Screening, identification and evaluation of an acidophilic strain of Bacillus velezensis B4-7 for the biocontrol of tobacco bacterial wilt.

Tobacco (Nicotiana tabacum L.) bacterial wilt, caused by Ralstonia solanacearum, is indeed a highly destructive plant disease, leading to substantial damage in tobacco production. While biological control is considered an effective measure for managing bacterial wilt, related research in this area has been relatively limited compared to other control methods. In order to discover new potential antagonistic bacteria with high biocontrol efficacy against tobacco bacterial wilt, we conducted an analysis of the microbial composition differences between disease-suppressive and disease-conducive soils using Illumina sequencing. As a result, we successfully isolated six strains from the disease-suppressive soil that exhibited antibacterial activity against Ralstonia solanacearum. Among these strains, B4-7 showed the strongest antibacterial activity, even at acidic conditions with a pH of 4.0. Based on genome analysis using Average Nucleotide Identity (ANI), B4-7 was identified as Bacillus velezensis. In greenhouse and field trials, strain B4-7 significantly reduced the disease index of tobacco bacterial wilt, with control efficiencies reaching 74.03% and 46.88% respectively. Additionally, B4-7 exhibited plant-promoting abilities that led to a 35.27% increase in tobacco production in field conditions. Quantitative real-time (qPCR) analysis demonstrated that strain B4-7 effectively reduced the abundance of R. solanacearum in the rhizosphere. Genome sequencing and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis revealed that strain B4-7 potentially produces various lipopeptide metabolites, such as microlactin, bacillaene, difficidin, bacilysin, and surfactin. Furthermore, B4-7 influenced the structure of the rhizosphere soil microbial community, increasing bacterial abundance and fungal diversity, while also promoting the growth of different beneficial microorganisms. In addition, B4-7 enhanced tobacco's resistance to R. solanacearum by increasing the activities of defense enzymes, including superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL), peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO). Collectively, these findings suggest that B. velezensis B4-7 holds significant biocontrol potential and can be considered a promising candidate strain for eco-friendly management of tobacco bacterial wilt.

Structure, Synthesis, and Catalytic Performance of Emerging MXene-Based Catalysts.

As traditional fossil fuel energy development faces significant challenges, two-dimensional layered materials have become increasingly popular in various fields and have generated widespread research interest. MXene is an exceptional catalytic material that is typically integrated into functional composite materials with other substances to enhance its catalytic-reaction performance. Improving the thermal stability, electrical conductivity, and electrochemical activity, as well as enhancing the specific surface structure, can make the material an excellent catalyst for photoelectrocatalysis and energy-regeneration reactions. The article mainly outlines the structural characteristics, preparation methods, and applications of MXene in the field of catalysis. This text highlights the latest progress and performance comparison of MXene-based catalytic functional materials in various fields such as electrochemical conversion, photocatalysis, renewable energy, energy storage, and carbon capture and conversion. It also proposes future prospects and discusses the current bottlenecks and challenges in the development of MXene-based catalytic materials.

Klebsiella michiganensis: a nitrogen-fixing endohyphal bacterium from Ustilago maydis.

Ustilago maydis is a pathogenic fungus in Basidiomycota causing corn smut disease. A strain of U. maydis YZZF202006 was isolated from the tumor of corn smut collected from Jingzhou city in China. The intracellular bacteria were confirmed inner hyphal of the strain YZZF202006 by PCR amplification and fluorescence in situ hybridization (FISH) and SYTO-9. An endohyphal bacterium YZUMF202001 was isolated from the protoplasts of the strain YZZF202006. It was gram-negative, short rod-shaped with smooth light yellow colony. The endohyphal bacterium was genomic evidenced as Klebsiella michiganensis on the basis of average nucleotide identity (ANI) analysis and the phylogram. Then K. michiganensis was GFP-Labeled and reintroduced into U. maydis, which confirmed the bacterium can live in hyphae of U.maydis. The bacterium can grow on N-free culture media. Its nitrogenase activity was reached av. 646.25 ± 38.61 nmol·mL- 1·h- 1 C2H4 by acetylene reduction assay. A cluster of nitrogen fixation genes (nifJHDKTXENXUSVWZMFLABQ) was found from its genome. The endohyphal K. michiganensis may play an important role to help nitrogen fixation for fungi in the future.

Evaluating the potential of Bacillus licheniformis YZCUO202005 isolated from lichens in maize growth promotion and biocontrol.

Lichens exist in an organismal organization of mycobiont, photobiont, and non-photoautotrophic bacteria. These organisms contribute to the growth of lichens even in poor nutrition substrates. However, studies on the isolation and application of non-photoautotrophic bacteria in plant growth and biocontrol are scanty. Therefore, a study was conducted to isolate and evaluate the potential of non-photoautotrophic bacteria from lichen tissues in maize plant growth promotion and biocontrol of plant pathogens (fungi and bacteria). Five bacterial strains were isolated and tested for their ability to produce indole-3-Acetic Acid (IAA). One bacterium named YZCUO202005 produced IAA, siderophores and biofilms, solubilized phosphate and potassium and exhibited extracellular enzymes (cellulases, proteases, amylase, and ß -1,3-Glucanase). Based on the 16S rRNA sequence analysis results, YZCUO202005 was identified as Bacillus licheniformis. The strain inhibited the growth of five pathogenic fungi with an inhibition percent of between 58.7% and 71.7% and two pathogenic bacteria. Under greenhouse conditions, YZCUO202005 was tested for its abilities to enhance maize seed germination, and vegetative growth. Compared with the control treatment, the strain significantly enhanced the growth of stem length (i.e. 18 ± 0.64 cm, 78 ± 0.92 cm), leaf length (i.e. 10 ± 0.36 cm, 57 ± 1.42 cm), leaf chlorophyll levels (i.e., 13 ± 0.40, 40 ± 0.43 SPAD), and root length (i.e, 9.8 ± 2.25 cm, 22.5 ± 6.59 cm). Our results demonstrated that B. licheniformis YZCUO202005 from lichens has the potential to promote plant growth and reduce fungal and bacterial pathogens' growth. Furthermore, the results suggest that lichens are naturally rich sources of plant growth promotion and biocontrol agents that would be used in agriculture.

Characterization of a Plant Growth-Promoting Endohyphal Bacillus subtilis in Fusarium acuminatum from Spiranthes sinensis.

Successful seed germination and seedling growth in orchids require an association with mycorrhizal fungi. An endophytic Fusarium fungal strain YZU 172038 exhibiting plant growth-promoting (PGP) ability was isolated from the roots of Spiranthes sinensis (Orchidaceae). The harboring endohyphal bacteria were detected in the hypha by SYTO-9 fluorescent nucleic acid staining, fluorescence in situ hybridization (FISH), and PCR amplification of the 16S rDNA gene's region. Consequently, one endohyphal bacterium (EHB) - a strain YZSR384 was isolated and identified as Bacillus subtilis based on morphology, phylogenetic analysis, and genomic information. The results indicated that the strain YZSR384 could significantly promote the growth of rice roots and shoots similar to its host fungus. Its indole acetic acid (IAA) production reached a maximum of 23.361 µg/ml on the sixth day after inoculation. The genome annotation revealed several genes involved in PGP traits, including the clusters of genes encoding the IAA (trpABCDEFS), the siderophores (entABCE), and the dissolving phosphate (pstABCS and phoABDHPR). As an EHB, B. subtilis was first isolated from endophytic Fusarium acuminatum from S. sinensis.

First Report of Leaf Spot on Ipomoea nil Caused by Alternaria alternata in China.

Ipomoea nil (Linnaeus) Roth, belonging to the Convolvulaceae family, is an ornamental and medicinal plant in China, which has the function of diuretic and expectorant, and it is also a common weed in the field. In October 2021, a leaf spot disease was observed on I. nil in a field as weed in Jingzhou (N 30° 21', E 112° 19'), Hubei Province, China. Symptoms began as small brown blotches, then developed into oval or irregularly shaped brown necrotic lesions. In severe cases, the leaves were completely necrotic and detached. In the surveyed area, the incidence was between 30% - 40%. To isolate the pathogen, twenty-one leaf pieces (5×5 mm) were cut from the lesion edges of seven symptomatic leaves, disinfected with 70% ethanol and 2% sodium hypochlorite (NaOCl), rinsed with sterile water five times, then placed on three potato dextrose agar (PDA) modified with 50 µg/mL kanamycin, and incubated at 25 °C in dark for 5 days. The isolates were subcultured by transferring mycelium tips. Sixteen fungal strains were isolated from the tissues, and nine of them showed similar morphological characteristics. After cultured 7 days on PDA at 25 °C, the nine colonies were initially white, then turned greenish brown to black in the center and had abundant fine villous aerial mycelia up to 61.5 mm in average diameter. To examine its conidial morphology, the fungi were cultured for 7 days on potato carrot agar (PCA) at 22°C with a light/dark period of 8/16 h. On PCA, conidia were brown or olive-brown, obclavate to obpyriform, with a short beak, one to five transverse and zero to three longitudinal septa. They formed chains of 1 - 8 conidia, with branches. Conidia were 16 - 46 µm long and 8 - 14 µm wide (n=50). These morphological features were similar to those described in Alternaria spp. (Simmons 2007). A single isolate "Q2" was selected for molecular identification because it was the most aggressive in preliminary leaf pathogenicity assays. The internal transcribed spacer (ITS) region of rDNA and histone 3 (H3) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990) and H3-1a/H3-1b (Zheng et al. 2015). BLAST analysis revealed that the sequences (ITS, ON360984; H3, ON375577) were 100% identical to Alternaria alternata (ITS, MK396607; H3, MN840996), respectively. Maximum likelihood analysis based on combined two gene sequences was conducted with an evolutionary model of GTR+I+G under 1000 bootstrap replicates. Phylogenetic tree showed that Q2 and Alternaria alternata 21-5 and BLH-YB-11 located in one clade supported with 99% bootstrap values. The pathogen was identified as A. alternata. To fulfill Koch's postulate, 10 ml conidia (106 spores/ml) of Q2 was sprayed on five healthy seedlings, with sterile distilled water as a control. All leaves were rinsed three times with sterile water before inoculation. All seedlings were placed in sealed plastic bags with air valves, and grown in a greenhouse (25 ± 2 ˚C, RH 65%). The test was repeated twice. After 10 days, symptoms typical of brown blotches similar to those observed in the field were observed on leaves of inoculated plants, while control remained healthy. A. alternata was re-isolated from the inoculated symptomatic leaves with a frequency of 100% based on morphological and molecular characters, thus Koch's postulate was confirmed. To the best of our knowledge, this is the first report of A. alternata causing leaf spot on I. nil in China. Our findings extended the host range of the pathogen A. alternata on characteristic plants.

First Report of stem rot in Cymbidium sinense Caused by Fusarium oxysporum (FOSC) in China.

Cymbidium sinense (Jackson ex Andr.) Willd is a perennial terrestrial plant in the orchid family mainly distributed in China, Japan, India and Southeast Asia that occupies a strong position in the flower market due to its bright green leaves and fragrant flowers (Zhang et al. 2013). Cymbidium sinense is not only valued by people for its ornamental and economic value, but its roots have antiasthmatic medicinal properties (Ke et al. 2004). In August 2020, about 15% stem rot on two-year old C. sinense with varying severity was observed in five nursery gardens located in Enshi city (N 30° 16', E 109° 29'), Hubei province, China. Typical symptoms of C. sinense included roots and inner part of the pseudobulbs changing from white to brown and rotting. Leaves became brown and withered from bottom to top, and there was an obvious blight yellow halo at the junction of diseased and healthy tissue, which eventually caused the whole plant to wilt and die (Fig. 1d). To isolate the pathogen, a total of 15 leaf tissues from the disease-health junction (3 × 3 mm) from 5 individual plants (3 leaves/plant) with symptoms were surface sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 3 min. The sterilized tissue was rinsed three times with sterilized water, and then placed on potato dextrose agar (PDA) for incubation at 28°C in the dark for 5 days. Isolated colonies were subcultured by a hyphal tip protocol. Thirteen fungal isolates were obtained. Through preliminary pathogenicity tests, we found that ten isolates induced leaf blight. These ten isolates with pathogenicity showed similar morphological characteristics, with initial white-flocculent aerial mycelium that secreted a lavender pigment and produced colonies with an irregular edge after 3 days on PDA. The ten strains were cultured on PDA plates at 28℃ for 5 and 15 days to observe colony and conidial characteristics. The ten strains were identified as Fusarium based on morphological characteristics (Leslie and Summerell 2006). Strain ML0303 was selected for further identification. Macroconidia were falciform, hyaline, slightly pointed at both ends with two to four septa, 24.0 ± 5.6 µm × 4.7 ± 0.8 µm (n = 50). Microconidia were hyaline, oval, globose, with zero to one septum, 5.5 ± 1.3 µm × 2.2 ± 0.5 µm (n = 50) (Fig. 1c). Total genomic DNA of strain ML0303 was extracted with a CTAB protocol (Stenglein and Balatti 2006). The translation elongation factor (EF-1α), RNA polymerase II second largest subunit (RPB2) and ß-tubulin (Tub2) genes were amplified respectively using primer pairs EF1/EF2, RPB2-5F2/RPB2-7cR and T1/T22 respectively (O'Donnell. et al. 2010, O'Donnell. et al. 1997). The EF-1α, RPB2 and Tub2 (accession numbers-MW719874, OL614838, OL689398, respectively) gene sequences were submitted to GenBank. EF-1α, RPB2 and Tub2 sequences of ML0303 showed 99.5% - 100% identity respectively with Fusarium oxysporum in the Genbank and FUSARIUM-ID databases. The multilocus sequence data was used to infer a phylogenetic tree via a Neighbor-joining (NJ), Maximum-likelihood (ML) and Maximum-Parsimony(MP) together with reference sequences from GenBank. The topology of the three trees was similar; only the NJ tree is presented here. Strain ML0303 and F. oxysporum formed a clade supported with high values (NJ/ML/MP: 96,95,97). The results indicated that the fungus was F. oxysporum based on the phylogenetic analysis and BLASTn queries. For pathogenicity tests, conidia of strain ML0303 were collected by rinsing PDA plates. Two-year-old C. sinense grown in plastic pots filled with sterilized autoclaved sandy loam soil were used for the tests. Three pots (two plants/pot) were included in each treatment. Spore suspensions (106spores/ml) of strain ML0303 were used to irrigate the stem-zone of the plants, and sterile water was used as control. The two treatments were placed in a greenhouse and incubated at 28±2℃ with a 14-hour light/10-hour dark cycle. The experiment was repeated twice. After three weeks, stem rot symptoms were observed on C. sinense inoculated with ML0303, that were the as same as observed in the nursery (Fig. 1e-h). No symptoms were observed on the negative control. Fusarium oxysporum was re-isolated from the infected plants to fulfill Koch's postulates. Partial EF-1α and RPB2 gene sequences were used for molecular identification. Members of the FOSC are notorious for causing many diseases, which includes stem rot of Sulcorebutia heliosa and root rot of Torreya grandis (Garibaldi et al. 2020; Zhang et al. 2016). To our knowledge, this is the first report of stem rot by F. oxysporum on C. sinense in China. The finding of this pathogen provides a clear target for stem rot control.

Plant Growth-Promoting Ability of Mycorrhizal Fusarium Strain KB-3 Enhanced by Its IAA Producing Endohyphal Bacterium, Klebsiella aerogenes.

Fusarium oxysporum KB-3 had been reported as a mycorrhizal fungus of Bletilla striata, which can promote the seed germination and vegetative growth. Endohyphal bacteria were demonstrated in the hyphae of the KB-3 by 16S rDNA PCR amplification and SYTO-9 fluorescent nucleic acid staining. A strain Klebsiella aerogenes KE-1 was isolated and identified based on the multilocus sequence analysis. The endohyphal bacterium was successfully removed from the wild strain KB-3 (KB-3-), and GFP-labeled KE-1 was also transferred to the cured strain KB-3- (KB-3+). The production of indole-3-acetic acid (IAA) in the culturing broths of strains of KE-1, KB-3, KB-3-, and KB-3+ was examined by HPLC. Their IAA productions were estimated using Salkowski colorimetric technique. The highest concentrations of IAA were 76.9 (at 48 h after inoculation), 31.4, 9.6, and 19.4 µg/ml (at 60 h after inoculation), respectively. Similarly, the three fungal cultural broths exhibited plant promoting abilities on the tomato root and stem growth. The results indicated that the ability of mycorrhizal Fusarium strain KB-3 to promote plant growth was enhanced because its endohyphal bacterium, Klebsiella aerogenes KE-1, produced a certain amount of IAA.

First Report of Anthracnose on Rubus rosaefolius Smith Caused by Colletotrichum boninense in China.

Raspberry (Rubus rosaefolius Smith), also called march bubble or milk bubble, is widely distributed and economically important in China. Raspberries are rich in nutrients such as essential amino acids, vitamin C, dietary fiber, superoxide dismutase (SOD) and minerals (Yang et al. 2019). In May 2019, a leaf spot disease was observed on raspberry in Enshi (N29°07'10', E108°23'12'), Hubei province of China. The symptoms were small dark-brown spots (Fig.1) on over 90% of observed plants. To isolate the pathogen, leaf sections (5 mm × 3 mm) from the border of the symptomatic tissue were cut and sterilized with 75% ethanol for 30 s, followed by 2% sodium hypochlorite (NaClO) for 2 min, and then rinsed three times with sterile water. Leaf sections were placed on potato dextrose agar (PDA) medium amended with 25 µg / ml ampicillin and incubated at 25 °C in the dark for 3 days. Isolated colonies were sub-cultured on PDA by hyphal tip transfer. Eight fungal isolates with similar morphology, abundant white aerial hyphae, were collected. Colonies on PDA grew up to 80 mm in diameter by 7 days at 25 °C. The center of each colony became black (Fig.2). Conidia were unicellular, oval and hyaline. Conidia ranged in size from 14.5 to 19.75 µm × 5.80 to 10.20 µm (n=50) in 20% (v/v) V8 vegetable juice medium. No appressoria were observed. Morphological characteristics are similar to those of Colletotrichum spp. (Moriwaki et al. 2003). Total genomic DNA of a representative isolate S1 was extracted with a CTAB method (Stenglein et al. 2006). Internal transcribed spacer (ITS) region of rDNA, actin (ACT) , beta-tubulin (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced with the primer pairs of ITS4 / ITS5, ACT512F / ACT783R, Bt-2a / Bt-2b and GDF1 / GDR1, respectively (Weir et al. 2012). BLAST results showed that ITS, ACT, TUB2 and GAPDH gene sequences (GenBank accession nos. MN498030, MT780498, MT780496 and MT780497, respectively) were 99% identical to those of Colletotrichum boninense Moriwaki, Sato & Tsukiboshi (GenBank accession nos. MF076598, JX009583, JQ005588 and JX009905, respectively). Concatenated sequences of the four genes were used to conduct a phylogenetic analysis using neighbor-joining method in MEGA7 (Toussaint et al. 2016). The isolate S1 clustered with above C. boninense strains retrieved from NCBI database. Therefore, the present isolate S1 was identified as C. boninense. Pathogenicity tests were performed using one-month-old raspberry plants, 24 controls and 30 inoculated. The plants were sprayed with conidial suspension ( 106 conidia / mL) cultured on 20% (v/v) V8 vegetable juice medium for 15 days. The control plants were sprayed with sterile distilled water. All plants were covered with plastic bags 24h to maintain the relative humidity in the field. Fifteen days after inoculation, typical symptoms of brown spots were observed on leaves similar to the disease on field plants, while the leaves from the control group remained asymptomatic. C. boninense was reisolated and identified from inoculated symptomatic leaves. Anthracnose on raspberry caused by Colletotrichum gloeosporioides (Dai et al. 2013) and C. fioriniae (Schoeneberg et al. 2020) has previously been reported. However, to the best of our knowledge, this is the first report of Colletotrichum boninense causing leaf spot on Raspberry in China. If more reports of this pathogen are found on raspberries, then it may be necessary to develop effective management strategies for controlling this disease.

First Report of Leaf Spot on Akebia trifoliata Caused by Phytophthora nicotianae in China.

Akebia trifoliata (Thunb.) Koidz. is a species in the family Lardizabalaceae, which belongs to deciduous woody lianas. It is an important species of plant used in Chinese medicine. In July 2019, a leaf spot disease was observed on A. trifoliata in a nursery garden in Jingzhou (N 30° 21', E 112° 19'), Hubei Province, China. Symptoms initially appeared as small brown spots and subsequently developed into subcircular or irregular-shaped brown necrotic lesions. In severe cases, the leaves became completely necrotic and abscised. The incidence of leaf symptoms on affected plants ranged was between 30% and 40%. To isolate the pathogen, pieces of symptomatic leaves were collected and excised at the margins of lesions, surface disinfected with 70% ethanol and 0.1% HgCl2, rinsed three times with sterile water, placed on potato dextrose agar (PDA) amended with 50 µg/ml kanamycin, and incubated at 28°C in the dark for 3 days. Isolated colonies were subcultured by transferring hyphal tips. Six fungal isolates were isolated from the collected tissues. All six isolates had similar colony morphologies on on PDA and were composed of white flocculent aerial hyphae. The average radial growth rate of colonies after 7 days was 11.2 mm/d. Isolates were later cultured on 20% V8 juice agar for 20 days to encourage sporulation. Sporangia were produced on V8 media and were colorless, inverted, pear-shaped, and terminal, with obvious mastoid, 22 to 34 × 28 to 46µm (n=50); Oospores were light brown, and suborbicular, with thick wall, 18 to 26µm (n=20); Globose chlamydospores were light brown, and suborbicular, 12 to 32µm (n=50). Antheridia were not observed suggesting homothallism. These morphological charactertistics were identical to those reported for Phytophthora nicotianae (Erwin and Ribeiro 1996). We selected a single isolate 'B2', for molecular identification because it was the most aggressive in leaf pathogenicity assays. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced using primers ITS1/ITS4 (White et al. 1990). BLAST analyis revealed that the ITS sequence (GenBank accession nos. MT472132) was 100% identical to other P. nicotianae strains (GenBank accession nos. KJ754387). To fulfill Koch's postulates, a 50 ml zoospores suspension (106 spores/ml) of B2 was sprayed on the foliage of three 1-year-old healthy seedlings. Sterile distilled water to inoculate control plants. After 10 days, typical symptoms of dark brown spots were observed on all the inoculated leaves, while the control leaves remained asymptomatic. P. nicotianae was re-isolated from the inoculated, symptomatic leaves, thus confirming Koch's hypothesis. The experiment was repeated three times. To the best of our knowledge, this is the first report of P. nicotianae causing leaf spot on A. trifoliata in China. P. nicotianae is a common stramenopile pathogen that infects many plant hosts. The presence of this pathogen in an A. trifoliata nursery should be carefully considered to mitigate possible outbreaks of this disease in other fields in this growing region.

Fusarium oxysporum KB-3 from Bletilla striata: an orchid mycorrhizal fungus.

Orchid mycorrhizal fungi are essential for the seed germination and vegetative growth of orchids. The orchid Bletilla striata has great medical value in China because its tuber is rich in mannan. Some endophytic fungi were isolated from the roots of B. striata. The isolate KB-3 was selected for experiments because it could promote the germination of B. striata seeds. Based on morphological characters and phylogenetic analysis, the isolate KB-3 was identified as Fusarium oxysporum. Co-cultivation experiments of KB-3 with B. striata and Dendrobium candidum were performed to demonstrate orchid mycorrhizal structures. Microscopic examination showed that KB-3 established colonization and produced coiled hyphal structures known as pelotons within the cortical cells of both orchid roots. The results confirm that F. oxysporum KB-3 can behave as an orchid mycorrhizal fungus.

Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm.

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16-22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16-22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16-22 somite stage embryos survived when cooled at -25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at -25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at -140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (-196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques.

Draft Genome Sequence of Bacillus amyloliquefaciens XK-4-1, a Plant Growth-Promoting Endophyte with Antifungal Activity.

Here, we report the draft genome sequence of a bacterial plant-growth-promoting endophyte, Bacillus amyloliquefaciens XK-4-1, which consists of one circular chromosome of 3,941,805 bp with 3,702 coding sequences (CDSs). The data presented highlight multiple sets of functional genes associated with its plant-beneficial characteristics.

Cyclodextrin glycosyltransferase encoded by a gene of Paenibacillus azotofixans YUPP-5 exhibited a new function to hydrolyze polysaccharides with ß-1,4 linkage.

The bacteria with hydrolysis activity to glucomannan were isolated from the rhizosphere of Amorphophallus konjac through enrichment cultivation. One strain with strong activity in degrading glucomannan was identified preliminarily as Paenibacillus azotofixans YUPP-5 according to the sequence analysis of 16S rDNA. This strain is able to hydrolyze many polysaccharide with ß-1,4 linkage, including glucomannan, galactomannan, xylan, carboxymethyl cellulose, and chitin. One hydrolytic enzyme band of approximately 70 kDa was examined from the supernatants of YUPP-5 by using zymogram with mixture polysaccharides as substrate. The encoding gene had an open reading frame of 2157 bp, which deduced cyclodextrin glycosyltransferase (CGTase), including 718 amino acids with a signal peptide in the N-terminal region. When the gene was expressed in Escherichia coli BL21, the recombinant CGTase exhibited strong activity in degrading polysaccharides with ß-1,4 linkage, and in forming cyclodextrin by using carboxymethyl cellulose as substrate. This CGTase exhibited some new functions. Finally, the hydrolytic oligosaccharides from galactomannan or glucomannan were detected by thin layer chromatography. Pentasaccharide, tetrasaccharide, trisaccharide, and disaccharide could be examined as reaction time went on.