Isolation of an endophytic fungus producing baccatin III from Taxus wallichiana var. mairei

The objective of this study was to isolate endophytic fungi producing baccatin III from yew for the purpose of baccatin III and paclitaxel manufacture. Surface sterilized bark of Taxus wallichiana var. mairei was used as source material with potato dextrose agar culture medium for isolation of endophytic fungi. Fungal cultures were extracted with a mixture of chloroform/methanol (1:1, v/v) and the baccatin III in the extracts was determined and authenticated with LC–MS. An endophytic fungus that produced baccatin III was identified by ITS rDNA and 26S D1/D2 rDNA sequencing. A total of 192 endophytic fungal strains were isolated from T. wallichiana var. mairei. Only one of the 192 strains produced baccatin III and it was identified as Diaporthe phaseolorum. The productivity of this strain cultured in PDA culture medium was 0.219 mg/l. The isolated endophytic fungus produced baccatin III at a relatively high level and shows promise as a producing strain for baccatin III and paclitaxel manufacture after strain improvement.     

Root Colonization and Phytostimulation by Phytohormones Producing Entophytic Nostoc sp. AH-12

Nostoc, a nitrogen-fixing cyanobacterium, has great potential to make symbiotic associations with a wide range of plants and benefit its hosts with nitrogen in the form of nitrates. It may also use phytohormones as a tool to promote plant growth. Phytohormones [cytokinin (Ck) and IAA] were determined in the culture of an endophytic Nostoc isolated from rice roots. The strain was able to accumulate as well as release phytohormones to the culture media. Optimum growth conditions for the production of zeatin and IAA were a temperature of 25 °C and a pH of 8.0. Time-dependent increase in the accumulation and release of phytohormones was recorded. To evaluate the impact of cytokinins, an ipt knockout mutant in the background of Nostoc was generated by homologous recombination method. A sharp decline (up to 80 %) in the zeatin content was observed in the culture of mutant strain Nostoc AHM-12. Association of the mutant and wild type strain with rice and wheat roots was studied under axenic conditions. The efficacy of Nostoc to colonize plant root was significantly reduced (P < 0.05) as a result of ipt inactivation as evident by low chlorophyll a concentration in the roots. In contrast to the mutant strain, wild type strain showed good association with the roots and enhanced several growth parameters, such as fresh weight, dry weight, shoot length, and root length of the crop plants. The study clearly demonstrated that Ck is a tool of endophytic Nostoc to colonize plant root and promote its growth.     

Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in Epiphytic and Endophytic Colonization of Leaves

The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen Pseudomonas syringae pv. syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The P. agglomerans strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the P. agglomerans strain formed aggregates that often extended over multiple epidermal cells. The P. syringae pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P. syringae pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic P. syringae pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic P. syringae pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the P. agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P. syringae pv. syringae strain on bean but not with growth of the P. syringae pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P. syringae pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic P. agglomerans strain were not.     

Paeonol produced by Chaetomium sp., an endophytic fungus isolated from Paeonia suffruticosa

In order to research the relationship between endophytic fungus and active ingredients in medicinal Paeonia suffruticosa, a total of 57 fungal isolates were isolated from the roots, stems, leaves and buds of medicinal plant Paeonia ostii; mycelium was collected after these fungal isolates were fermented on PDA medium for a few days; then the mycelium products were extracted; their extracts were analyzed by gas chromatography–mass spectrometry. With this method, a strain endophytic fungi named J1-2 which can produce paeonol was screened. Paeonol produced by J1-2 was analyzed by using a high resolution mass spectrometer (HRMS) and nuclear magnetic resonance (NMR). The potential paeonol-procucing named J1-2 was identified Chaetomium based on morphological characteristics and ITS sequence analysis. The current research initially indicates that endophytic fungi can affect the potency of peony. At the same time it also indicates that the numerous endophytic fungi inside the medicinal Paeonia suffruticosa are precious resource for the pharmaceutical natural products that are originally from the Paeonia suffruticosa.     

Isolation and identification of a novel endophytic bacterial strain with antifungal activity from wild blueberryVaccinium uliginosum

A strain of endophytic bacterium with a good anti-fungal ability was first isolated fromVaccinium uliginosum. It was identified asSerratia marcescens using the 16s rDNA sequence homology and through its physiological and biochemical characteristics. Through the alignment and cladistic analysis of homologous nucleotide sequences of knownSerratia marcescens, it was found to be a novel subspecies ofS. marcescens. An extensive suppressive spectrum of this endophytic strain ofS. marcescens against plant pathogenic fungi was observed. In particular, it inhibited the growth of the causal agents of blueberry leaf spot and of other ten plant fungal pathogens. The inhibition rate, among the different fungi, ranged from 22.7–70.5%. The study indicated the potential use of thisS. marcescens endophytic strain in the biological control of blueberry’s leaf spot as well as many other plant diseases. To our knowledge, this was the first time that an endophyticS. marcescens was isolated from a wild blueberry in China, and may also represent a novel introduction to the world.     

Predominant growth of α-amylase regulation mutant in continuous culture of Bacillus caldolyticus

The production of α-amylase in batch and continuous cultures of the strain SP of Bacillus caldolyticus was studied using a maltose-casitone medium. The three quantitative parameters of α-amylase production (maximum values of the specific production rate, volumetric productivity and concentration of α-amylase) increased in continuous culture by 2.5, 4.6 and 3.8 times respectively in comparison with a batch culture. It was found that a mutant strain M1 of α-amylase production was predominant in every run of the continuous cultures. The strain M1 differs from the strain SP in that it can produce α-amylase constitutively in a batch culture while the cells of strain SP require maltose for α-amylase production. On the contrary, α-amylase production by the strain M1 was repressed partly by maltose. Moreover, glucose repression on α-amylase production was not observed for the M1 strain while it was remarkable for the strain SP. The above-mentioned properties of the mutant M1 concerning the regulation of α-amylase production are not only advantageous for industrial use but also interesting from the viewpoint of basic microbiology.     

Horizontal Gene Transfer to Endogenous Endophytic Bacteria from Poplar Improves Phytoremediation of Toluene

Poplar, a plant species frequently used for phytoremediation of groundwater contaminated with organic solvents, was inoculated with the endophyte Burkholderia cepacia VM1468. This strain, whose natural host is yellow lupine, contains the pTOM-Bu61 plasmid coding for constitutively expressed toluene degradation. Noninoculated plants or plants inoculated with the soil bacterium B. cepacia Bu61(pTOM-Bu61) were used as controls. Inoculation of poplar had a positive effect on plant growth in the presence of toluene and reduced the amount of toluene released via evapotranspiration. These effects were more dramatic for VM1468, the endophytic strain, than for Bu61. Remarkably, none of the strains became established at detectable levels in the endophytic community, but there was horizontal gene transfer of pTOM-Bu61 to different members of the endogenous endophytic community, both in the presence and in the absence of toluene. This work is the first report of in planta horizontal gene transfer among plant-associated endophytic bacteria and demonstrates that such transfer could be used to change natural endophytic microbial communities in order to improve the remediation of environmental insults.     

Photosynthetic Bradyrhizobia Are Natural Endophytes of the African Wild Rice Oryza breviligulata

We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulata could significantly enhance cultivated rice production.     

Myrtucommulone production by a strain of Neofusicoccum australe endophytic in myrtle (Myrtus communis)

Myrtucommulones are acylphloroglucinol compounds reported from myrtle (Myrtus communis) and a few more plant species belonging in the Myrtaceae that have recently attracted the attention of pharmacologists for their anti-oxidant, anti-inflammatory and anti-tumor properties. An endophytic strain of Neofusicoccum australe recovered from a myrtle branch was selected based on the bioactivity of its culture extracts, and found to produce myrtucommulones A and D. A mixture of these compounds induced anti-proliferative effects on the human prostatic cancer cell lines DU145 and PC3, with a IC₅₀ of respectively 4.64 and 3.11 mg/l. Along the lines of recent evidences of the ability by endophytic fungi to produce bioactive compounds originally extracted from their host plants, this is the first report of myrtucommulones as secondary metabolites of an endophytic fungal strain. The availability of a microbial strain to be cultured in vitro may provide access to more substantial amounts of these products for further investigations in view of their possible pharmaceutical use.     

一株银杏内生真菌菌株的抑菌活性成分研究

从银杏叶柄分离筛选到具抗菌活性的内生真菌Colletotrichum.SP NTB-2菌株,利用硅胶柱色谱、制备高效液相色谱等方法在其发酵产物中分离到抗枯草芽孢杆菌、鼠伤寒沙门氏菌等具有广谱抑菌活性的化合物,经MS、NMR等波谱数据确认该活性成分为芹菜素-8-C-葡萄糖苷(apigenin-8-C-β-D-glucopyranoside),该化合物首次从真菌中分离得到。     

Influence of a bacteriophage on the population dynamics of toxic dinoflagellates by lysis of algicidal bacteria

A lytic phage (øZCW1) was isolated from an algicidal bacterium Pseudoalteromonas sp. strain SP48 that specifically kills the toxic dinoflagellate Alexandrium tamarense. We demonstrated that øZCW1 could trigger the growth of A. tamarense by inhibiting the growth of algicidal bacterium SP48. In contrast, the growth of A. tamarense was suppressed when cocultured with either SP48 or the øZCW1-resistant mutant of SP48. This study provides the first evidence of the indirect impact of bacteriophage on bloom-forming microalgae via phage lysis of alga-killing bacteria.     

一株产紫杉醇罗汉松内生真菌的分离和鉴定

[目的]紫杉醇是重要的抗癌药物,主要从罗汉松等植物中提取,为了保护罗汉松等种质资源,本文从罗汉松植株中分离产紫杉醇内生真菌,并对内生真菌所产紫杉醇的抗肿瘤活性进行了分析.[方法]采用组织块法自罗汉松的根、茎、叶等组织中分离内生真菌;通过四唑蓝(Methyl ThiazolylTetrazolium,MTT)比色法筛选有抗肿瘤活性的内生真菌菌株,通过薄层层析(Thin Layer Chro-matography,TLC)和高效液相色谱(High Performance Liquid Chromatography,HPLC)对内生真菌所产活性物质进行鉴定;采用抽提法抽提内生真菌所产紫杉醇,应用Vero细胞对抽提的紫杉醇的活性进行了分析.[结果]从罗汉松属(Podocrapus)植物中分离到155株内生真菌,其中28株内生真菌具有较高的抑癌活性.将其中一株菌株A2命名为EPTP-1,经形态学和分子分类学分析鉴定为烟曲霉(Aspergillus fumigatus).菌株EPTP-1中抽提的紫杉醇5.553μg/L～555.3 μg/L作用24h表现出明显的致细胞凋亡作用.菌株EPTP-1发酵5天时紫杉醇的产率为0.5578±0.0294 mg/L.[结论]从罗汉松中分离到了一株产紫杉醇内生真菌EPTP-1,可作为紫杉醇类药物工业化生产的候选菌株.     

Endophytic fungi from Peruvian highland and lowland habitats form distinctive and host plant-specific assemblages

Biodiversity and biogeography of leaf-inhabiting endophytic fungi have not been resolved yet. This is because host specificity, life cycles and species concepts, in this heterogeneous ecological guild of plant-associated microfungi, are far from being understood. Even though it is known that culture-based collection techniques are often biased, this has been the method of choice for studying fungal endophytes. Isolation of fungal endophytes only through culture-based methods could potentially mask slow growing species as well as species with low prevalence, preventing the capture of the communities’ real diversity and composition. This bias can be partially resolved by the use of cultivation-independent approaches such as direct sequencing of plant tissue by next generation techniques. Irrespective of the chosen sampling method, an efficient analysis of community ecology is urgently needed in order to evaluate the driving forces acting on fungal endophytic communities. In the present study, endophytic ascomyceteous fungi from three different plant genera (Vasconcellea microcarpa, Tillandsia spp., and Hevea brasiliensis) distributed in Peru, were isolated through culture-based sampling techniques and sequenced for their ITS rDNA region. These data sets were used to assess host preferences and biogeographic patterns of endophytic assemblages. This study showed that the effect of the host’s genetic background (identity) has a significant effect on the composition of the fungal endophytic community. In other words, the composition of the fungal endophytic community was significantly related to their host’s taxonomic identity. However, this was not true for all endophytic groups, since we found some endophytic groups (e.g. Xylariales and Pleosporales) occurring in more than one host genus. Findings from this study promote the formulation of hypotheses related to the effect of altitudinal changes on the endophytic communities along the Eastern Andean slopes. These hypotheses and perspectives for fungal biodiversity research and conservation in Peru are addressed and discussed.     

Colonization by endophytic Ochrobactrum anthropi?Mn1 promotes growth of Jerusalem artichoke

The Ochrobactrum anthropi Mn1 strain, taxonomically identified using 16S ribosomal DNA sequence, was isolated from roots of Jerusalem artichoke. Its endophytic colonization was investigated microscopically using green fluorescent protein introduced by vector pHC60. The strain entered Jerusalem artichoke tissues through the root, and was localized in the roots and stems. The plant growth-promoting (PGP) effects of O. anthropi Mn1 were assessed in greenhouse as well as field trials with different nitrogen supplies. Only under moderate to ample nitrogen supply, could O. anthropi Mn1 promoted growth of host plant. The PGP effects of the strain were symbiotic nitrogen fixation, root morphological optimization and enhanced nutrient uptake. We hypothesize that the symbiotic interspecies interaction might be quorum sensing related.     

An Endophytic Sanguinarine-Producing Fungus from Macleaya cordata,Fusarium proliferatum BLH51

Fermentation processes using sanguinarine-producing fungi other than Macleaya cordata may be an alternative way to produce sanguinarine (SA), which is a quaternary benzo[c]phenanthridine alkaloid possessing antibacterial, anthelmintic, and anti-inflammatory properties. In this study, a SA-producing endophytic fungus strain BLH51 was isolated from the leaves of M. cordata grown in the Dabie Mountain, China. Strain BLH51 produced SA when grown in potato dextrose liquid medium. The amount of SA produced by this endophytic fungus was quantified to be 178 μg/L by HPLC, substantially lower than that produced by the host tissue. The fungal SA—which was analyzed by thin layer chromatography and high-performance liquid chromatography—was shown to be identical to authentic SA. Strain BLH51 was identified as Fusarium proliferatum based on the morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. To the best of our knowledge, this is the first report concerning the isolation and identification of endophytic SA-producing fungi from the host plant, which further proved that endophytic fungi are valuable reservoirs of bioactive compounds.     

一株抗玉米纹枯病内生细菌的分离鉴定及其抗病促生作用

玉米纹枯病是玉米主要的真菌病害,从优良玉米品种川单13号中分离到一株对玉米纹枯病菌具有明显抑制作用并能够促进玉米生长的内生细菌.通过形态、生理生化特性分析以及16SrDNA序列同源性比较,鉴定该菌株为枯草芽孢杆菌.盆栽试验表明,菌株能够抑制玉米纹枯病的发生,抑菌率为 43.01%,促生试验表明,该菌能够显著促进玉米苗的生长,促生作用与植物生长刺激素(IAA)和效果相似,显示出良好的开发前景.     

Endophytic bacteria isolated from wild jojoba [<Emphasis Type="Italic">Simmondsia chinensis</Emphasis> L. (Schneider)] roots improve in vitro propagation

Endophytic bacteria promote plant growth, reduce stress caused by biotic and abiotic factors, and can trigger active defense reactions in plants. This study aimed to evaluate enzyme activity of in vitro jojoba (Simmondsia chinensis) plants inoculated with endophytic bacteria. In vitro shoots of female and male plants were inoculated with strains of Azospirillum brasilense (Cd), Methylobacterium aminovorans (JRR11), Rhodococcus pyridinivorans (JRR22) or co-inoculated with a mixture of JRR11?+?JRR22. A total of 10 treatments were performed to evaluate shoot and root length; changes in key enzymes involved in plant defense (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and phenylalanine ammonia lyase) after post-inoculation (45 days). All endophytic bacteria strains used promoted plant growth and rhizogenesis. Differences were found in enzyme activity between female and male plants. The plants inoculated with JRR22 strain, showed the highest enzyme activity suggesting an induced systemic response and a potential increase in plant resistance to pathogen attack.     

沙棘根瘤内生细菌中抑菌促生菌株的筛选和鉴定

【背景】植物内生细菌可产生具有抑菌和促生活性的物质,既能抑制植物病原菌对寄主植物的侵染,也能促进植物的生长。沙棘根瘤内生细菌是根瘤内除共生固氮的弗兰克氏菌外,与沙棘共生的一大类微生物。研究具有抑菌和促生活性的植物内生菌,可为微生物菌肥的研究提供理论基础。【目的】筛选具有优良抑菌和促生活性的沙棘根瘤内生细菌,初步研究其抑菌和促生活性,并对菌株进行鉴定。【方法】采用双层琼脂法、琼脂扩散法、双层平板对峙法、牛津杯法进行沙棘根瘤拮抗性内生细菌的筛选。选取抑菌活性较高的内生细菌,分别采用Salkowski比色法、ChromeAzurolS(CAS)平板检测法和钼锑抗比色法进行产吲哚乙酸、铁载体及溶磷能力的测定。采用发酵液灌根法测定沙棘根瘤内生细菌SR308对黄瓜促生作用的盆栽效果。通过形态和培养特征、生理生化试验及16S rRNA基因序列分析法对菌株TT201和SR308进行鉴定。【结果】从131株沙棘根瘤内生细菌中筛选出9株具有较强抑菌活性的内生细菌,其中菌株TT201抑菌性最佳、抑菌谱广;菌株SR308的促生活性最好,其发酵液对黄瓜的生长具有较强的促进作用。对具有较强抑菌和促生活性的菌株TT201和SR308进行鉴定的结果表明,菌株TT201为侧孢短芽孢杆菌(Brevibacilluslaterosporus),菌株SR308为蕈状芽孢杆菌(Bacillusmycoides)。【结论】获得2株具有优良抑菌和促生活性的沙棘根瘤内生细菌,为进一步开发微生物农药及菌肥提供了资源。