First report of Fusarium solani causing Fusarium wilt of Bletilla striata (hyacinth orchid) in Zhejiang province of China.

Bletilla striata, a member of the family Orchidaceae, is a perennial herbaceous plant used in Chinese medicine. It is a commonly cultivated economic crop in the Yangtze River Basin provinces of China, as its roots are used to treat bleeding and inflammation. In Zhejiang province, Bletilla striata has a planting area of 1400 hectares with a total production of approximately 2.6×106 kg. In October 2021, over 40% of B. striata plants showed severe wilt in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. In July, leaf curling, crinkling, and leaf-edge browning of the diseased plants were first noticed in the field. Then, necrotic streaks gradually spread to the roots. Stems displayed chlorosis and withering and when they were cut vertically, symptoms such as vascular bundle discoloration, appeared. After October, the individual plants slowly wilted and died, their aboveground parts became filamentous, and the epidermis detached from the corm's fibrous roots. Diseased plants were easily removed as the corm root had fractured. White mycelia were clearly seen in the stem. Three symptomatic leaves and three stems were cut, their surfaces disinfected, and plated on potato dextrose agar (PDA). Six strains were subsequently isolated from all samples. Fungal colonies with white to cream-colored mycelia from all tissues appeared after 3 d of incubation at 26 °C. Pure cultures obtained after monospore isolation were examined for their morphological characteristics. The colonies grew rapidly, were fluffy and appressed, and had cottony white to pale cream coloration. Microconidia were hyaline, oval to reniform, with zero or one-septate (4.0-12.0 × 1.0-5.5 µm), and usually formed on elongated monophialidic conidiogenous cells. Macroconidia were wide, fusiform, or slightly curved with one or three septa (23.0-36.0 × 4.5-7.0 µm). Chlamydospores were spherical and were abundant on carrot agar (CA) medium within 2 wk. Fresh mycelia and conidia that grew at 26 ℃ for 7 d were collected from PDA plates. Next, DNA was extracted using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). We amplified a portion of RNA polymerase II second largest subunit gene (RPB2) using primers 5f2/7cr (O'Donnell et al. 2010), the internal transcribed spacer (ITS) region using primers ITS1F/ITS4 (White et al. 1990), and the partial translation elongation factor-1α gene using primers EF1/ EF2 (O'Donnell et al. 1998) from the genomic DNA and sent the PCR amplicons for sequencing at Tsingke Biotechnology Co., Ltd., Wuhan, China. A BLAST search of the obtained sequences (GenBank accessions OP743920, OP913183, and OP913180) showed 99-100% homology with the respective sequences of the Fusarium solani reference isolate NRRL46702 (O'Donnell et al. 2008). Based on the morphological and molecular characteristics and BLAST search, the fungus was identified as F. solani (Leslie and Summerell 2006). Pathogenicity of the purified F. solani isolate was assessed by inoculateing a F. solani spore suspension of 1×106 conidia/mL (20 mL per seedling) on corm wounds made with a toothpick. Four inoculated and three non-inoculated seedlings (sterilized water as a negative control) were grown in a greenhouse at 26 °C under natural sunlight and covered with plastic bags to maintain humidity for 72 h. After 15 d, leaf browning on leaf edges, new leaf bases, and corm epidermis was observed. Symptoms, similar to those detected in the original sample, developed on the inoculated leaves, whereas the controls remained asymptomatic. Fusarium solani was successfully re-isolated from all four inoculated seedlings, and their identity confirmed by generating partial Tef1 and RPB2 sequences, thereby fulfilling the Koch's postulate. To our knowledge, F. solani has not been previously reported as a pathogen of B. striata.

First report of Calonectria pseudopteridis causing brown spot on Eriocaulon buergerianum in Zhejiang province, China.

Eriocaulon buergerianum is a traditional Chinese herb, used to treat eye diseases. In July 2020, a severe brown spot disease occurred on E. buergerianuim in Yongjia county (120°19'E, 27°58'N), Zhejiang province, China. Seventy-three plants from a survey of about 150 plants showed brown leaf spots. The spots were yellowish-brown to brown, and primarily affected leaves. As the disease progressed, the spots expanded, and fused. Sixty of the 150 plants wilted (Fig. 1 A-D). Diseased tissues were surface-sterilized in 75% ethanol (30 s), rinsed three times with sterile distilled water, air-dried (5 min), placed on potato dextrose agar (PDA) at 26°C (12-h light/dark cycle), and cultured for 4 days. Hyphal tip technique was used to obtain five isolates, which were transferred to malt extract agar (MEA), oatmeal agar (OA), and PDA. After seven days of growth at 26°C, the colonies had light, yellowish-brown centers with gray-white edges; the reverse sides had reddish- to yellowish-brown centers. Seven-day-old colonies grown on oatmeal agar (OA) produced a sparse aerial mycelium with yellowish-brown centers, and light yellowish-brown centers on the reverse side. Seven-day-old colonies grown on PDA exhibited yellowish-brown, floccose, aerial mycelia with reddish-brown to yellowish-brown pigment on the back (Fig. 1 E-G). The colonies also produced microsclerotia on OA and PDA media. On carnation leaf agar (CLA) and water agar (WA), only macroconidia were produced. Macroconidiophores comprised a stipe bearing penicillate, fertile branches, and a clavate vesicle. Macroconidia were cylindrical, rounded at both ends, colorless, hyaline, 1- to 2-septate, but mainly one, and 71.86 to 96.11 µm (mean = 82.27 µm, n = 50) × 4.13 to 5.21 µm (mean = 4.63 µm, n = 50) (Fig. 1 H-K). Morphological characteristics of the isolate on CLA or WA medium were similar to the Calonectria petridis species complex (Alfenas et al. 2015; Liu et al. 2020). The actin (Act), calmodulin (Cal), internal transcribed spacer (ITS), Histone3 (His3), a fragment of the large subunit nuclear ribosomal DNA (LSU), RNA polymerase II subunit 2 (Rpb2), translation elongation factor 1α (Tef1), and ß-tubulin 2 (Tub2) genes were sequenced. The sequences were deposited in GenBank as: His3: Not detected; Act, OM933603, MZ770831, OM933604-OM933606; Cal, OM933607, MZ770832, OM933608-OM933610; ITS, OM955624, MZ720794, OM955625-OM955627; LSU, OM955640, MZ720792, OM955641-OM955643; Rpb2, OM933615, MZ770834, OM933616-OM933618; Tef1, OM933611, MZ770833, OM933612-OM933614; and Tub2, OM933619, MZ770835, OM933620-OM933622. Sequences of the Act, Cal, ITS, LSU, Rpb2, and Tef1 genes of these strains show a 99% match to the ex-type strain Ca. pteridis CBS111793 and Ca. pseudopteridis CBS163.28 (Act, GQ280494, MT335112; Cal, GQ267413, MT335347; ITS, GQ280616, MT359808; LSU, GQ280738, MT359568; Rpb2, KY653438, MT412640; Tef1, FJ918563, MT412878). The Tub2 sequences showed 99% identity with sequences of the ex-holotype Ca. pteridis CBS111793 (Tub2, DQ190578) (Alfenas et al. 2015; Liu et al. 2020; Lombard et al. 2016). Phylogenetic analysis showed that these strains and Ca. pseudopteridis CBS163.28 were clustered in a high-support bootstrap value clade (bootstrap = 85) (Fig. 2). In August 2020, healthy E. buergerianum plants in the blooming-fruiting stage were used to test the pathogenicity of the isolates. The mycelium of isolate GJC3 was cultured on CLA medium at 26°C (12-h light/dark cycle) for ten days. Eight healthy plants were inoculated by spraying with a conidial suspension (10 mL of 1 × 105 conidia/mL). Another eight plants were sprayed with sterilized water 10 mL of sterilized water as controls. The experiment was repeated three times. Pathogenicity tests were performed in the greenhouse (60% relative humidity, 28 to 20°C day/night, temperature range, and natural sunlight). After 15 days, inoculated plants showed yellowish-brown to brown spots and withering of the leaves, whereas the control plants remained healthy (Fig. 1 L-N). Ca. pseudopteridis was reisolated from the inoculated plants, and its morphology and gene sequences were similar to the original isolate GJC3. Ca. pseudopteridis was not isolated from the control plants. Morphological characteristics, molecular data and pathogenicity test identified these organisms as Ca. pseudopteridis. This report provides a basis for further research on biology and management of this disease.

Reproductive and developmental toxicity assessments of LPM3480392, a novel mu opioid receptor biased agonist in rats.

Opioids remain the most powerful analgesics for moderate to severe pain but their clinical use, misuse and abuse has been an alarming medical problem, especially for those users at child-bearing age. Mu-opioid receptor (MOR) biased agonists have been suggested as superior alternatives with better therapeutic ratios. We recently discovered and characterized a novel MOR biased agonist, LPM3480392, which demonstrates robust analgesic effect, favorable pharmacokinetic performance, and mild respiratory suppression in vivo. To understand the safety profile of LPM3480392 on the reproductive system and embryonic development, this study evaluated the effects of LPM3480392 on the fertility and early embryonic development, embryo-fetal development, and pre- and postnatal development in rats. Results showed that LPM3480392 had mild effects on parental male and female animals, accompanied by subtle early embryonic loss and delayed ossification of fetal development during organogenesis period. In addition, although minor effects were found on normal developmental milestones and behaviors in the pups, there was no evidence of malformed effect. In conclusion, these results suggest that LPM3480392 has a favorable safety profile with only minor effects on the reproductive and developmental outcomes in animals, which support the development of LPM3480392 as a novel analgesic.

First report of Curvularia geniculata causing leave spot disease of Bletilla striata in Zhejiang of China.

Bletilla striata, a perennial herbaceous plant belonging to the family Orchidaceae, is native to China and is widely distributed in the Yangtze River basin. In China, B. striata is a popular medicinal plant that is typically used to reduce wound bleeding and inflammation. In September 2021, distinct leaf spot symptoms were observed in more than 50% of B. striata plants in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. Small, round, pale brown, necrotic spots were first observed on the leaves. Subsequently, these lesions became grayish brown in the center and dark brown with slight protuberances at the margins and eventually enlarged to 5-8 mm on the leaves. Over time, the small spots enlarged and coalesced into necrotic streaks (1-2 cm). Leaves with symptoms of disease were cut, surface-sterilized, and plated on potato dextrose agar (PDA). Fungal colonies (28×28 mm) with grayish-black mycelia from all tissues were produced after 3 days of incubation at 26 °C. The mature colonies eventually turned black in the center, with obvious rings appearing after 10 days of culture. Basal conidia ranged from pale to dark brown, whereas apical ones were pale brown, with central cells being larger and darker than basal cells. Conidia were smooth and either fusiform, cylindrical, or slightly curved with rounded tips. They ranged in length from 22.34 to 36.82 (mean = 28.63) µm with 2-4 septations and slight septal constrictions. Monospore isolation was performed to obtain a pure culture. Strain BJ2Y5 was subsequently stored in the strain Preservation Center of Wuhan University (Wuhan, China) and the strain preservation number CCTCC M 2023123 was obtained. Fresh mycelia and conidia that grew at 26 ℃ for 7 days were collected from PDA plates. DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Shanghai, China). The phylogenetic position of isolate BJ2-Y5 was clarified based on DNA sequence analysis of three loci, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Berbee et al. 1999), the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of the second largest subunit of RNA polymerase II (RPB2; O'Donnell et al. 2007). A BLAST search (GenBank accession nos. OP913168, OP743380, and OP913171) showed 99% homology to the reference isolate CBS 220.52. Based on the morphological and molecular information presented in this study these isolates were identified as C. geniculata (Hosokawa et al. 2003). Furthermore, we evaluated the pathogenicity of B. striata leaves by smearing a conidial suspension (106 conidia/mL) on both sides of leaves with and without wounds. Five inoculated and three non-inoculated leaves (smeared with sterile distilled water as a negative control) were kept in a greenhouse at 26 °C under natural sunlight and covered with plastic bags for 72 h to maintain humidity. After 7 days, small round spots appeared on the wounds. Fifteen days later, the symptoms of disease on the wounded inoculated leaves were similar to those observed in the original sample, whereas the control plants remained healthy. No symptoms of infection were observed in unwounded inoculated leaves. C. geniculata was successfully re-isolated from all five inoculated leaves and was confirmed based on Koch's postulates. To the best of our knowledge, C. geniculata infection has not been previously reported in B. striata.

First report of Calonectria ilicicola causing fruit rot on postharvest Prunus persica in Zhejiang Province, China.

Peach (Prunus persica) is an important economic tree fruit in China, with 15 million tons produced in 2020 (Xu et al. 2022). In September 2021, fruit rot on postharvest P. persica 'Yingqingtao' was observed in an orchard warehouse in Qixing district (120°41'E, 29°15'N), Zhejiang Province. Disease incidence was estimated at 25%, and yield loss was estimated at approximately 20% of the total yield. The naturally infected fruit had water-soaked, light brown lesions that fused, and produced a gray-white, dense mycelium (Fig. 1 A). The mycelia were transferred using a sterilized toothpick to potato dextrose agar (PDA) and cultured for 7 d. Macroconidia were used to produce five single-spore isolates, each from a different fruit. Six-day-old colonies grown on PDA at 26°C had light brown centers with gray-white edges; on the underside the centers were reddish brown and white towards the margin (Fig. 1 D). Isolate TGF2 was selected for further identification. Macroconidia were hyaline, straight, cylindrical, and one-to-three septae, 63.2 to 81.8 × 5.7 to 7.8 µm (mean = 73.9 ± 4.3 × 6.9 ± 0.5, n = 30) (Fig. 1 E). Chlamydospores were produced abundantly on PDA (Fig. 1 F), and measured 11.7 to 19.4 × 8.5 to 16.9 µm (n = 10). Perithecia were reddish orange, globose, and 329.9 to 417.1 µm in diameter on PDA (Fig. 1 G). Asci were hyaline and clavate, 61.2 to 91.8 × 14.4 to 20.7 µm (n = 10); ascospores were hyaline, slightly curved, 1- to 3-septate, mostly 1-septate, and 37.6 to 59.7 × 4.9 to 6.4 µm (mean = 49.9 ± 4.5 × 5.6 ± 0.4, n = 30) (Fig. 1 H-J). Morphological characteristics placed this organism within the Ca. kyotensis species complex (Liu et al. 2020). For molecular identification, the internal transcribed spacer (ITS: OP164807-OP164811), calmodulin (Cal: OP176049-OP176053), histone3 (His3: OP176054-OP176058), and translation elongation factor 1α (Tef1: OP176044-OP176048) genes were sequenced (Liu, et al., 2020). The twenty sequences were deposited in GenBank. A BLAST search of these sequences showed 99% identity with sequences of the ex-holotype Ca. ilicicola CMW 30998 (Liu et al. 2020). Bayes phylogenesis suggested that these strains and Ca. ilicicola CMW 30998 were clustered in the same clade (Bayesian posterior probability = 1) (Fig. 2). Integrating morphology and molecular data, these strains were identified as Ca. ilicicola. For pathogenicity tests, P. persica fruits were surface sterilized in 75% ethanol for 30 s and air-dried for 5 mins to allow the alcohol to volatilize. A conidial suspension (30 mL of 1 × 106 conidia/mL) of TGF2 was sprayed onto ten fruits, and ten fruits sprayed with sterilized water served as controls. The experiment was repeated three times. Fruits were kept on a mist bench at 26°C and 60% relative humidity. After 5 days, inoculated fruits showed necrotic lesions and a dense, gray-white mycelium, however, the control fruits showed no symptoms (Fig. 1 B, C). Ca. ilicicola was reisolated from lesions of inoculated fruits. Ca. ilicicola has been reported from Vaccinium sp., Glycine max, Medicago sativa (Farr and Rossman 2022; Kleczewski et al. 2019; Zhang et al. 2020). To our knowledge, this is the first report of Ca. ilicicola causing fruit rot of P. persica in China. In other research on Ca. ilicicola, we found that continuous light could inhibit its growth, suggesting a method to protect postharvest peaches.

First report of Diaporthe gardeniae causing branch blight of Gardenia jasminoides Ellis in Zhejiang Province, China.

Gardenia jasminoides Ellis, an important Chinese medicine, is cultivated on approximately 1,400 hectares in China. From August to October 2016, a severe disease affecting leaves, stems, and fruits of G. jasminoides, occurred in Cangnan (120°39'E, 27°48'N), Zhejiang province. Infected leaves or stems became shriveled, and in severe cases, young fruits presented red-brown or yellow necrotic lesions with numerous black spots. More than thirty diseased fruit and stem samples were collected, ten diseased fruits were surfacedisinfected (70% ethanol for 30 s, 1.5% sodium hypochlorite for 1 min) and kept at about 25℃ for 24 h with 80% humidity. The conidial suspension (105 conidia/ml) made of creamy drops secreted from the lesion black spots was spread onto PDA (Potato-Dextrose-Agar) and incubated at 25℃ in the dark for 7 d. Only one isolate (step01) was suspected to be the target pathogen, and other three isolates were Alternaria sp. The colony of step01 was white to grayish with an irregular edge on the front and a white to brown spiral grain on the back. Black pycnidia, produced after 20 d, were globose to subglobose, individual or overlapped, with an ostiole secreting a creamy conidial suspension. Alpha-conidia were aseptate, hyaline, oval to oblong with two oil balls, 7.4-15.9×2.4-4.5 µm (average 10.2×3.3 µm); beta-conidia were hyaline, aseptate, linetype, straight or slightly curled, 15.3-26.5×1.3-2.5 µm (average 20.8×1.6 µm). This isolate resembled Diaporthe sp. (Hansen and Barrett 1938). For species identification, DNA was extracted (Sangon Biotech Rapid Fungi Genomic DNA Isolation Kit - B518229)，and the internal transcribed spacer region (ITS), elongation factor (EF1-α), ß-Tubulin (TUB), and histone H3 (HIS) of step01 were amplified using the primer pairs ITS1/ITS4, EF1-728F/ EF1-986R, BT-2a /BT-2b and CYLH3F/CYLH3R, respectively (Udayanga et al 2014, Huang et al. 2015).These sequences were submitted to GenBank as KY797655 (ITS), MF158048 (EF1- α), MF158049 (TUB), and MF158050 (HIS). In comparison with the other sequence of Diaporthe sp. using MEGA7.0 (maximum likelihood, bootstrap replications=1,000), step01 showed 100% identity with D. gardeniae. Based on their morphology and molecular identification, step01 was identified as D. gardeniae (syn. Phomopsis gardeniae). Pathogenicity was tested on three one-year-old G. jasminoides plants by stem inoculation. Two or three stems per plant were inoculated by binding a mycelial plug (5 ×12 mm), covered by humid cotton and plastic film, to the tender stem. A total of two plants were treated. Plants were kept at about 25℃ for 4 weeks. Control plants were inoculated with PDA plugs. Leaf blight started from the apex, extended to the stalk, and the leaves finally fell off. Three months after inoculation, symptoms developed on the underlying leaves, the stem was withered with black spots, a pattern like that observed in the field. No symptoms appeared in the control leaves. Five identical colonies were re-isolated from symptomatic tissues and identified again as D. gardeniae, fulfilling the Koch's postulates. Several fungi are associated with canker, leaf spot, and fruit rot in Gardenia throughout China, including Pestalotiopsis sp. (Huang et al. 2006), Botryosphaeria dothidea (Dong et al. 2016), and Phoma sp. (Luo et al. 2016). To the best of our knowledge, this is the first report of D. gardenia infecting G. jasminoides Ellis in Zhejiang Province, China.

First report of Colletotrichum godetiae causing anthracnose fruit rot on Prunus pseudocerasus in Zhejiang province, China.

Prunus pseudocerasus (L.) G. Don is an economically important crop, with 8,420 hm2 of harvested area and 35,000 tons in 2020 (https://www.fao.org/faostat/zh/#data/QCL), and is one of the favorite fruits among consumers. A severe fruit rot disease of P. pseudocerasus cultivar "HeiZhenZhu" was observed in an orchard in Pujiang county (119°42'E, 29°21'N), Zhejiang province in April 2022. Sixty-three plants from a survey of about 200 plants showed anthracnose symptoms, with a disease incidence of more than 30%. Ten diseased fruits were collected from eight different trees. The naturally affected fruits during color changing stage showed initial light brown necrotic lesions, later, the lesions were depressed, and dark brown, the fruits were rotten, and pink conidial masses were produced (Fig. 1 A-F). Conidia were transferred using a sterilized needle into sterile water, diluted to approx. 10 conidia/µL, and spread onto potato dextrose agar (PDA) (Thermo Scientific™) (containing cephalosporin 50 µg/mL). After 24 h, single colonies were transferred, and six single-spore isolates were obtained from different plants. When the strains were grown at 26°C for 7 days, the colonies on PDA were flat with entire margin, surface medium gray-green to white, reverse salmon, light gray-green to white (Fig. 1 G). The conidia of the representative strain YTTJ-JHGS5 were unicellular, hyaline, smooth-walled, cylindrical to fusiform with one end round and one end acute, or both acute ends, 14.4 to 18.9 µm (mean = 16.4 ± 0.9 µm, n = 30) × 4.4 to 5.8 µm (mean = 5.1 ± 0.3 µm, n = 30) (Fig. 1 I). Conidiogenous cells were hyaline, smooth-walled, cylindrical to clavate, 6.5-16.8 µm × 2.6-5.3 µm (n = 30), opening 1.1-2.0 µm (Fig. 1 H). Appressoria were single, light brown to medium brown, elliptical or irregular in shape, the outline entire or undulate, 9.2 to 12.1 µm (mean = 9.2 ± 1.1 µm, n = 30) × 4.6 to 6.7 µm (mean = 5.6 ± 0.4 µm, n = 30) (Fig. 1 J). The morphological characteristics of YTTJ-JHGS5 were consistent with the Colletotrichum acutatum species complex (Damm et al. 2012). To further identify the species, the internal transcribed spacer (ITS), actin (ACT), beta-tubulin (TUB), chitin synthase (CHS-1), calmodulin (CAL) and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes were sequenced (Weir et al. 2012; O'Donnell et al. 2000). The thirty-six sequences had been deposited in GenBank (ITS: ON155427-ON155432; ACT: ON191542-ON191547; CAL: ON191548-ON191553; CHS-1: ON167522-ON167527; GPD: ON191554-ON191559; TUB2: ON191560-ON191565, respectively). A BLAST search of these sequences (ITS, ACT, CHS-1, GPD, and TUB2) showed 99% identity with the sequences of ex-holotype C. godetiae CBS133.44 (ITS: JQ948402; ACT: JQ949723; CHS-1: JQ949063; GPD: JQ948733; TUB2: JQ950053) (Damm et al. 2012); the sequences of CAL genes of these strains showed 99% identity with the sequence of C. godetiae VV-087 (CAL: MK416001) (Varjas et al. 2020). The Bayes phylogenesis showed that six strains and C. godetiae CBS133.44 were clustered in a robust branch (Bayesian posterior probability = 1). Based on the morphological characteristics and phylogenesis, these strains were identified as C. godetiae. To fulfill Koch's postulates, in April 2022, live plant pathogenicity tests were performed in the field, and color-changing-stage fruits of five-year-old trees were disinfected with 75% alcohol and air-dried for 10 min to volatilize excess alcohol. A conidial suspension (50 mL of 1 × 106 conidia/mL) was sprayed on fifteen fruits for each plant, fifteen fruits sprayed with sterilized water served as control. The experiment was repeated four times, each repeat contained ten trees. The daily average temperature and daily average RH in the orchard were 22°C and 67%, respectively. After 6 days, the fruit surfaces were depressed, dark brown, later, the lesions were expanded, and pink conidial masses were observed (Fig. 1, L). Control fruits remain healthy (Fig. 1, K). C. godetiae was re-isolated from the lesions of inoculated fruits. C. godetiae has been reported on Ceanothus sp., Fragaria × ananassa and so on worldwide (Farr and Rossman 2022), in addition, C. fructicola infected cherry (Zhao et al. 2022). To our knowledge, this is the first report of C. godetiae causing anthracnose fruit rot on Prunus pseudocerasus in China. This disease occurs mainly on immature fruits and leads to yield loss in the field, therefore it is necessary to take preventive measures in advance.

The preparation and properties of circularly polarized luminescent liquid crystal physical gels with self-supporting performance.

In recent years, great progress has been made in the preparation methods and performance regulation of host-guest doped CPL liquid crystal materials. However, there still exist some basic problems to be solved, such as complex packaging and unstable CPL properties. With the consideration of the above problems, in this study, we introduced gelators into the host-guest doped CPL liquid crystal materials to prepare CPL liquid crystal physical gels. The gelators can be assembled to form a nanofiber physical gel network, which limits the movement of the liquid crystals and enhances the stability of the CPL properties. Meanwhile, liquid crystal physical gels show self-supporting ability and the gel-sol phase transition temperature can reach 136 °C. The amplification of the glum value is achieved by self-assembly of chiral liquid crystals, and the glum value can reach -0.31. The phase structure changes with electric field and temperature, and the CPL properties can be regulated by changing the temperature and electric field. With the increasing applied voltage or the temperature, the glum value decreases. Therefore, we have successfully prepared a new type of CPL liquid crystal physical gels with self-supporting performance, stimulus response performance and large glum values.

First report of Colletotrichum scovillei causing anthracnose fruit rot on Eriobotrya japonicain in Zhejiang province, China.

Loquat (Eriobotrya japonica (Thunb.) Lindl.) is a fruit tree of high economic impotance in China. In May 2021, fruit rot on cv. "Baozhu" was observed in Yuhang district (119°40'E, 30°09'N), Zhejiang province, at an incidence of more than 30% within five orchards (the total affected area were about 121, 000 m2). Early symptoms of naturally affected fruits were dark brown, necrotic lesion (Fig. 1 A). Lesions subsequently expanded, and orange conidia were observed (Fig. 1 B). Conidia were transferred using a sterilized needle into sterile water, diluted to several conidia (approximately 10 conidia/µL) in the field of light microscope, and spread onto potato dextrose agar (PDA). After 24 h, single colonies were transferred, and six single-spore strain isolated from different fruits or locations were obtained. Six-day old colonies grown on PDA at 27°C had gray-green centers with white edges, and on the reverse side, the centers were brown (Fig. 1 C). Isolate PPGS2 was selected for further characterization. Conidia were unicellular, smooth-walled, hyaline, cylindrical with one rounded and one acute end, or with both ends rounded and they (n = 30) measured 11.1 to 16.0 µm (mean = 13.3 µm) × 3.0 to 4.6 µm (mean = 4.1) (Fig. 1 D). Appressoria were single or in smalln groups, light brown to dark brown, ovoid or elliptical with a smooth or undulate outline, and measured (n = 30) 4.6 to 8.7 µm (mean = 6.5 µm) × 4.0 to 5.6 µm (mean = 4.9 µm) (Fig. 1 H). These features of PPGS2 were consistent with species of the Colletotrichum acutatum species complex (Damm et al. 2012). For species identification, the internal transcribed spacer (ITS), beta-tubulin (TUB), chitin synthase (CHS-1), calmodulin (CAL), and actin (ACT) genes were sequenced (O'Donnell et al. 2000; Weir et al. 2012). The five sequences were deposited in GenBank (OK054581, OK077960, OK077987, OK077988 and OK077989, respectively). A BLAST search of these sequences showed 99% identity with sequences of the ex-holotype C. scovillei CBS126529 (Damm et al. 2012). The evolutionary tree shown that PPGS2 and C. scovillei CBS126529 were clustered in a branch (SH-aLRT/approximate Bayes test/bootstrap support = 88.6/0.999/96). Combining morphological characteristics with phylogenetic analysis, PPGS2 was identified as C. scovillei. To fulfill Koch's postulates, E. japonica fruit were disinfected with 75% alcohol and air-dried for 5 mins to allow the alcohol to volatilize. A conidial suspension (10 mL of 1 × 106 conidia/ml) of PPGS2 was sprayed onto six fruits, and six fruit sprayed with sterilized water served as controls. The experiment was repeated three times. Fruits were kept on a mist bench at 27°C and 80% relative humidity for 5 days. Inoculated fruit developed dark brown necrotic lesions that later fused and expanded (Fig. 1, E-G), whereas control fruit remained symptomless. C. scovillei was re-isolated from lesions of inoculated fruit. C. scovillei has been reported from fruit of Capsicum sp., Musa sp., Mangifera indica, and Clausena lansium in China (Farr and Rossman 2021). To our knowledge, this is the first report of C. scovillei causing anthracnose fruit rot of E. japonica in China. This disease results in severe economic losses both in the field and after harvest, and it is necessary to develop more effective prevention and control strategies.

First Report of Aureobasidium pullulans var. pullulans Causing Spot Blight of Pear (Pyrus pyrifolia) in Zhejiang Province of China.

Asian pear (Pyrus pyrifolia Nakai cv. cuiguan), is widely grown in Zhejiang province of China. In April 2019, symptoms consisting of small black, round leaf spots and blight flower petals were observed on over 30 % of 'Cuiguan' pear trees in an orchard (ca. 0.8 ha) near Cixi city, Zhejiang province, China. Initially, leaf spots were observed on leaf petioles, which, with time, enlarged and coalesced into necrotic streaks (1-2 cm) along the length of the petioles. Irregularly, reddish brown spots developed on flower petals, which hastened their senescence. Additional symptoms included round, black spots on leaves (2-3 mm in diameter) and necrosis of shoot tips. Symptomatic tissues from petals, petioles and leaves were plated onto potato dextrose agar (PDA). After five days of incubation at 26 °C, slimy fungal colonies (48×48 mm) with pinkish to orange-colored mycelia and with regular annulations were isolated from all tissues. After 10 days, cultured were shiny and dark brown in the center. The color of conidia ranged from hyaline to dark brown. Hyaline conidia were blastic, unicellular, ellipsoidal, smooth, with lengths that ranged from 11.03 to 27.14 (avg. 18.38) µm, and widths that ranged from 3.45 to 8.86 (avg. 6.04) µm (n = 50). Dark brown conidia were 1 to 2 celled, 10.89 to 26.03 (avg. 17.41) µm in length and 4.26 to 12.15 (avg. 6.94) µm in width (n = 50), and a slight septal constriction. Conidiogenous cells were clavate, hyaline, eseptate and top smoothly with 3-11 spores. Indistinct scars remained when the conidia dislodge from the conidiogenous cells. Single spore isolation was used to obtain pure cultures. Mycelia and conidia were scraped from cultures and DNA was extracted using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech). Amplified PCR products from the internal transcribed spacer (ITS) region ITS1F/ITS4 (White et al. 1990), the partial 28S rDNA (LSU) NL1/NL4 (Boekhout et al. 1995), the ß-tubulin (TUB) gene Bt2a/Bt2b (Glass & Donaldson 1995) and the partial elongase gene (ELO) ELO2-F/ELO2-R (Zalar et al. 2008) were sequenced (Tsingke Biotechnology Co., Hangzhou, Zhejiang). A blast search (GenBank Accession No. MT107050, OK485685, OK631951, OK631950) showed 99% Aureobasidium pullulans reference isolate CBS584.75 and EXF-150, which was consistent with the morphological data (Cene et al. 2014). Three-yr-old seedlings from 'Cuiguan' pears were spray with conidial suspension (106 conidia/ml) on the both sides of leaves without wounding. In a greenhouse (26 °C, natural light), six inoculated plants and three noninoculated plants (sprayed with sterile distilled water) enclosed in plastic bags to maintain humidity for 72 h. At 5 days after inoculation, shoot tips blackened and began to wilt. At 15 days after inoculation, symptoms similar to those on the original sample developed on inoculated petioles and leaves, whereas the control plants remained healthy. No symptoms were observed on leaves that were mature at the time of inoculation. Aureobasidium pullulans var. pullulans was reisolated from all inoculated plant. Overall, this disease shortened the life of pear flowers and reduced fruit set. To our knowledge, A. pullulans var. pullulans has not previously been reported as a pathogen of P. pyrifolia.

Engineering of a Biosensor in Response to Malate in Bacillus licheniformis.

Malate is an essential intermediate in the tricarboxylic acid (TCA) cycle; it also has valuable uses in medicine and food. The production of malate with a microbial synthesis method is still in its early stages. One of the key problems in metabolic engineering is that the dynamic and subtle changes in malate are difficult to detect. It remains critical to develop techniques with direct and precise detection of malate in microbial metabolism, which facilitates high-throughput screening of the engineered strains. In this study, a genetically encoded biosensor in response to malate was constructed in B. licheniformis. Key regulator MalR and the action site of the biosensor were first identified. Then, the output of the reporter gene expression was amplified by introducing a strong constitutive promoter and iteratively tuning the action sites. The engineered biosensor can respond to malate from 5 to 15 g/L; within this range, it shows a linear correlation between eGFP fluorescence and malate concentration. This biosensor enrich our toolbox of synthetic biology in pathway engineering for malate production in microorganisms.

First report of Colletotrichum fructicola and Colletotrichum nymphaeae causing leaf spot on Rubus corchorifolius in Zhejiang province, China.

Rubus corchorifolius is one of the most economically important fruit trees, (Tian et al. 2021). A severe leaf spot disease on leaves of R. corchorifolius was observed in Longquan county, Zhejiang province (118°42'E, 27°42'N) in 2019, with disease incidence of more than 20% on affected plants. The symptoms on leaves of the naturally affected plants were early necrotic lesion with white centers, surrounded by yellow halos (< 5 mm). Later, lesions were expanded with yellowish-brown centers, surrounded by yellow halos (< 5 mm). Putative pathogenic fungi were isolated as described by Fang (1998) and two pure single-colony fungal strains (FPZ1 and FPZ2) were selected for further analysis. The fungi was cultured on potato dextrose agar (PDA) medium for 6 days, at 25°C. The colonies had gray-green centers, white aerial mycelium and gelatinous orange conidial masses. The conidia were unicellular, smooth-walled, hyaline, cylindrical with obtuse to rounded ends, the size 10.15 to 14.09 µm (mean = 12.95 µm, n = 50) × 4.36 to 6.19 µm (mean = 5.19 µm, n = 50) were single, brown to dark brown, ovoid or irregular in shape, and 5.59 to 12.99 µm (mean = 8.77 µm, n = 50) × 4.68 to 10.36 µm (mean = 6.50 µm, n = 50). The characteristics of FPZ1 were consistent with the description of species in the Colletotrichum gloeosporioides complex (Weir et al. 2012). The conidia of FPZ2 were hyaline, smooth-walled, one-celled, fusiform, the size 9.34 to 14.09 µm (mean = 11.92 µm, n = 50) × 3.26 to 6.15 µm (mean = 4.89 µm, n = 50). Appressoria were single, darker brown, elliptical or irregular in outline, and 4.49 to 15.06 µm (mean = 9.88 µm, n = 50) × 3.23 to 7.42 µm (mean = 5.72 µm, n = 50) in size. The characteristics of FPZ2 were consistent with species of the Colletotrichum acutatum complex (Damn et al. 2012). For molecular identification of strains, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB), chitin synthase (CHS-1), and actin (ACT) genes were sequenced (Weir et al. 2012). For the strain FPZ1, the five sequences obtain were deposited in GenBank (MT846907, MT849313, MT849317, MT849315 and MT849319, respectively). A BLAST search of FPZ1 sequences showed 99% identity with the five loci sequences of type strain C. fructicola ICMP 18581 (JX010165, JX010033, JX010405, JX009866 and FJ907426) (Jayawardena et al. 2016). Similarly, for the strain FPZ2, the five sequences (MT846885, MT849314, MT849318, MT849316 and MT849320, respectively) had 99% identity with the five loci sequences of type strain C. nymphaeae CBS 515.78 (JQ948197, JQ948527, JQ949848, JQ948858 and JQ949518, respectively) (Jayawardena et al. 2016). Based on morphological characteristics and phylogenetic analysis, FPZ1 was identified as C. fructicola and FPZ2 as C. nymphaeae, respestively. For pathogenicity tests, 10 µL conidial suspension (1 × 106 conidia per ml) of FPZ1 was used to inoculate five healthy, non-wounded detached leaves, while five leaves inoculated with sterilized water served as control. The experiment was repeated three times, and all leaves were kept on a mist bench at 27°C and relative humidity 80% for 6 days. The inoculation sites of both FPZ1 and FPZ2 became brown and necrotic, while control leaves developed no symptoms. C. fructicola and C. nymphaeae were re-isolated from the lesions of inoculated leaves, fulfilling Koch's postulates. To our knowledge, this is the first report of C. fructicola and C. nymphaeae causing leaf spot on Rubus corchorifolius in China, and reports on the prevalence of C. gloeosporioides and C. acutatum species complexes will be beneficial to management of anthracnose in R. corchorifolius.

A new CcpA binding site plays a bidirectional role in carbon catabolism in Bacillus licheniformis.

Bacillus licheniformis is widely used to produce various valuable products, such as food enzymes, industrial chemicals, and biocides. The carbon catabolite regulation process in the utilization of raw materials is crucial to maximizing the efficiency of this microbial cell factory. The current understanding of the molecular mechanism of this regulation is based on limited motif patterns in protein-DNA recognition, where the typical catabolite-responsive element (CRE) motif is "TGWNANCGNTNWCA". Here, CRETre is identified and characterized as a new CRE. It consists of two palindrome arms of 6 nucleotides (AGCTTT/AAAGCT) and an intermediate spacer. CRETre is involved in bidirectional regulation in a glucose stress environment. When AGCTTT appears in the 5' end, the regulatory element exhibits a carbon catabolite activation effect, while AAAGCT in the 5' end corresponds to carbon catabolite repression. Further investigation indicated a wide occurrence of CRETre in the genome of B. licheniformis.

Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System.

Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. In this study, an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid was developed for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. By means of this design, the expression of the cas9 gene could be repressed without xylose, which significantly improved the transformation ratio from less than 0.1 cfu/µg to 2.42 cfu/µg DNA. Compared with this conditional system, a constitutive overexpression system led to significant growth retardation in bacterial cells. Both the biomass and specific growth rate decreased greatly. After transformation, successful genome editing could be triggered by 0.5% xylose. When the α-amylase gene amyL was used as a genomic target, the efficiencies of its disruption using three different protospacer-adjacent motif (PAM) sequences were 64.3%, 70.9%, and 47.1%, respectively. Moreover, temperature plays a pivotal role in the function of the constructed CRISPR system. The maximum success rate reached 97% at 20 °C, while higher temperatures negatively impacted the function of the system. These results suggested that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.

Construction of a novel sugar alcohol-inducible expression system in Bacillus licheniformis.

Bacillus licheniformis is an important industrial microorganism that can utilize a wide range of biomass. However, the lack of expression elements in B. licheniformis, especially regulated promoters, significantly restricts its applications. In this study, two promoters involved in the sugar alcohol uptake pathway, PmtlA and PmtlR, were characterized and developed as regulated promoters for expression. The results showed that mannitol, mannose, sorbitol, sorbose, and arabinose can act as inducers to activate expression from PmtlA at different levels. The induction by sorbitol was the strongest, and the optimal induction conditions were 15 g/L sorbitol during mid-logarithmic growth at 28 °C. In this work, the palindrome-like sequence 'TTGTCA-cacggctcc-TGCCAA' in PmtlA was identified as the binding site of the MtlR protein. This study helps to enrich the known inducible expression systems in B. licheniformis.

Preparation of immobilized lipase by modified polyacrylonitrile hollow membrane using nitrile-click chemistry.

The application of immobilized lipase in the enzymatic production of biodiesel has shown numerous advantages. In this study, surface of Polyacrylonitrile (PAN) hollow membrane was first modified using nitrile-click chemistry in order to fit for interaction with enzyme proteins. Then sodium alginate (SA) was introduced and the membrane was post-treated by CaCl2. When the prepared PAN-PEI-SA-CaCl2 was used for lipase immobilization, the protein loading was 36.90 mg/g, and the enzyme activity reached up to 54.47 U/g, which was 2.5 times as much as that of Novozym® 435. As a result, the constructed immobilized lipase obtained a maximum biodiesel yield of 78.5%, which was 2.4 times that of the Novozym® 435 in transesterification reactions. Moreover, the biodiesel yield decreased by only 11% after the immobilized enzyme was continuously used for 20 times. This study exhibits that this technic has broad application prospects in the field of conversion of biomass resources.

Earthquake distresses reproductive function but not fetal development in rats.

BACKGROUND: It is well known that earthquake severely threatens life-safety and physical damage. However, the empirical literature on the effects of nature disasters such as earthquake on the reproductive outcomes is limited. METHODS: On May 12th, 2008, a massive 8.0 magnitude earthquake occurred in Wenchuan, a city 92 km away from our animal facility [National Chengdu Center for Safety Evaluation of Drugs (NCCSED)]. To investigate whether this tremendous earthquake exerted adverse effects on the reproductive and developmental functions in Sprague-Dawley rats, we collected some relevant data from reproductive toxicity studies around the earthquake, and compared them with the background data, which were gathered before and after the earthquake. Copulation ratio, gestation ratio, and fertility of female rats, as well as uterine and fetal morphology, were examined. RESULTS: Our data demonstrated that the Wenchuan earthquake significantly decreased the copulation and gestation ratio, although it did not exhibit obvious side effects on some other reproductive and developmental parameters.

[Transformation of Didymella bryoniae mediated by Agrobacterium tumefaciens].

Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. The disease can seriously reduce melon yield and quality. However, little information is available on the genetics and functional genomics of the fungal pathogen. In this study, we developed an Agrobacterium-mediated transformation system for D. bryoniae by using a universal pathogenic isolate DB11 and the Agrobacterium tumefaciens strain C58C1 carrying plasmid pBIG2RHPH2 harboring the hygromycin B phosphotransferase gene (hph). Total 45 transformants could be obtained per 1 x 10(5) spores when 1 x 10(6) spores per milliliter of D. bryoniae spore suspension were cocultivated with Agrobacterium cells at OD600 = 0.15 for 48 h in the presence of induction medium (pH 5.2) containing acetosyringone at 200 microg/mL and selection medium contained 100 microg/mL of hygromycin B and 200 microg/mL of cefotaxime sodium, ampicillin and tetracycline, respectively. The transformants were stable when grown on PDA medium without hygromycin B for five times and were verified by PCR amplification with the hph primers and by Southern blot analysis with the hph probe. The transformation system will be useful for further studies of functional genes in D. bryoniae.