Anthracnose of Macleaya cordata Caused by Colletotrichum aenigma in China.

Macleaya cordata (Willd.) R. Br. is a perennial herbaceous medicinal plant (Papaveraceae) commercially cultivated in China which has been studied for detumescence, detoxification, and insecticidal effect (Lin et al. 2018). In August 2021, anthracnose was observed in 2-year-old M. cordata plants in Benxi county, northeast China (41°45'48″N, 123°69'15″E). Dozens of irregular reddish-brown spots (3-11 mm) were observed on each diseased leaf. The lesions were covered with a layer of gray-white mycelia. As the disease progressed, the spots became necrosis and perforation or they would merged into large lesions, ultimately resulting in wilted leaves (Fig. 1). More than 33% of the plants in a 16-ha field were infected in 2021. The diseased leaves were collected and cut into 3-8 mm pieces, surface-disinfested by immersing them into 1% NaOCl for 2 min, and rinsed three times with sterile distilled water. They were then dried with sterilized absorbent paper, placed on PDA medium amended with chloramphenicol (40 mg/L), and incubated in darkness at 25°C with a 12-h photoperiod. Twenty isolates (BLH1 to 20) were obtained and purified using a single-spore method. Isolate BLH12 was identified and used for the pathogenicity test. Colonies were sparsely fluffy with smooth edges, and gradually became gray to pale orange from the initial white. The underside of the colonies was pale orange towards the center. Conidia were single-celled, cylindrical, and transparent with broadly blunt ends, measuring (15.13 ± 1.14) × (5.80 ± 0.60) µm (n=50). Appressoria were single-celled, brown-to-dark brown, usually elliptical or irregular, and sometimes lobed. Setae were not observed. The isolate was initially identified as Colletotrichum gloeosporioides complex (Prihastuti et al. 2009). The identification was confirmed as described previously (Weir et al. 2012). The rDNA internal transcribed spacer region (OP415560), the glyceraldehyde-3-phosphate dehydrogenase (OP433642), chitin synthase (OP433643), calmodulin (OP433644), actin (OP433645), glutamine synthetase (OP433646), ß-tubulin (OP433647), and superoxide dismutase (OP433648) gene sequences were obtained (Carbone & Kohn 1999; Weir et al. 2012), and BLAST searches revealed 99-100% homology with the type culture ICMP 18608 (JX010244, JX010044, JX009683, JX009443, JX009744, JX010078, JX010389, and JX010311). A phylogenetic analysis of combining all loci indicated BLH12 and the type strain of C. aenigma were clustered in one group (Fig. 2). Based on the basis of morphological characteristics and phylogenetic relationships, BLH12 was identified as C. aenigma. For the pathogenicity test, healthy 2-year-old plants were sprayed with a BLH12 spore suspension (1 × 105/mL). Control plants were sprayed with sterile water.There were three replicates (five plants each) per treatment. All plants were incubated at 25°C (12-h photoperiod and 86% relative humidity) and examined after 7 days. The experiment was repeated twice. The inoculated plants showed lesions on the leaf surface, similar to those in the field, whereas the control plants were asymptomatic. The pathogen was successfully reisolated and identified as the methods mentioned above. This fungus reportedly infects the leaves of many woody plants in China (Wang et al. 2020; Zhang et al. 2021). This is the first report of C. aenigma causing anthracnose on M. cordata, which will provide an guideline for developing effective field control practices for the disease.

First Report of Calonectria montana Causing Leaf Spot on Ligularia fischeri in China.

Ligularia fischeri (Ledeb.) is a perennial herbal plant of Compositae that is cultivated commercially in China as a medicinal, ornamental, and edible plant. Leaf spots were observed in 2-year-old L. fischeri in Benxi County of northeast China, in August 2021. Irregular reddish brown spots ranging from 3 to 11 mm were observed on infected leaves, and each leaf had dozens of spots (Fig. 1). As the disease progressed, the diseased spots withered and the centers fell out, and multiple lesions merge into large diseased spots, causing leaf wilting. The roots and stem bases were not infected during the reproductive stage. More than 37% of the plants in a 18 ha field were infected in 2021. The ten diseased leaves were collected and cut into small (3-5 mm) pieces, which were surface-disinfested by immersing into 1% NaOCl for 2 min and rinsing with sterile distilled water three times. The leaf pieces were then placed on acidified potato dextrose agar (PDA) in petri plates and incubated in the dark at 25°C. Twenty isolates with the same morphological characteristics were obtained. Isolates were further purified by starting a new colony for each isolate from a single spore collected from water agar. Isolate TYTW7 was randomly selected for identification and pathogenicity testing. It grew rapidly and produced profuse aerial mycelia with a carmine red underside. The conidiophores had many fertile branches and formed an elongated stipe with a sphaeropedunculate vesicle at the tip. The one-septate conidia were cylindrical and almost straight with parallel walls and rounded ends. Their sizes ranged from 30.35 to 51.76 × 2.93 to 5.01 µm (n = 100) and the pathogens were initially identified as Calonectria sp. (Crous 2002; Crous et al. 2004; Lombard et al. 2015, 2016). Further confirmation of the identification was determined according to published method (Liu and Chen 2017; Shao and Li 2021). The partial gene regions including the translation elongation factor 1-alpha (GenBank accession no. OP290551), histone H3 (OP290552), calmodulin (OP290553) and ß-tubulin (OP290554) were obtained, and BLAST searches showed 99-100% homology with the ex-type culture CERC 8952 (MF527049, MF527065, MF527081 and MF527107) and phylogenetic analysis combining all loci revealed that the isolate TYTW7 and the type strain of Ca. montana clustered in one group (Fig. 2). Based on morphological characteristics and phylogenetic analysis, isolate TYTW7 was identified as Ca. Montana. Healthy 2-year-old plants were used for the pathogenicity test. A spore suspension (1×105 spores/mL water) was used to inoculate three host plants; sterile water was sprayed on the same number plants serving as a control. The experiment was repeated three times. All plants were incubated at 27±2°C (12h photoperiod) and were evaluated after seven days. The inoculated plants showed lesions on the leaf surface, similar to those in the field, and the control remained symptomless. The pathogens were successfully reisolated and identified by sequencing, and no pathogens were isolated from symptomless control plants. To our knowledge, this is the first report of Ca. montana causing L. fischeri leaf spot. The disease poses a threat to the production and more control strategies are needed on management options to minimize losses.

Anthracnose of Brachybotrys paridiformis Caused by Colletotrichum siamense in Northeast China.

Brachybotrys paridiformis Maxim. ex Oliv. (Boraginaceae) is a perennial medicinal plant and vegetable that is cultivated commercially in China. Anthracnose is a devastating disease of B. paridiformis, with annual production losses exceeding 33% based on our survey. In July 2021, anthracnose of B. paridiformis was observed on 2-year-old plants in Shenyang city, Northeast China, which is the most important region for B. paridiformis cultivation. Round or irregular-shaped black spots were exhibited on leaves, with the leaf edges most commonly infected. As the necrosis expanded, the leaves withered and dropped; young leaves were generally not infected (Fig. 1). More than 40% of the plants in a 21-ha sampling field were infected in 2021. Symptomatic leaves (n = 20) were collected and the diseased tissue was cut into small pieces, immersed in 1% NaOCl for 2 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes. After a 3-day incubation in darkness at 25 °C, 18 suspected single-pure morphologically identical Colletotrichum isolates were obtained and sequenced. Isolate SQZ9 was randomly selected and identified. Colonies on PDA were initially white, but gradually became pale brownish with a reverse side that was pale yellowish to pinkish. Aerial mycelia were grayish-white, dense, and cottony, with microsclerotia detected on some aging mycelia. The detected single-celled conidia (11.65-17.25 × 4.25-6.15 µm; n = 50) were fusiform to cylindrical with obtuse to slightly rounded ends. Appressoria were ovoid to clavate and medium brown. Setae were not observed. The morphological characteristics were similar to those of Colletotrichum spp. (Prihastuti et al. 2009; Weir et al. 2012). Initial BLAST searches of the GenBank database revealed the SQZ9 rDNA internal transcribed spacer region (OP389109, 566 bp), glyceraldehyde-3-phosphate dehydrogenase (OP407730, 260 bp), chitin synthase (OP407731, 301 bp), calmodulin (OP407732, 712 bp), actin (OP407733, 282 bp), glutamine synthetase (OP407734, 909 bp), ß-tublin (OP407735, 498 bp), and superoxide dismutase (OP407736, 396 bp) sequences were respectively 99%-100% similar to the C. siamense type strain JX010278, JX010019, JX009709, GQ856775, GQ856730, JX010100, JX010410, and JX010332 sequences (Carbone & Kohn 1999; Moriwaki & Tsukiboshi 2009; Stephenson et al. 1997). The SQZ9 identity was confirmed by constructing a phylogenetic tree combining all loci, which grouped the isolate and the C. siamense type strain in the same clade (Fig. 2). For pathogenicity tests, 15 healthy 2-year-old plants (3 plants per pot) were spray-inoculated with SQZ9 conidial suspension (1 × 105 conidia/mL) at 2 mL per plant. Same number of plants sprayed with water were used as control. This experiment was repeated twice. All plants were covered with clear plastic bags for 72 h to maintain high humidity and then placed in a greenhouse (29 °C, natural light, and 85% relative humidity). After six days, the inoculated leaves exhibited symptoms that were similar to those observed in the field, but the controls were symptomless. The same fungus was recovered from inoculated symptomatic leaves, and its identity was confirmed by sequencing and a phylogenetic analysis. This is the first report of C. siamense causing anthracnose on B. paridiformis in China. Future studies should assess the effectiveness of chemical and biological control measures for managing this disease.

Anthracnose of Tribulus terrestris Caused by Colletotrichum truncatum in Northeast China.

Tribulus terrestris L. is an annual herbaceous medicinal plant of Zygophyllaceae, which is cultivated commercially in China. Subrotund or irregular gray, sunken, necrotic spots ranging from 2 to 9 mm were observed on diseased leaves of T. terrestris landrace in Fushun County, Liaoning Province of northeast China in July 2021, with more than 32% of the plants being infected in a 18-ha field. The symptoms first appeared on older leaves and gradually spread to younger leaves. The lesions developed a white center gradually and became perforated; multiple lesions could coalesce (Fig. 1). Ten symptomatic leaves were collected and the diseased tissues were cut into small pieces, immersed in 1% NaOCl for 2 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes at 25°C in darkness. Fifteen suspected Colletotrichum single-spore fungal isolates (JL1 to JL15) with consistent morphological characteristics were obtained, and isolate JL6 was selected for identification and pathogenicity testing. Colonies on PDA were flat with an entire margin, dense and white at first, then became dark gray with numerous black microsclerotia and formed a concentric circular pattern with aging. Conidia were single-celled, sickle-curved with a tapered tip and truncate base, ranging from 16.46 to 20.26 µm in length and 2.81 to 3.96 µm in width (n=100). Setae were dark brown, septate, straight with a slightly acute tip, 75.45 to 135.63×3.19 to 4.95 µm in size. Appressoria were dark brown, round or irregular, mostly in groups. All characteristics were consistent with the descriptions of C. truncatum (Damm et al. 2009). Further confirmation of the identification was determined according to methods described previously (Damm et al. 2009). The rDNA internal transcribed spacer region (OP364400, 585 bp), and actin (OP380867, 290 bp), beta-tubulin (OP380868, 498 bp), chitin synthase 1 (OP380869, 277 bp), glyceraldehyde-3-phosphate dehydrogenase (OP380870, 280 bp), and histone (OP380871, 411 bp) genes were amplified by PCR and sequenced (Carbone and Kohn 1999; Glass and& Donaldson 1995; Guerber et al. 2003; O'Donnell and Cigelnik 1997). BLAST results showed 98-100% similarity at 85-97% coverage compared to the corresponding sequences of the type strain CBS 151.35 (GU227862, GU227960, GU228156, GU228352, GU228254, and GU228058). Phylogenetic analysis combining all loci revealed that the isolate JL6 and the type strains of C. truncatum clustered in one group (Fig. 2). One-year-old healthy seedlings of T. terrestris (cultivar: landrace) were used for pathogenicity test. Suspension (1×105 conidia/mL) of isolate JL6 was sprayed on ten seedlings, and ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. All plants were kept at 28±1°C (12 h photoperiod), and were evaluated after 7 days. The inoculated plants showed lesions on the leaf surface, similar to those in the field, and the control remained symptomless. The pathogen was successfully reisolated and identified using the methods mentioned above. To our knowledge, this is the first report of C. truncatum causing anthracnose on T. terrestris, which will provide valuable information for designing strategies to manage anthracnose on T. terrestris.

[Effects of magnesium supply level on growth, nutrient element absorption and distribution, and quality of Panax quinquefolium].

This study aims to investigate the effects of different magnesium supply levels on the growth, nutrient absorption and distribution, and quality of Panax quinquefolium, and to determine the optimum content of exchangeable magnesium in soil. Three-year-old plants of P. quinquefolium were used in this study, and eight magnesium supply gradients(CK, Mg1-Mg7) were designed for indoor pot experiment(cultivation in soil). The plant growth indexes, nutrient element content in soil and plant, and root saponin content were determined at the end of the growth period. The correlation analysis of nutrient element content in aboveground and underground parts of P. quinquefolium showed significantly negative correlations of magnesium-calcium, magnesium-potassium, and magne-sium-manganese. With the increase in magnesium supply level, the biological absorption coefficient of magnesium increased, while that of total nitrogen, potassium, iron, and manganese decreased; the biological transfer coefficient of magnesium decreased, while that of nitrogen, phosphorus, calcium, iron, and manganese increased. The saponin content was analyzed by principal component analysis, which showed the comprehensive score in the order of Mg4(2.537), Mg2(1.001), Mg3(0.600), Mg1(0), Mg7(-0.765), CK(-0.825), Mg6(-0.922), and Mg5(-1.663). The partial least squares-path modeling(PLS-PM) showed that the correlation coefficients of exchangeable magnesium and pH with quality were-0.748 and-0.755, respectively, which were significant. Magnesium-calcium, magnesium-potassium, and magnesium-manganese showed antagonism in the nutritional physiology of P. quinquefolium. Excessive application of magnesium can lead to the imbalance of nutrient elements in P. quinquefolium. The content of exchangeable magnesium in soil suitable for the quality formation of P. quinquefolium was 193.34-293.34 mg·kg~(-1). In addition to exchangeable magnesium, pH was also important to the quality formation of P. quinquefolium. Therefore, exchangeable magnesium and pH could be regarded as monitoring factors for the quality formation of P. quinquefolium.

[Research progress on pesticide residues of Panax ginseng].

Panax ginseng, a perennial herb, is prone to diseases and insect pests in the growth process, which are primarily prevented and treated by pesticides. However, due to the lack of standardization in the types, frequencies, and doses of pesticides, pesticide residues have become the main exogenous pollutants of P. ginseng. To explore the risk of pesticide residues in P. ginseng, this paper summarized and analyzed the common pesticide residues in P. ginseng, detection techniques, and pesticide residue limit stan-dards based on the published literature in recent years. The results revealed that the main pesticide residues in P. ginseng were organochlorine pesticides, such as tetrachloronitrobenzene, pentachloronitrobenzene, and hexachlorobenzene, and the detection techniques were dominated by gas chromatography(GC), liquid chromatography(LC), or those combined with mass spectrometry(MS). Because of the long half-life and difficulty in degradation, organochlorine pesticides have become the main factor affecting the export of P. ginseng. It is worth mentioning that P. ginseng has been classified as food in Japan, South Korea, the European Union, and other countries, and the standards of pesticide residues and limits are stricter than those in China. The quality and safety of P. ginseng are prerequisites for the efficacy of Chinese medicine and the development of traditional Chinese medicine. The formulation of scientific and effective standards for pesticide application and limits would promote the high-quality development of the P. ginseng industry.

[Research progress on pesticide residues of Panax notoginseng].

Panax notoginseng is a perennial Chinese medicinal plant, which has serious continuous cropping obstacles and is prone to a variety of diseases and insect pests during the growth process. At present, the prevention and control of pests and diseases is mainly carried out through chemical pesticides, and the consequent pesticide residues of P. notoginseng have attracted much attention. This study reviewed the types and detection methods of pesticide residues in P. notoginseng from 1981 to 2021, and compared the limits of pesticide residues in P. notoginseng in China and abroad to provide a reference for rational application of pesticides in P. notoginseng and quality control of medicinal materials, thereby promoting the sustainable development of the P. notoginseng industry in China. Currently, there are only 40 published papers on pesticide residues of P. notoginseng, which is indicative of a serious problem of insufficient research. At present, hundreds of pesticide residues in P. notoginseng can be detected simultaneously by using chromatography-tandem mass spectrometry. The pesticides detected have gradually changed from early prohibited ones, such as dichlorodiphenyl trichloroethane(DDT), benzene hexachloride(BHC), and parathion, to low toxic ones(e.g., dimethomorph, procymidone, propicona-zole, and difenoconazole). The dietary risk from pesticide residues in P. notoginseng is low, which would not cause harm to consu-mers. This study concluded that in the future, the development of the quality standard for pesticide residues of P. notoginseng should be actively carried out. To increase the pesticides used in actual production in the quality standard based on the existing ones and to guide farmers to use pesticides scientifically will be the focus of future work.

Incidence of extrauterine growth retardation and its risk factors in very preterm infants during hospitalization: a multicenter prospective study. / ä½é™¢æžæ—©äº§å„¿å®«å¤–ç”Ÿé•¿è¿Ÿç¼“åŠç›¸å ³å› ç´ çš„å¤šä¸­å¿ƒå‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To investigate the incidence of extrauterine growth retardation (EUGR) and its risk factors in very preterm infants (VPIs) during hospitalization in China. METHODS: A prospective multicenter study was performed on the medical data of 2 514 VPIs who were hospitalized in the department of neonatology in 28 hospitals from 7 areas of China between September 2019 and December 2020. According to the presence or absence of EUGR based on the evaluation of body weight at the corrected gestational age of 36 weeks or at discharge, the VPIs were classified to two groups: EUGR group (n=1 189) and non-EUGR (n=1 325). The clinical features were compared between the two groups, and the incidence of EUGR and risk factors for EUGR were examined. RESULTS: The incidence of EUGR was 47.30% (1 189/2 514) evaluated by weight. The multivariate logistic regression analysis showed that higher weight growth velocity after regaining birth weight and higher cumulative calorie intake during the first week of hospitalization were protective factors against EUGR (P<0.05), while small-for-gestational-age birth, prolonged time to the initiation of total enteral feeding, prolonged cumulative fasting time, lower breast milk intake before starting human milk fortifiers, prolonged time to the initiation of full fortified feeding, and moderate-to-severe bronchopulmonary dysplasia were risk factors for EUGR (P<0.05). CONCLUSIONS: It is crucial to reduce the incidence of EUGR by achieving total enteral feeding as early as possible, strengthening breastfeeding, increasing calorie intake in the first week after birth, improving the velocity of weight gain, and preventing moderate-severe bronchopulmonary dysplasia in VPIs.

[Response of root morphogenesis of medicinal plants to low phosphorus stress and molecular mechanism].

The content of available phosphorus in soil is generally low worldwide. Phosphorus, one of the necessary macroelements for plant growth and development, plays an important role in cell structure, material composition and energy metabolism, and signal transduction in plants. Phosphate transporter(PHT) genes are important for plant growth and development, root morphogenesis, secondary metabolism, hormone response, and phosphorus balance. Most of the active components in medicinal plants are secondary metabolites. Thus, it is essential to reveal the relationship between the regulation of phosphorus and the accumulation of active components in medicinal plants, especially the effect of phosphorus starvation on root morphogenesis of root medicinal materials and its coupling with hormone response. With advancement of molecular biology, scholars gradually emphasize the mechanism of PHT regulating the secondary metabolism of medicinal plants. This study summarized the strategies of plants to adapt to low phosphorus environment, such as changing root morphogenesis, inhibiting taproot growth, forming cluster root and changing physiological metabolism, PHT, its regulatory network, phenotypic biological characteristics and key genes in medicinal plants related to phosphorus starvation, and the response mechanism. The findings are expected to lay a basis for the cultivation of medicinal plants with high quality, excellent shape, and high price.

Occurrence of postharvest snow rot caused by Sclerotinia nivalis on Asian ginseng in China.

Asian ginseng (Panax ginseng) is a valuable medicinal plant that is commercially cultivated in China. A long postharvest storage period is required before ginseng is processed. From October 2019 to May 2020, snow rot was observed on the roots of 4- and 5-year-old fresh ginseng stored in three cold storage facilities located in Tonghua and Changbai cities in northeastern China, which are the most important regions for Asian ginseng production. We sampled 1,000 ginseng roots from the three cold storage facilities, and the average disease incidence was 21%. Initially, sparse hyphae and microsclerotia appeared on the root epidermis. Lesions gradually softened and the epidermis detached easily. Multiple infected sites slowly converged, resulting in the formation of a dense complex of multiple sclerotia and thick hyphae on the surface of the ginseng root as well as internal decay. The infection eventually spread to the adjacent ginseng roots (Fig. 1). Sixteen diseased ginseng roots were collected and then sclerotia were removed from the root surface, immersed in 1% NaClO for 2 min, rinsed three times with sterile water, and placed on potato dextrose agar (PDA) containing streptomycin (40 µg/mL) in Petri dishes. After a 3-day incubation at 20 °C in darkness, 22 suspected Sclerotinia isolates were obtained. Isolates SN1 and SN2 were randomly selected for identification. On PDA, fast-growing colonies produced white, sparse, powdery, and cotton-like aerial mycelia, and the reverse side showed the same color (Fig. 2). Small and white sclerotial primordia formed 3 days later and a ring of sclerotia was detected at the plate periphery. At 7 to 10 days after incubation, the mature sclerotia were black, spherical-to-subspherical, and elongated or fused to form irregular shapes. Each Petri dish produced 55-65 sclerotia, measuring 1.1 × 1.2 to 3.2 × 3.9 mm (n = 100). The sclerotia were firmly attached to the agar surface. The isolates were initially identified as Sclerotinia sp. (Saito 1997). After sequencing the nuclear ribosomal internal transcribed spacer region (MW927134 and MW927135) and the ß-tubulin gene (MW929179 and MW929180) (White et al. 1990; Glass and Donaldson 1995), BLAST searches revealed 100% homology with JX262268 and JX296007 of the published S. nivalis strain KGC-S0601, respectively. The pathogenicity of the two isolates was tested using detached ginseng roots. Briefly, healthy roots were washed, surface-disinfested with 75% alcohol, and rinsed with sterile water. Mycelial plugs (5 mm diameter) removed from the margin of actively growing colonies on PDA were placed on the ginseng roots. For each isolate, four roots were inoculated, with two plugs per root. Additionally, PDA plugs without mycelia were used as the negative control. The roots were placed in a fresh-keeping box at 20 °C in darkness and evaluated after 7 days. The pathogenicity test was repeated twice. The symptoms on the inoculated roots were the same as those observed on the roots during cold storage, whereas the control roots remained symptomless. The same fungus was reisolated consistently from all infected roots and its identity was confirmed by resequencing, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of S. nivalis causing postharvest snow rot on Asian ginseng in China. The occurrence of this disease threatens the postharvest storage of Asian ginseng. Hence, effective management strategies must be developed.

First Report of Anthracnose of American Ginseng caused by Colletotrichum sojae in Northeast China.

American ginseng (Panax quinquefolium) is a medicinal plant that is commercially cultivated in China. Anthracnose is a devastating disease of American ginseng, with annual production losses exceeding 20%. In July 2019, anthracnose of American ginseng was observed on 3-year-old plants in Fusong County, Jilin Province, China, the most important region of American ginseng. Round or irregular-shaped, brown, sunken and necrotic lesions (5 to 11 mm in diameter), occasionally with a concentric ring or surrounded by brown halos, were detected on leaves (Fig. 1). Multiple lesions gradually coalesced, eventually causing yellowing and wilting. More than 36% of plants in a 30-ha field were infected. Symptomatic leaves (n=16) were collected and the diseased tissue was cut into small pieces, immersed in 1% NaOCl for 2 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes. After incubation in darkness at 25°C for 4 days, 15 suspected Colletotrichum single-spore isolates purified in water agar were obtained. The isolate XTJ2 was randomly selected for identification. On PDA, colonies were white to gray, occasionally mixed with gray-black strips, and the reverse was similar to the surface. Colonies on nutrient-poor agar (SNA) were flat, thin, floccose, with an entire margin, whitish to pale gray with the same colors on the reverse. The conidia were hyaline, smooth-walled, straight with a rounded base and apex, ranging from 11.1 to 21.2 × 4.0 to 5.5 µm (n=100), with length/width =3.5. Conidia were initially aseptate, but became septate with age. Setae were dark brown with a slightly acute tip, 2 to 3-septa, and 31.5 to 81.6 µm long. Appressoria were rarely observed, brown, smooth-walled, oval, bullet-shaped or irregular. Chlamydospores were not observed. The isolate was initially identified as Colletotrichum sp. (Damm et al. 2019). Initial BLAST searches of XTJ2 sequences of the rDNA internal transcribed spacer region (GenBank accession no. MW048745), a partial glyceraldehyde-3-phosphate dehydrogenase (MW053381), chitin synthase 1 (MW053382), histone H3 (MW053383), actin (MW053384) and beta-tubulin (MW053385) in GenBank showed that the sequences were respectively 100% similar to Colletotrichum sojae sequences: NR_158358, MG600810, MG600860, MG600899, MG600954 and MG601016 (Carbone and Kohn 1999; Crous et al. 2004;Guerber et al. 2003). The identity of XTJ2 was confirmed by constructing a phylogenetic tree combining all loci, which grouped the isolate and the type strain of C. sojae into one clade (Fig. 2). The sequences of all isolates were genetically identical to the XTJ2 sequences. For pathogenicity tests, 15 healthy 3-year-old plants grown in five pots were spray-inoculated with the XTJ2 conidial suspension (1×105 spores/mL), and the same number of plants were sprayed with water as the control. This experiment was repeated twice. Plants were kept in a greenhouse (28°C, natural light, and 85% relative humidity) under clear plastic bags. After 10 days, inoculated leaves exhibited symptoms that were similar to those observed in the field, whereas the controls were symptomless. The same fungus was recovered and sequenced, and its identity was confirmed by a phylogenetic analysis. This is the first report of C. sojae causing anthracnose of American ginseng in China, being a potential threat to the production of this culture. More studies on the epidemiology of this disease are needed to improve disease management.

Occurrence of Dactylonectria torresensis Causing Root Rot on Astragalus membranaceus in northeastern China.

Astragalus membranaceus Bunge (Fabaceae) is a perennial medicinal herb widely cultivated in China. In June 2018, root rot was observed on two-year-old A. membranaceus plants in Chaoyangshan town (northeastern China). In a 40-ha field, over 40% of the plants exhibited root rot and the infected area ranged from 10 to 70% of the roots. The roots first exhibited circular or irregular brown, sunken and necrotic lesions, and finally multiple lesions coalesced. The infected root surface was destroyed, showing rusty and dry rot (Fig. 1). Symptoms were concentrated in the main roots (Carlucci et al. 2017). The aboveground parts of infected plants did not initially show symptoms but gradually wilted; 7.6% of the plants died when root decay became severe. Infected roots were not used for processing and were not marketable. Ten infected roots were collected from May to October 2018 from the above location. The diseased root tissue was cut into 25 mm3 pieces, immersed in 1% NaOCl for 2 minutes, rinsed three times with sterile water and placed on water agar in Petri plates. After 15 days of incubation at 20°C, 11 single-spore isolates were obtained. Isolates HQ1 and HQ2 were randomly selected for morphological and molecular identification. Colonies grown for 10 days produced yellow, cottony to felty aerial mycelium on potato dextrose agar. Conidiophores originating laterally or terminally from the mycelium were solitary to loosely aggregated and unbranched or sparsely branched. Macroconidia predominated and were cylindrical, with a tendency to gradually widen towards the tip; 1- to 3-septate; and 20.2 to 31.0 × 3.0 to 6.7 µm (n=100). Microconidia had mostly 0¬- to 1-septate and 8.6 to 16.7 × 1.9 to 5.1 µm (n=100) (Fig. 1). Chlamydospores were rare, but occasional chlamydospore chains were observed. The isolates were tentatively identified as Dactylonectria torresensis (Cabral et al. 2012a). Further confirmation of the two isolates was conducted by DNA sequencing of the internal transcribed spacer (ITS, GenBank accession no. MN558983 and MN558984), ß-tubulin (TUB, MN561692 and MN561693), histone 3 (HIS3, MN561694 and MN561695), and translation elongation factor (TEF, MN561696 and MN561697) genes (Cabral et al. 2012b). These sequences had 99 to 100% match with D. torresensis (JF735362 for ITS, JF735492 for TUB, JF735681 for HIS3 and JF735870 for TEF). Phylogenetic trees based on analyses of a concatenated alignment of all loci grouped these isolates into the D. torresensis clade (Fig. 2). The same two isolates were tested for pathogenicity. Healthy two-year-old plants were taken from the field, and their roots were disinfected with 75% alcohol for 3 minutes, rinsed with sterile water three times, immersed in a 1×105/ml spore suspension or sterile water (control) for 10 minutes, transferred to a tray filled with sterile sand and placed in a greenhouse (12 h photoperiod, 25°C). Twelve plants grown in three pots were used for each isolate, and the same number of plants were inoculated as a control. This experiment was repeated three times. After one month, inoculated plant roots showed the same symptoms as those observed in the field, while the controls remained symptomless and no pathogen was recovered. The same fungus was reisolated from all the infected plants and confirmed by sequencing all of the above genes. This is the first report of D. torresensis causing root rot in A. membranaceus in China. The occurrence of this disease poses a threat, and management strategies need to be developed.

Multi-Locus Phylogeny and Taxonomy of the Fungal Complex Associated With Rusty Root Rot of Panax ginseng in China.

Panax ginseng rusty root rot caused by the Ilyonectria species complex is a devastating disease, and it is one of the main factors contributing to the difficulty in continual cropping. Rusty root rot occurs in all ginseng fields, but little is known about the taxonomy of the fungal pathogen complex, especially Ilyonectria and Ilyonectria-like species. Rusty root rot samples were collected from commercial ginseng cultivation areas of China, and the pathogens were isolated and purified as single spores. Based on the combination analysis of multiple loci (rDNA-ITS, TUB, HIS3, TEF, ACT, LSU, RPB1, RPB2, and SSU) and morphological characteristics, the pathogens causing ginseng rusty root rot were determined. Fungal isolates were obtained from infected roots in 56 locations within main cultivation areas in China. A total of 766 strains were identified as Ilyonectria, Ilyonectria-like and Rhexocercosporidium species, including I. robusta (55.0%), I. communis (21.7%), I. mors-panacis (10.9%), I. pseudodestructans (2.0%), I. changbaiensis (1.3%), I. qitaiheensis (1.3%), Neonectria obtusispora (2.0%), Dactylonectria torresensis (0.5%), D. sp. (0.5%), and R. panacis (1.5%), and four novel species, Thelonectria ginsengicola (1.0%), T. jixiensis (1.0%), T. mulanensis (0.8%) and T. fusongensis (0.5%), with a total of 14 species. As the pathogen present in the highest proportion, I. robusta was the most prevalent and damaging species, unlike the pathogens reported previously. All of the examined strains were proven to cause ginseng rusty root rot. Our results indicate that the taxonomy of the fungal complex associated with ginseng rusty root rot includes Ilyonectria, Ilyonectria-like genera (Dactylonectria, Neonectria, and Thelonectria) and Rhexocercosporidium.

Seed-Associated Fungal Diversity and the Molecular Identification of Fusarium with Potential Threat to Ginseng (Panax ginseng) in China.

The utility of traditional methods for detecting seed-borne fungi is limited by the fact some fungi are unculturable or difficult to isolate. The seed-borne pathogens affecting Panax ginseng cultivation have not been fully characterized. Seed-borne fungi can be identified based on the high-throughput sequencing of internal transcribed spacer (ITS) amplicons. A hierarchical clustering tree diagram analysis based on operational taxonomic units revealed a relationship between the seed-borne fungi and the region from which the seeds were collected. This study analyzed the fungal diversity on 30 ginseng seed samples from the main ginseng-producing areas of China. The 50 most abundant genera were identified including those responsible for ginseng diseases, Fusarium, Alternaria, Nectria, Coniothyrium, Verticillium, Phoma, and Rhizoctonia. Fusarium species, which are the primary causes of root rot, were detected in all seed samples. The results of a phylogenetic analysis indicated that the seed-borne fungal species originating from the same region were closely related. Fungi on ginseng seeds from eight different regions were divided into eight clades, suggesting they were correlated with the local storage medium. A total of 518 Fusarium isolates were obtained and 10 species identified, all of which can be detrimental to ginseng production. Pathogenicity tests proved that seed-borne Fusarium species can infect ginseng seedlings and 2-year-old ginseng root, with potentially adverse effects on ginseng yield and quality.

[Photosynthetic characteristics and active ingredients differences of Asarum heterotropoides var. mandshuricum under different light irradiance].

Chlorophyll content,leaf mass to per area,net photosynthetic rate and bioactive ingredients of Asarum heterotropoides var. mandshuricum,a skiophyte grown in four levels of solar irradiance were measured and analyzed in order to investigate the response of photosynthetic capability to light irradiance and other environmental factors. It suggested that the leaf mass to per area of plant was greatest value of four kinds of light irradiance and decreasing intensity of solar irradiance resulted in the decrease of leaf mass to per area at every phenological stage. At expanding leaf stage,the rate of Chla and Chlb was 3. 11 when A. heterotropoides var. mandshuricum grew in full light irradiance which is similar to the rate of heliophytes,however,the rate of Chla and Chlb was below to 3. 0 when they grew in shading environment. The content of Chla,Chlb and Chl( a+b) was the greatest value of four kinds of light irradiance and decreasing intensity of solar irradiance resulted in its decreasing remarkably( P<0. 05). The rate of Chla and Chlb decreased but the content of Chla,Chlb and Chl( a+b) increased gradually with continued shading. The maximum value of photosynthetically active radiation appeared at 10: 00-12: 00 am in a day. The maximum value of net photosynthetic rate appeared at 8: 30-9: 00 am and the minimum value appeared at 14: 00-14: 30 pm at each phenological stage if plants grew in full sunlight. However,when plants grew in shading,the maximum value of net photosynthetic rate appeared at about 10: 30 am and the minimum value appeared at 12: 20-12: 50 pm at each phenological stage. At expanding leaf stage and flowering stage,the average of net photosynthetic rate of leaves in full sunlight was remarkably higher than those in shading and it decreased greatly with decreasing of irradiance gradually( P < 0. 05). However,at fruiting stage,the average of net photosynthetic rate of leaves in full sunlight was lower than those in 50% and 28% full sunlight but higher than those in 12% full sunlight. All photosynthetic diurnal variation parameters of plants measured in four kinds of different irradiance at three stages were used in correlation analysis. The results suggested that no significant correlation was observed between net photosynthetic rate and photosynthetically active radiation,and significant negative correlation was observed between net photosynthetic rate and environmental temperature as well as vapor pressure deficit expect for 12% full sunlight. Positive correlation was observed between net photosynthestic rate and relative humidity expect for 12% full sunlight. Significant positive correlation was observed between net photosynthetic rate and stomatal conductance in the four light treatments. Only,in 12% full sunlight,the net photosynthetic rate was significantly related to photosynthetically active radiation rather than related to environmental temperature,vapor pressure deficit and relative humidity. In each light treatment,a significant positive correlation was observed between environmental temperature and vapor pressure deficit,relative humidity as well as stomatal conductance. Volatile oil content was 1. 46%,2. 16%,1. 56%,1. 30% respectively. ethanol extracts was 23. 44%,22. 45%,22. 18%,21. 12% respectively. Asarinin content was 0. 281%,0. 291%,0. 279% and 0. 252% respectively. The characteristic components of Asarum volatile oil of plant in different light treatments did not change significantly among different groups.

[Relationship between R236C site in exon 7 of SP-B gene and respiratory distress syndrome in Han newborns in western Inner Mongolia].

OBJECTIVE: To detect and analyze the genetic variation in exon 7 of lung surfactant protein B (SP-B), and to investigate the relationship between the genetic variation and the incidence of neonatal respiratory distress syndrome (NRDS) in Han populations in western Inner Mongolia. METHODS: In the case-control study, 47 Han infants with NRDS were assigned to case group. All the 47 patients had the last three generations of their ancestors reside in western Inner Mongolia. Forty-seven Han newborns without NRDS were assigned to control group. PCR-based gene analysis was used to determine the mutation in exon 7 of SP-B gene and genotype and allele frequencies of the R236C site in exon 7 of SP-B gene. RESULTS: In Han newborns in western Inner Mongolia, there was no mutation in exon 7 of SP-B gene; two genotypes, CC and CT, were identified in the R236C site in exon 7 of SP-B gene. No TT genotype was found in the two groups. There were no significant differences in the genotype frequency of CC or CT as well as the allele frequency of C or T between the case and control groups (CC: 72% vs 85%, P>0.05; CT: 28% vs 15%, P>0.05; C: 85% vs 93%, P>0.05; T: 15% vs 7%, P>0.05). CONCLUSIONS: There is no mutation in exon 7 of SP-B gene in Han infants with NRDS in western Inner Mongolia. There is no significant association between the gene polymorphism of the R236C site in exon 7 of SP-B gene and the incidence of NRDS in Han populations in that region.