Effect of hot water treatment on development of anthracnose (Colletotrichum gloeosporioides) and quality of mango fruit at Jimma southwest Ethiopia

Anthracnose is the major postharvest disease of mango and occurs throughout mango producing areas of the world including Ethiopia. Evaluating effect of hot water treatment on development of anthracnose and quality of mango fruit is imperative. A total of three hot water levels 48, 52 and 56 °C at two time interval (5 and 10 min) were tested with factorial arrangement in completely randomised design. The study indicated that hot water treatment at different temperatures and time interval significantly (p < 0.001) affects disease development and shelf life and postharvest quality of mango fruits. Hot water treatments reduced the incidence and severity of anthracnose disease significantly (p < 0.001) in mango fruits as compared to control. There was a highly significant difference (p < 0.0001) on weight loss, total soluble solids, titratable acidity and fruit firmness of mango fruits due to treatment. The present study reviled that hot water treatment has a potential in reducing the postharvest loss due to anthracnose and improving the shelf life and quality of mango fruits. However, the reduction of disease pressure on fruits was not at applicable level, which call ups future effort on developing on integrated disease management strategies for reduction of postharvest loss of mango fruits.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用

枯草芽孢杆菌（Bacillus subtilis）作为食品级安全菌株,因其具有理化特征清晰、培养发酵方便等特点,广泛应用于异源蛋白质的高效表达以及高附加值物质的合成。传统的B. subtilis遗传转化方法存在操作流程繁琐、效率低等缺点,因此,开发方便高效的遗传转化系统具有重要意义。转录因子ComK被证实能调控B. subtilis感受态的形成,并在B. subtilis高效转化中有重要作用。构建1个含有木糖诱导启动子P_xyl调控comK表达的穿梭质粒pUBC01?P_xyl?comK的菌株B. subtilis K1,经木糖诱导条件优化后,质粒pHY300?p43?egfp的转化效率达到4.8×10³ CFU·μg^-1。此外,质粒pUBC01?P_xyl?comK可在无胁迫条件下连续培养及消除。木糖诱导感受态体系及质粒消除极大地提高了芽孢杆菌基因编辑和菌株改造的便捷性,同时增强了菌株尤其是生产菌株的性状稳定性。     

套袋与未套袋苹果果实表皮及心部真菌种群结构与变化动态

2011年在山东海阳,2013年在蓬莱和栖霞选取当地常规管理的苹果园,采用传统组织分离法对套袋苹果、未套袋苹果的果实表皮及心部组织进行真菌分离并进行形态学和分子生物学鉴定。对不同时期采集的套袋及未套袋果实表皮与心部的真菌种类、菌落数量、组织分离率、多样性、相似性系数、相对分离频率等指标进行统计分析。结果表明,两个年度内从果实表皮共分离获得真菌43属,从心部获得真菌31属,相对分离频率较高的真菌包括：链格孢Alternaria spp.、枝状枝孢Cladosporium cladosporioides、镰孢菌Fusarium spp.、青霉菌Penicillium spp.等。研究结果表明：果实套袋后,在果实表皮定殖真菌的种类减少,多样性降低,而数量增加,组织带菌率升高;随果实生长,套袋果实表皮上的真菌种类与未套袋果实表皮真菌种类的相似度逐渐降低;7、8月份,套袋果实表皮上真菌种类明显减少,至8月底,套袋果实表面只有链格孢等少数几种优势真菌定殖。果实套袋后,定殖于果实心部的真菌种类和数量有所波动,套袋果和未套袋果上所分离真菌的相似度有所降低;5月底自果实心部分离真菌的种类和数量与9月底所分离真菌的种类和数量差异不大。     

Relationship between tomato fruit growth and fruit osmotic potential under salinity

To investigate the relationship between fruit growth and fruit osmotic potential (Ψ_s) in salty conditions, a sensitive tomato cultivar (Lycopersicon esculentum Mill.) and a tolerant accession of the wild species Lycopersicon pimpinellifolium Mill. were grown in a greenhouse with 0 and 70 mM NaCl, and the growth of the fruit studied from 15 to 70 days after anthesis (DAA). L. pimpinellifolium did not reduce significantly fruit weight in salty conditions throughout the growth period, whereas L. esculentum fruit weights decreased significantly with salinity from 45 DAA. L. esculentum fruit fresh weight reductions resulted from both less dry matter and water accumulation, although the fruit water content was affected by salinity before the fruit weight. In both species, fruit osmotic potential (Ψ_s) decreased significantly with salinity during the rapid fruit growth phase, although the changes were different. Thus, fruits from L. pimpinellifolium salt treated plants showed a Ψ_s reduction at the beginning (15 DAA) twice as high as that found in L. esculentum. As the advanced growth stage (from 15 to 55 DAA), the Ψ_s reduction percentages induced by salinity were quite similar in L. pimpinellifolium fruits, while increased in L. esculentum. Under saline conditions, the solutes contributing to reduce the fruit Ψ_s during the first 55 DAA were the inorganic solutes in both species, while in the ripe fruits they were hexoses. L. esculentum fruits accumulated K⁺ as the main osmoticum in salty conditions, while L. pimpinellifolium fruits were able to use not only K⁺ but also the Na⁺ provided by the salt.     

Enhanced stability of the cloned Bacillus subtilis alkaline protease gene in alginate-immobilized B. subtilis cells

Expression and stability of the cloned Bacillus subtilis alkaline protease (aprE)gene was monitored throughout the growth of free and alginate-immobilized B. subtilis cells. The time as well as the level of expression of the aprE gene in alginate-immobilized cells was found to be close to that of free cells. The multicopy plasmid that carries the aprE gene was stably maintained in alginate-immobilized cells. Plasmid stability was greatly enhanced, it reached 83% and 8% after ten growth cycles for alginate-immobilized and free cells in the absence of stress, respectively. Data presented demonstrate that immobilization of B. subtilis recombinant cells would partially solve the problem of plasmid instability in B. subtilis.     

香蕉抗(感)病品种根系分泌物对枯萎病菌和枯草芽孢杆菌的生物效应

为明确香蕉根系分泌物对枯萎病菌及其生防枯草芽孢杆菌的生物效应,采用离位溶液培养法收集抗枯萎病香蕉品种(南天黄)和感枯萎病香蕉品种(桂蕉6号)的根系分泌物,研究根系分泌物对土壤微生物种群数量、香蕉枯萎病菌和枯草芽孢杆菌生长的影响。结果表明: 抗病品种根系分泌物能显著减少土壤真菌的数量,抑制枯萎病菌孢子的萌发;而感病品种根系分泌物能显著促进病菌菌丝生长和孢子的萌发,两个品种根系分泌物均能显著促进枯草芽孢杆菌的生长和生物膜形成。经抗(感)病香蕉品种根系分泌物处理,病菌菌丝生长速率分别为11.28和12.28 mm·d^-1,病菌孢子的萌发率分别为34.6%和79.5%;枯草芽孢杆菌培养12 h后菌体生长量的OD₆₀₀分别为1.27和1.14,生物膜形成量在静置培养72 h后OD₅₇₀分别达1.11和1.30,两个品种处理间的差异均达显著水平。枯草芽孢杆菌在香蕉感病品种根际中定殖的菌量显著高于抗病品种。通过对两个品种根系分泌物中可溶性总糖、游离氨基酸和有机酸的含量和组成分析,发现抗病品种根系分泌物中有机酸和游离氨基酸的含量明显高于感病品种,在各组成成分中,以乙酸和脯氨酸在抗(感)病香蕉品种根系分泌物中含量比值较高,分别达3.7和2.4倍。综上所述,抗病品种根系分泌物能抑制病菌生长,感病品种根系分泌物则会显著促进病菌生长,而两个品种根系分泌物均能显著促进枯草芽孢杆菌的生长和生物膜的形成。     

Cloning and production of Xylanase B from Paenibacillus barcinonensis in Bacillus subtilis hosts

Xylanase B from Paenibacillus barcinonensis was cloned in shuttle vectors for Escherichia coli and Bacillus subtilis, and expressed in Bacillus hosts. Several recombinant strains were constructed, among which B. subtilis MW15/pRBSPOX20 showed the highest production. This recombinant strain consists of a protease double mutant host containing P. barcinonensis xynB gene under the control of a phage SPO2 strong promoter. Maximum production was found when the strain was cultured in nutrient broth supplemented with xylans. Analysis of xylanase B location in B. subtilis MW15/pRBSPOX20 showed that the enzyme remained cell-associated in young cultures, consistent with its intracellular location in its original host, P. barcinonensis, and the lack of a signal peptide. However, when cultures reached the stationary phase, xylanase B was released to the external medium as a result of cell lysis. The amount of enzyme located in the supernatants of old cultures could account for 50% of total xylanase activity. Analysis by SDS-PAGE showed that xylanase B is an abundant protein found in the culture medium in late stationary phase cultures.     

yhcZ基因在苏云金芽胞杆菌生长中的功能研究

在枯草芽胞杆菌和蜡样芽胞杆菌中,yhcZ基因和yhcY基因组成双组分系统调控细菌生长,但yhcZ基因在苏云金芽胞杆菌中发挥的生物学功能尚未明确。本研究通过基因功能注释、上下游基因排列分析和氨基酸序列比对,证实苏云金芽胞杆菌库斯塔克亚种HD73中HD73_5824基因为yhcZ基因,推测其与HD73_5825基因(yhcY基因)共同组成双组份系统调控细菌生长。利用同源重组技术敲除HD73菌株中的yhcZ基因获得缺失突变体HD (ΔyhcZ),其在LB和SSM培养基中生长均慢于野生型HD73,而互补菌株HD(ΔyhcZ::yhcZ)菌株则能够部分恢复生长,表明yhcZ基因的缺失影响了该菌株细胞的生长。在以0.4%葡萄糖为唯一碳源的M9培养基中,HD (ΔyhcZ)生长速度快于HD73,表明yhcZ基因在该菌株吸收利用葡萄糖的过程中发挥重要作用。Biolog实验显示HD (ΔyhcZ)的单孔颜色变化率低于HD73,且对D/L-丝氨酸、甲酸、D-葡糖酸、L-组胺,D-乳酸甲酯以及柠檬酸等的吸收利用能力低于HD73,表明yhcZ基因能显著影响HD73菌株对碳源的利用。同时,HD(ΔyhcZ)对8% NaCl的耐受能力弱于HD73,表明该基因可能参与细菌细胞应力响应相关基因的表达与调控。以上结果表明yhcZ基因在HD73菌株生长过程中对葡萄糖及其他碳源的利用具有重要的促进作用。本研究结果为解析yhcZ基因调控葡萄糖及碳源利用的分子机制奠定基础,且为进一步研究细菌生长及发酵提供参考。     

Control of preharvest and postharvest fruit rot in Strawberry by Metschnikowia fructicola

The yeast Metschnikowia fructicola was tested as a preharvest treatment to control preharvest and postharvest rots of strawberry fruit in Turkey and Israel. In greenhouse trials, the efficacy of the yeast antagonist against preharvest rots was equal to that of a chemical control (fenhexamid) in two growing seasons. In an open-field experiment, the yeast reduced the incidence of rot to commercially acceptable levels. M. fructicola reduced the incidence of fruit rot by 56-69% in greenhouses, open-field culture, and in low plastic tunnels. The yeast suppressed postharvest incidence of fruit rot significantly better than fenhexamid. Among fruit from greenhouses, open-field culture, or tunnels, M. fructicola treatment reduced the incidence of fruit rot during postharvest storage by 70, 64, and 72%, respectively. When applied weekly in the greenhouse or in the field, the population density of M. fructicola was about 1×10⁵ cfu/fruit. Similar population density of the antagonist was also observed during storage of the fruit at 0° C.     

不同比例盐生小球藻-枯草芽孢杆菌共生体对砷酸盐耐性及富集差异

采用盐生小球藻与枯草芽孢杆菌为供试材料,构建不同藻菌比(1∶0、1∶1、1∶2、1∶3、1∶4)的共生体,在不同浓度砷酸盐[As(Ⅴ)]处理7 d后,测定藻菌共生体生长及其对砷的富集、吸附、吸收和形态转化。结果表明: 在As(Ⅴ)处理下,随着枯草芽孢杆菌比例的增加,藻菌共生体叶绿素含量、干重、比增长率显著提高,在750 μg·L^-1As(Ⅴ)处理时,1∶4的藻菌共生体分别达到1.81 mg·L^-1、125.0 mg、0.28 mg·L^-1·d^-1。藻菌比例由1∶0变为1∶4时,藻菌共生体对砷的富集和吸收呈下降趋势;砷的富集方式随砷浓度的增加而变化,在75～150 μg·L^-1As(Ⅴ)处理下以吸收为主,在300～750 μg·L^-1 As(Ⅴ)处理下以吸附为主。藻菌共生体内存在As(Ⅴ)和As(Ⅲ)两种形态,且随枯草芽孢杆菌比例的上升,As(Ⅴ)还原率增大(最高达到12.6%)。综上,枯草芽孢杆菌的添加提升了藻菌共生体对As(Ⅴ)的耐性和还原,但减少了对As(Ⅴ)的富集。     

生物药剂滴施对棉花黄萎病及根际土壤微生物数量和多样性的影响

生物农药是当前棉花黄萎病绿色防治的重要途径.本研究设置田间小区试验,研究了4种生物药剂不同滴施用量(枯草芽孢杆菌粉剂15、30、45 kg·hm^-2,“施倍健”哈茨木霉菌剂15、18、24 kg·hm^-2,渝峰“99植保”15、22.5、30 kg·hm^-2,中农绿康30、45 和60 kg·hm^-2)对棉花黄萎病的防治效果及土壤微生物群落组成和多样性的影响.结果表明: 各施药处理均不同程度降低了棉花黄萎病发病率和发病指数,全生育期平均防病效果达到50.0%~77.4%;木霉菌剂施药量18 kg·hm^-2的防效显著高于15 和24 kg·hm^-2,其他3种药剂防病效果均与施药量呈正相关.施药后土壤中病原菌大丽轮枝菌的相对丰度显著降低,且与防病效果呈极显著负相关.土壤细菌的数量与物种丰度随着施药量的增加而显著增加.施药后土壤放线菌的数量与物种丰度也显著增加,但放线菌数量在不同施药量间相差较大.土壤真菌的数量与物种丰度除木霉菌剂随施药量增加而增加外,其他3种药剂随着施药量的增加而减少.Pearson相关性分析显示,施药组的防病效果与细菌和放线菌数量呈正相关关系,而与真菌数量呈显著负相关;与放线菌、硝化螺旋菌门细菌、子囊菌门真菌、壶菌门真菌丰度呈现显著正相关,而与半知菌门真菌丰度呈显著负相关.建议在滴施用量上渝峰“99植保”30 kg·hm^-2、中农绿康60 kg·hm^-2、枯草芽孢杆菌可湿粉剂45 kg·hm^-2、“施倍健”哈茨木霉菌剂18 kg·hm^-2.     

Comparison and functional characterisation of three homologous intracellular carboxylesterases of Bacillus subtilis

Enzymatic hydrolysis of racemic mixtures may provide an attractive method for the enantiopure production of chiral pharmaceuticals. For example, the carboxylesterase NP of Bacillus subtilis Thai I-8 is an excellent biocatalyst in the kinetic resolution of NSAID esters, such as naproxen and ibuprofen methyl esters. Two homologues of this enzyme were identified when the genome sequence of B. subtilis 168 was revealed in 1997. We characterised one of the homologous, YbfK, as a very enantioselective 1,2-O-isopropylidene-sn-glycerol caprylate esterase, while only modest enantioselectivity towards the naproxen ester was observed. The other homologue, the carboxylesterase NA has not been characterised yet. The purpose of the present study was to fully characterise these three highly homologous esterases with respect to their applicability towards the enantiospecific hydrolysis of a wide range of compounds. The esterase genes were cloned and expressed in B. subtilis using a combination of two strong promotors in a multi-copy vector. After purification of the enzymes from the cytoplasm of B. subtilis, the biochemical and enantioselective properties of the enzymes were determined. Although all carboxylesterases have similar physico-chemical properties, comparison of their specific activities and enantioselectivities towards several compounds revealed rather different substrate specificities. We conclude that carboxylesterase NP and carboxylesterase NA are particularly suited for the enzymatic conversion of naproxen esters, while YbfK offers enantiopure (+)-IPG from its caprylate ester. Given the carboxylesterase activities of the esterases it has been proposed to rename the nap gene of B. subtilis 168 into cesA and the ybfK gene into cesB.     

PVA-biocatalyst with entrapped viable Bacillus subtilis cells

Bacillus subtilis cells were entrapped in polyvinyl alcohol (PVA)-cryogel beads without decay in their viability and capability of secretion of proteolytic enzymes (metalloproteinase and subtilisin). Conditions for preparation of the PVA-biocatalyst with suitable stability and viability of B. subtilis cells were optimized. Diffusion of various compounds into the cryogel (sliced beads) has been monitored on-line using image analysis system. Optimal working conditions and kinetic constants for hydrolysis of proteins catalyzed by the PVA-biocatalyst containing whole B. subtilis cells were estimated. The PVA-biocatalyst was applied in the hydrolysis of casein. The productivity of the biocatalyst (expressed as an amount of liberated aromatic amino acids) reached a maximal level of 12 mg g⁻¹ h⁻¹. Composition of mixture of peptides was dependent on pH, concentrations of Na⁺ and glucose, and in the reaction milieu. Protein hydrolysates of desired composition can be obtained using B. subtilis viable cells immobilized in PVA-gel. Incubation of the immobilized cells in a nutrient medium with casein successfully regenerated proteolytic activity of the biocatalyst.     

套袋对番石榴果实品质的影响

以新世纪番石榴品种为试材,研究套袋对番石榴果实品质的影响。结果表明,套袋可加速番石榴果实发育,明显增加果实大小和重量;透明和红色套袋对果实可溶性固形物含量影响不大,黑色套袋降低了果实可溶性固形物含量;套袋使果皮光滑,病虫害显著减少,并改善了果实着色。     

不同材质果袋春夏季节套袋对黄瓜果实发育和品质的影响

以“冬冠3号”黄瓜品种为试材,于春夏生长季节（4—7月份）在日光温室中研究了白膜袋、鲜膜袋、白纸袋和黄纸袋4种套袋处理对黄瓜果实微环境、果实生长发育和营养品质及农药残留的影响。结果表明：不论晴天还是阴天,所有套袋处理的袋内光照强度降低,相对湿度增大,温度提高;白纸袋增温效果最好,鲜膜袋内相对湿度最高,黄纸袋内光照最弱。单株单瓜套袋和单株果实连续套袋试验均表明,套袋后果实鲜重增长加快,瓜长度增加,瓜皮色显著变浅。连续套袋后,单瓜重普遍提高,大头瓜率降低,但化瓜率、弯瓜率和尖头瓜率增加;游离氨基酸含量提高;维生素C含量无显著变化;叶绿素和类胡萝卜素的含量普遍降低;可溶性蛋白质含量白纸袋和鲜膜袋的提高,黄纸袋和白膜袋的降低,但与CK间的差异均未达到5％显著水平。套袋可有效降低果实中氧化乐果的残留量,其中黄纸袋效果最好,其次为鲜膜袋、白膜袋和白纸袋。综合考虑各指标,认为春夏季节黄瓜果实套袋栽培应优先选用白纸袋,鲜膜袋和白膜袋不适宜在该季节使用。     

Purification, characterization and synergistic activity of β-1,3-glucanase and antibiotic extract from an antagonistic Bacillus subtilis NSRS 89-24 against rice blast and sheath blight

Antifungal compounds in the culture filtrate from Bacillus subtilis NSRS 89-24 that inhibited the growth of Pyricularia grisea and Rhizoctonia solani were mainly heat stable as the filter sterilized culture filtrate showed higher activity than an autoclaved one. The heat stable and labile components were due to an antibiotic and a β-1,3-glucanase, respectively. This β-1,3-glucanase was purified and characterized. Glucanase activity in the culture medium of B. subtilis NSRS 89-24 was inducible in the presence of 0.3% chitin, reaching a maximum on day 5. After purification, activity was associated with a protein of molecular mass of approximately 95.5 kDa by both gel filtration and native PAGE. Two major bands of M_r 64.6 and 32.4 kDa were revealed by SDS–PAGE. The enzyme had a K_m of 0.9 mg/ml, and V_max of 0.11 U, the optimal pH was 6.5–9.5 and was stable up to 50 °C. Both the pure enzyme and the antibiotic extract from the culture filtrate of the B. subtilis separately inhibited R. solani and P. grisea with MIC values of 12.5 and 6.25 mU/ml and 3.13 and 1.56 μg/ml, respectively. The glucanase enzyme in combination with the antibiotic showed a strong synergistic inhibitory effect on the hyphal growth of both fungi.     

Development and morphological features of biofilms formed by transgenic and wild type strains of Bacillus subtilis

The study addressed the ability of the transgenic strain (TM) B. subtilis 2335/pBMB105 (Km^rInf⁺) to form biofilms on the surface of liquid media of various compositions, inoculated with vegetative cells and spores. The morphological features of these biofilms do not differ from those of the films formed by the recipient strain (WT) B. subtilis 2335 (Km^s). However, the TM and the natural one differ in the dynamics of biofilm formation and the cellular composition of the films. Biofilms of the TM are formed earlier, develop at a higher rate, but decompose later than the films of the WT. When the medium is inoculated with vegetative cells, sporulation in the biofilms of both strains undergoes glucose repression; no such effect is observed when the medium is inoculated with spores. The TM does not form films when the medium is inoculated with spores and supplemented with glycerin and kanamycin.     

外源脱落酸对富士苹果果实膨大后期光合产物向果实运输的影响

为了明确脱落酸(ABA)对苹果果实糖分积累的影响机理,本试验以5年生‘烟富3’/M26/平邑甜茶为试材,采用¹³C同位素标记技术,研究在果实膨大后期用不同浓度(0、50、100和150 mg·L^-1)脱落酸溶液及氟啶酮(ABA生物合成抑制剂)处理果实对光合产物向果实运输及糖代谢的影响.结果表明: 随着ABA浓度的增加,糖代谢相关酶活性、蔗糖转运蛋白基因MdSUT1、MdSUT2.2和山梨醇转运蛋白基因MdSUT3相对表达量呈先升高后降低趋势,均以100 mg·L^-1ABA处理时最高.氟啶酮处理显著抑制了糖代谢酶活性和糖转运蛋白基因相对表达量.与其他处理相比,100 mg·L^-1ABA处理显著减少了叶片¹³C含量,增加了果实¹³C含量,提升了光合产物由叶片向果实的运输速率.表明外源ABA通过增强果实库强,促进更多的光合产物向果实运输,提高了成熟期果实可溶性糖含量.