弹性蛋白酶基因(PAE)的克隆及在毕赤酵母中的表达

以1株产弹性蛋白酶的铜绿假单胞菌(Pseudomonas aeruginosa)基因组DNA为模板,经PCR扩增得到的铜绿假单胞菌弹性蛋白酶(P.acruginosa elastase,PAE)基因,与GenBank中的序列对比发现同源性为99%.成功地构建了重组表达载体pPIC3.5K/PAE,莺组质粒Sac Ⅰ线性化后转化毕赤酵母(Pichia pastoris)菌株KM71中,通过PCR和表型鉴定表明,PAE基因已经整合到毕赤酵母染色体上.经大量筛选获得48株含高拷贝的重组毕赤酵母转化子.在甲醇诱导下,经过毕赤酵母高密度发酵进行PAE的表达,经SDS-PAGE分析.结果表明,在培养基上清中含有一明显特异性蛋白条带,大小为34kD.活性检测结果,酶活为1 060 U/mL,是出发菌株的26倍.     

河道淤泥和堆肥蛭石混合发酵制备基质及其育苗效果

农村河道清淤产生的淤泥,体量大、有机物浓度高,处置不当会造成二次污染。现代农业的工厂化育苗需求大量的营养土,就地取土导致耕地退化。该研究利用功能微生物发酵淤泥制备育苗基质,研究不同菌株发酵基质的物理和生物学性状,基质培育西瓜苗的生长、生理参数和抗逆性能。结果表明:微生物处理均能够提升淤泥基质物理和生物学性能,同时能够提升育苗质量。其中Trichoderma harzianum T83(T83)、Bacillus amyloliquefaciens IAE(BIAE)菌株发酵基质性能最好。相较于对照处理基质的最大持水量、总孔隙度、毛管孔隙度、通气孔隙度,T83处理分别增加了64.25%、52.65%、45.05%、56.11%;BIAE处理分别增加了101.17%、45.43%、61.43%、38.14%。相较于对照处理西瓜苗的株高、鲜质量、干质量、叶绿素含量、根系活力、根际真菌、细菌数量,T83处理分别增加了66.85%、52.07%、72.16%、43.13%、54.93%、110.62倍、1.63倍;BIAE处理分别增加了80.40%、57.34%、82.37%、54.88%、46.40%、67.26%、2.60倍、2.94倍。T83和BIAE处理西瓜苗叶片过氧化氢酶和超氧化物歧化酶酶活显著增加,根系丙二醛含量显著降低。真菌菌株T. harzianum T83和细菌菌株B. amyloliquefaciens IAE发酵淤泥,能够显著提升其农用品质,为淤泥高附加值化农用提供一条可行的途径。     

Molecular identification and genetic analysis for 24 turf-type Cynodon cultivars by Sequence-Related Amplified Polymorphism markers

Bermudagrass (Cynodon ssp.) germplasm is genetically diverse and widely distributed in the world. The study was conducted to identify and assess the molecular variation and relationship among 24 cultivars developed in China, Australia and the USA. Sequence-Related Amplified Polymorphism (SRAP) was applied to cultivars identification in this study for the first time. Thirty of the 90 SRAP primer combinations generated a total of 274 clearly bands encompassing 249 (91%) polymorphic. Each bermudagrass cultivar has its unique binary code and can be distinguished from the others. Three distinct clusters were obtained by unweighted pair-group method with arithmetic averages based on the polymorphic markers. Coefficients of genetic distance among the genotypes ranged from 0.57 to 0.97. The results demonstrated that SRAP marker is a stable molecular marker technique for the identification of bermudagrass cultivars and their genetic relationships.     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

喷施乙烯利对‘HAES900’澳洲坚果果实脱落和品质的影响

为解决澳洲坚果采收难和采收成本高的关键问题,以‘HAES900’澳洲坚果成龄结果树为材料,在采收期分别喷施低浓度梯度(0.40、0.65、0.90 g/L)和高浓度梯度(0.80、1.30、1.80 g/L)的乙烯利溶液,对喷施乙烯利溶液后的落果率、果柄附着力和果实品质进行了调查和分析。结果表明,低浓度乙烯利对果实脱落有促进作用,但落果率达不到生产要求;喷施1.80 g/L的乙烯利溶液2周和4周后的落果率分别达到98.75%和100.00%,可满足生产要求;喷施浓度为0.80、1.30和1.80 g/L的乙烯利会使果柄附着力显著降低,且果柄附着力与落果率呈极显著负相关;果柄附着力可作为判断澳洲坚果适宜采收期的指标之一。采用本方法采收对壳果含水量、单壳果重、壳果平均横径、单果仁重、出仁率和一级果仁率等品质参数无显著影响,且可缩短澳洲坚果采收周期,降低采收成本。     

Trichoderma species associated with green mould disease of Pleurotus ostreatus and their sensitivity to prochloraz

Green mould disease causes serious economic losses in Pleurotus ostreatus crops worldwide, including in Italy, where prochloraz is the only chemical fungicide allowed to control the disease. The effectiveness of the doses 0.01, 0.05, 0.25 and 1.25 μL L⁻¹ (field dose) of prochloraz (Sponix Flow, 450 g L⁻¹), against colony growth rate and spore germination of Trichoderma pleuroti, T. pleuroticola and T. guizhouense strains on wheat straw extract agar plates were evaluated. Complete inhibition of Trichoderma pleuroti and T. pleuroticola growth was shown by the field dose of prochloraz, and also by the 0.25 μL L⁻¹ dose for T. pleuroti. Complete inhibition of spore germination occurred for all Trichoderma strains at field dose, and at 0.25 μL L⁻¹ for T. pleuroti strains. In in vivo assays, the effect of prochloraz doses 0.05, 0.25 and 1.25 μL L⁻¹ on colonization of straw substrate by T. pleuroti, T. pleuroticola and T. guizhouense inoculated at two spore densities (10² and 10⁵ spores mL⁻¹) immediately after P. ostreatus spawn was studied. Trichoderma pleuroti and T. pleuroticola were both responsible for green mould disease, whereas T. guizhouense was not pathogenic. Trichoderma pleuroti was more aggressive than T. pleuroticola. Prochloraz was effective against T. pleuroti at the field dose, and against T. pleuroticola at 0.25 and 1.25 μL L⁻¹. The study on Trichoderma × Pleurotus interaction type showed that Trichoderma species were active against the mycelial growth of P. ostreatus by competition for space and nutrients, and neither hyphal interaction nor effect by volatile or nonvolatile metabolites occurred.     

Differential responses of chickpea (Cicer arietinum L.) — Rhizobium combinations to saline soil conditions

Summary Chickpea cultivars (Cicer arietinum L.) and their symbiosis with specific strains of Rhizobium spp. were examined under salt stress. The growth of rhizobia declined with NaCl concentrations increasing from 0.01 to 2% (w : v). Two Rhizobium spp. strains (F-75 and KG 31) tolerated 1.5% NaCl. Of the 10 chickpea cultivars examined, only three (Pusa 312, Pusa 212, and Pusa 240) germinated at 1.5% NaCl. The chickpea — Rhizobium spp. symbiosis was examined in the field, with soil varying in salinity from electrical conductivity (EC) 4.5 to EC 5.2 dSm^-1, to identify combinations giving satisfactory yields. Significant interactions between strains and cultivars caused differential yields of nodules, dry matter, and grain. Four chickpea — Rhizobium spp. combinations, Pusa 240 and F-75 (660 kg ha^-1), Pusa 240 and IC 76 (440 kg ha^-1), Pusa 240 and KG 31 (390 kg ha^-1), and Pusa 312 and KG 31 (380 kg ha^-1), produced significantly higher grain yields in saline soil.     

Phosphate-solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid coastal lagoon

 The phosphate-solubilizing potential of the rhizosphere microbial community in mangroves was demonstrated when culture media supplemented with insoluble, tribasic calcium phosphate, and incubated with roots of black (Avicennia germinans L.) and white [Laguncularia racemosa (L.) Gaertn.] mangrove became transparent after a few days of incubation. Thirteen phosphate-solubilizing bacterial strains were isolated from the rhizosphere of both species of mangroves: Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus atrophaeus, Paenibacillus macerans, Vibrio proteolyticus, Xanthobacter agilis, Enterobacter aerogenes, Enterobacter taylorae, Enterobacter asburiae, Kluyvera cryocrescens, Pseudomonas stutzeri, and Chryseomonas luteola. One bacterial isolate could not be identified. The rhizosphere of black mangroves also yielded the fungus Aspergillus niger. The phosphate-solubilizing activity of the isolates was first qualitatively evaluated by the formation of halos (clear zones) around the colonies growing on solid medium containing tribasic calcium phosphate as a sole phosphorus source. Spectrophotometric quantification of phosphate solubilization showed that all bacterial species and A. niger solubilized insoluble phosphate well in a liquid medium, and that V. proteolyticus was the most active solubilizing species among the bacteria. Gas chromatographic analyses of cell-free spent culture medium from the various bacteria demonstrated the presence of 11 identified, and several unidentified, volatile and nonvolatile organic acids. Those most commonly produced by different species were lactic, succinic, isovaleric, isobutyric, and acetic acids. Most of the bacterial species produced more than one organic acid whereas A. niger produced only succinic acid. We propose the production of organic acids by these mangrove rhizosphere microorganisms as a possible mechanism involved in the solubilization of insoluble calcium phosphate. Received: 21 April 1999     

Response of spring wheat (Triticum aestivum) to interactions between Pseudomonas species and Glomus clarum NT4

The effects of interactions between pseudomonads (Pseudomonas cepacia strains R55 and R85, P. aeruginosa strain R80, P. fluorescens strain R92, and P. putida strain R104) and the arbuscular mycorrhizal fungus Glomus clarum (Nicol. and Schenck) isolate NT4, on spring wheat (Triticum aestivum L. cv. Laura), grown under gnotobiotic and nonsterile conditions, were investigated. Although plant growth responses varied, positive responses to pseudomonad inoculants generally were obtained under gnotobiotic conditions. Shoot dry weight enhancement ranged from 16 to 48%, whereas root enhancement ranged from 82 to 137%. Shoot growth in nonsterile soil, however, was unaffected by pseudomonad inoculants, or reduced by as much as 24%. Shoot growth was unaffected or depressed by G. clarum NT4 whereas early root growth was enhanced by 38%. Significant interactions between the pseudomonad inoculants and G. clarum NT4 were detected. Typically, dual inoculation influenced the magnitude of response associated with any organism applied alone. The effect of these pseudomonads on G. clarum NT4 spore germination was investigated. Germination was inhibited when spores were incubated either on membranes placed directly on bacterial lawns of strains R85 and R104 (i.e., direct assay), or on agarose blocks separated from the bacteria by membranes (i.e., diffusion assay). When the agarose blocks were physically separated from the pseudomonad (i.e., volatile assay), there was no evidence of inhibition, suggesting that a nonvolatile, diffusible substance(s) produced by both strains R85 and R104 may inhibit G. clarum NT4 spore germination. Received: 11 December 1995     

柱花草炭疽病菌的鉴定及生物学特性研究

对柱花草炭疽病菌的鉴定及生物学特性测定结果表明：柱花草炭疽病菌为盘长孢状刺盘孢菌[Colletotrichumgloeosporioides（Penz）Sacc]．病菌生长温度15～36℃;适宜生长温度25～30℃;分生孢子萌发的温度6～40℃,最适萌发温度为28℃;分生孢子在饱和湿度下萌发率为83．1％,在饱和湿度加水滴中达87．9％;分生孢子的致死温度为49℃10min;病菌生长的pH值是2．5～14．0,最适pH值为6．4～7．2;病菌对碳源的利用较氮源好。