Identification of Lactobacillus delbrueckii subsp. bulgaricus in starter culture in production of cultured milk products

Synthetic oligonucleotide Lbul4F and Lbul5R primers were developed for the Lactobacillus delbrueckii subsp. bulgaricus DNA detection in a polymerase chain reaction; these primers have specificity and allow us to carry out efficient identification of strains and cultures of these bacteria that are used in starter cultures in production of cultured milk products.     

产胞外多糖副干酪乳杆菌HCT对酸乳品质的影响

在嗜热链球菌单菌发酵剂或保加利亚乳杆菌和嗜热链球菌混合发酵剂2种常规发酵剂的基础上添加产胞外多糖（EPS）副干酪乳杆菌HCT共同发酵,研究HCT对酸乳理化、质构以及流变学特性的影响。结果表明：与不加HCT的对照相比,在混合发酵剂中添加质量分数为1%的HCT菌株,凝乳时间可缩短0.5h,酸乳成品的硬度和黏着性分别提高了0.23N和3.81N;而在单菌发酵剂中添加则产酸较慢,延长发酵时间,酸乳成品的硬度和黏着性分别降低0.12N和1.0N,但内聚性增加了0.15。此外,在2种发酵剂中添加HCT均能显著提高酸乳成品的黏度,具有一定的抗剪切破坏作用,同时赋予酸乳良好的色泽、口感和组织状态,对持水力的影响也不显著。     

Conidia of one Fusarium solani isolate from a soybean-production field enable to be virulent to soybean and make soybean seedlings wilted

Fusarium is usually thought to cause soybean root rot,which results in a large quantity of annual yield loss in soybean production,by its secretions including Fusarium toxins and cell wall degrading enzymes,but not by the conidia themselves that do not underlie any virulence so far.Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone.We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis.Of them,except for one isolate that showed diversity of virulence to different soybeans(virulent to one soybean whereas avirulent to another soybean),the others were all virulent to the two tested soybeans:both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection,and their virulence had dosage effects that only conidia cultures of at least 5×10~6 conidia mL~(-1) could show virulence to soybean;however,the sole conidia of the F.solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted.Finally,we developed the specific cleaved amplified polymorphic sequences(CAPS)markers to easily differentiate Fusarium isolates.The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of Fusarium to soybean.     

Locus TUTOU2 determines the panicle apical abortion phenotype of rice (Oryza sativa L.) in tutou2 mutant

Rice panicle apical abortion (PAA) is a detrimental agronomic trait resulting in spikelet number reduction and yield loss. To understand its underlying molecular mechanism, we identified one recessive PAA mutant tutou2 from the offspring of tissue cultures. The mutation locus was finely mapped to a 75-kb interval on the long arm of chromosome 10. Sequence analysis revealed a single nucleotide substitution of A to T at the 941 position of LOC_Os10g31910 in tutou2, resulting in an amino acid change from isoleucine to phenylalanine. Complementation analysis showed that the degenerated panicle phenotype in tutou2 was rescued in the transgenic lines. A phenotype similar to tutou2 can also be obtained by LOC_Os10g31910 knockout in wild-type rice. These results suggested that LOC_Os10g31910 is the causative locus TUTOU2 responsible for the tutou2 PAA phenotype and probably also the locus of DEL1, previously documented as a leaf senescence gene. The significant phenotypic differences between del1 and tutou2 suggest that the locus DEL1/TUTOU2 plays roles in both leaf and panicle development which were not considered fully in previous studies.     

Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

An E3 ubiquitin ligase gene(Genbank accession no.: MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.). Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp, encoding 424 amino acids. Phylogenetic tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×bretschneideri. The predicted protein structural domain of MdPUB29 showed that it contained a U-box domain. qRT-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species, and was highly expressed in the root, while the expression of MdPUB29 was significantly inhibited by exogenous NaCl. Immunoblotting assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress conditions. Three-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with AtPUB29. The growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions, suggesting that MdPUB29 may positively regulate salt tolerance.     

氮磷比率对两种蓝藻和两种绿藻生长的影响

基于千岛湖2009-2010年春夏硅藻不占优势,蓝藻和绿藻发生相互演替的现象,选取千岛湖两种优势蓝藻和两种优势绿藻进行室内生态实验来比较氮磷比对于蓝藻和绿藻生长的影响,得到的主要结果如下:室内条件下,以BG-11培养基为基础设置了5组氮磷比(原子比:N/P=76.77、N/P=307.06、N/P=153.53、N/P=15.35、N/P=3.84)的培养液,研究氮磷比变化对两种蓝藻(水华微囊藻Microcystis flos-aquae和蓝纤维藻Dactylococcopsis sp.)和两种绿藻(小球藻Chlorella sp.和四尾栅藻Scenedesmus quadricauda)的影响,利用logistic生长方程描述四种藻生长曲线且拟合度均较高,水华微囊藻和四尾栅藻随着氮磷比的降低,最大生物量K值和内禀增长率r值均升高,蓝纤维藻则在接近于Redfield比值的培养基中r值最高,小球藻在接近于或低于Redfield比值的培养基中出现暴发性增殖。水华微囊藻在高氮磷比组(N/P=307.06和N/P=153.53)实验结束时藻体累积的总氮含量显著低于其他组(P0.05),四尾栅藻低氮磷比组(N/P=3.84)单细胞氮含量也显著低于其他各组(P0.05),四种藻类藻体中总磷的变化则是随着氮磷比的降低而显著升高(P0.05)。     

Understanding the metabolism of Mycoplasma mycoides subsp. capri in vitro by a transcriptomic analysis

It is generally known that the culture for mycoplasma is time-consuming and a variety of nutrients are needed in the culture medium. This brings a lot of difficulties to mycoplasma research and application, including Mycoplasma mycoides subsp. capri (Mmc). Furthermore, little research on the characteristics of Mmc metabolism has been reported. In this study, Mmc PG3 strain cultures were investigated for dynamic gene expression. Culture samples were harvested during logarithmic phase (PG3-1), stationary phase (PG3-2), decline phase (PG3-3) and late decline phase (PG3-4). Twelve RNA samples (three replicates for each of the four growth stages considered) from these cultures were collected and sequenced. Paired comparison between consecutive growth phases in the four growth stages showed 45 significant differentially expressed genes (P<0.01) were linked to PG3 metabolism. The enzymes these genes coded were mainly involved in ATP synthase, pyrimidine metabolism, nicotinate and nicotinamide metabolism, arginine and proline metabolism. Among these, cytidylate kinase, fructose 1,6-bisphosphate aldolases Class II, nicotinate-nucleotide adenylyltransferase and dihydrolipoamide dehydrogenase play a key role in Mmc metabolism. These results provide a baseline to build our understanding of the metabolic pathway of Mmc.     

Biotechnological testing of the regulator activity of products of mechanochemical treatment of peat and wood waste

The biological activity of substances obtained by mechanochemical treatment methods from natural raw material (peat, fir needles, larch bark) is tested by means of rape and alfalfa tissue cultures. The character of action and effective doses for regulating in vitro plant growth and development are determined.     

In-vitro assessment for the control of Fusarium species using a lactic acid bacterium isolated from yellow pitahaya(Selenicereus megalanthus(K. Schum. Ex Vaupel Moran))

The fungistatic activity of a lactic acid bacterium, which had been isolated from yellow pitahaya cultures, against fungi associated with basal rot(Fusarium oxysporum and Fusarium fujikuroi) was measured in the present study. Its activity was assessed in three fractions: fermented(S1), metabolic products(S2), and biomass(S3), using two fermentation substrates: Man Rogosa Sharpe agar(MRS) and potato dextrose agar(PDA). The bacterium was molecularly identified as Lactobacillus plantarum. S3 reduced F. fujikuroi growth by 100% over 48 h of fermentation, which occurred during the stationary phase of bacterial growth. The three fractions' fungistatic activity against F. fujikuroi depended on the substrate employed. The fermentation kinetic parameters for L. plantarum indicated that its specific growth rate was 0.46 h~(–1), with 93.63% substrate consumption, 0.045 kg kg~(–1) cell yield, and 0.54 kg kg~(–1) product yield. The kinetic parameters calculated will allow for bacteria production scaling. These in-vitro trials reveal L. plantarum's possible application as a biocontrol agent for diseases associated with Fusarium. However, further ex-vivo and in-vivo researches are required to demonstrate its behavior in crops.     

Mycoplasma leachii causes polyarthritis in calves via the blood route but is not associated with pneumonia

Mycoplasma leachii was initially isolated from arthritic calves in South Queensland, Australia, and its ability to cause clinical polyarthritis in calves was demonstrated by experimental infection. However, the source of M. leachii infection in calves and its means of spreading are not well known. In this study, one-month-old calves were inoculated with cultures of M. leachii or joint fluid (collected from M. leachii-infected calves) through the intraarticular, intravenous, intratracheal, intranasal or oral routes. Multidisciplinary procedures, including clinical assessment, etiology assessment, pathology and immunohistochemistry (IHC), were used to evaluate the pathogenicity of M. leachii in calves and to elucidate the transmission route of M. leachii infection in calves. The results showed that all calves inoculated intraarticularly with cultured GN407 or joint fluid and two-thirds of the calves inoculated intravenously with joint fluid developed severe polyarthritis, and the M. leachii antigen was detected in the joints of all infected calves by IHC and PCR. In contrast, calves inoculated with cultured M. leachii or joint fluid through the intratracheal, intranasal or oral routes did not show any M. leachii infection in the clinical assessment, etiology assessment, or pathology and IHC results. These results indicated that polyarthritis caused by M. leachii in calves is transmitted via the blood route; however, this disease is not transmitted through the respiratory or digestive routes. In addition, the M. leachii antigen was not detected in the lungs of all the inoculated calves using IHC and PCR, indicating that M. leachii is not associated with pneumonia, even in the calves inoculated through the respiratory duct. These findings are important information for the prevention and control of calf polyarthritis caused by M. leachii.     

Optimization of Transformation Efficiency of Suspension Cultured Vitis vinifera cv. Chardonnay Embryogenic Cells

Vitis vinifera cv. Chardonnay suspension cultures were established from proembryogenic mass and employed for optimizing Agrobacterium-mediated transformation system. One-factor-at-a-time experiment revealed that OD₆₀₀ of Agrobacterium, time of inoculation, co-cultivation, and cell-drying before inoculation significantly affected the transformation efficiency which reached maximum 21.5% at the following conditions: 0.8 of OD₆₀₀, 25 min of inoculation, 2 d of co-cultivation, and 10 min of cell drying. Response surface methodology experiments based on a five-level, four-factor central-composite rotatable design were then used to optimize these selected factors. The optimized conditions for Chardonnay grape transformation were: 0.8711 of OD₆₀₀, 28.9 min of inoculation, 2.25 d of co-cultivation and 11.76 min of cell drying. After optimization, transformation efficiency was 26.2% and there were no interactions among different factors.     

Identification of commercial cultivars of Agaricus bisporus in China using genome-wide microsatellite markers

Agaricus bisporus is one of the most widely cultivated mushrooms in the world. Commercial cultivars are usually phenotypically alike and easy to be copied by isolating tissue cultures. This brings great challenges to distinguish different cultivars and to protect new varieties. Thus, techniques for the accurate identification of cultivars are essentially required. In this study, we accurately identified 11 commercial cultivars of A. bisporus released in China by using microsatellite (SSR, simple sequence repeat) markers. SSR markers were developed by mining the genome sequence. A total of 3 134 SSRs were identified, of which 1 490 SSRs were distributed in gene models, and 1 644 in the intergenic regions. A total of 17 polymorphic primer pairs were developed and SSR fingerprints were constructed for all the commercial cultivars. These SSR markers generated a total of 73 alleles, with an average of 4.29 per locus. Specifically, the primer combination of AB_SSR_2341 and AB_SSR_2590 could distinguish all the 11 commercial cultivars. The similarity coefficients of the 11 commercial cultivars were between 0.56 and 0.95 indicating that some of them were close related. Our results provide an efficient technique for the identification of A. bisporus cultivars in China, which can also facilitate the marker-assisted breeding in the future.     

PGC-1α differentially regulates the mRNA expression profiles of genes related to myofiber type specificity in chicken

Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α) played a prominent role in regulating muscle fiber type transition and composition. However, the role of PGC-1α in chicken muscle has seldom been explored. To investigate the effect of PGC-1α on chicken skeletal muscles in this study, the PGC-1α gene was overexpressed or silenced in chicken primary myoblasts by using lentivirus, and then the effects of the PGC-1α gene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation. The results showed that overexpression of PGC-1α from proliferation to differentiation was accompanied by the up-regulated expression of Pax7, MyoD, and CnAα, which was significantly(P0.01) increased after one day of transfection(1 I). The enhancement of MyoG, MEF2 c, and MyHC SM expression lagged, which was improved significantly(P0.01) after four days of transfection(1 I3 D). Overexpression of PGC-1α decreased(P0.01) the MyHC FWM expression after four days of transfection(1 I3 D), and it had no significant impact(P0.05) on the expression of CnB1, NFATc3, and MyHC FRM during myofiber formation. The effective silence(P0.01) of PGC-1α by lentivirus mediating short hairpin RNA(shRNA) was detected after four days of transfection(1 I3 D) in cultures, and the lack of its function in chicken primary myoblasts significantly(P0.01) down-regulated the expression of Pax7, MyoD, CnAα, MyoG, MEF2 c, and MyHC SM, significantly(P0.01) up-regulated the expression of MyHC FWM, and had no significant impact(P0.05) on the expression of CnB1, NFATc3, and MyHC FRM. These results indicated that the role of PGC-1α in regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals, which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.     

Involvement of the autophagy-related gene BdATG8 in development and pathogenicity in Botryosphaeria dothidea

Botryosphaeria dothidea is a destructive fungal pathogen that causes Botryosphaeria canker and fruit ring rot on apple worldwide. Autophagy is a process of self-degradation that maintains intracellular homeostasis via lysosomal pathway. To date, the biological role of autophagy in B. dothidea remains unknown. In this study, we identified and characterized the autophagy-related gene BdATG8 in B. dothidea. BdATG8 was able to restore the defect in nitrogen starvation tolerance of Saccharomyces cerevisiae ATG8 deletion mutant. GFP-BdAtg8 was shown to be a useful marker for monitoring autophagy in B. dothidea. Target deletion of BdATG8 (ΔBdAtg8) blocked autophagy and significantly impaired mycelial growth, conidiation and perithecium formation. In addition, ΔBdAtg8 showed significantly increased sensitivity to phytoalexin and oxidative stress, suggesting that BdATG8 plays critical roles in overcoming phytoalexin and reactive oxygen species (ROS)-mediated plant immunity. Pathogenicity assays revealed that ΔBdAtg8 almost lost ability to infect hosts. Overall, our results indicate that BdATG8 plays an important role in fungal development, stress responses and pathogenesis in B. dothidea.     

黑龙江省药用植物根际土壤真菌多样性

为了解黑龙江省药用植物根际土壤真菌的种群结构和区系分布特点,于2010年7月份和10月份,在黑龙江省的伊春、铁力、绥化、哈尔滨、牡丹江和佳木斯6个中药材产区共采集土壤样品220份,所采集的药用植物种类主要有五味子、平贝母、刺五加、党参、防风、柴胡、桔梗、黄芩等14种.经稀释平板法和土壤颗粒平板法分离共获得1016株真菌,经形态鉴定归为35属86种,其中接合菌7属10种,占7.78％;子囊菌1属2种,占0.69％;无性型真菌27属74种,占70.76％,其余20.77％的菌株为不产孢真菌.试验结果表明,黑龙江省药用植物根际土壤真菌的种群多样性丰富,其中青霉属Penicillium、曲霉属Aspergillus、木霉属Trichoderma、镰孢菌属Fusarium是优势种群,粘帚霉属Gliocladium、金孢属Chrysosporium、毛霉属Mucor、枝孢属Cladosporium、枝顶孢属Acremonium、根霉属Rhizopus是亚优势种群.不同药用植物根际土壤真菌区系的结构和组成存在一定的差异.除无孢类群外,青霉属Penicillium、曲霉属Aspergillus、木霉属Trichoderma和镰孢菌属Fusarium是14种药用植物根际土壤真菌的优势菌群.五味子、平贝母和柴胡是黑龙江省种植的主要中药材,它们在6个采样地点间的真菌种群的多样性水平存在差异,其中伊春地区的多样性指数(H’=2.9574)和丰富度指数(R=5.6683)最高,而佳木斯地区的均匀度指数(J=0.9200)最高.不同地区的相似性水平也存在差异,其中牡丹江与绥化的药用植物根际土壤真菌种群组成之间的相似性系数最高(Cj=0.6315),牡丹江与哈尔滨的相似性最低(Cj=0.3704).     

Molecular characterization of the SAUR gene family in sweet cherry and functional analysis of PavSAUR55 in the process of abscission

Small auxin up RNA(SAUR) is a large gene family that is widely distributed among land plants. In this study, a comprehensive analysis of the SAUR family was performed in sweet cherry, and the potential biological functions of PavSAUR55 were identified using the method of genetic transformation. The sweet cherry genome encodes 86 SAUR members, the majority of which are intron-less. These genes appear to be divided into seven subfamilies through evolution. Gene duplication events indicate that fra...     

Neopestalotiopsis eucalypti,a causal agent of grapevine shoot rot in cutting nurseries in China

Grapevine (Vitis vinifera L.) is an economically important fruit crop in the world, and China ranks first in the production of grapes with approximately 15% of the world's total yield. However, diseases that cause the death of grapevine shoots pose a severe threat to the production of grapes. In this study, the fungus Neopestalotiopsis eucalypti was identified as a causal pathogen of grapevine shoot rot based on the morphology of conidia and a phylogenetic analysis. The phylogenetic analysis was performed with three isolates based on the combined sequence of internal transcribed spacer (ITS) region of ribosomal DNA, part of the translation elongation factor 1-alpha (Tef) and the β-tubulin (Tub2) genes. The three isolates were all identified as N. eucalypti. Pathogenicity tests of the three fungal isolates were conducted on grapevines shoots in vitro and in vivo. The results showed that all three fungal isolates caused severe rot lesions on the inoculated grapevine shoots, and N. eucalypti was re-isolated from the inoculated grapevine shoots. Therefore, N. eucalypti was confirmed as a causal agent of the grapevine shoot rot. This is the first report of N. eucalypti causing grapevine shoot disease in China.