et al. 2015; Marsberg et al. 2017; Xu et al. 2023). In addition, B. dothidea also reported causing Botryosphaeria canker disease on plum (Lin et al. 1994). But to our knowledge, this is the first documentation of B. dothidea causing postharvest fruit rot on plum in China. This discovery imparts critical insights into the management of this high-risk disease affecting plum in China.

DUSP4 modulates RIG-I- and STING-mediated IRF3-type I IFN response.

Detection of cytosolic nucleic acids by pattern recognition receptors, including STING and RIG-I, leads to the activation of multiple signalling pathways that culminate in the production of type I interferons (IFNs) which are vital for host survival during virus infection. In addition to protective immune modulatory functions, type I IFNs are also associated with autoimmune diseases. Hence, it is important to elucidate the mechanisms that govern their expression. In this study, we identified a critical regulatory function of the DUSP4 phosphatase in innate immune signalling. We found that DUSP4 regulates the activation of TBK1 and ERK1/2 in a signalling complex containing DUSP4, TBK1, ERK1/2 and IRF3 to regulate the production of type I IFNs. Mice deficient in DUSP4 were more resistant to infections by both RNA and DNA viruses but more susceptible to malaria parasites. Therefore, our study establishes DUSP4 as a regulator of nucleic acid sensor signalling and sheds light on an important facet of the type I IFN regulatory system.

First Report of Leaf Spots Caused by Pestalotiopsis nanjingensis on Photinia × fraseri in China.

Photinia × fraseri Dress, belonging to the Rosaceae family, is widely cultivated as an ornamental plant in China. In July 2022, the leaf spot symptoms were observed on over thirty P. × fraseri plants in an approximately 2-hectare park in Xinjian District, Nanchang City, Jiangxi Province, China (28°43'02″ N, 115°44'01″ E), with a disease incidences of roughly 10% . At first, small, grayish-white lesions appeared on the leaf edges, later expanding into 2 to 10 mm circular or irregular spots. These spots turned grayish-white to brown, with dark brown margins. Eventually, some lesions' centers dried and died. For fungal isolation, ten symptomatic leaves were randomly collected. The edges between the diseased and healthy tissues were cut into small pieces (4 × 4 mm). These pieces were then surface-sterilized by dipping in 70% ethanol for 30 s and 1% NaClO for 30 s. Subsequently, they were rinsed three times with sterile distilled water. Leaf pieces were then transferred to potato dextrose agar (PDA) medium and incubated at 25 °C for 3-4 days. Eight isolates with similar colony morphology were collected from diseased leaves. Colonies of this fungus on PDA were nearly round, white, and had sparse aerial mycelium on the surface with black, gregarious conidiomata. The conidia were nearly cylindrical, smooth, hyaline, and 4-septate, measuring 16.7 to 24.3 × 4.2 to 6.6 µm (mean 20.9 × 5.3 µm, n=50). The three middle cells were smooth, doliiform, and brown, with concolorous septa that were darker than the rest of the cell. They measured 11.8 to 17.0 µm long (mean 14.1 µm, n=50). The basal and apical cells were triangular and transparent. The basal cells had a mean length of 4.7 µm and were equipped with a basal appendage, while the apical cells had two appendages with a mean length of 17.7 µm(n=50). The characteristics of these isolates match those of Pestalotiopsis species (Maharachchikumbura et al. 2014). To identify them accurately, three representative isolates, namely JFRL 03-161, JFRL 03-162, and JFRL 03-226, were selected for further analysis. The internal transcriptional spacer (ITS) region, ß-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1-α) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), BT2a/BT2b (Glass and Donaldson 1995), and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. All sequences (ITS: OR342044-OR342046, TUB2: OR343299-OR343301, and TEF1-α: OR343302-OR343304) were deposited in GenBank. A BLASTn homology search revealed 99-100% identity to Pestalotiopsis nanjingensis CSUFTCC16 (ex-type). The sequences included ITS (OK493602, 486/486 nucleotides), TUB2 (OK562377, 438/439 nucleotides), and TEF1-α (OK507972, 478/478 nucleotides). The maximum likelihood analyses were performed for the combined ITS, TUB2 and TEF1-α data sets using IQtree web server (Trifinopoulos et al. 2016). The resulting phylogenetic tree demonstrated a strong association: the three isolates clustered tightly with P. nanjingensis forming a clade with robust 99% bootstrap support. This clustering, consistent with both morphological and molecular characteristics, confirmed the identity of the fungus as P. nanjingensis. To evaluate its pathogenicity, we obtained 3-year-old P. × fraseri 'Red Robin' plants, which were purchased then potted in a controlled climate chamber. We surface sterilized six healthy leaves of P. × fraseri with 70% ethanol and created wounds using a sterile needle. Subsequently, we inoculated a 50 µL conidial suspension (1 × 106 conidia/mL) of the isolate JFRL 03-161 on these wounded leaves. In parallel, another six leaves from P. × fraseri were inoculated with sterile distilled water, serving as the control group. All potted plants were incubated under conditions of 26 °C and 80% humidity. After seven days, all leaves inoculated with isolate JFRL 03-161 displayed symptoms similar to those observed in the field, whereas the control leaves remained unaffected. To fulfill Koch's postulates, we re-isolated P. nanjingensis plants from the symptomatic leaves and identified it based on morphological and molecular characteristics. It has been reported that two species of Pestalotiopsis, namely P. microspora and P. trachicarpicola can caused damage to the leaves of P. × fraseri in China (Xu et al. 2022; Zhu et al. 2021). However, to our best knowledge, this is the first report on leaf spot caused by P. nanjingensis on P. × fraseri in China. Therefore, it is necessary to pay more attention to the leaf spot disease of P. × fraseri caused by Pestalotiopsis species and develop appropriate control strategies.

Real-Time Monitoring of the Level and Activity of Intracellular Glutathione in Live Cells at Atomic Resolution by 19F-NMR.

Visualization and quantification of important biomolecules like glutathione (GSH) in live cells are highly important. The existing methods are mostly from optical detection and lack of atomic resolution on the activity of GSH. Here, we present a sensitive 19F-NMR method to quantify real-time variations of GSH in live cells in a reversible manner. This NMR method prevents extracellular leakage and irreversible consumption of intracellular GSH during the detection. The high performance of the reactive 19F-probe enables accurate determination of intracellular GSH content at atomic resolution, from which information on GSH variations with respect to the extracellular and intracellular conditions can be inferred. In addition, we demonstrate the applicability of this NMR method to quantify the GSH levels between different live cell lines and to disclose the distinct differences between the intracellular environment and cell lysates. We foresee the application of 19F-NMR to monitor real-time variations of intracellular GSH levels in relation to GSH-involved central cellular processes.

First Report of Lasiodiplodia pseudotheobromae Causing Postharvest Fruit Rot on Indian Jujube in China.

Indian jujube (Ziziphus mauritiana Lamarck), is one of the most popular fruit crops in South China. In March 2023, a fruit rot of indian jujube with about 5% disease incidence was observed in two supermarkets of Nanchang City, Jiangxi Province, China. Initially, the symptoms appeared as slightly brown spots on the fruit surface, with disease progression, the lesions gradually expanded and covered with a layer of hyphae. Small pieces (3 to 4 mm2) from the periphery of 15 diseased fruit were surface disinfected using 1% sodium hypochlorite for 30 s, rinsed three times in sterilized distilled water, air dried, and then aseptically placed onto potato dextrose agar (PDA) media and incubated at 25°C for three days. A total of ten single spore isolates with similar morphology were obtained. Colonies of these consisted of initially white, gradually turning gray and eventually becoming black, and aerial hyphae were dense and fluffy. Conidiogenous cells were smooth, hyaline, cylinder-shaped, and holoblastic. Conidia were ellipsoidal, top and base-rounded, and thick-walled, immature conidia were colorless, hyaline, and aseptate, compared with dark brown color of the mature conidia, which were one-septate with longitudinal striations, ranging in size from 22.8 to 31.8 (mean 27.6) µm in length and 12.2 to 20 (mean 14.6) µm in width. The morphological characteristics were consistent with the characteristics of the Lasiodiplodia species (Phillips et al. 2013). To accurately identify the strain, three representative isolates, namely JFRL 03-1147, JFRL 03-1148, and JFRL 03-1149, were selected for further identification. The internal transcribed spacers (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB2) genes/regions were amplified and sequenced using primers ITS1/ITS4, EF1-688F/EF1-1251R, and Bt2a/Bt2b, respectively (Chen et al. 2021). These nucleotide sequences were deposited in GenBank with accession numbers OQ804425-OQ804427 (ITS), OQ818097-OQ818099 (TEF1-α), and OQ818100-OQ818102 (TUB2). A BLASTn homology search for these nucleotides showed 99-100% identity to ITS (EF622077, 487 nt/487 nt), TEF1-α (EF622057, 306 nt/307 nt) and TUB2 (EU673111, 434 nt/434 nt) sequences of Lasiodiplodia pseudotheobromae CBS 116459 (ex-type). The maximum likelihood analyses were performed for the combined ITS, TEF1-α and TUB2 data set using IQtree web server (Trifinopoulos et al. 2016). The phylogenetic tree showed that the three isolates clustered with Lasiodiplodia pseudotheobromae in a clade with 99% bootstrap support. Therefore, the fungus was identified as L. pseudotheobromae based on morphological and molecular characteristics. To evaluate pathogenicity, 4 healthy fruits of indian jujube were surface sterilized with 75% ethanol and wounded by sterile needle, and a 5-mm-diameter agar with 5-days-old mycelium of the isolate JFRL 03-1148 cultured on PDA at 25°C was put on the wound. Another set of 4 fruits was inoculated with sterile agar plugs as controls. The fruits were cultured at 25℃ and 85% relative humidity, and the test was repeated twice. These fruits inoculated with L. pseudotheobromae showed similar rot symptoms after 3 days, while the control group remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as L. pseudotheobromae by morphological and molecular analysis. L. pseudotheobromae has previously been reported causing fruit rot on citrus, mango and papaya (Alam et al. 2021; Chen et al. 2021; Netto et al. 2014). But to our knowledge, this is the first report of L. pseudotheobromae caused postharvest fruit rot on indian jujube in China. Therefore, managers should pay more attention to postharvest fruit rot disease caused by L. pseudotheobromae, and formulate appropriate disease control measures to reduce its losses.

First Report of Anthracnose Caused by Collectotrichum higginsianum on Rumex crispus in China.

Rumex crispus L. is a perennial herb with medicinal properties derived from its roots and whole plant (Bhandari et al. 2022). In December 2022, symptoms of anthracnose were observed in approximately 40% of naturally occurring R. crispus plants in Longquan Reservior, Nanchang city (115°53' N, 28°43' E), Jiangxi Province, China. Initially, red lesions appeared randomly on various parts of the leaf blade, which gradually became dry and brown at the center, eventually leading to leaf death. To isolate the fungal pathogen responsible, ten symptomatic leaves were randomly collected and their lesions were cut into small pieces (4 × 4 mm). The leaf fragments were surface-sterilized in 70% ethanol for 45 s and then in 1% NaClO for 45 s. The leaf pieces were rinsed three times with sterile distilled water. The surface-sterilized leaf pieces were then placed onto potato dextrose agar (PDA) and incubated at 28 ℃, dark condition for 3 days. Twelve isolates were obtained, characterized by a milky white and uneven growth pattern with a white root-like structure branching out at the edge, along with scattered black deposits on the bottom of the plate. Conidiogenous cells cylindrical, smooth-walled, hyaline, 9.3-23.2 × 3.6-4.2 µm. Conidia elliptical, aseptate, smooth-walled, with one end blunt and the other truncate, ranging in size from 10.4 to 22.3 (mean 16.7) µm in length and 3.2 to 5.0 (mean 4.1) µm in width (n = 50), which are consistent with the characteristics of the members of Colletotrichum destructivum species complex (Damm et al. 2014). To accurately identify the strain, three representative isolates, namely JFRL 03-930, JFRL 03-931, and JFRL 03-935, were selected for further identification. The internal transcribed spacer (ITS) region, actin (ACT), chitin synthase (CHS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GADPH), histone3 (HIS3), and beta-tubulin (TUB2) genes were amplified and sequenced using specific primer pairs, including ITS5/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, GDF1/GDR1, GYLH3F/CYLH3R, and T1/Bt2b (Damm et al. 2014). All sequences were deposited in GenBank with accession numbers OQ560476-OQ560478 (ITS), OQ576154-OQ576156 (ACT), OQ576157-OQ576159 (CHS), OQ576160-OQ576162 (HIS3), OQ576163-OQ576165 (GADPH), and OQ576166-OQ576168 (TUB2). A maximum likelihood phylogenetic tree was constructed using IQtree v1.5.6 based on the combined ITS, ACT, CHS, GAPDH, HIS3 and TUB2 data set (Nguyen et al. 2015), The phylogenetic tree showed that the three isolates clustered with C. higginsianum in a clade with 91% bootstrap support. Based on morphology and molecular characters, the isolates were identified as C. higginsianum of the C. destructivum species complex. To confirm the pathogenicity, One-year-old R. crispus were collected from the wild and potted in an climate chamber. Six healthy leaves of R. crispus were surface sterilized with 70% ethanol and wounded by sterile needle, and a 20-µl conidial suspension (3×105 conidia/ml) of the isolate JFRL 03-931 was inoculated on the wound. Another set of six leaves of R. crispus was inoculated with distilled water as controls. The potted plants were incubated under conditions of 25 ℃ and 80% humidity. After 10 days, reddish brown spots were observed on all inoculated leaves, while the control leaves remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated leaves and confirmed as C. higginsianum by morphological and molecular analysis. It has been reported that C. higginsianum caused anthracnose disease on several cruciferous vegetables, Boehmeria nivea and Rumex acetosa in China (Damm et al. 2014; Wang et al. 2011; Patel et al. 2014; Zhang et al. 2018). But to our knowledge, this is the first report of C. higginsianum casued anthracnose on Rumex crispus in China. Therefore, we should pay more attention to this pathogen and develop appropriate control strategies.

First Report of Leaf Blotch Caused by Phyllosticta capitalensis on Daphne odora in China.

Daphne odora Thunb. an evergreen shrub with scented flowers, is used for ornamental purposes but it also has medicinal benefits (Otsuki, et al. 2020). In August 2021, leaf blotch symptoms were observed on roughly 20% of leaves of D. odora var. marginata plants in Fenghuangzhou Citizen Park, Nanchang city (28°41'48.12″ N, 115°52'40.47″ E), Jiangxi Province, China. Brown lesions first appeared on the edges of leaves, which eventually dried and died (Fig. 1A). For fungal isolation, 12 symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface-sterilized by dipping in 70% ethanol for 10 s and 1% sodium hypochlorite for 30 s, and then rinsed three times with sterile distilled water. Leaf pieces were then plated on potato dextrose agar (PDA) and incubated at 28 ℃ for 3-4 days. A total of 10 isolates were recovered from the diseased leaves. The pure colonies of all fungal isolates had similar characteristics, and three isolates were randomly selected (JFRL 03-249, JFRL 03-250 and JFRL 03-251) for further study. Colonies of this fungus were gray and uneven, with a granular surface, and irregular white edges, finally turning black on PDA (Fig. 1B, C). Pycnidia were black, globose and 54-222 µm in diameter (Fig. 1D). Conidia were hyaline, single-celled, and nearly elliptical, which ranged from 7 to 13 × 5 to 7 µm (n=40) (Fig. 1E). These morphological characteristics were consistent with those described for the fungus Phyllosticta spp. (Wikee et al. 2013a). To confirm the fungal identity, the internal transcribed spacer (ITS) region, actin (ACT), translation elongation factor 1-alpha (TEF1-a), glyceradehyde-3-phosphate dehydrogenase (GPD) and RNA polymerase II second largest subunit (RPB2) genes were amplified using primers ITS5/ITS4, ACT-512F/ACT-783R, EF-728F/EF2, Gpd1-LM/Gpd2-LM and RPB2-5F2 /fRPB2-7cR, respectively (Wikee et al. 2013b). The sequences of the selected isolates were 100% identical. Hence, sequences of one representative isolate JFRL 03-250 were deposited in GenBank (OP854673, ITS; OP867004, ACT; OP867007, TEF1-a; OP867010, GPD; and OQ559562, RPB2). BLAST search analysis in GenBank showed 100% similarity with those of P. capitalensis (GenBank accession nos. ITS, MH183391; ACT, KY855662; TEF1-a, KM816635; GPD, OM640050 and RPB2, KY855820). From a phylogenetic perspective, a maximum likelihood phylogenetic tree was constructed by using IQtree V1.5.6 based on multiple sequences (ITS, ACT, TEF1-a, GPD and RPB2) (Nguyen et al. 2015), and the cluster analysis resulted the representative isolate JFRL 03-250 within a clade comprising Phyllosticta capitalensis (Fig. 2). Based on morphological and molecular characters, the isolate was identified as P. capitalensis. To confirm pathogenicity and fulfill Koch's postulates, 6 healthy potted plants were inoculated with 1× 106 conidia/ml suspension of isolate JFRL 03-250 by spraying on the leaves, whereas 6 plants were sprayed with sterile distilled water to serve as control. All potted plants were incubated at 28°C, 80% relative humidity and 12-h light/12-h dark alternating conditions in a climate cabinet. After 15 days, similar symptoms were observed in the inoculated leaves as in the field (Fig. 1F), whereas control leaves remained asymptomatic (Fig. 1G) and P. capitalensis was successfully re-isolated from the symptomatic leaves. Previously, P. capitalensis has been reported to cause brown leaf spot disease of various host plants around the world (Wikee et al. 2013b). However, to our knowledge, this is the first report of brown leaf spot caused by P. capitalensis on D. odora in China.

Stabilizing Nitroxide Spin Labels for Structural and Conformational Studies of Biomolecules by Maleimide Treatment.

Nitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the solo thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long-time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron-electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu.

First Report of Postharvest Fruit Rot on Gannan Navel Orange Caused by Diaporthe unshiuensis in Jiangxi Province, China.

The Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) is one of the most widely planted citrus fruit cultivars in Ganzhou City, Jiangxi Province, China. A Gannan navel orange was harvested from an orchard in Yudu County, Ganzhou City, Jiangxi Province, China, in October 2022 (25.95N, 115.41E). Approximately 5% of the fruit rotted after being stored at room temperature for about two weeks.Infected fruits appear brown and rotted with slightly indented edges. Initially symptoms of infected fruits was small circular, light brown, which the rot expands, slightly water-stained halo circle with slightly indented edges. The surface of 10 infected fruits was sterilized with 75% ethanol, and the lesion edge was cut into 5-mm-diameter pieces, and the pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for five days. A total of eight morphologically similar isolates were obtained. PDA results showed dense white and fluffy aerial mycelia in the center of colonies with sparser edges. Two types of conidia were produced; the alpha conidia were hyaline, ellipsoidal or clavate, and aseptate, with 2 oil drops, 4.8 to 7.5 × 2.1 to 2.7 µm (n=30). The beta conidia were hyaline, aseptate, filiform, smooth, straight to sinuous, 16.9 to 27.5 × 1.3 to 1.6 µm (n=30). These isolates exhibit morphological characteristics similar to those of the Diaporthe genus. Genomic DNA of two representative isolates (JFRL-03-1130 and JFRL-03-1131) was extracted for further confirmation. The internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS3) genes were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b, respectively (Udayanga et al. 2015). These nucleotide sequences were deposited into the GenBank database with accession numbers OQ691637-OQ691638 (ITS), OQ701022-OQ701023 (TUB), OQ701016-OQ701017 (CAL), OQ701018-OQ701019 (TEF1-α) and OQ701020-OQ701021 (HIS3). The maximum likelihood analyses were performed for the combined ITS, TEF1-a, TUB, HIS3, and CAL data set using Phylosuite V1.2.2 (Zhang et al. 2020). The phylogenetic tree showed that the two isolates clustered with D. unshiuensis in a clade with 100% bootstrap support. Therefore, the fungus was identified as D. unshiuensis based on morphological and molecular characteristics. To evaluate pathogenicity, a sterile scalpel was used to wound 10 surface-sterilized fruits, and a 5-mm-diameter mycelial plug of the isolate JFRL 03-1130, cultured on PDA at 25℃ for 7-days, was put on the wound. Another set of 10 fruits was similarly inoculated with sterile agar plugs as controls. The fruits were cultured at 25°C and 85% relative humidity, and the test was repeated twice. These fruits inoculated with D. unshiuensis showed similar rot symptoms after 10 days, while the control group remained symptomless. In order to prove Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as D. unshiuensis by molecular techniques, but never from the control fruits. Diaporthe unshiuensis has been reported as an endophyte associated with citrus and a pathogen that causes melanose disease in citrus (Chaisiri et al. 2020; Huang et al. 2015). However, to the best of our knowledge, this is the first reported case of D. unshiuensis causing postharvest fruit rot on Citrus sinensis. In the past, D. sojae has also been reported causing postharvest fruit brown rot disease on Citrus sinensis in China (Xiao, et al. 2023); Therefore, managers should pay more attention to postharvest fruit rot disease caused by Diaporthe species and implement storage strategies to control and reduce losses.

First Report of Diaporthe fusicola Causing Postharvest Fruit Rot of Peach (Prunus persica) in China.

In May 2022, rot symptoms were observed on postharvest peach (Prunus persica [L.] Batsch) fruits in a market in Nanchang, Jiangxi province (28°44' N; 115°50' E), China. A total of 80 samples were collected from three different fruit stalls through the market survey. The incidence of this disease was 10 to 15%, and severity varies from approximately 30 to 50% of fruit surface coverage. The symptom of infected fruits was circular, pale brown to brown, rotten, necrotic lesions, covered with white hyphae and small spore masses. Eight symptomatic peach fruits were surface disinfected with 75% ethanol for 30 sec and incisions were made with a sterile scalpel. Small pieces from symptomatic tissues were placed on a potato dextrose agar (PDA) medium and incubated at 25℃ for 7 days. Six isolates were obtained in total. Colonies on PDA were initially white, aerial, fluffy at first, and darkened with age. Alpha conidia were fusoid, hyaline, aseptate, guttulate, tapering towards ends, and ranged in size from 9.8 to 5.1 µm × 3.2 to 2.1 µm (x ̅=7.1 ± 1.0 × 2.6 ± 0.3 µm, n=60). Beta conidia were not seen. For further confirmation, genomic DNA was extracted from three isolates (04-10, 04-11, and 04-12), the internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α) and histone H3 (HIS) genes were amplified by using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, CYLH3F/H3-1b (Udayanga et al. 2015), respectively. Sequences were deposited in GenBank (Accession Nos. ON994257 to ON994259 for ITS, OP076824 to OP076826 for TUB, OP076827 to OP076829 for CAL, OP076821 to OP076823 for TEF1-α, OP076830 to OP076832 for HIS). BLAST results showed that ITS and TEF1-α have 99.8% pairwise identity to Diaporthe fusicola (MN816432, KF576256), and the TUB, CAL, and HIS sequences also have 100% pairwise identity to D. fusicola (KF576287, MT978147, MT978142). Phylogenetic analyses of concatenated sequences using Bayesian inference and the maximum likelihood confirmed the identity. To verify Koch's postulates, the pathogenicity of three isolates was tested on harvested healthy peach fruits. Five surface-sterilized fruits were wounded by a sterile scalpel and inoculated with 5-mm-diameter mycelial plugs from 10-day-old PDA plates. Another set of five fruits was inoculated with sterilized PDA plugs as controls. All fruits were incubated at 26℃ with 80% relative humidity. The experiment was repeated three times. After 5 days, the fruit inoculated with mycelial plugs showed pale brown lesions with whitish mycelium mass, similar to the previous rot symptoms, whereas the control fruit remained symptomless. The same pathogen was reisolated from the inoculated fruit with symptoms and identified as D. fusicola by molecular techniques, but never from the control. Diaporthe fusicola (Diaporthe amygdali complex) was first described on leaves of Lithocarpus glabra in China (Gao et al. 2015) and reported as an agent causing leaf blotch on Osmanthus fragrans (Si et al. 2020) and pear shoot canker (Guo et al. 2020). However, this is the first report of D. fusicola causing postharvest fruit rot on peach. The managers involved must consider the impact of this disease and develop an effective fruit storage strategy.

First Report of Brown Rot Caused by Phytophthora nicotianae on Navel Orange Fruit in China.

Navel orange (Citrus sinensis Osbeck cv. Newhall) is widely planted in southern China. From September to November 2021, severe outbreaks of Phytophthora brown rot were observed on navel orange fruit in three local orchards in Ganzhou City (28.80N, 115.53E), Jiangxi Province, China, with a disease incidence of 25 to 35%. Symptomatic fruit was mostly observed 1-m from the ground. Initial symptoms on infected fruit were circular, pale-brown to brown, water-soaked, slightly sunken lesions, covered with sparse white mycelia-like growth. As the disease progressed, the lesions turned dark brown and enlarged on the fruit surface. Three to four infected fruits were randomly collected from each orchard, placed in transparent plastic bags and immediately brought back to the laboratory for isolations. Infected fruits were surface-disinfested with 70% ethanol for 60 sec, and rinsed three times with sterile water. Symptomatic tissues from the margin between necrotic and healthy tissues were cut into 5 mm × 5 mm pieces, placed onto potato dextrose agar and incubated at 28°C for 5 days. Nine isolates were obtained. Colonies of three isolates (JFRL 03-16, 03-18, 03-19) in 10-day-old 20% V8 juice agar consisted of abundant, white, cottony aerial mycelia. Hyphal swellings and coenocytic mycelium were observed. Sporangia were ovoid, ellipsoid to spherical, papillate, and ranged in size from 17.2 to 60.1 µm × 15.8 to 48.6 µm (x ̅=46.2 ± 5.5 × 32.4 ± 4.8 µm, n=50). Chlamydospores were spherical, suborbicular, and ranged from 17.8 to 45.9 µm diam (x ̅=30.5 ± 3.5 µm, n=50). Oospores were not observed in pure cultures. These morphological characteristics were consistent with those of P. nicotianae (LaMondia et al. 2014). Genomic DNA was extracted from a representative isolate, JFRL 03-18, using the NuClean Plant Genomic DNA kit (CWBIO, China). The internal transcribed spacer (ITS) region, ras-related protein ypt1 (YPT), ß-tubulin (TUB) gene were amplified by Polymerase Chain Reaction using primers ITS1/ITS4 (White et al. 1990), Yph1F/Yph2R (Schena et al. 2008), and TUBUF2/TUBUR1 (Kroon et al. 2004), respectively. All sequences were deposited in GenBank (Accession Nos. ON231777 for ITS, ON246910 for YPT, ON246908 for TUB). BLASTN homology search for these nucleotide sequences showed 100% identical to the ITS (MH341621), YPT (MK058408), TUB (MH760160) sequences of P. nicotianae. Sequences of twelve Phytophthora species and Pythium ostracodes were downloaded from GenBank. The phylogenetic tree of combined ITS, YPT, TUB sequences showed that the isolate JFRL 03-18 clustered with P. nicotianae. To complete Koch's postulates, zoospore suspensions were prepared from the cultures grown on 10-day-old V8 juice agar of isolates (JFRL 03-16, 03-18, 03-19). Pathogenicity tests were performed on healthy and surface-disinfested navel orange fruit. Nine fruits were gently wounded with a needle, inoculated with 10 µl zoospore suspension (104 zoospores/ml) of three isolates separately, and three fruit treated with sterilized water as controls. All fruit were incubated at 25℃ with 80% relative humidity and the test was repeated three times. After 7 days of incubation, the fruit inoculated with P. nicotianae showed similar brown rot symptoms and the control fruit remained symptomless. The pathogen was re-isolated from all inoculated fruits and confirmed as P. nicotianae by morphological and molecular analysis. Phytophthora nicotianae was previously reported on Hamlin sweet orange (Citrus sinensis (L.) Osbeck) fruit causing Phytophthora brown rot in Florida (Graham and Timmer 1995; Hao et al. 2018). To our knowledge, this is the first report of P. nicotianae causing Phytophthora brown rot of navel orange fruit in China. Based on the severity of this disease, local growers should develop and implement integrated disease management strategies for control.

First Report of Postharvest Fruit Brown Rot Disease on Navel Orange Caused by Diaporthe sojae in China.

In October 2020, a postharvest fruit brown rot symptom was observed on navel orange (Citrus sinensis Osbeck cv. Newhall) fruits in a local fruit market in Ganzhou, Jiangxi Province, China. The disease incidence increased up to 15% in 40 fruits with a 7-day-long storage at room temperature. The disease symptoms on the infected fruit were circular, light brown to brown, slightly sunken lesions, covered with whitish mycelium mass, and brown rot in the center. To isolate the causal organism, infected fruits were surface sterilized with 1% NaClO solution for 30 sec, and rinsed thrice with sterilized water. Symptomatic tissues at the margins were cut into 5-mm2 pieces, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. Thirteen morphologically similar single-spore fungal isolates were obtained from the isolation experiment. Fungal colonies were white, fluffy, cottony texture, reverse buff to light yellow, with black stromata at maturity. Alpha conidia were hyaline, aseptate, ellipsoid to clavate, tapering towards the ends, often biguttulate, and ranged in size from 6.8 to 9.8 µm × 2.7 to 4.5 µm (n=50). Beta conidia were hyaline, aseptate, smooth, straight to sinuous, and with size ranging from 12.1 to 21.3 µm × 0.9 to 2.2 µm (n=50). Morphological features were consistent with those of Diaporthe sojae (Dissanayake et al. 2015). For molecular identification, DNA was extracted from the representative isolate JFRL 03-13, the internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS) genes were amplified by using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b (Udayanga et al. 2015), respectively. The resulting sequences were deposited in GenBank (Accession Nos. OM281710 for ITS, OM289961 for TUB, OM289964 for CAL, OM289963 for TEF1-α, and OM289962 for HIS). BLAST analysis revealed that these sequences were 100% similar to the sequences of ITS (MN816426), TUB (MK941336), CAL (MN894375), TEF1-α (MN894447), HIS (MN894409) published for D. sojae. Phylogenetic analysis was conducted based on the concatenated sequences (ITS, TUB, CAL, TEF1-α, and HIS) by Maximum likelihood analysis (ML) and Bayesian inference (BI) using IQtree v.1.6.11 and MrBayes v.3.2.7 (Guo et al. 2020). The phylogenetic tree showed that the isolate clustered with D. sojae. To confirm pathogenicity, mature and healthy harvested fruits of navel orange (Citrus sinensis Osbeck cv. Newhall) were surface sterilized. Ten fruits were wounded by a sterile scalpel and put a 7-mm-diamter agar plug with 7-day-old mycelium of the isolate JFRL03-13 cultured on PDA at 25°C, noncolonized PDA plugs were used as the control. Inoculated fruits were incubated at 25℃ with 80% relative humidity. After 10 days, the similar symptoms were observed on the inoculated sites and spread on the surface of fruits, whereas the control remained symptomless. The pathogen was re-isolated from the lesions of inoculated fruits and confirmed as D. sojae via morphological and molecular analysis. The assays were repeated twice, fulfilling the Koch's postulates. Although D. sojae is known as the major causative agent of pod and stem blight, and has been reported as an endophyte in the twigs and leaves of citrus (Huang et al. 2015; Santos et al. 2011), but to our knowledge, this is the first report of postharvest fruits brown rot disease on navel orange caused by D. sojae in China. However, further investigation of the specific causes of this disease is necessary to help the local fruit farmers develop effective disease management strategies.

First Report of Brown Spot of Dekopon Fruit Caused by Cladosporium tenuissimum in China.

Dekopon citrus (Citrus reticulata Shiranui) is a three-way hybrid (Citrus unshiu Marcov. × C. sinensis Osbeck × C. reticulata Blanco) developed in Japan in 1972. This citrus is popular in China due to its sweet and tender taste (Lim 2012). In November of 2021, a brown spot disease on Dekopon fruits with about 20% disease incidence was observed in an orchard of the Institute of Citrus Research in Ganzhou, Jiangxi Province, China. Initially, the symptoms appeared as slightly sunken deep red to purple spots on the fruit surface, with the disease progression, lesions became brown to brown-black large necrotic regions covered with a fluffy layer of gray spores. Infected fruits were surface sterilized with 70% ethanol for 30 sec and rinsed three times with sterile distilled water. Diseased tissues from the edge of lesions were cut into small segments, placed onto potato dextrose agar and incubated at 25℃ for 7 days. Ten single-spore isolates were obtained in total. Fungal colonies were olive green to dark green, velvet-like in texture and sporulated abundantly, surrounded by grayish-white hyphae. Conidiophores were subcylindrical, straight, septate, solitary or in clusters of two to three, and ranged in size from 65 to 550 × 3.8 to 6.3 µm (x ̅= 261.7 ± 60.5 × 5.2 ± 0.4 µm, n=50). Ramoconidia were cylindrical，aseptate, and 10 to 22 × 2.8 to 4.5 µm (x ̅= 15.5 ± 1.4 × 4.0 ± 0.9 µm, n=50). Conidia were lemon-shaped to oval-shaped, smooth-walled, and 1.8 to 5.0 × 1.4 to 2.5 µm (x ̅= 3.9 ± 0.4 × 2.2 ± 0.2 µm, n=50). The morphological characteristics of the pathogen were consistent with those of Cladosporium tenuissimum Cooke (Li et al. 2021). For further identification, DNA was extracted from two representative isolates. The internal transcribed spacer (ITS) region, translation elongation factor (EF1-α), and actin (ACT) were amplified by using primers ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R (Bensch et al. 2012), respectively. ITS (OM232067, OM232068), EF1-α (OM256525, OM256526) and ACT (OM256529, OM256530) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, EF1-α and ACT) phylogenetic analysis was conducted using the Maximum Likelihood method (Nguyen et al. 2015). Based on the morphological characteristics and the molecular data, two fungal isolates were identified as C. tenuissimum. To evaluate pathogenicity, fifteen fruits were surface sterilized with 1% NaClO solution for 30 sec, rinsed twice with sterile distilled water and dried. Dekopon fruits (n=10) were wounded with a sterile needle and inoculated with a 10 µL drop of conidial suspension (1 × 106 conidia/mL) of isolate GZCJ-1, followed by incubation at 25℃ and 80% relative humidity. The controls (n=5) were treated with sterile water and maintained under the same conditions. Five days after inoculation, small brown sunken spots were observed on the wounded and inoculated fruits. After 7 days, lesions were coated by a layer of brown conidia that were similar to those described above, whereas control remained symptomless. Pathogenicity test was repeated twice. Cladosporium tenuissimum was consistently re-isolated from inoculated fruits and confirmed by morphological and molecular data, fulfilling the Koch's postulates. To our knowledge, this is the first report of C. tenuissimum causing the brown spot of dekopon fruit in China and perhaps the world. The disease may become the potential risk for fruit production, making fruits unfit for marketing purposes, and the appropriate management actions will be necessary.

The cis-2-Dodecenoic Acid (BDSF) Quorum Sensing System in Burkholderia cenocepacia.

It has been demonstrated that quorum sensing (QS) is widely employed by bacterial cells to coordinately regulate various group behaviors. Diffusible signal factor (DSF)-type signals have emerged as a growing family of conserved cell-cell communication signals. In addition to the DSF signal initially identified in Xanthomonas campestris pv. campestris, Burkholderiadiffusible signal factor (BDSF) (cis-2-dodecenoic acid) has been recognized as a conserved DSF-type signal with specific characteristics in both signal perception and transduction from DSF signals. Here, we review the history and current progress of the research on this type of signal, especially focusing on its biosynthesis, signaling pathways, and biological functions. We also discuss and explore the huge potential of targeting this kind of QS system as a new therapeutic strategy to control bacterial infections and diseases.

Simultaneous Quantification of Biothiols and Deciphering Diverse GSH Stability in Different Live Cells by 19F-Tag.

GSH, Cys, Hcy, and H2S are important biothiols and play important roles in the living systems. Quantitative and simultaneous determination of these biothiols under physiological conditions is still a challenge. Herein, we developed an effective 19F-reactive tag that readily interacts with these four biothiols for the generation of stable thioether products that have distinguishable 19F-chemical shifts. These thioester compounds encode the characteristic fingerprint profiles of each biothiols, allowing one to simultaneously quantify and determine these biothiols by 1D 19F NMR spectroscopy. The intra-/extracellular GSH in live cells was assessed by the established strategy, and remarkable variations in the GSH stability were determined between the normal mammalian cells and cancer cells. It is notable that GSH hydrolyzes efficiently in the out-membrane of the cancer cells and the lysates. In contrast, GSH remains stable in the tested normal cells.

Simultaneous identification and quantification of amino acids in biofluids by reactive 19F-tags.

A robust method to identify and quantify amino acids close to physiological conditions by 1D 19F NMR was established. Each 19F-derivatized amino acid has its characteristic chemical-shift profile that is readily identified in the mixture of amino acids or in biofluids including fetal bovine serum and cell lysates. The method shows great potential in metabolomics and biochemical analysis.

Rigid, Highly Reactive and Stable DOTA-like Tags Containing a Thiol-Specific Phenylsulfonyl Pyridine Moiety for Protein Modification and NMR Analysis*.

Site specific installation of a paramagnetic ion with magnetic anisotropy in a biomolecule generates valuable structural restraints, such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs). These paramagnetic effects can be used to characterize the structures, interactions and dynamics of biological macromolecules and their complexes. Two single-armed DOTA-like tags, BrPSPy-DO3M(S)A-Ln and BrPSPy-6M-DO3M(S)A-Ln, each containing a thiol-specific reacting group, that is, a phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein modification by formation of a reducing resistant thioether bond between the protein and the tag. The two tags present high reactivity with the solvent exposed thiol group in aqueous solution at room temperature. The introduction of Br at the meta-position in pyridine enhances the reactivity of 4-phenylsulfonyl pyridine towards the solvent exposed thiol group in a protein, whereas the ortho-methyl group in pyridine increases the rigidity of the tag in the protein conjugates. The high performance of these two tags has been demonstrated in different cysteine mutants of ubiquitin and GB1. The high reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high-resolution NMR measurements of biological macromolecules and their complexes.