内生Bacillus svelezensis HBB5菌株发酵盾叶薯蓣产薯蓣皂苷元的研究

目的探讨内生Bacillus svelezensis HBB5菌株发酵宿主植物盾叶薯蓣产薯蓣皂苷元的能力。方法接种内生B.svelezensis HBB5及B.subtilis ATCC 6633菌株(0.35×10~8 CFU/mL)至含盾叶薯蓣地下茎组织的液体培养基,32℃、165~185 r/min连续发酵108 h,检测发酵液细菌、pH、淀粉、麦芽糖、葡萄糖、淀粉酶(α-amylase)及薯蓣皂苷元溶出率等指标。结果内生B.svelezensis HBB5菌株有较强的酸、碱耐受力[pH(4.8±0.2)~(8.4±0.2)],相比B.subtilis ATCC 6633[pH(5.2±0.2)~(8.7±0.2)]差异不明显;前者达峰值生长量(60×10~(8±2) CFU/mL)明显高于后者(32×10~(8±2) CFU/mL)。发酵36~60 h时,B.svelezensis HBB5、B.subtilis ATCC 6633菌株发酵液的淀粉、麦芽糖、葡萄糖浓度达峰值,分别为(37.41±3.12)、(27.83±2.14)ng/mL,(21.06±1.25)、(16.54±1.08)ng/mL,(54.33±3.12)、(36.65±2.10)ng/mL,前者均高于后者。同时,B.svelezensis HBB5菌株维持高的α-amylase酶活性及薯蓣皂苷元溶出率。结论内生B.svelezensis HBB5菌株拥有较强的耐酸碱、降解淀粉、提高薯蓣皂苷元溶出的能力,为工业生产薯蓣皂苷元提供了一个新的方法。     

难培养菌的多条带PCR产物产生原因分析

【目的】分析严重威胁柑橘产业发展的毁灭性病害——柑橘黄龙病的强致病性病原亚洲种“Candidatus Liberibacter asiaticus”的种群多样性研究中出现多条带PCR产物的原因,为难培养菌的分子生物学研究提供参考。【方法】通过使用PCR、聚丙烯酰胺凝胶电泳(PAGE)和测序相结合分析“Ca. L. asiaticus”基因组上2个短串联重复(Short tandem repeats,STR)基因的PCR产物。【结果】单个菌株可扩增获得多个PCR条带且其扩增产物多态性受寄主品种影响;这2个STR基因的PCR产物在菌株间呈明显的多态性;扩增所获得的序列可来自“Ca. L. asiaticus”本身,也可来自其寄主或内生菌。【结论】难培养菌的STR基因PCR产物多态性产生的主要原因是该基因内部的串联重复序列数目存在差异,但目的菌及外源物种(寄主或内生菌等)基因组的非特异性扩增也是影响其多态性的因素。     

Botryorhodines A-D, antifungal and cytotoxic depsidones from Botryosphaeria rhodina, an endophyte of the medicinal plant Bidens pilosa

An endophytic fungus (Botryosphaeria rhodina) was isolated from the stems of the medicinal plant Bidens pilosa (Asteraceae) that is known for its anti-inflammatory, antiseptic and antifungal effects. The ethyl acetate extract of the fungal isolate exhibits significant antifungal activity as well as potent cytotoxic and antiproliferative effects against several cancer cell lines. Activity-guided fractionation resulted in the isolation of a complex of four depsidones, botryorhodines A-D and the auxin indole carboxylic acid. Botryorhodine A and B show moderate to weak cytotoxic activities against HeLa cell lines with a CC₅₀ of 96.97 μM and 36.41 μM, respectively. In addition, they also show antifungal activity against a range of pathogenic fungi such as Aspergillus terreus (MIC 26.03 μM for botryorhodine A and 49.70 μM for B) and the plant pathogen Fusarium oxysporum (MIC 191.60 μM for botryorhodine A and 238.80 μM for B). A potential role of the endophyte in modulating fungal populations living within or attacking the host plant is discussed.     

Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils

Technogenic activities (industrial—plastic, textiles, microelectronics, wood preservatives; mining—mine refuse, tailings, smelting; agrochemicals—chemical fertilizers, farm yard manure, pesticides; aerosols—pyrometallurgical and automobile exhausts; biosolids—sewage sludge, domestic waste; fly ash—coal combustion products) are the primary sources of heavy metal contamination and pollution in the environment in addition to geogenic sources. During the last two decades, bioremediation has emerged as a potential tool to clean up the metal-contaminated/polluted environment. Exclusively derived processes by plants alone (phytoremediation) are time-consuming. Further, high levels of pollutants pose toxicity to the remediating plants. This situation could be ameliorated and accelerated by exploring the partnership of plant-microbe, which would improve the plant growth by facilitating the sequestration of toxic heavy metals. Plants can bioconcentrate (phytoextraction) as well as bioimmobilize or inactivate (phytostabilization) toxic heavy metals through in situ rhizospheric processes. The mobility and bioavailability of heavy metal in the soil, particularly at the rhizosphere where root uptake or exclusion takes place, are critical factors that affect phytoextraction and phytostabilization. Developing new methods for either enhancing (phytoextraction) or reducing the bioavailability of metal contaminants in the rhizosphere (phytostabilization) as well as improving plant establishment, growth, and health could significantly speed up the process of bioremediation techniques. In this review, we have highlighted the role of plant growth promoting rhizo- and/or endophytic bacteria in accelerating phytoremediation derived benefits in extensive tables and elaborate schematic sketches.     

Fungal diversity in galls of baldcypress trees

The baldcypress midge (Taxodiomyia cupressi and Taxodiomyia cupressiananassa) forms a gall that originates from leaf tissue. Female insects may inoculate galls with fungi during oviposition, or endophytes from the leaf tissue may grow into the gall interior. We investigated fungal diversity inside of baldcypress galls, comparing the gall communities to leaves and comparing fungal communities in galls that had successful emergence versus no emergence of midges or parasitoids. Galls of midges that successfully emerged were associated with diverse gall fungal communities, some of which were the same as the fungi found in surrounding leaves. Galls with no insect emergence were characterized by relatively low fungal diversity.     

植物内生菌及其活性代谢产物

植物内生茵是一种新的微生物资源,具有潜在的应用价值。近年来,从植物内生茵中寻找新的生物活性物质的研究方兴未艾。对近年来从植物内生茵中发现的抗肿瘤、抗茵、抗病毒、杀虫、免疫抑制、抗氧化、降糖等活性化合物及其相应的产生茵的研究作一简要综述。     

Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites of root‐knot and potato cyst nematodes

The effects of a host plant on reproduction/abundance of fungal populations in relation to soil nutrients released by plants in the rhizosphere were studied. Abundance in the soil and potato rhizosphere of the fungi Paecilomyces lilacinus, Monographella cucumerina (CABI 380408) and Pochonia chlamydosporia var. chlamydosporia (Pc280, potato cyst nematode biotype) and P. chlamydosporia var. catenulata (Pc392, root‐knot nematode biotype) were assessed. The different ability of break crops (oilseed rape, sugarbeet and wheat) in the potato rotation to support Pa. lilacinus, Pochonia isolates Pc280 and Pc392 and abundance of the latter two isolates in soil and rhizosphere of potato plants infected with Meloidogyne incognita were also studied. Potato chits and crop seedlings were planted into boiling tubes containing 5000 chlamydospores or conidia g^?1 in acid washed sand (pH 6) and kept in a growth chamber at 20°C, and 16 h of light for up to 9 weeks. The abundance of the fungi in sand (fallow) differed significantly between fungal species, being in general less abundant in the absence than in the presence of the plant, although there was no interaction between plant species and fungal isolate. There was evidence of a different response to Me. incognita for Pc392 than for Pc280 but there was no significant effect of the presence of the nematode on the rate of increase of the fungus.     

Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis

Undifilum oxytropis (Phylum: Ascomycota; Family: Pleosporaceae) is a slow growing endophytic fungus that produces a toxic alkaloid, swainsonine. This endophyte resides in locoweeds, which are perennial flowering legumes. Consumption of this fungus by grazing animals induces a neurological disorder called locoism. The alkaloid swainsonine, an α-mannosidase inhibitor, is responsible for the field toxicity related to locoism. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi. Genetic manipulation of endophytic fungi is important to better understand biochemical pathways involved in alkaloid synthesis, but no transformation system has been available for studying such enzymes in Undifilum. In this study we report the development of protoplast and transformation system for U. oxytropis. Fungal mycelia required for generating protoplasts were grown in liquid culture, then harvested and processed with various enzymes. Protoplasts were transformed with a fungal specific vector driving the expression of Enhanced Green Florescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. In all cases, resistance to antibiotic hygromycin B was maintained. Such manipulation will open avenues for future research to decipher fungal metabolic pathways.     

一株多花黄精内生真菌的鉴别及其抗菌代谢产物

【目的】药用植物内生真菌是一类重要的微生物资源,能代谢产生多种生物活性物质。本研究从浙江庆元百山祖自然保护区多花黄精(Polygonatum cyrtonema)分离获得1株具有抗菌活性的菌株zjqy610。【方法】通过形态和ITS rDNA序列分析,鉴定为变灰青霉(Penicillium canescens)。采用正相硅胶柱层析和凝胶(Sephadex LH-20)柱层析,以紫外光或碘蒸汽显迹,配合活性追踪等,从zjqy610发酵液中分离获得3个具抗真菌活性的化合物。【结果】通过质谱和核磁共振波谱技术分别将其结构鉴定为:乙基氧苯氨基亚胺乙酸(o-acetylbenzeneamidinocarboxylic acid)、灰黄霉素(griseofulvin)和[1,2-b]呋喃2-甲基3-羧甲基4-羟基-5-甲氧基萘(naphtho[1,2-b]furan-3-carboxylic acid,4-hydroxy-5-methoxy-2-methyl-)。抑菌活性测试表明,3个化合物对多种植物病原真菌具有抑制活性,其中化合物zjqy610D-4对番茄灰葡萄孢、圆形炭疽菌、泻根亚隔孢壳和核盘菌4种病原真菌的活性最强,半抑制浓度EC50分别为0.68、0.38、0.91和0.61mg/L。【结论】该化合物具有开发成农用抗生素的价值。     

Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The entomopathogenic fungus Beauveria bassiana was established in coffee seedlings after fungal spore suspensions were applied as foliar sprays, stem injections, or soil drenches. Direct injection yielded the highest post-inoculation recovery of endophytic B. bassiana. Establishment, based on percent recovery of B. bassiana, decreased as time post-inoculation increased in all treatments. Several other endophytes were isolated from the seedlings and could have negatively influenced establishment of B. bassiana. The recovery of B. bassiana from sites distant from the point of inoculation indicates that the fungus has the potential to move throughout the plant.