First Report of Alternaria Leaf Blight Caused by Alternaria alternata on Convallaria majalis in China.

Convallaria majalis is native to temperate zones in the northern hemisphere, Europe, Asia, North America and China, and has excellent ornamental properties and medicinal value. In July 2021, leaf blight was observed on nearly 70~90% of C. majalis plants growing in Heilongjiang University of Chinese Medicine campus (45.72°N, 126.68°E) from Harbin City, China. The typical symptom on the leaves is irregular dark brown lesions. As the brown lesions expanded and eventually coalesce, they form large necrotic areas, with chlorosis, curling, and wilting at the apical edge of the diseased leaf. Ten symptomatic leaves were randomly collected from twenty different plants at the site. Several fragments of diseased tissues (5×5mm) were disinfected in 75% ethyl alcohol for 30 s and 7% NaOCl for 60 s, rinsed three times in sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 25 °C in the dark for 7 days. Twenty purified fungal isolates were obtained by single spore isolation. The morphology of all the twenty isolates was similar, and two isolates were randomly selected (LL, LL01) for further study. Colonies of these isolates on PDA were off-white to black with abundant cotton-like aerial hyphae, and the diameter of the colony is 72 to 85 mm. On potato carrot agar (PCA) medium, these isolates produced light brown and solitary conidiophore with septum. Conidia were ovate to pear-shaped, brown to black in color, with 1-4 transverse septa and 0-2 longitudinal septa, and measured 20.5 to 38.5 × 7 to 13.5 µm (n=100). The isolates were identified as Alternaria alternata according to their morphological characteristics (Simmons 2007). Two representative isolates LL and LL01 were used for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS4/ITS5(White et al. 1990), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nawashima 2020) and Alt-for/Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS, OM319508, OP799847; RPB2, OM649830, OP830846; GAPDH, OM296234, OP830845; TEF1, OM393717, OP830844; Alta1, OM171258, OP830847). Phylogenetic analyses showed 100% identity between LL and LL01 and the type strain CBS 118815. Thus, the fungus was identified as A. alternata based on morphology and molecular analysis. Pathogenicity tests were done by spraying conidial suspensions containing 106 conidia/ml of A. alternata isolates LL and LL01 on leaves of six healthy C. majalis plants, separately. Another six plants were sprayed with sterile distilled water as control and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25° C. Plastic bags were removed from plants after 48 h. After 15 days inoculation, the similar symptoms were observed on the inoculated plants, whereas control plants remained healthy. The pathogenicity tests were conducted three times. A. alternata was reisolated and identified based on morphological and molecular traits, thus fulfilling Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf blight on C. majalis in China and worldwide. The result will serve as the foundation for management leaf blight of C. majalis.

First report of Alternaria alternata causing leaf blight on Trollius chinensis in China.

Trollius chinensis is widely distributed in east Asian countries that include China, Siberia, and Japan, with antibacterial, antiviral, anti-inflammatory and analgesic activity for medical applications. In August 2021, leaf blight was observed on nearly 80~95% of T. chinensis plants growing in Daxinganling (51.43°N, 126.39°E) from Heilongjiang Province, China. Initial symptoms were gray-black necrosis, wilting progressing from the leaf margin, and eventual defoliation. Six T. chinensis plants with typical symptoms were randomly collected, and three fresh leaf samples were collected from each plant. Diseased leaf pieces that measured 5 mm square were disinfected in 75% ethyl alcohol for 30 s and 7% NaClO for 60 s, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA). Twelve fungal isolates, obtained by single-spore isolations, were selected for further. These isolates produced colonies that measured 63 to 73 mm in diameter after 7 days growth on PDA. Colonies were black to brown in color with gray-white aerial hyphae on their surfaces, neat edges, olive green on the back. The isolates produced conidia that were ovate to pear-shaped, brown to black in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, smooth surfaced, parietal cells extending into the beak, and measured 12.5 to 37.5 × 5.0 to 12.5 µm(n=150). Conidiophores were dark, erect or curved, branched, with pronounced spore marks, and measured 35.0 to 50.0 × 4.0 to 5.0 µm(n=150). All twelve fungal isolates were morphologically similar to Alternaria alternata (Simmons 2007). Two representative isolates jlh01 and jlh02 were used for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS4/ITS5, RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nakashima 2020) and Alt-for/Alt-rev (Woudenberg et al.2015). The resulting sequences were deposited in GenBank (ITS, OM095427, OM108099; RPB2, OM131213, OM131214; GAPDH, OM201165, OM201166; TEF1, OM131211, OM131212; Alta1, OM201167, OM201168). Phylogenetic tree results showed 100% similarity between jlh01, jlh02 and the type strain CBS 118812. Morphological and molecular analysis results confirmed the identity of the fungus as A. alternata. Pathogenicity tests were done by spraying water-spore suspensions containing 106 spores per ml of A. alternata isolates jlh01 and jlh02 on leaves of six healthy T. chinensis plants, separately. Six control plants were sprayed with distilled water and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25° C. Plastic bags were removed from all plants after 48 h. Black brown lesions and concentric rings developed on spore-inoculated plants after 15 days and control plants remained symptomless. The pathogenicity tests were conducted three times. A. alternata was reisolated and identified based on morphological and molecular traits, thus fulfilling Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf blight on T. chinensis in China. Based on the plant's medicinal value, this report provides the basis for further research and control of T. chinensis leaf blight.

First report of leaf spot caused by Curvularia muehlenbeckiae on Fraxinus rhynchophylla Hance in China.

Fraxinus rhynchophylla Hance, is a deciduous trees cultivated on a commercial scale focused on medicinal and wood production. In September 2021, leaf spot was observed on F. rhynchophylla in Heilongjiang Province (127.34°E, 45.19°N), China. These symptoms were observed on 100% F. rhynchophylla plants and the incidence of diseased leaves per plant reached 70% in fields measuring 90 ha. Disease symptoms were small yellow flecks initially, and then turned to gray necrotic spot. Ten diseased leaves were collected randomly from 5 plants and surface disinfested. Tissue samples (2 × 2 mm) were cut at the disease-health junction of the leaves, surface sterilized in 75% ethanol for 30 s, submerged in a 7% NaOCl solution for 3 mins, and rinsed three times with sterile water. Leaf segments were placed onto potato dextrose agar (PDA) and incubated at 26℃ for 5 days. After isolation and purification of monospore, the colonies of the all isolates were inky black, with aerial fluffy mycelium, and concentric whorls on PDA. The conidiophore is septate, single-branched, brown, smooth and 35 - 313 × 2 - 5 µm in size (n = 50), while the conidia are brown, bow-shaped, mostly four cells, with three septa and 12 - 385 × 5 - 20 µm in size (n = 150). The morphological characters matched those of Curvularia muehlenbeckiae (Madrid et al. 2014). DNA was extracted from isolates HQLa and HQLb and used for PCR amplification of RNA polymerase II gene (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequences using the primer fRPB2-SF/fRPB2-7Cr (Schoch et al. 2009), gpd1/gpd2 (Berbee et al. 1999), respectively. The RPB2 (OM984674, OM984675) and GAPDH (OM984672, OM984673) were deposited in GenBank. The phylogenetic tree was constructed by combining other published sequences of RPB2 and GAPDH genes using the maximum likelihood method, and the results showed that the obtained isolates clustered into the same clear branch as C. muehlenbeckiae CBS 144.63 (HG779180, HG779108), with 100% bootstrap support. Combining morphological characteristics and phylogenetic analysis of the fungus, the obtained isolates were identified as C. Muehlenbeckiae. To fulfill the Koch's postulates, pathogenicity tests were carried out on newly grown leaves of F. rhynchophylla. Conidia of the selected isolates grown on PDA plates were flooded with sterile distilled water. Spore suspension was adjusted to 105 spores/mL with the hemocytometer. Three leaves of each plant were disinfected with 1% NaOCl for 2 min, washed with sterilized distilled water three times, and dried with sterile paper towels. Three plants were randomly selected for inoculation under field conditions and each leaf was sprayed with 2 mL of the spore suspension for a total of nine leaves, then the plants were bagged and moistened for 48 h. However, control leaves were sprayed with distilled water. Symptoms were observed nine days after inoculation. No symptoms were observed on control leaves. The same fungus was successfully re-isolated from the lesions. The experiment was replicated three times with the same results and C. muehlenbeckiae identification was confirmed by morphological observations and RPB2 and GAPDH sequencing, indicating that the fungus is the causal pathogen of leaf spot disease on F. rhynchophylla. This is the first report of C. muehlenbeckiae determined as fungal pathogens on F. rhynchophylla plant in China. The results of the study laid the foundation for the future occurrence and epidemiological pattern of the disease and scientific control.

Advances in mitochondrial DNA base editing technology.

Mitochondria, the energy factories of higher eukaryotes, provide energy (ATP) for life activities through aerobic respiration. They possess their own genome, mitochondrial DNA (mtDNA), which encodes 37 genes. Mutations in mtDNA cause mitochondrial diseases, and more than 100 pathogenic mutations have been identified in human mtDNA, with a total incidence rate of about 1/5000. In recent years, advances in CRISPR-based base editing technology have enabled accurate editing of nuclear genes. However, it remains a challenge to achieve precise base editing on mtDNA due to the difficulty of guide RNA in the CRISPR system passing through the mitochondrial double-membrane. In 2020, David R. Liu's group at Harvard University reported a double-stranded DNA deaminase DddA from Burkholderia cenocepacia, which was fused with the programmable transcription activator-like effector (TALE) and uracil glycosylase inhibitor (UGI) to develop DddA-derived cytosine base editors (DdCBEs). Using DdCBEs, they were able to achieve specific and efficient C?G to T?A conversion on mtDNA for the first time. In this review, we summarize the recent progress of mitochondrial base editing technology based on DddA and prospect its future application prospects. The information presented may facilitate interested researchers to grasp the principles of mitochondrial base editing, to use relevant base editors in their own studies, or to optimize mitochondrial base editors in the future.

First report of Alternaria alternata causing leaf spot of Cynanchum atratum Bunge in China.

Cynanchum atratum Bunge belongs to Asclepiadaceae, and is distributed in North Korea, Japan and China. Its roots and rhizomes have antibacterial, antiviral, anti-inflammatory and anti-tumor effects. In July 2021, a leaf spot was observed in a 1.3 ha plantation of C. atratum in Harbin, Heilongjiang Province in China. The incidence was more than 85%. Initial symptoms were yellowing leaves with circular, or ellipsoid brown spots forming on leaf apexes or leaf margins. Small spots expanded and coalesced to form large circular or irregular, pale to light brown lesions, and leaves finally withered. Thirty, 5 × 5 mm, leaf pieces excised from the junction of symptomatic and healthy tissues were collected from different leaves with typical symptoms on ten plants, sterilized in 75% ethanol for 30s, then in 2% NaClO for 30s, rinsed in sterile water three times, placed on potato dextrose agar (PDA) plates, incubated for 5 days at 28°C in the dark, further purified by single spore method and transferred to new PDA and potato carrot agar (PCA) plates. Finally, 12 fungal isolates, most with similar morphology, were selected. After a 7-day incubation in the dark, colonies on PDA were 53 to 70 mm in diameter, circular and grayish brown. A total of 150 conidia were evaluated for morphology. Conidia were single or in chains, ovoid to inverted pear-shaped, with 2 to 6 transverse septa, 0 to 4 longitudinal or oblique septa, and measured 16.5 to 56.5µm × 9.0 to 16.5 µm. Beaks and supposititious beaks were mostly columnar, rarely conical, 0 to 22.5 µm × 2.5 to 4.0 µm. Conidiophores were solitary or clustered, pale brown, erect or bent, branched or unbranched, separated, 53.5 to 120.5 µm × 2.5 to 6.0 µm (Fig 1). Based on morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007). Two representative isolates (BW and BW2) were used for molecular identification. Internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit (RPB2), Alternaria major allergen (Alt a 1), translation elongation factor 1-alpha (TEF-1 α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced with the primers ITS1/ITS4 (White, et al. 1990), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou. 2013), Alt-F /Alt-R (Hong et al. 2005), TEF-F/TEF-R (Carbone and Kohn. 1999) and GDF/GDR (Templeton et al. 1992). The sequences obtained were deposited in GenBank (ITS: OM317915, ON534349; RPB2: OM296253, ON550475; Alt a 1: OM171248, O550474; TEF: OM238096, O550473; GAPDH: OM296217, ON550472). The phylogenetic analysis of maximum likelihood tree by MEGA 7 showed that the two isolates had 98% similarity with A. alternata CBS 916.96 (Fig 2). To test pathogenicity, 40-day-old plants were sprayed with spore suspensions (1×106 spores /mL) from 7-day-old cultures of BW and BW2. Each isolate was inoculated onto 3 leaves on 3 separate plants. Three other plants were sprayed with sterile distilled water as a control. The plants were incubated in the greenhouse (natural light, T: 25℃, H: 50%). After 15 days, the leaves turned yellow and irregular grayish spots appeared. The fungi reisolated from the inoculated leaves shared the same morphological and molecular features as A. alternata, fulfilling Koch's postulates. No fungi were isolated from the control group. This is the first time to report A. alternata causing leaf spot on C. atratum. Leaf spot can reduce the yields of C. atratum and this study provides a basis for the prevention and control of the disease.

First report of Alternaria alternata causing leaf blight on Actaea dahurica in China.

In July 2019, leaf blight on Actaea dahurica, a plant with high value in Chinese traditional medicine, was discovered in a 2 ha planting area in Heilongjiang Province (129.6°E, 44.6°N), China. Disease incidence was 90% in the field. Symptoms consisted of irregular black spots with gray margins on both sides of the leaf, often at the leaf margin, mostly on the older leaves. To isolate the pathogen, ten diseased leaves were randomly collected, surface disinfested, and 5 x 5 mm segments were removed from the margin of the lesions. Leaf segments were placed onto potato dextrose agar (PDA) and incubated at 25 ℃ for 7 days. Ten pure cultures with the same morphological characteristics were obtained from three leaves showing typical symptoms. Cultures on PDA initially had a cottony mycelium, white-gray to gray. After two to three weeks of growth, mycelium color changed from gray to black. Conidiophores were clustered, dark at the base, tapering to the apex, born from simple sublates, unbranched, with 1 to 5 septa, and 70.4-530.3 × 5-7.5 µm in size. Conidia were 12.5-82.5 × 5.2-20.3 µm, usually in chains, had 2 to 8 transverse septa, 0 to 4 longitudinal or oblique septa, and a smooth brown surface. Simple, pale, vimineous or verrucous beaks developed from the apical cells with 0 to 4 septa. The morphological characteristics were consistent with Alternaria species (Simmons, 2007). To fulfill Koch's postulates, pathogenicity tests were carried out on three-month-old A. dahurica plants. A spore suspension was prepared from PDA cultures of isolates SM0101 and SM0102 and adjusted to 105 spores/mL using a hemocytometer. Each leaf was sprayed with 2 mL of the spore suspension, then incubated at 25 ℃ for 7 days. The same number of healthy A. dahurica plants were sprayed with sterile water as a control. After 7 days, small brown necrotic spots appeared on inoculated plants, but the control group showed no symptoms. A fungus with the same characteristics as that used for inoculation was re-isolated from the lesions. This experiment was replicated three times, and the results of each experiment were consistent. Genomic DNA was extracted from isolates SM0101 and SM0102 and used for PCR amplification of the rDNA internal transcribed spacer regions (ITS), RNA polymerase II gene (RPB2) and Alternaria allergen a 1 (Alt a 1) gene sequences using the primer pairs ITS1/ITS4 (White et al. 1990), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou, 2013) and Alt-for/Alt-rev (Hong et al. 2005), respectively. The ITS (OL703042, OL616086), RPB2 (OL703043, OL898416), and Alt a 1 sequences (OL616087, OL898415) were deposited in GenBank. The sequences obtained in this study had the highest match to corresponding sequences of Alternaria alternata CBS 916.96 (AF347031, KC584375, AY563301). For isolate SM0101 the matches were ITS (461/461 bp), RPB2 (897/985 bp), and Alt a 1 (488/488 bp). For isolate SM0202 the matches were ITS (457/457 bp), RPB2 (893/985 bp), and Alt a 1 (484/484 bp). A phylogenetic analysis was performed using MEGA7 software. The alignment included sequences from 16 ex-type Alternaria species and the two isolates causing leaf blight on A. dahurica. Branch supports were calculated with 1,000 bootstrap replicates, and phylogenetic inference was performed using the maximum likelihood estimation. The fungus isolated from A. dahurica clustered with A. alternata. This is the first report of A. alternata on A. dahurica in the world. This report will help to identify the disease symptoms in the field and provides a basis for research into the occurrence, distribution, and control of leaf blight on A. dahurica.

First report of leaf blight on Ligusticum jeholense (Nakai et Kitagawa) Nakai et Kitagawa caused by Alternaria alternata in China.

Ligusticum jeholense (Nakai et Kitagawa) Nakai et Kitagawa is one of the sources of Chinese herb "Gao-Ben". It is widely distributed in the Northeastern China. L. jeholense has antipyretic, antibacterial and anti-inflammatory effects (Zhang et al. 2021). In September 2021, a serious leaf blight was found in a 1.2 ha plantation of L. jeholense in Harbin, Heilongjiang Province, and the incidence was about 85%. The foliar symptoms were grayish-brown lesions, surrounded by a yellow margin at the edge of the leaf. In serious cases, the lesions extended into the middle of the leaf, and finally the whole leaf withered. A total of 12 samples (5×5mm) from symptomatic and healthy junction of 12 infected leaves from 6 different plants of L. jeholense with typical symptoms were cut and surface disinfected in 75% ethanol, and with 7% NaClO for 1 min, then rinsed three times with sterilized water. These tissues were placed onto Potato dextrose agar (PDA) plates at 28℃ in the dark. The colonies cultured for 7 days were obtained and transferred onto new PDA and potato carrot agar (PCA) plates by single spore method to further purify. After 7 days, the colonies on PDA were 63 to 75 mm in diameter, circular, grayish, with white aerial hyphae on the edge, the back of the colonies were grayish green. A total of 150 conidia on PCA were single or in chains, ovoid, inverted pear, 2 to 6 transverse septa, 0 to 3 longitudinal or oblique septa, 16.5 to 67.5 × 8.5 to 20.5 µm. The beaks were conical or cylindrical, 2.5 to 25.3 × 2.0 to 3.0 µm. Conidiophores were grayish brown, erect or bent, separated, 57.0 to 137.0 × 5.1 to 13.7 µm. Morphological characteristic showed the 12 isolates were the same fungus and similar to Alternaria sp. (Simmons 2007). Two typical strains (LGB and LGB2) from twelve isolates were randomly selected for molecular identification. Genomic DNA was extracted from mycelia of two isolates on PDA by modified CTAB method, and internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit (RPB2) and Alternaria major allergen (Alt a 1), translation elongation factor 1-alpha (TEF) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified and sequenced with the primers ITS1/ITS4, RPB2-5F2/RPB2-7CR, Alt-F /Alt-R, TEF-F/TEF-R and gpd-F/gpd-R (Woudenberg et al. 2015). The obtained sequences were deposited in GenBank (ITS: OM319506, OM943431; RPB2: OM393721, OM984854; Alt a 1: OM649816, OM984853; TEF: OM238108, OM984852; gpd: OM296228, OM984851). The phylogenetic analysis of maximum-likelihood tree by MEGA7 showed the LGB and LGB2 had 100% identity with A. alternata CBS 916.96. For pathogenicity test, conidial suspension (1 × 106 spores/mL) of the strain LGB and LGB2 was sprayed on 10 healthy 40-day-old L. jenholense plants and five plants with sterile water as control. The plants were incubated at 25℃. After 28 days, grayish withering appeared on the leaves. The test was repeated three times. The same fungi were re-isolated from the inoculated leaves and with the same morphological and molecular characteristics as A. alternata, fulfill the Koch's postulates. No symptoms and fungi were found in the control group. This is the first report of leaf blight on L. jenholense caused by A. alternata. Leaf blight could reduce the yields of L. jenholense. This study provides a reference for the prevention and treatment to the leaf blight of L. jenholense.

First report of leaf spot on Clematis brevicaudata DC. caused by Alternaria alternata in China.

Clematis brevicaudata DC. is distributed in China, Korea, Mongolia, Russia and Japan. This plant is both ornamental and medical, used in the treatment of nervous disease, dyskinesia and other diseases. In September, 2019, a leaf spot on C. brevicaudata was first found in a 5 ha C. brevicaudata plantation in Harbin, Heilongjiang Province, China. The incidence was about 80%. The symptoms were elliptical, circular, or irregular brown to black necrotic lesions in leaf apex and leaf margin. Ten fresh sample leaves with typical symptoms were collected from ten C. brevicaudata plants. The tissues (5mm×5mm) between symptomatic and healthy junction were cut and surface disinfected in 75% ethanol, and with 7% NaClO for 1 min, then rinsed three times with sterilized water, 30s each time. The sterilized tissues were inoculated on potato dextrose agar (PDA) plates for 7 days at 25℃. The colonies were obtained and transferred onto new PDA and potato carrot agar (PCA) plates by single spore method to further purify. After 7 days, the colonies on PDA were 50 to 63 mm in diameter, circular, grayish brown, with white aerial hyphae. A total of 150 conidia on PCA were single or in chains, ovoid, inverted pear, 2 to 7 transverse septa, 0 to 3 longitudinal or oblique septa, 17.5 to 57.5 × 7.5 to 17.5 µm. Beaks and supposititious beaks were mostly columnar, rarely conical, 2.5 to 6.0 × 2.0 to 3.0 µm. Conidiophores were solitary or clustered, pale brown, erect or bent, branched or unbranched, separated, 112.0 to 151.0 × 5.1 to 14.7 µm. Ten isolates purified on PDA were obtained. Morphological identification showed the ten isolates were similar and appeared to be Alternaria alternata (Simmons, 2007). Two strains from ten isolates were selected for molecular identification. Genomic DNA was extracted from mycelia of two isolates (LD2020520 and LD2020521) on PDA using a modified CTAB method. Internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit gene (RPB2), Alternaria major allergen (Alt a 1), endopolygalacturonase (endoPG) and glyceraldehyde 3-phosphate dehydrogenase (gpd) were amplified and sequenced using two directional sequencing with the primers ITS1/ITS4, RPB2-F/RPB2-R, Alt-F/Alt-R, end-F/end-R and gpd-F/gpd-R (Woudenberg et al. 2015). The sequences obtained were deposited in GenBank (ITS: MT501762, OK571395; RPB2: MT506027, OK631891; Alt a 1: MT506026, OK631890; endoPG: ON054189, ON054188; gpd: ON054191, ON054190). The phylogenetic analysis of maximum-likelihood tree by MEGA 7 software showed that the two isolates had 99% identity with the A. alternata CBS 916.96. For pathogenicity testing, eighteen leaves of six 5-week-old plants were sprayed with spore suspensions (1×106 spores /mL) of the 7 days-old isolates LD2020521 and LD2020520 (Each isolate infected three plants and each infected three leaves). Three plants were sprayed with sterile distilled water as a control group. The plants were incubated at 25℃. After 15 days, taupe irregular spots appeared on the leaves. The pathogenicity test was repeated three times. The same fungi were re-isolated from the inoculated leaves and with the same morphological and molecular characteristics as LD2020520 and LD 2020521, fulfilling Koch's postulates. No fungi were isolated from the control group. This is the first report of leaf spot on C. brevicaudata caused by A. alternata. Leaf spot can reduce the yields of C. brevicaudata. This study provides a reference for the prevention and treatment to the leaf spot of C. brevicaudata.

First Report of Leaf Blight Caused by Alternaria alternata on Leonurus japonicus in China.

Leonurus japonicus is cultivated throughout China and is commonly used for medicinal, cosmetic, ornamental and culinary purposes. A leaf blight on L. japonicus was first observed in September 2021 in a field at a research and development farm in Liupu Town, Zhuji City (120.23°N, 29.72°E), Zhejiang Province, China. Disease incidence was more than 90% across the 30 ha. Symptoms included nearly round black to brown spots on the leaf margins that gradually enlarged causing leaves to wither. To isolate and identify the causal organism, 12 L. japonicus leaves from four different plants with typical symptoms were collected, and 5×5 mm tissues were excised at the junction of the diseased and healthy tissue. Samples were surface-sterilized in 75% ethanol for 30s, followed by 7% NaOCl for 1 min, and rinsed three times with sterile distilled water (Sun et al. 2022), and placed on potato dextrose agar (PDA) at 25℃. After 7 d, single-spore isolations were conducted. (Zhu et al. 1992) After 8 d, the colonies on PDA were 75 to 86 mm diam, dark brown, with an irregular shape. A total of 150 conidia on PDA were an inverted rod shape or oval, dark brown, 20 to 45 × 7.5 to 11.3 µm, with a short beak and no septa; or columnar or conical, 2.5 to 20 × 2.5 to 5 µm, with 0 to 6 transverse septa, 0 to 3 longitudinal or oblique septa. The conidiophores were dark or branched, with multiple conidial scars, 15 to 62.5 × 3.0 to 5.0 µm. According to morphological characteristics observation, the 12 isolates were most similar to A. alternata (Simmons 2007). To further identify the fungal species, internal transcribed spacer (ITS) rDNA regions, and the following genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen (Alt a 1), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF) were amplified and sequenced using the primers ITS4/ITS5, RPB2-5F/RPB2-7CR, gpd1/gpd2, EF1-728F/EF1-986R, and Alt-for/Alt-rev (Woudenberg et al. 2015). Sequences were uploaded (ITS: OM095432, OM095433; RPB2: OM275409, OM275410; GAPDH: OM275411, OM275412; TEF1: OM160771, OM160772; Alta1: OM160773, OM160774). The similarity of YMCLZL, YMCLZL01 and the type strain CBS 59593 T (KP124320, KP124175, KP125096, KP124788, JQ646399) on the phylogenetic tree was 97%. To evaluate pathogenicity, a conidial suspension (106 conidia/ml) of isolates YMCLZL or YMCLZL01 was sprayed on the leaves of six 15-day old healthy plants. The same number of plants were also sprayed with only distilled water as non-inoculated controls. Plants were covered with plastic bags at 25℃ for 48 h. After 8 d, inoculated plants had round, gray and black spots on leaves, while the control plants did not. The experiment was repeated three times. The fungus was reisolated from all diseased leaves fulfilling Koch's postulates. To our knowledge, this is the first report of L. japonicus leaf blight caused by A. alternata on L. japonicus worldwide. The occurrence of leaf blight will be challenging for the commercial production of L. japonicus.

First Report of Alternaria Leaf Blight Caused by Alternaria alternata on Phedimus aizoon in China.

Phedimus aizoon is native to east Asian countries that including China, Siberia, Korea, Mongolia, and Japan. In China, the plant is highly valued for use in folk medicine, for detoxification and analgesia, blood pressure, hemostasis, and used as an ornamental. In August 2021, a leaf spot and blight disease were observed on P. aizoon in a 120-ha field in Pizhou, Jiangsu Province, China where disease incidence reached 90%, and almost every leaf was withered. Early symptoms appeared as dark brown lesions on leaf margins that enlarged and coalesced to form large necrotic areas. In efforts to determine the cause of the disease, ten symptomatic leaves were randomly collected from ten different plants at the site. Diseased leaf pieces that measured 5 mm2 were disinfected in 75% ethyl alcohol for 30 s and 7% NaOCl for 60 s, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA). Ten fungal isolates obtained by single-spore isolations were selected for further study. These isolates produced colonies that measured 70 to 82 mm in diameter after 7 days growth on PDA. Colonies were black to brown in color with gray-white aerial hyphae on their surfaces. The isolates produced conidia that were ovate to pear-shaped, brown to black in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, smooth surfaced, parietal cells extending into the beak, and measured 10 to 35.5 × 5.0 to 12.5 µm. Conidiophores were brown, erect or curved, branched, with pronounced spore marks, and measured 7.5 to 37.5 × 2.5 to 5.0 µm. All ten fungal isolates were morphologically similar to Alternaria alternata (Simmons 2007). Two representative isolates FC01 and FC02 were used for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS4/ITS5, RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nakashima 2020) and Alt-for/Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS, ON584560, ON564492; RPB2, ON729984, ON703241; GAPDH, ON652866, ON652867; TEF1, ON652868, ON652869; Alta1, ON652870, ON652871). Phylogenetic analyses showed 100% identity between FC01 and FC02 and the type strain CBS 916.96. Thus, the fungus was identified as A. alternata based on morphology and molecular analysis. Pathogenicity tests were done by spraying conidial suspensions containing 106 conidia per ml of A. alternata isolates FC01 and FC02 on leaves of five healthy P. aizoon plants, separately. Five control plants were sprayed with distilled water and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25° C. Plastic bags were removed from plants after 48 h. Dark brown lesions developed on inoculated plants after 16 days and control plants remained symptomless. The pathogenicity tests were conducted three times. A. alternata was reisolated and identified based on morphological and molecular traits, thus fulfilling Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf blight on P. aizoon in China and worldwide. Based on the plant's medicinal value, further studies should be directed toward control of this disease.

Four new fatty acid derivatives from Diaporthe sp. T24, an endophytic fungus isolated from Ligularia fischer.

The endophytic fungus Diaporthe sp. is known to contain many secondary metabolites, but fatty acid derivatives have rarely been found. In this study, four new fatty acid derivatives (1-4), together with four known compounds (5-8), were isolated from Diaporthe sp., which was obtained from the stem of Ligularia fischeri. The absolute configurations of the new compounds 1-4 were deduced based on spectroscopic technique and J-based coupling constant analysis. Moreover, compound 1 exhibited cytotoxic activities against HCT-8 and MCF-7 cancer cells, and compounds 3 and 4 showed modest selectivity for HCT-8 cells by MTT assay.

Response of Bioactive Metabolite and Biosynthesis Related Genes to Methyl Jasmonate Elicitation in Codonopsis pilosula.

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L-1 MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L-1 MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by ¹H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in ß-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula.

Comparison of Two Different Astragali Radix by a ¹H NMR-Based Metabolomic Approach.

Astragali Radix (AR) is a commonly used herbal drug in traditional chinese medicine and is widely used for the treatment of diabetes, cardiovascular diseases, nephropathy, and neuropathy. The main source of AR in China is the dried root of Astragalus membranaceus var. mongholicus (Bge.) Hsiao, and both cultivated and wild ARs are used clinically. A systematic comparison of cultivated AR (GS-AR) and wild AR (SX-AR) should be performed to ensure the clinical efficacy and safety. In this study, the chemical composition of the two different ARs, which were collected in the Shanxi (wild) and Gansu (cultivated) provinces, were compared by NMR-based metabolic fingerprint coupled with multivariate analysis. The SX-AR- and GS-AR-induced metabolic changes in the endogenous metabolites in mice were also compared. The results showed that SX-AR and GS-AR differed significantly not only in the primary metabolites but also in the secondary metabolites. However, alterations among the endogenous metabolites in the serum, lung, liver, and spleen were relatively small. This study provided a novel and valuable method for the evaluation of the consistency and diversity of herbal drugs, and further studies should be conducted on the difference in polysaccharides as well as the biological effects between the two kinds of AR.

[Effect of adjuvant therapy with Dingguier umbilical paste on intestinal flora of diarrhea infants and its action characteristics].

The applicator therapy is a unique method to treat infant diarrhea in traditional Chinese medicines and widely applied in clinical practice. Currently, many researchers have proved the rationality of the therapy based on the traditional Chinese medicine mechanism and on the data from clinical practice, but its action mechanism is uncertain at present. In this study, with the assistance of pediatric practitioners, the automated ribosomal intergenic-spacer analysis (ARISA) was adopted to study the effect of the adjuvant therapy with Dingguier umbilical paste on intestinal flora of diarrhea infants, in which Dingguier umbilical paste served as the adjuvant therapy in oral traditional Chinese medicines and fecal samples of infants with different diarrhea symptoms were collected and used as the study materials. The results showed that the adjuvant therapy had a significant effect on the shift of intestinal flora, which was associated with the decrease in the similarity difference to the normal control group and the increase in the number of operational taxonomic units (OTUs) shared with the normal control group. Additionally, adjuvant therapy with Dingguier umbilical paste also showed long action duration and increased OTUs number. These results indicated that Dingguier umbilical paste has the effect in restoring the micro-ecosystem of unbalanced intestinal bacteria. Intestinal flora may be one of major targets for the applicator therapy for the infant diarrhea, but not for the single oral traditional Chinese medicine for infant diarrhea.

[Application of methyl in drug design].

The methyl group plays an important role in the rational drug design. Introducing methyl into small molecules has become an important strategy of lead compound optimization. The application of methyl in drug design is reviewed in this paper. Methyl can modulate the physicochemical, pharmacodynamic, and pharmacokinetic properties by ortho effect, inductive effect, and conformational effect. It also improves the metabolic stability as a soft metabolic point. In addition, introducing methyl into drug molecules can also be applied as a strategy in new uses of old drugs and generate me-too drugs.

[Status and analysis of Astragali Radix resource in China].

To explore the status of the resources of Astragali Radix, a survey on its germplasm resources was carried out. Some conclusions can be drawn for Astragali Radix: the major source is the cultivated Astragalus mongolicus. The new major cultivation areas for A. mongolicus and A. membranaceus are Shandong and Gansu province. The semi-wildly planting model in Shanxi province maintains the genuine trait of Astragali Radix, but its yield is limited, and now a combination model has been developed. The major problems for Astragali Radix are the selection of planting sites, the rot root and difficulty in collecting and processing. Several developmental proposals for Astragali Radix were put forward including rational distribution of planting areas, establishment of standard system, development and standardization of producing technologies.

Comparison of the effects of DC031050, a class III antiarrhythmic agent, on hERG channel and three neuronal potassium channels.

AIM: This study was conducted to test the selectivity of DC031050 on cardiac and neuronal potassium channels. METHODS: Human ether-à-go-go related gene (hERG), KCNQ and Kv1.2 channels were expressed in CHO cells. The delayed rectifier potassium current (I(K)) was recorded from dissociated hippocampal pyramidal neurons of neonatal rats. Whole-cell voltage patch clamp was used to record the voltage-activated potassium currents. Drug-containing solution was delivered using a RSC-100 Rapid Solution Changer. RESULTS: Both DC031050 and dofetilide potently inhibited hERG currents with IC(50) values of 2.3 ± 1.0 and 17.9 ± 1.2 nmol/L, respectively. DC031050 inhibited the I(K) current with an IC(50) value of 2.7 ± 1.5 µmol/L, which was >1000 times the concentration required to inhibit hERG current. DC031050 at 3 µmol/L did not significantly affect the voltage-dependence of the steady activation, steady inactivation of I(K), or the rate of I(K) from inactivation. Intracellular application of DC031050 (5 µmol/L) was insufficient to inhibit I(K). DC031050 up to 10 µmol/L had no effects on KCNQ2 and Kv1.2 channel currents. CONCLUSION: DC031050 is a highly selective hERG potassium channel blocker with a substantial safety margin of activity over neuronal potassium channels, thus holds significant potential for therapeutic application as a class III antiarrhythmic agent.

Urinary metabonomic study using a CUMS rat model of depression.

Chronic unpredictable mild stress (CUMS) is a well-validated model of depression. In this study, a urinary metabonomics method based on the NMR spectrometry was used to study the metabolic perturbation in CUMS-induced rat depression model. With pattern recognition analysis, a clear separation of CUMS rats and healthy controls was achieved, and nine endogenous metabolites contributing to the separation were identified. CUMS-treated rats were characterized by the increase of glycine, pyruvate, glutamine, and asparagines, as well as the decrease of 2-oxoglutarate, dimethylglycine, citrate, succinate, and acetate. The urinary biochemical changes related to the metabolic disturbance in CUMS induced depression, and the possible correlations with live qi stagnation in traditional Chinese medicine are discussed. The work shows that CUMS is a reliable model for studying depression, and the noninvasive urinary metabolomic method is a valuable tool to investigate the biochemical pertubations in depression as an early diagnostic means.

Metabolic fingerprinting of Tussilago farfara L. using ¹H-NMR spectroscopy and multivariate data analysis.

INTRODUCTION: The flower bud of Tussilago farfara L. is widely used for the treatment of coughs, bronchitis and asthmatic disorders in traditional Chinese medicine. In Europe, the plant has been used as herbal remedies for virtually the same applications, but the leaves are preferred over flowers. OBJECTIVE: To systematically evaluate the chemical profiles of Tusssilago farfara leaves and flowers along with the identification of the polar and non-polar metabolites. METHODOLOGY: Metabolic profiling carried out by means of ¹H-NMR spectroscopy and multivariate data analysis was applied to crude extracts from flowers and leaves. Metabolites were identified directly from the crude extracts through one-dimensional and two-dimensional NMR spectra. RESULTS: A broad range of metabolites were detected without any chromatographic separation. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) of ¹H-NMR data provided a clear separation between the samples. The corresponding loadings plot indicated that higher levels of phenylpropanoids, amino acids, organic acids and fatty acids, as well as lower levels of sugars, terpenoids and sterols were present in the leaves, as compared with flowers. For the flowers, more phenylpropanoids were present in fully open flowers, while more sugars and fatty acids were present in flower buds. CONCLUSION: NMR spectra (one- and two-dimensional) are useful for identifying metabolites, especially for the overlapped signals. The NMR-based metabolomics approach has great potential for chemical comparison study of the metabolome of herbal drugs.

[Metabolomic study of flower buds of Tussilago farfara in different development stages by GC-MS].

OBJECTIVE: Plant metabolomics combined with GC-MS was used to investigate metabolic fingerprinting of Tussilago farfara at different growth stages. METHOD: Dried Samples were extracted by two-phase solvent system to obtain polar and nonpolar parts, which were subjected to GC-MS analysis. Metabolites were identified by NIST data base search and comparison with the authentic standards. The data were introduced into SIMCA-P 11.0 software package for multivariate analysis after pretreatment. RESULT: Fifty-four metabolites were identified, including 35 polar metabolites and 19 nonpolar compounds. The score plot for PCA showed clear separation of the different development stages of flower buds of T. farfara, showing a trend of gradual change. Samples of October, November, December were in close proximity on the plot, indicating that the metabolome of these three periods was similar, samples from September (early development) and March (after flowering) were far away, showing big chemical differences. Content comparison results of some representative metabolites reveals that, the content of proline, lysine and linoleic acid increased gradually to the highest in the medium term, but sharply decreased to the lowest after flowering; the content of malic acid and citric acid were the lowest in the medium term; sucrose content decreased gradually, and then reached the lowest level after blooming. CONCLUSION: It is obvious that metabolites of the early development and flowering stage were quite different with those of the traditional harvest time, suggesting that they can not be used as traditional medicine. This study will provide a research basis for harvest time determination and bioactive compounds of T. farfara.