9568D镰孢霉作用于死态龙血树形成血脂的研究

1　引　　言剑叶龙血树血竭来自于剑叶龙血树 (Ｄｒａｃａｅｎａｃｏｃｈｉｎｃｈｉｎｅｎｓｉｓ)的树脂 ,早年的研究[[2 ] 发现人工割伤可以促进树脂的积累 .江东福等[4] 证实剑叶龙血树血竭的形成与真菌相关 ,用特异性真菌接种活体龙血树 ,激活龙血树的防御反应 ,产生含植物防卫素的血竭[1,3 ,6] .但人们注意到野外自然环境下一些衰老枯死的剑叶龙血树的材质上亦有血竭形成 ,其与真菌的作用是否有关联 ?本文采用分离自剑叶龙血树根部的内生真菌 95 6 8Ｄ镰孢霉接种于剑叶龙血树材质(经灭活处理 ) ,保湿培养 4～ 5个月后 ,在接种部位有红色…     

云南血竭的化学成分及抗真菌活性

云南血竭为剑叶龙血树（Ｄｒａｃａｅｎａ ｃｏｃｈｉｎｃｈｉｎｅｎｓｉｓ（Ｌｏｕｒ．）Ｓ．ｃ．Ｃｈｅｎ）树脂,从中分离到５个芳香族化合物：对羟基苯甲酸乙酰（１）,７,４＾′－二羟基黄烷（２）,７－羟基－４＾′－甲氧基黄烷（３）,７,４＾′－二羟基黄酮（４）和ｌｏｕｒｅｉｒｉｎ Ａ（５）以及１个甾体皂甙（６）,并对其中３个酚性成分进行了抗真菌活性检测。另外,用薄层层析法对云南血竭、广西血竭、海南血竭及     

龙血树真菌群及其对血竭形成的影响

从柬埔寨龙血树（Ｄｒａｃａｅｎａｃｏｃｈｉｎｃｈｉｎｅｎｓｉｓ）茎杆中分离到３０３株真菌,其中镰刀菌属（Ｆｕｓａｒｉｕｍ）菌株占总分离频率的５２％,其次是短梗霉（Ａｕｒｅｏｂａｓｉｄｉｕｍ）和枝孢霉（Ｃｌａｄｏｓｐｏｒｉｕｍ）。通过活体接种对血竭产生的影响试验表明,对血竭形成起重要作用的真菌主要是禾谷镰刀菌龙血树变种（Ｆ．ｇｒａｍｉｎｕｍｖａｒ．ｄｒａｃａｅｎａ）等４株红色镰刀菌,可使血竭形成量提高６６％－１２０％。     

中国血竭基源植物的研究与利用

血竭是我国传统名贵中药,目前商品血竭主要来源于棕榈科黄藤属(Daemonorps)植物的果实和百合科龙血树属(Dracaerta)植株茎干的红色树脂.在我国境内发现的、有野生分布的血竭基源植物只为海南龙血树和剑叶龙血树两种.系统综述了我国血竭基源植物的资源状况、血竭形成机理、血竭基源植物的开发利用、人工繁育及栽培技术等方面的研究进展,并对野生血竭基源植物资源的保护、开发与利用、血竭产生机制的研究等进行了展望.     

3种龙血树PAL基因克隆及分子鉴定

该研究以3种龙血树(剑叶龙血树、海南龙血树和岩棕)的DNA和总RNA为模板,采用同源克隆法克隆龙血竭3种基源植物的苯丙氨酸解氨酶(phenylalanine ammonia-lyase)基因PAL,利用DNAMAN、MEGA7等软件进行生物信息学分析,以探讨龙血竭3种基源植物的分子鉴定方法,为生产实际中对不同来源的龙血竭原料的鉴定提供理论依据。结果表明:(1)在龙血竭3种基源植物中分别获得长度为718 bp的PAL片段,序列分析发现,3种龙血树PAL片段高度一致(99.69%),仅存在5处碱基突变;系统进化分析表明,龙血树PAL片段与百合科植物PAL基因聚为一类。(2)RT-PCR分析发现,PAL基因在3种龙血树的根、茎、叶中均有表达,同种龙血树不同组织的表达量差异较小,且PAL基因在岩棕中的表达量较高,在剑叶龙血树和海南龙血树中表达量较低。(3)利用保守引物进行分子鉴定发现,同种龙血树PAL序列一致,3种龙血树PAL片段存在5处碱基突变,且这些碱基突变为稳定遗传,表明3种龙血树PAL基因高度保守。(4)根据突变位点建立了龙血竭3种基源植物的分子鉴定方法,进一步分子鉴定结果表明,剑叶龙血树的碱基位置为GCGGG,海南龙血树的碱基位置为CTGGC,岩棕的碱基位置为GTTTG。该研究结果为龙血竭原材料溯源和质量控制奠定了理论与技术基础。     

黄藤属植物的药用价值

血竭素有“活血圣药”之称,原药系由天然分布于热带雨林中的棕榈科黄藤属（Daemonorops）的常绿攀缘植物麒麟血藤（D．draco）及该属其他物种的果实的分泌树脂萃取而成。我国商品血竭,传统上原由东南亚进口。由于热带森林面积税减,野生植物资源枯竭,血竭货源匮乏,价格昂贵,科研人员通过实验研究,寻找开发代用植物资源生产血竭。目前,国内市场供应的血竭的大部分提取于龙舌兰科龙血树属（Dracaena）的一些植物,如箭叶龙血树（D．cochinensis）,柬埔寨龙血树（D．cambodiana）。初步研究表明,黄藤（Dae－monoropsmarcaritae）的…     

腔孢纲真菌的三个新种

在调查香龙血树Ｄｒａｃａｅｎａ ｆｒａｇｒａｎｓ（Ｌ．）Ｋｅｒ－Ｇａｎｌ．的病害中,发现了腔孢纲真菌的３个新种,即：Ｐｈｌｍｏｐｓｉｓ ｄｒａｃａｅｎｉｃｏｌａＺ．Ｄ．Ｊｉａｎｇ,Ｐ．Ｇ．Ｘｉ ｅｔ Ｐ．Ｋ．Ｃｈｉ,Ｂａｒｔａｌｉｎｉａ ｄｒａｃａｅｎａｅ Ｐ．Ｇ．Ｘｉ,Ｚ．Ｄ．Ｊｉａｎｇ ｅｔ Ｐ．Ｋ．Ｃｈｉ及Ｓｐｈａｅｒｏｐｓｉｓ ｄｒａｃａｅｎａｅ Ｐ．Ｇ．Ｘｉ ｅｔ Ｐ．Ｋ．Ｃｈｉ,模式标     

龙血树真菌群及其对血褐形成的影响

从柬埔寨龙血树茎杆中分离到３０３株真菌,其中镰刀菌属菌株占总分离频率的５２％,其次是短梗霉和枝孢霉。通过活体接种对血竭产生的影响试验表明,对血竭形成起重要作用的真菌主要是禾谷镰刀菌龙血树变种等４株红色镰刀菌,可使血竭形成量提高６６％－１２０％。     

剑叶龙血树内生真菌的分离及体外抑菌活性筛选

为研究剑叶龙血树内生真菌资源多样性,初步探讨和筛选具有抑菌活性的特异性菌株以及进一步开发剑叶龙血树内生真菌的抗菌活性化合物。该文采用植物组织分离法从剑叶龙血树茎和叶中分离内生真菌,对内生真菌进行液体发酵7 d,经乙酸乙酯萃取后制得粗提物,并采用牛津杯扩散法,以10种常见病原菌和5种临床耐药菌为靶标检测其发酵粗提物的抑菌活性,对有较好抑菌活性的内生真菌进行分子鉴定。结果表明:(1)从剑叶龙血树茎、叶中共分离得到345株内生真菌,294株对一种以上指示菌有抑制活性;(2)其中84株内生真菌对5株临床耐药菌均有不同程度的抑制活性,占所分离菌株总数的24.35%,75%的内生真菌对金黄色葡萄球菌有抑制活性。这说明剑叶龙血树中存在多种有抑菌活性的内生真菌,为剑叶龙血树内生菌抗菌活性成分挖掘及新型抗菌药物筛选奠定了基础。     

血竭及其原植物

血竭,又名麒麟竭,是名贵的传统中药,有活血祛瘀、消肿止痛、收敛止血的功效。提取血竭的原植物已知包括龙舌兰科的龙血树属(Dracaena)、棕榈科的黄藤属(Daemonoropus)、大戟科巴豆属(Crofon)、以及豆科的青龙木属(Pterocarpus)等属中的十余种植物。在国外,血竭多称为龙血(Dragon’s blood),非洲用龙舌兰科的龙血树属的树干割取血竭已有两千余年的历史,而用棕榈科植物黄藤果实     

In Vitro Induction of Haploid Plantlets from Unpollinated Young Ovaries of Lily and Its Embryo logical Observations

Unpollinated young ovaries of lily (Lilium davidii var. willmottiae (Wilson) Roffill) were inoculated on modified MS medium and N6 medium. Ovary cultures were incubated at 25–28℃, and illuminated with a fluorescent light of about 800–1200 Lux. Cultured ovaries gradually became thicker and longer after 10 days. The calli (about 6–12 mm in size) were produced after 40 days. The calli were then transferred to the differentiation medium. After 50 days, regeneration plantlets were formed. Embryoids were directly produced from some ovaries, which then developed into plantlets. Observation of chromosome number of regeneration plants shows: 65.71% regeneration plants are haploid plants, 34.29% are diploid. Embryological observation of ovary culture shows that haploid plants are from megaspore tetrad, while diploid plants are probably from nucellus cell.     

In vivo measurement of phytochrome in tomato fruit

Presence of phytochrome in two kinds of tomatoes (Lycopersicon esculentum Mill.), the yellow lutescent strain and cherry tomatoes (L. esculentum Mill. var. cerasiformecv. Red Cherry), was established by measuring the absorption difference spectra of the whole fruit after irradiation with red and with far red light. Phytochrome content was determined in yellow lutescent tomatoes and decreased gradually during the ripening period.     

Variants of Aspergillus alutaceus var. alutaceus (formerly Aspergillus ochraceus) with altered ochratoxin A production.

The present studies, using Aspergillus alutaceus var. alutaceus Berkeley et Curtis (formerly A. ochraceus Wilhelm) NRRL 3174 along with three other wild-type strains, were undertaken in an attempt to understand the effects of irradiation and other treatments on mycotoxin production in grain. Bedford barley was inoculated with spores of NRRL 3174, gamma irradiated, and incubated at 28 degrees C and 25% moisture. After 10 days of incubation, two colony types, ochre (parental) and yellow (variant), were isolated from the grain. Further culturing of the yellow variant resulted in the spontaneous appearance of a white variant that exhibited greatly enhanced fluorescence under UV light. In subsequent work, we have also isolated variants producing a soluble red pigment. In addition, in model experiments involving irradiation (1 kGy) of pure cultures, induction frequencies ranging between 2 and 4% (survival basis) were observed for the yellow and red variants. Inoculation of these variants into wheat and incubation for 14 days at 28 degrees C and 32% moisture resulted in ochratoxin A production in the relative amounts of 0.09:1:4.6:9.3 for the red, ochre (parental), yellow, and white variants, respectively. Additional characteristics of these isolates are described. Confirmation that the white high-ochratoxin-A-producing variants were derived from the parental strain was demonstrated by obtaining revertant sectors in monoclonal cultures of the variants.     

Variants of Aspergillus alutaceus var. alutaceus (formerly Aspergillus ochraceus) with altered ochratoxin A production.

The present studies, using Aspergillus alutaceus var. alutaceus Berkeley et Curtis (formerly A. ochraceus Wilhelm) NRRL 3174 along with three other wild-type strains, were undertaken in an attempt to understand the effects of irradiation and other treatments on mycotoxin production in grain. Bedford barley was inoculated with spores of NRRL 3174, gamma irradiated, and incubated at 28 degrees C and 25% moisture. After 10 days of incubation, two colony types, ochre (parental) and yellow (variant), were isolated from the grain. Further culturing of the yellow variant resulted in the spontaneous appearance of a white variant that exhibited greatly enhanced fluorescence under UV light. In subsequent work, we have also isolated variants producing a soluble red pigment. In addition, in model experiments involving irradiation (1 kGy) of pure cultures, induction frequencies ranging between 2 and 4% (survival basis) were observed for the yellow and red variants. Inoculation of these variants into wheat and incubation for 14 days at 28 degrees C and 32% moisture resulted in ochratoxin A production in the relative amounts of 0.09:1:4.6:9.3 for the red, ochre (parental), yellow, and white variants, respectively. Additional characteristics of these isolates are described. Confirmation that the white high-ochratoxin-A-producing variants were derived from the parental strain was demonstrated by obtaining revertant sectors in monoclonal cultures of the variants.     

Experimental histoplasmosis capsulati in athymic nude mice

Granuloma formation in nude (nu/nu) mice and their heterozygous littermates (nu/+ mice) against Histoplasma capsulatum var. capsulatum infection was studied.A culture of H. capsulatum var. capsulatum, isolated from a granuloma in the nasal cavity of a Japanese patient, was used in this experiment. Sixteen specific-pathogen-free male nu/nu and 32 nu/+ mice were used in this study.The nu/+ mice were divided into two groups. Sixteen nu/+ mice in one group and 16 nu/nu mice were inoculated intraperitoneally with 10⁶ yeast cells of the fungus, those in the other group of nu/+ mice were inoculated intravenously with the same number of the yeast cells. Two mice out of each group were sacrificed 2, 3, 7, 11, 14, 18, 25 and 30 days after inoculation, and each of their organs was examined histopathologically. In addition, pieces of these tissues were cultured on Sabouraud's dextrose agar slants.In the nu/+ mice inoculated intraperitoneally, although the fungus was recovered from the spleen, kidney and lymph nodes during the initial course of the infection, lesions were not detected in their histopathological sections. In the nu/+ mice inoculated intravenously, colonies were recovered from all of the organs examined, other than the brain and thymus, 7 days after inoculation.Histopathologically, a few microfoci consisting chiefly of mononuclear cells with or without yeast cells were found in the liver sections 4 days after inoculation. Seven and 11 days after inoculation the number of lesions had increased. They had large accumulations of mononuclear cells. From day 14 on, almost all of the yeast cells had lost most of their staining affinity or were destroyed in the granuloma. From day 25 on, the granulomatous lesions changed gradually to fibrous tissue.In the nu/nu mice the fungus was readily recovered from the spleen, liver, kidney and lymph nodes. Histopathologically, a few microfoci consisting of mononuclear cells were present in the liver sections 4 days after inoculation. That is to say, during the initial course of infection granulomas were formed. In the liver, from day 7 on, the lesions were large and their number increased. However, there was a definite difference between the nu/nu and nu/+ mice. In the former, the yeast cells were not killed, and they continued to multiply within the granulomas. These granulomas were never transformed into fibrous tissue.     

Control of Gaeumannomyces graminis Var. tritici in Wheat by Isolates of the G. graminis var. graminis/Phialophora sp. (Lobed Hyphopodia) Complex under Field Conditions

In an experiment carried out under field conditions, wheat inoculated with Gaeumanmyces graminis var. tritics had less take-all and yielded significantly more grain when simultaneously inoculated with G. graminis var. graminis and a lobed hyphopodiate phialophora sp. then when unprotected with these fungi.     

Development and validation of a binomial sequential sampling plan for the greenbug (Homoptera: Aphididae) infesting winter wheat in the southern plains

From 1997 to 1999, Schizaphis graminum (Rondani), intensity (number per tiller) was estimated on 115 occasions from hard red winter wheat fields located throughout the major wheat growing regions of Oklahoma. A total of 32 and 83 fields was sampled during the fall and spring, respectively. The parameters of linear regressions relating the mean number of greenbugs per tiller (m) and the proportion of infested tillers (PT) differed significantly between fall and spring infestations. The PT-m linear model provided a good fit for data on S. graminum for fall and spring infestations at tally thresholds of 0, 1, 2, and 3. A tally threshold (T) represents the number of greenbugs present on a tiller before the tiller is classified as infested by >T greenbugs. A regression model with a tally threshold of 2 was the most precise for classifying S. graminum populations during fall growth of winter wheat because it explained a greater amount of the variation in the PT-m relationship (97%) than models with other tally thresholds. A separate spring model with a tally threshold of 1 was the most precise for classifying S. graminum populations during spring growth of winter wheat. Sequential sampling stop lines based on sequential probability ratio tests were calculated for economic thresholds of 3 or 6 greenbugs per tiller for fall infestations and 6 or 9 greenbugs per tiller for spring infestations. With the newly developed parameters, the average sample number required to classify greenbug populations near economic thresholds (as above or below the economic threshold) varied from 69 to 207. We expect that the sampling plans for greenbugs in winter wheat developed during this study will be efficient and useful tools for consultants and producers in the southern plains.     

Experimental inoculation of Terminalia catappa seedlings with an environmental isolate of Cryptococcus neoformans var. gattii serotype C

In 1997, our laboratory reported for the first time the isolation of Cryptococcus neoformans var. gattii serotype C associated with almond tree (Terminalia catappa) detritus. This finding led to a more detailed follow up of the association between the plant and the yeast. Preliminary data have shown that survival of the yeast in almond trees seedlings goes beyond 100 days. The aim of the present study was to establish if under the conditions previously studied, C. neoformans var. gattii would remain viable for longer periods. A total of 83 almond tree seedings, 20-40 cm high, were inoculated with C. neoformans var. gattii serotype C (INS-755). Assays were carried out inoculating the stem or the soil where the seedlings were planted. Observations were undertaken for a period of up to 12 months. As processing techniques we employed the endophytic fungi procedure (stems), maceration (roots, leaves) and standard suspension method (soils). Additionally, microscopic visualization of the yeast in plant tissues was done with trypan blue plus lactophenol. C. neoformans var. gattii was recovered from the inoculated plants for a period of up to 12 months post-inoculation; additionally, the fungus had the capacity to migrate from the stem to the soil and viceversa, without causing macroscopic or microscopic alterations in the plant tissues. This finding suggests that there appears to be an association between the host plant and C. neoformans var. gattii in the environment.     

The impact of transgenic wheat expressing GNA (snowdrop lectin) on the aphids Sitobion avenae, Schizaphis graminum, and Rhopalosiphum padi

This study investigated the impact of transgenic wheat expressing Galanthus nivalis agglutinin (GNA), commonly known as snowdrop lectin, on three wheat aphids: Sitobion avenae (F.), Schizaphis graminum (Rondani), and Rhopalosiphum padi (L.). We compared the feeding behavior and the life-table parameters of aphids reared on GNA transgenic wheat (test group) and those aphids reared on untransformed wheat (control group). The results showed that the feeding behaviors of S. avenae and S. graminum on GNA transgenic wheat were affected. Compared with the control group, they had shorter initial probing period, longer total nonprobing period, shorter initial and total phloem sap ingestion phase (waveform E2), shorter duration of sustained ingestion (E (pd) > 10 min), and lower percentage of phloem phase of the total observation time. Moreover, S. graminum made more probes and had a longer total duration of extracellular stylet pathway (waveform C). The fecundity and intrinsic rate of natural increase (r(m)) of S. avenae and S. graminum on the transgenic wheat were lowered in the first and second generations, however, the survival and lifespan were not affected. The effects of the GNA expressing wheat on S. graminum and S. avenae were not significant in the third generation, suggesting rapid adaptation by the two aphid species. Despite the impact we found on S. avenae and S. graminum, transgenic GNA expressing wheat did not have any effects on R. padi.