Development of methods for detection and Agrobacterium-mediated transformation of the sterile,endophytic fungus Muscodor albus

Symbiotic endophytes, unlike plant pathogens, do not usually induce visible host response. This may constraint the researcher's decision whether a plant has been successfully infected by the endophyte. In order to properly study the establishment, development and progress of an endophyte in the host plant and host-endophyte interactions, methods for the identification and localization of endophytic microorganisms are needed. Towards this aim, we focused at two levels: (A) We constructed M. albus-specific primers for polymerase chain reaction (PCR). In vitro, these primers specifically detected only M. albus strains and not isolates of related fungi (such as Daldinia sp. and a Xylariaceae sp.). (B) For direct visualization of the fungi, we inserted a reporter gene (gfp) into M. albus hyphae using Agrobacterium-mediated transformation. Since M. albus is a sterile fungus (i.e., without spores or fungal fruiting bodies), we used chopped fungal mycelium for the transformation procedure. We transformed three different isolates of M. albus using Agrobacterium-mediated transformation. Fifty-nine different transformants were collected with a transformation efficacy of 0.0004–0.0026%. Although PCR-based detection and direct visualization of the transformants in planta were unsuccessful, all tested transformants (with one exception) exhibited similar biological activity to their cognate wild type. This work provides a significant step forward in molecular research of the relationships between this endophytic genus and their hosts.     

哈尔滨市室外空气真菌组成和分布的初步研究

采用曝皿法(孟加拉红培养基)连续12个月对哈尔滨市实验林场、东北林业大学家属区和哈尔滨市二环路、兴路区间3个地点的空气真菌进行取样,初步确定了哈尔滨市室外空气真菌的种类及其动态变化。试验共得到9 908个真菌菌落,经鉴定为22属(或群)49种。试验结果表明:室外空气真菌的优势菌属依次为枝孢属、链格孢、曲霉属、青霉属、附球菌属、木霉属和无孢菌群,分别占总菌落数的21.54%、15.16%1、4.70%、12.20%、11.35%、7.86%和7.80%;室外空气真菌的种类在一天中基本没有变化,但菌落的数量有一定变化,总菌落数从早至晚也逐渐增加;不同季节室外真菌的种类和菌落数有明显变化,秋季最多,冬季最少;不同采样地点的空气真菌的种类变化不大,但菌落数量有差异,菌落数量依次为公路林场家属区。     

Distribution,occurrence and natural invertebrate hosts of indigenous entomopathogenic fungi of Central India

We report here that, during periodical surveys of insects inhabiting diverse habitats for the collection of entomopathogenic fungi; a large number of isolates were recovered belonging to seven species, from various regions of Madhya Pradesh and Chhattisgarh forest areas and agricultural fields. The most common entomopathogenic fungi such as Beauveria bassiana, Nomuraea rileyi, Paecilomyces farinosus and Paecilomyces fumosoroseus were found to infect various insect hosts species naturally viz. Hyblaea puera, Eutectona machaeralis, Diachrysia orichalcea, Spodoptera litura, and few new insect hosts of these fungal pathogens among Indian insect population were collected for the first time from Central India, such as beetles of Agrilus species, hairy caterpillars of Lymantria species. The isolation, identification, maintenance and pathogenicity assay of these isolates was performed prior to deposition in culture collection center.     

Genetic characterization of Mortierella alpina by sequencing the 18S‐28S ribosomal gene internal transcribed spacer region

Aims: The aim of this study was to establish a genetic background of Mortierella alpina, which is a cosmopolitan, soil‐inhabiting Zygomycete that also has important biotechnology potential. Methods and Results: A total of 44 18S‐28S ribosomal gene internal transcribed spacer (ITS1‐5·8S rDNA‐ITS2 DNA) regions of M. alpina from three diverse locations (Far East Asia, North America and West‐Central Europe) were sequenced and investigated. The sequences between M. alpina and the three closely related species (Mortierella macrocystis, Mortierella gamsii and Mortierella humilis) showed 74–84% sequence identity. When a phylogenetic tree was constructed with a neighbour‐joining algorithm, four clades of M. alpina isolates were clearly distinct with high bootstrap values. In addition, isolates from the West‐Central Europe were found to have the highest gene and nucleotide diversities. Conclusions: The ITS region was a suitable tool for distinguishing M. alpina from other closely related species. The region also provided information of the diversity of M. alpina. Significance and Impact of the Study: The study established a genetic background of M. alpina for identification and the diversity of M. alpina provided information for further isolation and screening of the fungus.     

Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase

Ischnoderma resinosum produced extracellular ligninolytic enzymes laccase and MnP. The activity of laccase achieved the maximum on day 10 (29.4 U L⁻¹), the MnP on day 14 (34.5 U L⁻¹). Laccase and Mn-peroxidase were purified from the culture liquid using gel permeation and ion-exchange chromatographies. Purified Mn-peroxidase performed decolorization of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22, while all dyes were decolorized after addition of the redox mediators violuric acid (VA) and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and HBT. The highest decolorization rates were detected in acidic pH (3–4).     

Purification and mechanistic characterisation of two polygalacturonases from Sclerotium rolfsii

Sclerotium rolfsii (strain CBS 350.80) was found to produce extraordinary high amounts of polygalacturonases (PGs). Two of these extracellular enzymes were purified by a recently introduced preparative electrophoretic device (isoelectric focusing mode of free flow electrophoresis). PG 1 (39.5 kDa, pI 6.5) and PG 2 (38 kDa, pI 5.4) exhibited quite similar properties, they were found to be both endo-acting enzymes. Both PGs cleaved penta- and trigalacturonic acid while tetragalacturonic acid was only cleaved when trigalacturonic acid was present. The latter substrate was hydrolysed much faster by PG 2. Both enzymes were active on pectins with different degrees of esterification, they were sensitive towards Ca-cations and not glycosylated. The kinetic properties were measured by viscosimetry with polygalacturonic acid as a substrate. NMR experiments on a model substrate revealed an inverting mechanism of carbohydrate hydrolysis for both enzymes.     

Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.     

栎金钱菌活性提取物抗衰老研究

用栎金钱菌活性提取物灌胃衰老模型小鼠,观察其对小鼠血清SOD活性、肝组织MDA和脂褐素的含量、脾指数、胸腺指数和肾指数的影响。结果表明,栎金钱菌活性提取物能使雄性小鼠血清SOD活性提高40.16%,肝MDA含量减少47.12%,肝脂褐素含量减少89.48%,脾指数增加35.13%,胸腺指数增加48.92%,肾指数增加11.26%,表明栎金钱菌活性提取物具有良好的抗衰老作用。栎金钱菌活性提取物800mg/kg·d的剂量具有最好的抗衰老效果,且具有性别的差异,对雄性小鼠的抗衰老作用略优于对雌性小鼠的。     

Biodiversity of Fusarium species in Mexico associated with ear rot in maize, and their identification using a phylogenetic approach

Fusarium proliferatum, F. subglutinans, and F. verticillioides are known causes of ear and kernel rot in maize worldwide. In Mexico, only F. verticillioides and F. subglutinans, have been reported previously as causal agents of this disease. However, Fusarium isolates with different morphological characteristics to the species that are known to cause this disease were obtained in the Highland-Valley region of this country from symptomatic and symptomless ears of native and commercial maize genotypes. Moreover, while the morphological studies were not sufficient to identify the correct taxonomic position at the species level, analyses based in the Internal Transcribed Spacer region and the Nuclear Large Subunit Ribosomal partial sequences allowed for the identification of F. subglutinans, F. solani, and F. verticillioides, as well as four species (F. chlamydosporum, F. napiforme, F. poae, and F. pseudonygamai) that had not previously been reported to be associated with ear rot. In addition, F. napiforme and F. solani were absent from symptomless kernels. Phylogenetic analysis showed genetic changes in F. napiforme, and F. pseudonygamai isolates because they were not true clones, and probably constitute separate sibling species. The results of this study suggest that the biodiversity of Fusarium species involved in ear rot in Mexico is greater than that reported previously in other places in the world. This new knowledge will permit a better understanding of the relationship between all the species involved in ear rot disease and their relationship with maize.