Leaf Spot Caused by Didymella glomerata on Chaihu (Bupleurum falcatum L.) in China.

Bupleurum falcatum is a Apiaceae family herbal medicinal plant, which has the functions of soothing liver, relieving depression, relieving fever, dispelling stagnation, and regulating menstruation. B. falcatum roots have been used in Chinese herbal formbulary for at least 2000 years (Ahmadimoghaddam et al. 2021). In June 2021, infected leaves of B. falcatum that had dark brown, circular, elliptical or irregular shaped lesions or severely withered were obtained in Yichang (30.75 ° N，111.24 ° E), Hubei, China. Disease incidence was approximately 40% in the 20 hm2 B. falcatum plantation base. Fifteen small pieces (3 mm) were cut from the junction between disease and health of surface sterilized (with 75% alcohol) leaves and then plated on potato dextrose agar (PDA). After 3 days incubation, eight isolates with the same colony morphology were sub-cultured and purified by hyphal tip isolation. Isolate CHYB1 cultured on potato dextrose agar (PDA) was selected for identification. The colony was initially white and later producing gray and brown. Pycnidia were dark, spherical or flat spherical, and 78.3 to 137.4 µm in diameter. Conidia were oval mostly, smooth, aseptate, and 18 the size was 3.7 to 5.1 × 1.6 to 2.5 µm. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were the internal transcribed spacer (ITS) and beta-tubulin gene (TUB2) using ITS1/4 (White et al. 1990) Btu-F-F01/Btu-F-R01 primers (Wang et al. 2014), respectively. BLAST analysis of the ITS sequence (MZ818334.1) had 99% similarity to a 498 bp portion of D. glomerata sequence in GenBank (KR709012.1) and TUB2 sequence (OL439060) had 100% similarity to a 323 bp portion of D. glomerata sequence in GenBank (LT592974.1). All isolates (CHYB1-8) were taken for a pathogenicity test in laboratory on surface-disinfested leaves of B. falcatum. Mycelial plugs (5 mm) were excised from the margin of colony cultured for 5 days, and placed on surface-disinfested leaves of potted B. falcatum which involved creating small wounds. The potted plants were placed in a closed bucket to keep 80% relative humidity. Controls were inoculated with non-colonized PDA plugs (5 mm). All treatments had three replicates. On the inoculated B. falcatum, the leaves of B. falcatum appeared brown spot and been covered with off-white hyphae 7 DPI. By comparision, the control leaves had no symptoms. The pathogen was reisolated from the inoculated leaves and exhibited same morphological characteristics and ITS sequence as those of D. glomerata. D. glomerata was reported to cause round leaf spot on Sophora tonkinensis Gagnep and black spot disease of Actinidia chinensis in China (Pan et al. 2018; Song et al. 2020). To our knowledge, this is the first report of leaf spot caused by D. glomerata on B. falcatum in China.

Pseudomonas cichorii causing leaf spot disease on Banxia (Pinellia ternata) in China.

Banxia (Pinellia ternata) is an important Chinese medicinal material in the family Araceae and is a widely grown herb in China. In September 2021, a leaf spot disease was observed on Banxia field, with an incidence rate of 35 to 40 % in a 4-ha field, in Zhongxiang City, Hubei Province of China. Symptoms were observed as yellow-white centers, water-soaked edges, irregular lesions, and gradually developed into a yellowish-brown center and a dark-brown edge. Necrotic spots gradually increased, leading to leaf chlorosis and plant death. Margins of leaf lesions were excised form diseased tissue and were plated on nutrient agar (NA) using serial dilution. Growth on NA was predominantly cream-colored circular bacterial colonies with undulated margins. Characterization of three randomly chosen bacterial isolates (JYB1, JYB7 and JYB8) suggests they are Gram-negative, levan negative, arginine dihydrolase negative, oxidase positive, potato soft rot positive, and tobacco hypersensitive positive. Isolates were identified as Pseudomonas cichorii based on the LOPAT scheme (Cottyn et al. 2009). Taxonomic positioning was confirmed genetically by PCR analysis using primers set: 16S rRNA gene universal primers 27F/1492R (Weisburg et al. 1991) and hrcRST gene specific primers Hcr1/Hcr2 (Cottyn et al. 2011). Homology search of 16S rRNA gene sequences (GenBank accessions: JYB1, MZ749668; JYB7, MZ823822; and JYB8, MZ823823) indicated 99.93 % (1396 bp) identity with P. cichorii strains (GenBank accessions: MK356431, JX913785, MZ723344). Similarly, comparison of the hrcRST locus (GenBank accessions: JYB1, MZ977010; JYB7, MZ977011; and JYB8, MZ977012) shared 99.38% (812 bp) with P. cichorii strains (GenBank accessions: CP007039, CP074349, GU324131). Koch's postulate was performed on healthy 30-day-old Banxia plants to confirm pathogenicity of the isolated strains. Leaves were injected with 50 µL bacterial suspensions (1x108 cfu/ml) by sterile syringe. The negative control was inoculated with sterile water. The inoculated Banxia plants were incubated at 28 °C, 70 to 80 % relative humidity, and exhibited water-soaked lesions on the leaf surface within two days around the inoculation sites. Within seven days, all leaves withered and plants died. In contrast, control plants remained healthy and symptomless. The pathogen was consistently reisolated from diseased plants and morphologically and molecularly identified as P. cichorii, while no bacterial colonies were isolated from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of bacterial leaf spot caused by P. cichorii on Banxia in China. As one of the main producing areas of Banxia in China, Jianghan Plain of Hubei Province has a planting area of nearly 20 square kilometers. The occurrence of this bacterial disease has the potential threat to the Banxia industry, more research is needed for breeding disease resistance and for developing chemical control.

First Report of Anthracnose Caused by Colletotrichum jinshuiense on Goldthread in China.

Goldthread (Coptis chinensis Franch) is one of the most widely used Chinese traditional medicine plants with remarkable medicinal properties (Mizrahi et al. 2014). In July 2019, a new anthracnose-like leaf spot disease was observed in Banqiao Town, Enshi, Hubei, China. The incidence rate ranged from 10% to 20%. Infected leaves firstly showed oil-like dots, further gradually expanded to irregular whorls with a pale center and dark-brown edge. Petiole infection led to leaves dropping when severe occurrence. Black acervuli were developed on the infected leaves with abundant setae, especially near veins. To identify the causal agent, 4-mm2 tissues were derived from the disease-health junction and surface-disinfected with 0.1% mercury dichloride for 1 min and 75% ethanol for 30 s respectively. They were placed on a PDA plate and incubated at 25°C after being rinsed with sterile water three times. Isolates were purified by single spore isolation. Colonies on PDA were white to pale-gray with dense aerial mycelia, and the underside was yellowish to olive. Colonies grow 77.5 to 81.5 mm in 1 week. No conidia were observed during vegetable growth, but conidiomatal acervuli were found on infected leaves. Setae were 1-3 septate, dark-brown, 78.0 to 134.5 µm (mean = 108 ± 23.4) long, 4.1 to 9.1 µm (mean = 6.1 ± 1.1) diameter, cylindrical to conical, apices acute. Conidiophores hyaline to pale brown, septate. Conidia were hyaline, unicellular, aseptate, curved, cylindrical, often guttulate, measuring 20.1 to 28.0 × 3.5 to 5.4 µm (mean = 25.4 ± 1.7 × 4.5 ± 0.5 µm), L/W ratio = 5.6. Hyphae septate branched, hyaline to pale brown, 1.6 to 4.5 in diameter. Hyphopodial appressoria pale to medium brown, smooth-walled, globose or obovoid, 6.3 to 9.9 × 4.1 to 7.6 µm (mean = 8.3 ± 0.9 × 7.6 ± 0.7 µm), L/W ratio = 1.1. Morphological features were similar to the description of C. jinshuiense (Fu et al. 2019). To identify its phylogenetic position, maximum-likelihood (ML) analyses of two isolates (Esh8 and Esh 11) were implemented with a concatenation of multiple sequences of the internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin (TUB2), and chitin-synthase 1 (CHS-1) using MEGA 7. The sequences were amplified using primers ITS1/ITS4, GDF1/GDR1, ACT-512F/ACT-783R, T1/Bt2b, CHS-79F/CHS-354R (Weir et al. 2012) and deposited in GenBank with accession numbers MW440484 - MW440485 (ITS), MW676256 - MW676257 (GAPDH), MW676252 - MW676253 (ACT), MW676254 - MW676255 (TUB2) and MW676258 - MW676259 (CHS-1). Results indicated they were clustered with C. jinshuiense in the C. dematium species complex. Isolates were inoculated onto injured healthy leaves (20 leaves) with mycelial plugs, ten leaves being inoculated with blank plugs were used as control. Disease symptoms were consistent with those observed in the field after five days post-inoculation with a 100% incidence rate, while no symptom was observed on the control leaves. And same isolates were isolated from six inoculated leaves with 100% re-isolation frequency. These results fulfilled Koch's postulates. In a previous study, C. boninense was identified as the causal agent of goldthread anthracnose in Chongqing, China (Ding et al. 2020). To our knowledge, this study is the first report of anthracnose on goldthread caused by C. jinshuiense in China.

Full-Length Transcriptomic Sequencing and Temporal Transcriptome Expression Profiling Analyses Offer Insights into Terpenoid Biosynthesis in Artemisia argyi.

Artemisiae argyi Folium is a traditional herbal medicine used for moxibustion heat therapy in China. The volatile oils in A.argyi leaves are closely related to its medicinal value. Records suggest that the levels of these terpenoids components within the leaves vary as a function of harvest time, with June being the optimal time for A. argyi harvesting, owing to the high levels of active ingredients during this month. However, the molecular mechanisms governing terpenoid biosynthesis and the time-dependent changes in this activity remain unclear. In this study, GC-MS analysis revealed that volatile oil levels varied across four different harvest months (April, May, June, and July) in A. argyi leaves, and the primarily terpenoids components (including both monoterpenes and sesquiterpenes) reached peak levels in early June. Through single-molecule real-time (SMRT) sequencing, corrected by Illumina RNA-sequencing (RNA-Seq), 44 full-length transcripts potentially involved in terpenoid biosynthesis were identified in this study. Differentially expressed genes (DEGs) exhibiting time-dependent expression patterns were divided into 12 coexpression clusters. Integrated chemical and transcriptomic analyses revealed distinct time-specific transcriptomic patterns associated with terpenoid biosynthesis. Subsequent hierarchical clustering and correlation analyses ultimately identified six transcripts that were closely linked to the production of these two types of terpenoid within A. argyi leaves, revealing that the structural diversity of terpenoid is related to the generation of the diverse terpene skeletons by prenyltransferase (TPS) family of enzymes. These findings can guide further studies of the molecular mechanisms underlying the quality of A. argyi leaves, aiding in the selection of optimal timing for harvests of A. argyi.

[Identification, biological characteristics, and control of pathogen causing Pinellia ternata soft rot in Hubei province].

This study was designed to identify the pathogen causing soft rot of Pinellia ternata in Qianjiang of Hubei province and screen out the effective bactericides, so as to provide a theoretical basis for the control of soft rot of P. ternata. In this study, the pathogen was identified based on molecular biology and physiological biochemistry, followed by the detection of pathogenicity and pathogenicity spectrum via plant tissue inoculation in vitro and the indoor toxicity determination using the inhibition zone method to screen out bactericide with good antibacterial effects. The control effect of the bactericide against P. ternata soft rot was verified by the leave and tuber inoculation in vitro. The phylogenetic tree was constructed based on the 16 S rDNA, dnaX gene, and recA gene sequences, respectively, and the result showed that the pathogen belonged to the same branch as the type strain Dickeya fangzhongdai JS5. The physiological and biochemical tests showed that the pathogen was identical to D. fangzhongdai, which proved that the pathogen was D. fangzhongdai. The pathogenicity test indicated that the pathogen could obviously infect leaves at 24 h and tubers in 3 d. As revealed by the indoor toxicity test, 0.3% tetramycin, 5% allicin, and 80% ethylicin had good antibacterial activities, with EC_(50) values all less than 50 mg·L~(-1). Tests in tissues in vitro showed that 5% allicin exhibited the best control effect, followed by 0.3% tetramycin and 10% zhongshengmycin oligosaccharide, and their preventive effects were better than curative effects. Therefore, 5% allicin can be used as the preferred agent for the control of P. ternata soft rot, and 0.3% tetramycin and 10% zhongshengmycin oligosaccharide as the alternatives. This study has provided a certain theoretical basis for the control of P. ternata soft rot.

Occurrence of Stem Blight Caused by Pseudomonas extremorientalis on Pinellia ternata in China.

Pinellia ternata is a perennial herbaceous plant, which tubers can be used for anti-inflammatory and has a significant position in Traditional Chinese Medicine (Marki et al. 1987). In April 2020, bacterial stem blight first occurred on P. ternata in Jingmen City (30°32'N, 111°51'E), Hubei Province, China. In the follow-up investigation, the disease also appeared in plantations of P. ternata in Qianjiang City, Tianmen City. Initial symptoms showed orange-red streak on the stem, then progressed into chlorotic and water-soaked lesions, which caused roots to be necrotic and leaves to stunting, fading, and wilting. In the end, the leaves withered, the stems rotted completely, and the incidence of plant collapse reached 20~30%. To isolate the plant pathogenic bacteria, twenty P. ternata plant samples with distinct chlorotic stem symptoms were obtained from two fields in Jingmen City. Symptomatic samples were cut to 1-cm-long pieces by sterile scalpel, then were streaked onto nutrient agar medium and grow at 28℃ for 48 h. Four pure typical aerobic, gram-negative bacteria were isolated by characterized with transparent, smooth, round, convex surfaces. The isolated colonies did not produce fluorescent pigments on King's B medium. In addition, the isolates were positive for nitrate reduction, arabinose, mannitol, D-ribose, sucrose, D-sorbitol, and were negative for gelatin liquefaction, rhamnose, D-glucose, D-melibiose. These characteristics were identified as Pseudomonas extremorientalis (Ivanova et al. 2002). One representative colony ZJH1 was selected randomly for further verification. The 16s rRNA, gyrB, and rpoD regions were obtained with primers 27F/1492R (Weisburg et al. 1991), gyrB-Fps/ gyrB-Rps, and rpoD-Fps/ rpoD-Rps, respectively (Sarkar and Guttman. 2004). These sequences were deposited in GenBank as accession nos. MT459234.1, MT469887.1 and MT469886.1, which revealed 99% homology with P. extremorientalis strain BS2774 (accession nos. LT629708.1). The pathogenicity of P. extremorientalis strain ZJH1 was confirmed by using 3-month-old, healthy, greenhouse-grown P. ternata plants. The stems were stabbed and inoculated 10 µL of the bacterial suspension (108 CFU / ml), inoculating the same amount of sterile water as a control, repeated 5 times for each treatment. The plants were cultivated in a greenhouse at 28 °C and a humidity of 80%. Three days later, the stems showed necrosis, followed by the withered leaves and died plants, whereas the control had no symptoms. P. extremorientalis were reisolated and verified again from symptomatic plants, which was consistent with Koch's postulates. This experiment was repeated thrice to get the same result. To our knowledge, this is the first report of bacterial stem blight caused by P. extremorientalis on P. ternata in China. Stem blight caused by P. extremorientalis poses a significant threat to yield and marketability of P. ternata. Further research on selecting resistant variety and effective chemical control is needed. References: Ivanova, E. P., et al. 2002. Int J Syst Evol Micr. 2113:2120. https://doi.org/10.1099/00207713-52-6-2113 Marki, T., et al. 1987. Planta Med. 53:412. Sarkar, S. F., Guttman, D. S. 2004. Appl. Environ. Microbiol. 70:1999. https://doi.org/10.1128/AEM.70.4.1999-2012.2004 Weisburg, W. G., et al. 1991. J. Bacteriol. 173:697. https://doi.org/10.1128/jb.173.2.697-703.1991 F. F. Wang and Y. J. You contributed equally to this work. The author(s) declare no conflict of interest. Funding: National Modern Agricultural Industrial Technology System (grant no. CARS-21), Technology R&D Program of Enshi Tujia and Miao Autonomous Prefecture (grant no. D20190015), Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ01), Key R&D Program of Hubei Province (grant no. 2020BCA059), Key Technology R&D Projects of Hubei Agricultural Science and Technology Innovation Center (grant no. 2020-620-000-002-04).

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal rot caused by Fusarium redolens in China.

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal stem rot caused by Fusarium redolens in China Tao Tang1, Fanfan Wang1, Jie Guo1, Xiaoliang Guo1, Yuanyuan Duan1,Jingmao You1* 1 Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences, Enshi, 445000, China. Duohua huangjing (Polygonatum cyrtonema Hua), a herbal medicine, that is mostly planted in several provinces in China. In April 2020, severe diseases with about 40% seedling losse was found in the Huangjing seedling base in Shiyan city, Hubei province. The symptoms included softening and decay of the roots and stem bases, a progressive yellowing and wilting of leaves, and finally being completely rotted. Small pieces of symptomatic stems (0.5 cm in length) and leaves (0.5 × 0.5 cm in size) were surface sterilized with 75% ethanol for 30 s, followed by 0.1% HgCl2 for 1 min, rinsed three times with sterile water, and then dried with sterilized absorbent paper. The sections were placed on potato dextrose agar (PDA) medium containing 10 µg/ml of ampicillin and incubated at 25°C in the dark. After 3 days incubation, eight isolates with the same colony morphology were sub-cultured and purified by hyphal tip isolation. Macroconidia were sickle-shaped, 15.8 - 32.3 × 3.1 - 5.6 µm (n = 25), and three to five septate. Microconidia were oval or kidney-shaped, 5.2 - 11.4 × 2.0 - 3.2 µm (n = 25), and zero to one septate. To confirm the identity of the pathogen, molecular identification was performed with strain HJCD1. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were the internal transcribed spacer (ITS) and translation elongation factor 1α (TEF-1α) using ITS1/4 (White et al. 1990) , EF1/EF2 (Taylor et al. 2016), respectively. Following BLAST searches and phylogenetic reconstruction, the ITS region (GenBank MW485770.1) showed 99% identity with those of Fusarium redolens in GenBank (KU350713.1) and the TEF-1α (GenBank MW503930.1) showed 100% identity with F. redolens GenBank (MK922537.1). Pathogenicity tests were performed to fulfill Koch's postulates. Huangjing seedlings were rinsed with sterile water, wiped clean with sterile absorbent paper, and transferred to a tray covered with wet filter paper to maintain high humidity. The mycelial piugs of F. redolens HJCD1 were inoculated onto the surface of leaves and basal stems. Controls were inoculated with sterile PDA plugs. The inoculated seedlings were sealed with plastic wrap, and then cultivated in a 25 ℃ growth chamber with 16 h of light per day. The pathogen-inoculated plants exhibited etiolation and typical wilt symptoms after 4 days, whereas no symptoms were observed in the control plants. F. redolens was reisolated from the infected tissues, and colony morphology and ITS sequence of re-isolates were same as that of HJCD1. The pathogen has been reported previously in american ginseng in China (Fan et al. 2021), lentil in Pakistan (Rafique et al. 2020), and wild rocket in United Kingdom (Taylor et al. 2019). However, to the best of our knowledge, this is the first report of F. redolens causing seelding basal rot on Duohua huangjing in China. References: White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Taylor, A., et al. 2016. Mol. Plant Pathol. 17:1032. https://doi.org/10.1111/mpp.12346 Fan, S. H., et al. 2021. Plant Dis. https://doi.org/10.1094/PDIS-11-19-2519-PDN Rafique, K., et al. 2020. Plant Dis. 9:104. https://doi.org/10.1094/PDIS-11-19-2519-PDN Taylor, A., et al. 2019. Plant Dis.6:103. https://doi.org/10.1094/PDIS-12-18-2143-PDN Funding: Science Funds for Young Scholar of Hubei Academy of Agricultural Science (grant no. 2020NKYJJ20), National Modern Agricultural Industrial Technology System (grant no. CARS-21), Technology R&D Program of Enshi (grant no. D20190015), Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ03), Key Laboratory of Integrated Management of Crops of Central China, Ministry of Agriculture, P. R. China / Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control (grant no.2020ZTSJJ6).

Occurrence of Dickeya fangzhongdai Causing Soft Rot of Banxia (Pinellia ternata) in China.

Banxia [Pinellia ternata (Thunb.) Breit., Araceae] is a perennial herbaceous plant, where the tuber is commonly used in traditional Chinese herbal medicine. In the summer of 2020, an outbreak of soft rot of Banxia was observed in Zhugentan Town (30°50'N, 112°91'E), Qianjiang City, Hubei Province, with about 56% percentage of infected plants. Symptomatic plants initially appeared with small water-soaked spots on leaves that progressed into extensive translucent spots when facing a light source. The bacteria further spread to the stems and tubers. Infected tubers appeared normal, but inner macerated inclusions exuded when touched. The whole plant was macerated and collapsed within a few days. Ten leaves with typical symptoms were obtained from a diseased field, by surface sterilizing in 75% ethanol for 30 s and 0.3% NaClO for 5 min, washing the tissue sections three times in sterile water. Small pieces of tissue (5 × 5 mm) were removed from lesion borders, plated on nutrient ager medium, and cultivated at 37 ℃ for 48 h. Five representative isolates were selected for further identification. Colonies were all smooth and transparent. In addition, these strains were Gram-negative, and had the ability to reduce D-arabinose, melibiose, galactose, raffinose, rhamnose, inositol, and mannitol, but not reduce 5-keto-D-gluconate, L-xylose, amygdalin, and sorbitol. Genomic DNA was extracted from isolate stain ZG5. The 16S rDNA gene, recombinase A (recA) gene, and DNA polymerase III subunits gamma and tau (dnaX) were amplified by PCR with the primers 27f/1492r (Weisburg et al. 1991), recF/recR (Waleron et al. 2002), and dnaXf/dnaXr (Slawiak et al. 2009), respectively. The PCR products were sequenced, then submitted to GenBank (GenBank MW332472, MW349833, MW349834, respectively). BLAST search showed that the sequences of 16S rDNA, recA, and dnaX respectively matched ≥99% with D. fangzhongdai strains DSM 101947 (CP025003), QZH3 (CP031507), and PA1 (CP020872). Pathogenicity tests were performed on 10 healthy, 3-month-old P. ternate plants. Five plants were injected with 20 µl of bacterial suspension (108 CFU/ml) of isolate ZG5, and other plants were injected with sterile water as a negative control. All tested plants were incubated at 28 ℃ and individually covered with a plastic bag. After 24 h, soft rot symptoms all appeared on the pathogen-inoculated leaves, whereas no symptoms on the control leaves. The pathogenicity test was repeated three times and obtained same results. Koch's postulates were fulfilled by reisolating D. fangzhongdai from inoculated plants. Meanwhile, PCR were performed on the reisolated bacteria as above described, and the pathogen was identified and confirmed as D. fangzhongdai. Here we report that D. fangzhongdai causes soft rot of P. ternata in China. The disease progressed very rapidly, and reduced the yield and quality of tubers. Thus, more research is needed to implement effective strategies to manage this disease.

Baseline Sensitivity and Control Efficacy of Strobilurin Fungicide Pyraclostrobin Against Sclerotium rolfsii.

Sclerotium rolfsii is a fungi pathogen of southern blight with broad host range. The quinone outside inhibitor fungicide pyraclostrobin was officially approved for controlling many diseases in 2015. In this study, baseline sensitivity of S. rolfsii to pyraclostrobin was established by measuring the 50% effective concentration (EC50) values of 155 isolates of S. rolfsii. The EC50 values ranged from 0.0291 to 1.0871 µg/ml, with a mean EC50 of 0.4469 ± 0.2490 µg/ml (mean ± standard deviation). In a preventive fungicide in vitro experiment and a field experiment, pyraclostrobin preventive efficacy reached 90% and 80%, respectively, which were much higher than that of the control agent carbendazim. Curative efficacy of pyraclostrobin was significantly lower than its preventive efficacy. Pyraclostrobin at 0.1, 0.5, and 2 µg/ml significantly reduced the number of sclerotia produced on potato dextrose agar medium, but had no significant influence on their total weight. Pyraclostrobin had no significant influence on mycelial cell membrane permeability, but it significantly reduced oxalate secretion and protein synthesis of S. rolfsii. Our findings are of great significance for resistance monitoring of S. rolfsii and also provide new insight into the action mechanism of pyraclostrobin against S. rolfsii.

Comparative transcriptome analysis of rhizome nodes and internodes in Panax. japonicus var. major reveals candidate genes involved in the biosynthesis of triterpenoid saponins.

The rhizome of P. japonicus var. major, one of the important herbs in Traditional Chinese medicine (TCM), has been used as tonic and hemostatic drugs in Tujia and Miao ethnic groups of China for thousand years. In this study, comparative metabolite and transcriptome analysis of rhizome nodes and internodes of wild P. japonicus var. major was performed to reveal their different roles in the biosynthesis of triterpene saponins. The results showed that the node was the crucial section for the synthesis of ginsenosides in the rhizome. The content of oleanane-type ginsenosides in the node was much higher than those in the internode. Most isoprenoid biosynthesis-related genes were highly expressed in the node. And, candidate UDP-glycosyltransferase (UGT) genes were also found to be differentially expressed between node and internode. Our study will provide a better understanding of the metabolism of ginsenosides in the rhizome of P. japonicus var. major.

Full-length transcriptome sequencing and modular organization analysis of oleanolic acid- and dammarane-type saponins related gene expression patterns in Panax japonicus.

The saponins found in Panax japonicus, a traditional medicinal herb in Asia, exhibit high degrees of structural and functional similarity. In this study, metabolite analysis revealed that oleanolic acid-type and dammarane-type saponins were distributed unevenly in three tissues (rhizome_Y, rhizome_O, and secRoot) of P. japonicus. Single-molecule real-time (SMRT) sequencing and next generation sequencing (NGS) data revealed distinct and tissue-specific transcriptomic patterns relating to the production of these two types of saponins. In the co-expression network and hierarchical clustering analyses, one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and two 1-deoxy-D-xylulose-5-phosphate synthase (DXS) etc. transcripts were found to be key genes associated with the biosynthesis of oleanolic acid and dammarane-type saponins in P. japonicus, respectively. In addition, cytochrome p450 (CYP) and UDP-glucuronosyltransferase (UGT) family proteins that serve as regulators of saponin biosynthesis-related genes were also found to exhibit tissue-specific expression patterns. Together these results offer a comprehensive metabolomic and transcriptomic overview of P. japonicus.

Fusarium acuminatum Associated with Root Rot of Ophiopogon japonicus (Linn. f.) in China.

Ophiopogon japonicus (Linn. f.) is a perennial evergreen in the Liliaceae family that is cultivated in many provinces of China due to its high medicinal and economic value . In April 2019, an unknown root rot disease was observed on the rhizomes of O. japonicus in a commercial production field in Xiangyang City (30.83° N, 112.53° E), Hubei Province. Disease incidence was approximately 10-20%. Symptoms included chlorosis, drooping and rolling of the leaves followed by rapid death of entire plant. Infected roots appeared to be softened, necrotic, and shriveled with reddish fungal growth. Infected tissues were disinfested on surface with 75% ethanol for 30 s and 0.1% HgCl2 for 1 min, rinsed with sterile distilled water, and dried. Small pieces (2 mm × 2 mm) were then excised from disinfested tissue and incubated on potato dextrose agar (PDA) medium at 25 ℃ in the dark. After 3 days of incubation, six isolates with 75% of isolation rate and same colony morphology were sub-cultured and purified by hyphal tip isolation. Purified cultures grew rapidly and media plates (70×70 mm ) were covered with hyphae after 3 to 4 days. Cultures were initially white and became pink or red over 5 days. Microconidia were not observed. Macroconidia were produced from monophialides on branched conidiophores, which were slender, equilaterally curved, and measured 32.5 to 53.5 µm in length and 3.5 to 5.1 µm in width, with three to five septa. All strains were preliminarily identified as Fusarium acuminatum (Eslie and Summerell 2006) on the basis of morphology. To confirm the identity of the pathogen, molecular identification was performed with strain MD1. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were internal transcribed spacer (ITS), RNA polymerase second largest subunit (RPB2) and beta-tubulin gene (TUB2) regions of rDNA, using ITS1,4 (Yin et al. 1990) , RPB2-5f2/7cr (O'Donnell et al. 2010)and Btu-F-F01, Btu-F-R01 primers(Wang et al. 2014), respectively. Nucleotide sequences were deposited in NCBI (GenBank MT525360.1; MW164629; MT588110.1). BLAST analysis of the ITS sequence had 100% similarity to a 517 bp portion of F. acuminatum sequence in GenBank (MK764994.1) ；RPB2 sequence had 100% similarity to a 687 bp portion of F. acuminatum sequence in GenBank (HM068330.1) and TUB2 sequence had 99% similarity to a 964 bp portion of F. acuminatum sequence in GenBank (KT965741.1). A pathogenicity test was performed in laboratory on O. japonicus roots with isolate MD1. Mycelial plugs (5 mm) were excised from the margin of colony cultured for 5 days, and placed on three-years-old tuberous roots covered with wet sterile cotton and kept at 25℃, under 80% relative humidity. Controls were inoculated with non-colonized PDA plugs (5 mm). All treatments had three replicate plants. On incolated plants, white hyphae covered on O. japonicus roots 3 DPI became pink and by 5 DPI, roots had rot symptoms. By comparision, the control plants had no symptoms. The pathogen was reisolated from the inoculated roots and exhibited same morphological characteristics and ITS sequence as those of F. acuminatum. F. acuminatum was reported to cause fruit rot on postharvest pumpkin and Vaccinium corymbosum in China (Li et al. 2019; Wang et al. 2016).To our knowledge, this is the first report of root rot caused by F. acuminatum on O. japonicus in China.

First report of Sclerotinia blight caused by Sclerotinia sclerotiorum on Pinellia ternata in China.

Pinellia ternata is a perennial herb that belongs to the Araceae Family. The tuber of the P. ternata plant contains active ingredients such as alkaloids and pinellia starch, and can be used as a Chinese medicine for antineoplastic and anti-inflammatory disorders in humans (Marki et al. 1987). In April 2019, lesions with rotting were observed during the flowering phase on spathes of P. ternata in a field in Qianjiang City (30°50'N, 112°92'E), Hubei Province, which is one of the main production areas of P. ternata in China. Approximately 10% of P. ternata plants were symptomatic. The initial symptoms of infection were reddish-brown lesions, followed by the appearance of white cottony mycelia. Subsequently, lesions became gray-brown, and rotted with white mycelium that eventually formed on the lesions after 2 to 3 days. In the later phases, spathes were completely rotted and mycelia began to spread to the stems, until the plant wilted and died. Ten spathes at the initial stage of infection were collected in Zhugentan Town, Qianjiang City disinfested with 0.5% sodium hypochlorite for 1 min and 75% alcohol for 20 sec, then washed with sterile distilled water three times, dried, and placed on Petri plates with potato dextrose agar (PDA) and incubated at 22℃ for two days. Six fungal isolates were obtained and purified by hyphal tip isolation in fresh culture, respectively. Culture media was covered with white hyphae after 3 to 4 days of incubation, and dark-gray, rough, irregular sclerotia (1.5 to 5.5 mm in length × 1.0 to 3.5 mm in width) formed on the margins of the media, followed by the melanization as the culture aged. Eventually, black sclerotia were formed and wrapped by white hyphae. All isolates were preliminarily identified as Sclerotinia sclerotiorum (Lazarovits et al. 2000). To further identify the pathogens, molecular identification was performed with one of the six isolates (BXH1). Polymerase chain reaction was performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers SSasprF/SSasprR for the aspartyl protease gene (Abd-Elmagid et al. 2013). BLAST search analysis revealed that the 456-bp ITS sequence (GenBank MT436756.1) was ≥99% similar to S. sclerotiorum (MT177216.1, MN105884.1, MG931017.1, etc.), and the 173-bp aspartyl protease gene sequence (GenBank MT584031.1) was too (MK028159.1, MK028161.1, AF271387.1, etc.). Pathogenicity tests were carried out by inoculating disease-free, surface-disinfested spathes of thirty 30-day-old P. ternata plants in plastic pots with a sterilized mixture of peat moss and vermiculite (3:1). Five mycelial plugs (6 mm) were excised from the margin of a colony cultured for 5 days. The plugs were placed on five spathes covered with wet sterile cotton at 22±1℃, and 80% relative humidity, with a 12-h photoperiod. Five control plants were inoculated with noncolonized PDA plugs. Lesions formed on the second day, then rot and white hyphae began to appear on the third day, while the controls had no symptoms. Similar results were obtained in three repeated experiments with S. sclerotiorum being re-isolated from all diseased plants, in accordance with Koch's postulates. This disease is an emerging problem in P. ternata fields in Qianjiang, leading to extensive yield reduction and significant economic losses. To our knowledge, this is the first report of Sclerotinia blight on P. ternata in China. References: Abd-Elmagid, A., et al. 2013. J. Microbiol. Methods 92:293. https://doi.org/10.1016/j.mimet.2012.12.020 Marki, T., et al. 1987. Planta Med. 53:412. Lazarovits, G., et al. 2000. Pestic Biochem Phys. 54:62. https://doi.org/10.1006/pest.2000.2474 White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Funding: National Modern Agricultural Industrial Technology System (grant no. CARS-21), Technology R&D Program of Enshi (grant no. D20190015), Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ01), Key Laboratory of Integrated Management of Crops of Central China, Ministry of Agriculture, P. R.China / Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control (grant no.2019ZTSJJ6).

[Effects of biocontrol strain BZJN1 and streptoprofen on physicochemical properties and bacteria structure of rhizosphere soil of Atractylodes macrocephala].

Soil microorganisms are one of the important biological indictors of soil quality and can reflct the comprehensive ecological environment characteristics of the soil. The research of soil microbial diversity is the key to know the ecological functions and balance with soil. In this paper, high-throughput sequencing on PCR-amplified 16 S rRNA gene V3-V4 fragments was used to determine the bacterial diversity in rhizosphere soil of A. macrocephala under the treatment with BZJN1 or streptoprofen. The results showed that there were no significant differences of the bacteria in A. macrocephala rhizosphere soil of the streptoprofen treatment group and the biocontrol BZJN1 treatment group. All the soil bacteria was classified into 25 categories,67 classes, 108 orders, 167 families and 271 generas, except some unidentified bacteria. Proteobacteria(30.7%-34.8%) was the dominant phylum, of which Alphaproteobacteria(16.8%-18.5%) was the dominant subgroup. Compared with the control group, the relative abundance of multiple phylums bacteria in the rhizosphere soil of A. macrocephala was significantly changed in the streptoprofen treatment group and the biocontrol BZJN1 treatment group. In addition, RDA analysis showed that there was connection with different environmental factors and microbial communities. The abundance of the three genera in the rhizosphere soil of A. macrocephala was significantly positively correlated with Invertase, Urease and AP. PICRUSt function prediction results showed that BZNJ1 could enhance some bacterial functions and promote the plant growth. Biocontrol is a new type of green and safety control pest method. BZNJ1 significantly enhances some bacterial functions on the basis of effectively preventing root rot of A. macrocephala and promoting plant growth, and has no significant effect on the soil bacterial community structure. All the results can provide theoretical support for popularization of BZNJ1.

[Isolation and identification of pathogen of Dendrobium officinale gray mold and its prevention and control].

Through investigation,it was found that the main disease of leaves was grey mold on Dendrobium officinale in Hubei province,which has a great impact on the yield and quality of D. officinale. The identification of morphological and molecular biological was used to prove that the pathogen was Botrytis cinerea. Through test the effect of 5 plant source fungicides and 4 antibiotic fungicides on mycelial growth of strain HS1,which proved 0. 3% eugenol had the best inhibitory effect,EC50 was 0. 29 mg·L-1,the second was1% osthol and EC50 was 1. 12 mg·L-1,the EC50 of 0. 5% matrine was 9. 16 mg·L-1,the EC50 of the other six fungicides was higher than 10 mg·L-1. The field control effect test proved that 0. 3% eugenol had the best control effect,reaching 89. 44%,secondly for 1%osthole,which was 77. 17%,0. 5% matrine was in the third place with 62. 37% of effective rate. However,the control effect of the other fungicides was less than 60%. The three plant-derived fungicides were safe for the produce of D. officinale and showed no phytotoxicity. The effect of these fungicides on the growth of D. candidum was tested,and proved that all the fungicides were safe and harmless to D. candidum. This study provides a research basis for the safe and effective prevention and control gray mold of D. officinale.

[Identification of endophytic bacteria BZJN1 and research on biological control of root rot of Atractylodes macrocephala].

In this study， an endophytic bacteria strain BZJN1 was isolated from Atractylodes macrocephala， and identified as Bacillus subtilis by physiological and biochemical tests and molecular identification. Strain BZJN1 could inhibit the growth of mycelia of Ceratobasidium sp. significantly， and the inhibition rate was more than 70%. The mycelium growth deformity with bulge as spherical and partially exhaustible in apex or central with microscopic observation. The inhibitory rates under 3% and 6% concentrations of the cell free fermentation were 22.7% and 38.7% expectively. The field test proved that the control efficacy of treatment of 1×108 cfu·mL⁻¹ is 75.27% and 72.37% after 10 and 20 days. All the treatments of strain BZJN1 was able to promote the growth of A. macrocephala， the treatment of 1×108 cfu·mL⁻¹ could able to increase the yield to 14.1%.

[Study on Dry Matter Accumulation and Nutrient Uptake of Shiyao Angelica sinensis].

OBJECTIVE: To study the dry matter accumulation and nutrient uptake of Shiyao Angelica sinensis, in order to provide theoretical basis for reasonable fertilization. METHODS: The aerial part(stems and leaves) and root were collected in different growth periods, and the fresh weight, dry weight and contents of nitrogen, phosphorus and potassium were measured. RESULTS: The dry matter accumulation and nutrient uptake were well fitted a Logistic equation with the increase of the number of days after sowing. In the early stage of Shiyao Angelica sinensis growth, the dry matter accumulation and nutrient uptake were very slow. Beginning in late July, the dry matter accumulation and nutrient uptake of stems and leaves sped up. Starting from the occurrence peak in August, the roots of nutrient uptake sped up. In late September, the dry matter accumulation of root reached a peak. In early October, the dry matter accumulation and nutrient uptake slowed down significantly. The middle of October was the harvest time. CONCLUSION: Some phosphorus should be applied as base fertilizer in the plantation of Shiyao Angelica sinensis, and nitrogen should be applied as top fertilizer in different growth periods.