人参(Panax ginseng)根系分泌物成分对人参致病菌的化感效应

采用室内培养结合生物学测定的试验方法,研究了不同浓度人参(Panax ginseng)根系分泌物成分苯甲酸、邻苯二甲酸二异丁酯、十六酸和2,2-二(4-羟苯基)丙烷对人参立枯丝核菌(Rhizoctonia solani)、黑斑菌(Alternaria panax)、疫病菌(Phytophthora cactorum)、菌核菌(Sclerotinia schinseng)、锈腐菌(Cylindrocarpon destructans)和绿色木霉菌(Trichoderma viride)菌落生长及孢子萌发的化感效应.结果显示,不同浓度人参根系分泌物成分对人参致病菌及绿色木霉菌的化感效应存在显著差异.苯甲酸浓度与人参立枯丝核菌、菌核菌和锈腐菌菌落生长以及人参黑斑菌、锈腐菌孢子萌发呈负相关,与人参黑斑菌、绿色木霉菌菌落生长呈正相关;对人参疫病菌菌落生长的化感效应表现为低浓度和高浓度抑制,中浓度促进.邻苯二甲酸二异丁酯浓度与人参立枯丝核菌、黑斑菌、菌核菌和绿色木霉菌菌落生长以及人参黑斑菌孢子萌发呈负相关;对人参锈腐菌菌落生长和孢子萌发表现为低浓度和高浓度抑制,中浓度促进;对人参疫病菌菌落生长表现为低浓度和中浓度抑制,高浓度促进.2,2-二(4-羟苯基)丙烷浓度与人参立枯丝核菌、黑斑菌、疫病菌、绿色木霉菌菌落生长以及人参黑斑菌、锈腐菌孢子萌发呈负相关;对人参菌核菌、锈腐菌菌落生长表现中浓度促进,高浓度抑制.十六酸浓度与人参锈腐菌、疫病菌和绿色木霉菌菌落生长呈正相关,与人参锈腐菌孢子萌发呈负相关,对黑斑菌孢子萌发表现为中浓度抑制.4种根系分泌物的等量混合物浓度与人参致病菌及拮抗木霉菌菌落生长速率呈负相关.     

The mode of action of cell wall-degrading enzymes and their interference with Nod factor signalling in <Emphasis Type="Italic">Medicago sativa</Emphasis> root hairs

Medicago sativa L. (alfalfa) root hairs respond to Nod factors [NodRm-IV(C16:2,S)] in a host-specific manner with depolarization and rapid ion fluxes. Protoplasts prepared from these cells using the cell wall-digesting enzymes pectolyase and cellulase do not, or to a rather small extent, respond to Nod factors. In an effort to understand this activity loss we analyzed the mode of action of both enzymes with respect to their effects on the root hairs as well as their interference with the Nod factor response. (i) In the presence of the enzymes, Nod factor at saturating concentrations neither depolarized the plasma membrane of root hairs nor caused ion fluxes. Even after removal of the enzymes, Nod factor responses were strongly refractory. (ii) After a lag-phase of 12-18 s, pectolyase depolarized the plasma membrane, alkalized the external space, acidified the cytosol and increased the cytosolic Ca(2+) activity. (iii) Cellulase, without a lag-phase, depolarized the plasma membrane, acidified the cytosol, but only marginally increased the cytosolic Ca(2+) activity. Unlike pectolyase, the cellulase response was only weakly refractory to a second addition. (iv) Neither enzyme increased the membrane conductance, but pectolyase inhibited the H(+)-pump. (v) Pectolyase shows all the signs of an elicitor, while cellulase yields a mixed response. (vi) Denatured enzymes yielded strong effects similar to those of untreated enzymes. We conclude that the effects shown do not originate from enzymatic activity, but from interactions of the proteins with cell wall or plasma membrane constituents. It is further concluded that these enzymes (pectolyase more so than cellulase) trigger defense-related signal pathways, which makes protoplasts prepared with such enzymes unsuitable for studies of symbiotic or defense-related signalling.     

Morphological and Molecular Characterization of Pomegranate Fruit Rot Pathogen, <Emphasis Type="Italic">Chaetomella raphigera</Emphasis>, and its Virulence Factors

A new fungal pathogen was isolated from rotten pomegranates collected from the orchards of different parts of Maharashtra. The pathogen was morphologically identified as Chaetomella raphigera followed by sequencing of ITS and D1/D2 hypervariable region of LSU (28S) of rRNA gene. The pathogen produced pectinase, cellulase, xylanase and protease in liquid medium at a concentration of 71, 13.8, 54.3 and 7 U/ml respectively. Enzyme activity was also determined during pathogenesis in the tissues artificially infected by C. raphigera. Xylanase activity was maximum (25.1 U/g) followed by pectinase (19.2 U/g) and cellulase (1.5 U/g), whereas, protease activity was unnoticed. There was significant correlation (P < 0.05) between disease rating scale and pectinase, xylanase and cellulase activity in infected tissues. This indicates the simultaneous production of hydrolytic enzymes that aids in necrosis of fruit tissues. The elevated levels of these enzymes in infected tissues as compared with control suggest their possible role in pathogenesis. Thus, pectinase, cellulase and xylanase produced by C. raphigera acts as major virulence factors in the development of fruit rot in pomegranates. This is a first report of fungal fruit rot caused by C. raphigera in pomegranate.     

Production of Pectinase and Cellulase by Cladosporium cucumerinum with Dissolved Carbohydrates and Isolated Cell Walls of Cucumber as Carbon Sources

The scab fungus Cladosporium cucumerinum can use pectins and polygalacturonic acid as sole sources of carbon. Cellulose and Ca-polygalacturonate are not available carbon sources for the fungus. When growing on sucrose or pectin, pectinase is produced. In these cases the production of cellulase is insignificant. On a mixture of pectin and carboxymethylcellulose also cellulase is produced. Both pectinase and cellulase are released into the culture filtrate when the fungus grows on cell walls without ionic proteins, whereas only cellulase is released when cell walls with ionic proteins are the carbon source. Pectinase produced by the pathogen can bind to isolated cell walls. The bound pectinase can be extracted with 1 M NaCl from cell walls without ionic proteins, but not from cell walls with ionic proteins. A water-extract or 1 M NaCl-extract of cucumber hypocotyls with visible disease symptoms contains cellulase but no pectinase activity. Lack of pectinase activity in the 1 M NaCl-extract may be due to inhibition by a component that could be extracted by NaCl from the cucumber cell walls.     

一株产纤维素酶真菌的筛选、鉴定及酶学性质初步研究

经过初筛和复筛从土样中分离出1株高产纤维素酶真菌SNB9,经形态学和ITS序列分析。鉴定为黑曲霉（Aspergu Uusniger）。生长条件的测定显示该菌生长范围偏酸。发酵后纤维素酶的最适作用pH在4．0—5．0,最适作用温度在45—55℃。滤纸酶活为9．29U／mL,C,酶活为23．69U／mL,CMCase酶活为38．23U／mL,β-葡萄糖苷酶活为65．52U／mL。发酵液中除了纤维素酶,还发现有辅助酶,包括木聚糖酶、淀粉酶、果胶酶、蛋白酶。     

Pectinase and cellulase enhance the control of Abutilon theophrasti by Colletotrichum coccodes

Inundative mycoherbicidal biocontrol agents are typically insufficiently virulent to be commercially competitive with herbicides in row crop agriculture, and require enhancement. Pectinase and cellulase are typically used by pathogens during infection. Thus, it was hypothesized that adding exogenous cell wall degrading enzymes might enhance fungal infection. Pectinase or cellulase was added to inocula of aqueous chopped mycelial suspensions of a strain of Colletotrichum coccodes for control of Abutilon theophrasti. Plants treated with 5.3×10⁶ C. coccodes propagules mL^-1 and 1.65 U mL^-1 pectinase had more rapid and complete disease development. Similar trend was achieved when 10 U mL^-1 of cellulase were added to 2.2×10⁶ C. coccodes propagules mL^-1. Adding pectinase or cellulase did not increase the host range of the wild-type fungus. The results suggest that there might be value to transforming biocontrol agents to overproduce these enzymes.     

Occurrence of a cobalt-induced and cobalt-containing nitrile hydratase in Rhodococcus rhodochrous J1

The formation of nitrile hydratase required cobalt ions in Rhodococcus rhodochrous J1. No other transition-metals could replace the cobalt ion. The Rhodococcus nitrile hydratase was purified to homogeneity and found to contain a cobalt atom. The occurrence of a cobalt-induced and cobalt-containing nitrile hydratase, different from the nitrile hydratases in Pseudomonas chlororaphis B23 and Brevibacterium R312 containing a ferric ion in their active center, has been demonstrated here for the first time.     

白腐真菌血红密孔菌漆酶复合酶预处理烟梗丝的响应面法优化

烟梗是烟草工业的重要副产物,也是宝贵的自然资源。本研究首先利用白腐菌漆酶对烟梗丝进行预处理,提升了添加烟梗丝的卷烟品质;然后分别以木质素、纤维素、半纤维素和果胶的降解率为响应值,采用Box-Behnken设计建立方程模型,对漆酶、纤维素酶、半纤维素酶和果胶酶组成的复合酶预处理烟梗丝条件进行了优化。结果表明：每100g烟梗丝加入30U漆酶,在料液比为35%、温度为30℃、酶解pH为5处理48h的条件下预处理的烟梗丝对提升卷烟品吸效果最佳,烟梗丝中木质素、纤维素、半纤维素和果胶的降解率分别为20.16%、15.10%、7.20%和12.40%;为获得与之相同的各组分降解率,响应面法优化漆酶复合酶最佳处理条件为：每100g烟梗丝加入漆酶14.72U、纤维素酶1.00U、半纤维素酶1.00U、果胶酶8.45U。验证发现烟梗丝各组分降解率实测值与理论值无显著性差异,且显微结构观察显示复合酶处理后的烟梗丝表面致密结构被破坏,孔洞数量明显增加。本研究获得的白腐菌漆酶预处理后的烟梗丝在卷烟中的添加能有效改善卷烟品质,且漆酶复合酶的使用大幅减少了漆酶的用量,降低了漆酶预处理烟梗丝的成本,为废弃烟梗生物质的资源化利用提供了重要依据。     

Anaerobic degradation of pectin by mixed consortia and optimization of fermentation parameters for higher pectinase activity

Samples from biogas digesters, sewage ponds, animal house effluents and food processing wastes were used in enrichment systems seeking anaerobic bacteria producing pectinases. Among the 46 anaerobic consortia developed from various samples, four showed high pectinase activity under static anaerobic conditions. Investigation of fermentation variables showed the optimum conditions for pectinase activity were pH 7.0, 45°C and 72 h of growth with 0.5% pectin in the cultivation medium. A 1.4- to 1.6-fold increase in the pectinase activity was achieved under these conditions. The maximum yield of enzymes (62.72 U ml-1 of pectinase, 4.74 U ml^-1 of polygalacturonase, 113.30 U ml^-1 of pectin lyase, 2.10 U ml^-1 of pectinesterase, 0.75 U ml^-1 of total cellulase and 9.27 U ml^-1 of xylanase) was recorded with the consortia C-S2 developed from decomposed plant samples collected from a pond.     

Isolation of a ribonuclease from sanchi ginseng (Panax pseudoginseng) flowers distinct from other ginseng ribonucleases

A single-chained ribonuclease was isolated from the aqueous extract of sanchi ginseng (Panax pseudoginseng) flowers. It exhibited a molecular mass of 23 kDa, an N-terminal sequence with some similarity to other enzymes involved in RNA metabolism but different from known ribonucleases, and considerably higher activity toward poly U than poly C and only slight activity toward poly A and poly G. The purification protocol entailed ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on carboxymethyl (CM)-cellulose, and gel filtration on Superdex 75. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. Maximal activity of the ribonuclease was attained at pH 7. On either side of this pH the enzyme activity underwent a drastic decline. The enzyme activity was at its highest at 50 degrees C and dropped to about 20% of the maximal activity when the temperature was decreased to 20 degrees C or elevated to 80 degrees C. The characteristics of sanchi ginseng flower ribonuclease were different from those of the ribonucleases previously purified from sanchi ginseng and Chinese ginseng roots including ribonuclease from Chinese ginseng flowers which are morphologically very similar to sanchi ginseng flowers.     

Production of Polysaccharidases in Different Carbon Sources by Leucoagaricus gongylophorus Möller (Singer), the Symbiotic Fungus of the Leaf-Cutting Ant Atta sexdens Linnaeus

Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens, produces polysaccharidases that degrade leaf components by generating nutrients believed to be essential for ant nutrition. We evaluated pectinase, amylase, xylanase, and cellulase production by L. gongylophorus in laboratory cultures and found that polysaccharidases are produced during fungal growth on pectin, starch, cellulose, xylan, or glucose but not cellulase, whose production is inhibited during fungal growth on xylan. Pectin was the carbon source that best stimulated the production of enzymes, which showed that pectinase had the highest production activity of all of the carbon sources tested, indicating that the presence of pectin and the production of pectinase are key features for symbiotic nutrition on plant material. During growth on starch and cellulose, polysaccharidase production level was intermediate, although during growth on xylan and glucose, enzyme production was very low. We propose a possible profile of polysaccharide degradation inside the nest, where the fungus is cultured on the foliar substrate.     

栽参对土壤微生物生态及土壤酶活性的影响

我国人参种植历史最久,面积最大,总产量最高。现在,随着国内外市场对人参需求的增加,植参面积还在迅速扩大。但是,人参最忌连作,凡栽种过人参的土地,一般在三、四十年内不能再植参,否则人参大部分乃至全部烂掉。这种土地一般俗称为“老参地”。建国以后,曾对老参地形成和改造进行过许多试验研究,但均未奏效。迄今为止,我国还在沿用     

Effects of ginseng on Pseudomonas aeruginosa motility and biofilm formation

Biofilm-associated chronic Pseudomonas aeruginosa lung infections in patients with cystic fibrosis are virtually impossible to eradicate with antibiotics because biofilm-growing bacteria are highly tolerant to antibiotics and host defense mechanisms. Previously, we found that ginseng treatments protected animal models from developing chronic lung infection by P. aeruginosa. In the present study, the effects of ginseng on the formation of P. aeruginosa biofilms were further investigated in vitro and in vivo. Ginseng aqueous extract at concentrations of 0.5-2.0% did not inhibit the growth of P. aeruginosa, but significantly prevented P. aeruginosa from forming biofilm. Exposure to 0.5% ginseng aqueous extract for 24 h destroyed most 7-day-old mature biofilms formed by both mucoid and nonmucoid P. aeruginosa strains. Ginseng treatment enhanced swimming and twitching motility, but reduced swarming of P. aeruginosa at concentrations as low as 0.25%. Oral administration of ginseng extracts in mice promoted phagocytosis of P. aeruginosa PAO1 by airway phagocytes, but did not affect phagocytosis of a PAO1-filM mutant. Our study suggests that ginseng treatment may help to eradicate the biofilm-associated chronic infections caused by P. aeruginosa.     

Preparation of single cells from aggregated Taxus suspension cultures for population analysis

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism.     

Enhanced co-production of pectinase,cellulase and xylanase enzymes from Bacillus subtilis ABDR01 upon ultrasonic irradiation

The effect of low-intensity ultrasound irradiation was studied to improve the co-production for pectinase, cellulase, and xylanase enzymes using Bacillus subtilis ABDR01. Different parameters such as ultrasonic irradiation at the different growth phases of the bacterial strain, ultrasound power, irradiation duration, and irradiation duty cycle were assessed. Sonication with 90 W ultrasound power, 25 kHz frequency with 70 % duty cycle for 5 min at 6 h of bacterial growth phase gave the maximum productions of 87.82 U/ mL pectinase 22.17 U/ mL cellulase and 137.95 U/ mL xylanase respectively. The enzyme activity of pectinase, cellulase, and xylanase was enhanced by about 38.15 %, 53.77 %, and 24.59 %, respectively, compared to non-sonicated control cultivation. This optimized low-frequency ultrasound irradiation to bacterial cells enhanced the nutrient uptake rate and increased the cell wall permeability, which results in higher enzyme productivity. Our results signify the effectiveness of low-frequency ultrasound irradiation for improved enzyme yields and hyperactivation during microbial fermentation.     

Production of Cell Wall-Degrading Enzymes by the Phytopathogenic Fungus Sclerotinia sclerotiorum

The range of polysaccharide-degrading enzymes and glycosidases formed by the phytopathogenic fungus Sclerotinia sclerotiorum was monitored following growth on 16 carbohydrate substrates. Endo- and exoenzymes capable of degrading cellulosic, hemicellulosic, and pectinolytic polysaccharides were secreted. Pectinolytic activities were produced constitutively on all of the substrates tested. Cellulolytic enzymes were not induced in simple sugar (i.e., glucose or xylose) media. Polysaccharide growth substrates and cellulase inducers increased all of the enzyme activities tested. Gel filtration analysis revealed the appearance of new molecular forms of pectinase, β-xylosidase, and cellobiosidase during induction on pectin and carboxymethyl cellulose media.     

The screening, characterization, and use of omega-laurolactam hydrolase: a new enzymatic synthesis of 12-aminolauric acid

Several omega-laurolactam degrading microorganisms were isolated from soil samples. These strains were capable of growing in a medium containing omega-laurolactam as sole source of carbon and nitrogen. Among them, five strains (T7, T31, U124, U224, and U238) were identified as Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, Rhodococcus sp. U224, and Sphingomonas sp. U238, respectively. The omega-laurolactam hydrolyzing enzyme from Rhodococcus sp. U224 was purified to homogeneity, and its enzymatic properties were characterized. The enzyme acts on omega-octalactam and omega-laurolactam, but other lactam compounds, amides and amino acid amides, cannot be substrates. The enzyme gene was cloned, and the deduced amino acid sequence showed high homology with 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12) from Arthrobacter sp. KI72 and Pseudomonas sp. NK87. Enzymatic synthesis of 12-aminolauric acid was performed using partially purified omega-laurolactam hydrolase from Rhodococcus sp. U224.     

放线菌制剂对人参生长及根域土壤微生物区系的影响

以小兴安岭地区人参为研究对象,探索放线菌制剂对人参的促生效应及对人参根区、根表土壤微生物区系的影响.结果表明： 经放线菌制剂Streptomyces pactum（Act12）处理后,人参药用部分产量增加,品质改善;叶片诱导酶活性提高,根系活力增强;土壤中细菌、放线菌的数量和比例显著增加,真菌的数量和比例减少.与对照相比,土壤微生物区系结构改变:优势菌荧光假单胞菌、韩国假单胞菌和氧化微杆菌在根区、根表土壤中的数量大幅提高;病原真菌烟色织孢霉在根区土壤中减少,在根表土壤中消失.表明施用放线菌制剂Act12能够改善土壤微生物区系,提高人参植株的抗性和根系活力,增加产量并改善品质.     

Isolation and Identification of Viable Embryo Sacs in Several Angiosperm Species

Since the enzymatic technique for isolating embryo sac (ES) has been established on fixed materials of several angiosperms as well as on fresh ovules of Antirrhinum majus in our lab, further works on isolation of viable ESs were carried on. Fresh ovules were macerated in a solution of enzymes, sucrose with or without potassium dextran sulphate by a microshaker at 28–30℃ for several hours. The enzymes included pectinase, cellulase, snailase and pectolyase Y-23, the combination and concentration of which varied with the plant species and the developmental stages of ESs. To date the mature ESs of Helianthus annuus, A. majus and Nicotiana tabacum and the ESs after fertilization with proembryo and endosperm cells in the two former species were well isolated. Nomarski interference contrast and Hoechst 33253 fluorescence microscopical observations revealed that the ESs retained their cell structure and were rich of ergastic substances. Fluorochromasia induced by fluorescein diacetate further proved that they were really viable.     

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

Abstract

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2?mM CaCO_3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55?U/ml, 79.22?U/ml, 12.43?U/ml, 64.66?U/ml, and 21.12?U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.