Use of Native Promoter-eGFP as a Gene Reporter on Onion Epidermis to Analyze Gene Expression of AVR-Pia, an Avirulence Effector of Rice Blast Pathogen

Rice blast disease, caused by Magnaporthe oryzae, is a major rice disease over the world. Recent studies have identified avirulence effectors from the blast fungus that trigger rice immune against the pathogen invasion after specific interaction with resistance (R) proteins in rice. AVR-Pia is one of avirulence effectors that correspond to Pia-resistant protein, inducing hypersensitive response (HR). Enhanced Green fluorescent protein (eGFP) was used as a reporter of AVR-Pia expression in this study. We synthesized expression vector containing native promoter of AVR-Pia (PRR) fused to eGFP gene. Rice sheath assay was done to observe the fluorescence and the signal was found in appressoria and invasive hypha of M. oryzae, suggesting that AVR-Pia is expressed in appressorium and invasive hyphae after penetration. Although, rice sheath assay is a reliable way to study rice-pathogen interaction, it is a consuming-time method. Onion epidermis was tested to check the availability to use as model system instead of rice sheath. After inoculation M. oryzae containing PPR::eGFP on onion epidermis, fluorescence was observed in appressoria and invasive hypha of transformants similar to observation on rice sheath. Therefore, onion epidermis can be used as plant cell model to study M. oryzae effectors expression by fluorescence observation

Biodesulfurization of the mixture of dibenzothiophene and its alkylated derivatives by Sphingomonas subarctica T7b

Organosulfur compounds classified as dibenzothiophenes (DBTs) and their derivatives are contained in petroleum. When used as fuel, these substances release SOx emissions, thus contributing to air pollution, acid rain, and climate change. Therefore, it is necessary to reduce the content of these organic sulfur compounds in fuels and one way to achieve this is through bacterial desulfurization. This study reports the biodesulfurization process of a mixture of DBT, 4-hexyl DBT, 4,6-dibutyl DBT, and various organosulfur compounds in light gas oil (LGO). The experiment was conducted by treating 1 mL of aromatic organosulfur compounds with 100 mg/L in \textit{n}-tetradecane or 1 mL LGO with 5 mL mineral salts in sulfur-free medium, incubated at 27 °C for 5 days with shaking at 273 rpm. Gas chromatography analyses revealed that the growing Sphingomonas subarctica T7b cells desulfurized and converted 88.29% of DBT to 2-hydroxybiphenyl as a metabolite while a mixture of DBT and 4,6-dibutyl DBT was desulfurized at 86.40\% and 7.00%, respectively. Furthermore, the mixture of DBT, 4-hexyl DBT, and 4,6-dibutyl DBT had a desulfurization percentage of 84.40%, 41.00%, and 6.66%, respectively, after five days of incubation. The compounds were observed to desulfurize slightly better as single compounds compared to when mixed with other aromatic sulfur compounds

Burkholderia insecticola sp. nov., a gut symbiotic bacterium of the bean bug Riptortus pedestris

A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64(T), was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei Glade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314(T) (100%), Burkholderia turbans LMG 29316(T) (99.52%) and Burkholderia ptereochthonis LMG 29326(T) (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome BLAST Distance Phylogeny confirmed B. peredens LMG 29314(T), B. ptereochthonis LMG 29326(T) and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA-DNA hybridization experiments unambiguously demonstrated that strain RPE64(T) represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C-16:0, C-18:1 omega 7c and C-17:0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64(T) from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64(T) is classified as representing a novel species of the genus Burkholderia, for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64(T) (=NCIMB 15023(T)=JCM 31142(T))

Microflora and Selected Metabolites of Potato Pulp Fermented with an Indonesian Starter Ragi Tapé

When potato pulp was mixed with Indonesian starter ragi tapé and incubated, both lactic acid and ethanol were gradually formed and attained certain concentrations during 2 days of fermentation. Viable counts of fungi in fresh weight matter, yeasts and lactic acid bacteria after fermentation were 105, 107 and 105 CFU/g, respectively. Denaturing gradient gel electrophoresis of the PCR-amplified internal transcribed spacer of 18S–28S rRNA genes detected Amylomyces rouxii-Rhizopus oryzae, Mucor indicus, Candida tropicalis and Saccharomycopsis fibuligera and revealed that Amylomyces rouxii-Rhizopus oryzae dominated throughout the fermentation period. Amylomyces rouxii cannot be discriminated from the lactic acid-accumulating group of Rhizopus oryzae because the amplified sequences of these fungi were shown to be identical. Morphological characteristics were then studied for Rhizopus-like fungi isolated from fermented potato pulp. Those strains that had produced an enormous number of chlamydospores in the aerial and substrate mycelium were identified as Amylomyces rouxii. The microflora of fermented potato pulp was similar to that made from glutinous rice, namely tapé ketan

Pengaruh Sumber Karbon Terhadap Produksi Enzim Inulinfruktotransferase Dari Nonomuraea SP

The aims of this research is to produce inulinfructotransferase enzyme with potentially benefit of bacteria Actinomycetes which was isolated from Indonesia soils to produce Difructoanhydride-Ill (DFA III), a compound has physiology activities to enhance calsium absorption of rats. cows and human colons so that the materials can be applied as antiosteoporosis. The processing of inulinfructotransferase enzyme from Nonomuraea sp by usingshakeflask level and study the influence of carbohydrate source were conducted. This enzyme has optimum temperature at 65rtC,higher than optimum temperature which has found before and the activities was stable at 70dc for 20 minutes. So that the enzyme has potentially applicable for industry scale. The results indicated that optimation condition for processing inulinfructotransferase enzyme of Nonomuraea sp : temperature sore. incubation time 48 hours.carbon source of inulin 20 g/L, nitrogen source 5g/L, noyeast extract added, the enzyme activities was 7,3 Unit/ml; However,when medium contains yeast extract 0,2 gIL,the inulinfructotransferase enzyme activity was 14,6 Unit/mL

Insight into the population dynamics of pathogenic bacteria causing grapevine crown gall in snowfall areas: snow cover protects the proliferation of pathogenic bacteria

Grapevine crown gall (GCG) is a significant bacterial disease caused by tumorigenic Allorhizobium vitis (TAV) and is prevalent worldwide. TAV infects grapevines through wounds such as freezing injuries. Although grapevines typically avoid being wounded under snow cover, GCG occurs in many commercial vineyards in snowy regions. This study investigated the TAV population in GCG gall tissues, grapevine skins, and snow on grapevine skins from six infected vineyards located in Hokkaido, Japan, an area known for heavy snowfall. TAV was isolated not only from gall tissues but also from skins and snow on skins throughout the year. Hierarchical Bayesian model (HBM) analysis revealed that the number of TAV cells in gall tissues was affected by cultivar and low temperature, while those in skins were affected by location and low temperature. Additionally, Bayesian changepoint detection (BCD) showed that the number of TAV cells in gall and skin tissues increased during winter, including the snowfall season. Furthermore, the TAV population in grapevine skins under the snow was significantly higher than those above the snow, indicating that TAV under the snow is protected by the snow and can survive well during the snowfall season. This study highlights the ability of TAV to overwinter on/in galls and skins under the snow and act as inoculum for the next season

Genomic Analysis of the Basal Lineage Fungus Rhizopus oryzae Reveals a Whole-Genome Duplication

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called “zygomycetes,” R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99–880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin–proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14α-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments

Construction of pDESTR, a GATEWAY Vector for Gene Disruption in Filamentous Fungi

We have constructed pDESTR, a destination vector of gateway system especially for gene targeting and disruption in filamentous fungi. The vector was constructed by removing the multicloning site of pGEM-T easy vector, and inserting hygromycin phosphotransferase gene construct from pCB1004, and a gateway vector conversion cassette. In order to construct a DNA for gene disruption, only an inverse-polymerase chain reaction (PCR) amplification of the restricted, target sequence is needed. After the amplification with a 5′CACC-tagged primer and an ordinary primer, the DNA fragment will be inserted into pENTR/D-TOPO vector and then transferred into pDESTR through LR-recombination reaction. The resulting vector has the disruption construct, after being digested with the restriction enzyme used for the inverse-PCR. The effectiveness of this vector was assessed in Neurospora crassa. The use of pDESTR will therefore simplify the construction of a targeting vector, where multiple ligation steps are usually neede

Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers

The zygomycete Rhizopus oryzae currently is identified by sporangiophore morphology and growth temperature, but heterogeneity of the species has been reported. We examined the suitability of organic acid production as an effective taxonomic character for reclassification of the species. Strains were divided into two groups, LA (lactic acid producer) and FMA (fumaric-malic acid producers) according to organic acid production. These grouping were confirmed as phylogenetically distinct because analyses of rDNA ITS, lactate dehydrogenase B, actin, translation elongation factor-1α and genomewide AFLP resolved the same two exclusive clusters, corresponding with the organic acid grouping. Reclassification of strains in the FMA group as R. delemar was proposed

The molecular phylogeny of the genus Rhizopus based on rDNA sequences

In order to establish the molecular phylogeny of the genus Rhizopus, three molecules of the ribosomal RNA-encoding DNA (rDNA), complete 18S, internal transcribed spacer (ITS)1-5.8S-ITS2, and 28S D1/D2 regions of all the species of the genus were sequenced. Phylogenetic trees showed three major clusters corresponding to the three groups in the current morphological taxonomy, microsporus-group, stolonifer-group, and R. oryzae. R. stolonifer var. lyococcos was clustered independently from the major clusters. R. schipperae clustered differently in all trees. Strains of R. sexualis had multiple ITS sequences. A. rouxii clustered with R. oryzae. These results indicate the possibility of molecular identification of species groups using rDNA sequencing. Reclassification of the genus might be appropriate