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 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.

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 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 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.

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 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 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.

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.

Tumor promoting effects of glucagon receptor: a promising biomarker of papillary thyroid carcinoma via regulating EMT and P38/ERK pathways.

Glucagon is a crucial hormone involved in the maintenance of glucose homeostasis. Large efforts to define the role of glucagon receptor (GCGR) have been continuously made in recent years, but it is still incomplete about its function and mechanism. We performed this study to verify its potential impacts on papillary thyroid carcinoma (PTC) progression. Correlation between GCGR expression and PTC was elaborated using The Cancer Genome Atlas (TCGA) database. The Kaplan-Meier method was used to analyze the connection between GCGR expression and prognosis of PTC patients. GCGR expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis; simultaneously, cell viability was elucidated using cell proliferation and colony formation assays following siRNAs strategy. Transwell analyses were conducted to measure the invasion and migration of PTC cells. Flow cytometry analysis was conducted to examine apoptotic ability. The cAMP ELISA kit was employed to measure the cAMP level in PTC cells. Our data determined that the expression level of GCGR was increased in PTC tissues and cells in contrast to normal tissues and Nthy-ori 3-1, respectively. Up-regulated GCGR expression was linked with the lower survival rate in patients with PTC. Functional analysis in vitro suggested that GCGR knockdown attenuated PTC cell proliferation, colony formation, invasion, and migration whilst intensified apoptosis. Down-regulated GCGR was able to increase cAMP level. Furthermore, reduction of GCGR could result in the inactivation of epithelial-mesenchymal transition (EMT) and P38/ERK pathways. In conclusion, the findings of this study disclosed that GCGR promoted PTC cell behaviors by mediating the EMT and P38/ERK pathways, serving as a potential diagnostic and prognostic biomarker as well as therapeutic target for PTC.

Quality Analysis of Different Specification Grades of Astragalus membranaceus var. mongholicus (Huangqi) from Hunyuan, Shanxi.

Background: Huangqi is a famous Chinese medicinal material whose Dao-di producing area is Hunyuan, Shanxi. Huangqi produced in Hunyuan, Shanxi, were divided into several different specifications and grades according to the diameters and different positions of root system. Objective: This article investigates the quantitative characteristics of chemical compositions in different specifications and grades of Astragalus membranaceus var. mongholicus roots, aiming to elucidate the correlation between specifications and/or grades and chemical compositions in Huangqi. Methods: Based on the field investigation, samples of Huangqi collected from Hunyuan, Shanxi, were divided into different specifications and grades. The content of seven flavonoids and five saponins in Astragalus membranaceus var. mongholicus roots of different specifications and grades were determined simultaneously by HPLC-diode-array detection-evaporative light-scattering detection (HPLC-DAD-ELSD). Results: Huangqi was processed by traditional methods, and its commercial specification was classified by different parts of the root system, such as ge-da-tou, hong-lan-qi, zheng-bai-qi, fu-bai-qi, mao-wei-zi, and qi-jian. The total content of seven flavonoids and five saponins in ge-da-tou, qi-jian were lower. The total content of seven flavonoids in hong-lan-qi was much higher, while that of five saponins was much lower. The total content of seven flavonoids in lateral roots or fibrous roots were higher, and that of five saponins was lower, such as zheng-bai-qi, fu-bai-qi, and mao-wei-zi. According to the root diameters, Huangqi was classified to special grade, grade I, grade II, grade III, grade IV, or grade V. Among six grades of Huangqi, the total content of seven flavonoids in grade III, grade IV, and grade V were lower, while the total content of five saponins in them were much higher. Conclusions: There is an obvious difference on the distribution pattern of contents of seven flavonoids and five saponins in Huangqi of different specifications and grades, which provide a certain scientific basis for the quality evaluation of Huangqi. Highlights: The content of seven flavonoids and five saponins in Huangqi were determined by HPLC-DAD-ELSD. The relationship between the commercial specification grades and chemical components of Dao-di herbs Astragalus membranaceus var. mongholicus (Huangqi ) from Hunyuan, Shanxi were revealed, which provided a chemical basis for the classification of commercial specification grades of dao-herbs Astragalus membranaceus var. mongholicus (Huangqi ) from Hunyuan, Shanxi.

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.

Delisheng induces antiproliferation and apoptosis effects in Hep3B cells via modulation of angiogenic proteins.

Delisheng is a widely used antineoplastic agent in China. Although previous studies revealed that Delisheng exhibits numerous pharmacological effects including the inhibition of cancer cell differentiation and enhancement of immune function with the lowest toxicity, the precise anticancer mechanisms of Delisheng in human hepatocellular carcinoma (HCC) cells remains largely unknown. The present study investigated the potential mechanisms underlying the anticancer properties of Delisheng on Hep3B cells. Delisheng demonstrated a strong anti-proliferation effect on Hep3B cells compared with normal liver HL-7702 cells, as detected by MTT assays. In addition, Delisheng arrested the cells in G/G1 phase. Furthermore, it exhibited a pro-apoptotic effect on Hep3B cells, as detected by flow cytometry. When exposed to Delisheng, Hep3B cells demonstrated decreased vascular endothelial growth factor (VEGF) and osteopontin (OPN) and increased endostatin (ES) protein expressions, as detected using immunocytochemistry staining and western blotting. These data suggest that Delisheng induces antiproliferation and apoptosis of Hep3B cells via modulation of VEGF, OPN and ES protein expression. It is hypothesized that Delisheng may be used as a novel anticancer therapeutic in HCC.

The clinical use of the platelet/lymphocyte ratio and lymphocyte/monocyte ratio as prognostic predictors in colorectal cancer: a meta-analysis.

BACKGROUND: Conflicting evidence exists regarding the effects of platelet/lymphocyte ratio (PLR) and lymphocyte/monocyte ratio(LMR) on the prognosis of colorectal cancer (CRC) patients. This study aimed to evaluate the roles of the PLR and LMR in predicting the prognosis of CRC patients via meta-analysis. METHODS: Eligible studies were retrieved from the PubMed, Embase,andChina National Knowledge Infrastructure (CNKI) databases, supplemented by a manual search of references from retrieved articles. Pooled hazard ratios (HR) with 95% confidence intervals (95% CI) were calculated using the generic inverse variance and random-effect model to evaluate the association of PLR and LMR with prognostic variables in CRC, including overall survival (OS), cancer-specific survival (CSS) and disease-free survival (DFS). RESULTS: Thirty-three studies containing 15,404 patients met criteria for inclusion. Pooled analysis suggested that elevated PLR was associated with poorer OS (pooled HR = 1.57, 95% CI: 1.41 - 1.75, p< 0.00001, I2=26%) and DFS (pooled HR = 1.58, 95% CI: 1.31 - 1.92, p< 0.00001, I2=66%). Conversely, high LMR correlated with more favorable OS (pooled HR = 0.59, 95% CI: 0.50 - 0.68, p< 0.00001, I2=44%), CSS (pooled HR = 0.54, 95% CI: 0.40 - 0.72, p< 0.00001, I2=11%) and DFS (pooled HR = 0.82, 95% CI: 0.71- 0.94,p=0.005, I2=29%). CONCLUSIONS: Elevated PLR was associated with poor prognosis, while high LMR correlated with more favorable outcomes in CRC patients. Pretreatment PLR and LMR could serve as prognostic predictors in CRC patients.

Polycomb complex protein BMI-1 promotes invasion and metastasis of pancreatic cancer stem cells by activating PI3K/AKT signaling, an ex vivo, in vitro, and in vivo study.

Cancer stem cell theory indicates cancer stem cells are the key to promote tumor invasion and metastasis. Studies showed that BMI-1 could promote self-renew, differentiation and tumor formation of CSCs and invasion/metastasis of human cancer. However, whether BMI-1 could regulate invasion and metastasis ability of CSCs is still unclear. In our study, we found that up-regulated expression of BMI-1 was associated with tumor invasion, metastasis and poor survival of pancreatic cancer patients. CD133+ cells were obtained by using magnetic cell sorting and identified of CSCs properties such as self-renew, multi-differentiation and tumor formation ability. Then, we found that BMI-1 expression was up-regulated in pancreatic cancer stem cells. Knockdown of BMI-1 expression attenuated invasion ability of pancreatic cancer stem cells in Transwell system and liver metastasis capacity in nude mice which were injected CSCs through the caudal vein. We are the first to reveal that BMI-1 could promote invasion and metastasis ability of pancreatic cancer stem cells. Finally, we identified that BMI-1 expression activating PI3K/AKT singing pathway by negative regulating PTEN was the main mechanism of promoting invasion and metastasis ability of pancreatic CSCs. In summary, our findings indicate that BMI-1 could be used as the therapeutic target to inhibiting CSCs-mediated pancreatic cancer metastasis.

Prognostic value of Notch-1 expression in hepatocellular carcinoma: a meta-analysis.

Association of Notch-1 expression with prognosis of patients with hepatocellular carcinoma (HCC) remains controversial. We conducted a meta-analysis to reevaluate the association of Notch-1 expression with clinicopathological characteristics and prognosis of HCC. PubMed, Embase, Web of Science, and China National Knowledge Infrastructure were searched to look for relevant studies. The association between Notch-1 expression and clinicopathological parameters and overall survival (OS) was then reassessed using the meta-analysis for odds ratio (OR) or hazard ratio (HR) and 95% confidence interval (CI). A total of seven studies, including 810 HCC patients, were eligible for the meta-analysis. Our data showed that high Notch-1 expression was able to predict poor OS (HR 1.50, 95% CI 1.17-1.83, P=0.0001). The pooled OR showed that high Notch-1 expression was significantly associated with tumor metastasis (OR 0.37, 95% CI 0.16-0.86, P=0.02) and tumor size >5 cm (OR 0.48, 95% CI 0.26-0.88, P=0.02). In contrast, there was no association between high Notch-1 expression and tumor differentiation, late TNM stage, tumor number, and portal vein invasion of HCC. In conclusion, Notch-1 overexpression might predict poorer survival and more aggressive behavior in patients with HCC.

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.

Transcriptome sequencing of Codonopsis pilosula and identification of candidate genes involved in polysaccharide biosynthesis.

BACKGROUND: Codonopsis pilosula (Franch.) Nannf. is one of the most widely used medicinal plants. Although chemical and pharmacological studies have shown that codonopsis polysaccharides (CPPs) are bioactive compounds and that their composition is variable, their biosynthetic pathways remain largely unknown. Next-generation sequencing is an efficient and high-throughput technique that allows the identification of candidate genes involved in secondary metabolism. PRINCIPAL FINDINGS: To identify the components involved in CPP biosynthesis, a transcriptome library, prepared using root and other tissues, was assembled with the help of Illumina sequencing. A total of 9.2 Gb of clean nucleotides was obtained comprising 91,175,044 clean reads, 102,125 contigs, and 45,511 unigenes. After aligning the sequences to the public protein databases, 76.1% of the unigenes were annotated. Among these annotated unigenes, 26,189 were assigned to Gene Ontology categories, 11,415 to Clusters of Orthologous Groups, and 18,848 to Kyoto Encyclopedia of Genes and Genomes pathways. Analysis of abundance of transcripts in the library showed that genes, including those encoding metallothionein, aquaporin, and cysteine protease that are related to stress responses, were in the top list. Among genes involved in the biosynthesis of CPP, those responsible for the synthesis of UDP-L-arabinose and UDP-xylose were highly expressed. SIGNIFICANCE: To our knowledge, this is the first study to provide a public transcriptome dataset prepared from C. pilosula and an outline of the biosynthetic pathway of polysaccharides in a medicinal plant. Identified candidate genes involved in CPP biosynthesis provide understanding of the biosynthesis and regulation of CPP at the molecular level.