ß-Aminobutyric Acid and Powdery Mildew Infection Enhanced the Activation of Defense-Related Genes and Salicylic Acid in Cucumber (Cucumis sativus L.).

Powdery mildew disease, caused by Sphaerotheca fusca, is a major disease affecting cucumbers cultivated in greenhouses. This study was conducted to find defense genes induced by ß-aminobutyric acid (BABA) and powdery mildew in cucumber. Disease severities of 25% and 5% were exhibited by the 2000 and 5000 mg/L BABA-treated cucumber, respectively. BABA did not affect the spore germination of the powdery mildew pathogen, showing that BABA is not an antifungal agent against the pathogen. In quantitative real-time PCR analysis, BABA-treated cucumber upregulated the transcriptional levels of the defense genes CsPAL, CsPR3, CsPR1, CsLOX1, CsLOX23, Cs LecRK6.1, CsWRKY20, and Cupi4 in cucumber to maximum levels at 48 h, whereas CsLecRK6.1 reached maximum expression after 24 h, and further, salicylic acid (SA) levels were significantly increased in BABA-treated cucumber plants. In addition, the cucumber infected with powdery mildew underwent a 1.6- to 47.3-fold enhancement in the defense genes PAL, PR3, PR1, Lox1, Lox 23, LecRK6.1, WRKY20, and Cupi4 compared to heathy cucumber. These results suggest that the BABA-induced defense response is associated with SA signaling pathway-dependent systemic acquired resistance (SAR) in cucumber, which is involved in plant resistance mechanisms.

Artificial Cultivation Characteristics and Bioactive Effects of Novel Tropicoporus linteus (Syn. Phellinus linteus) Strains HN00K9 and HN6036 in Korea.

Phellinus strains were collected from different areas in Korea. Of them, the fast mycelial growing strains were artificially cultivated on the oak logs to produce fruiting body. The varieties, Phellinus linteus ASI26099 (Korea Sanghwang) and P. baumii PBJS (Jangsoo Sanghwang) were grown under the same conditions as controls. Their cultivating characteristics including mycelial colonization, pinhead formation, and fruiting body formation rate were investigated on the logs. Basidiocarps of Phellinus strains HN00K9, HN6036, and ASI26099 were concentrically zonate and shallowly sulcate, and dark chestnut showing typical characteristics of Tropicoporus linteus (synonyum: P. linteus, Inonotus linteus, polyporus linteus), which is distinguishably different to PBJS. HN00K9 showed the highest yield of fruiting body among the mushroom strains. The ß-glucan content in fruiting bodies of HN00K9 was 20% higher than those of other strains. Bioactive effects of polysaccharide samples from fruiting bodies of Phellinus strains, HN00K9, HN6036, ASI26099, and PBJS were assessed on cell viability and cytokine (IL-6 and TNF-α) inhibition and finally on anticancer to different human cancer cells.

Defense Response and Suppression of Phytophthora Blight Disease of Pepper by Water Extract from Spent Mushroom Substrate of Lentinula edodes.

The spent mushroom substrate (SMS) of Lentinula edodes that was derived from sawdust bag cultivation was used as materials for controlling Phytophthora blight disease of pepper. Water extract from SMS (WESMS) of L. edodes inhibited mycelial growth of Phytophthora capsici, suppressed Phytophthora blight disease of pepper seedlings by 65% and promoted growth of the plant over 30%. In high performance liquid chromatography (HPLC) analysis, oxalic acid was detected as the main organic acid compound in WESMS and inhibited the fungal mycelium at a minimum concentration of 200 mg/l. In quantitative real-time PCR, the transcriptional expression of CaBPR1 (PR protein 1), CaBGLU (ß-1,3-glucanase), CaPR-4 (PR protein 4), and CaPR-10 (PR protein 10) were significantly enhanced on WESMS and DL-ß-aminobutyric acid (BABA) treated pepper leaves. In addition, the salicylic acid content was also increased 4 to 6 folds in the WESMS and BABA treated pepper leaves compared to water treated leaf sample. These findings suggest that WESMS of L. edodes suppress Phytophthora blight disease of pepper through multiple effects including antifungal activity, plant growth promotion, and defense gene induction.

Isolation and identification of phytochemicals and biological activities of Hericium ernaceus and their contents in Hericium strains using HPLC/UV analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Hericium ernaceus has been traditionally used for the treatment of dyspepsia, gastric ulcer and enervation in traditional Chinese medicine for a long time. AIM OF THE STUDY: To examine the effect of Hericium strains on their ability to inhibit LPS and interferon-γ induced NO production in cell culture and the bioassay correlation of hericenone C, D, F, isolated from H. ernaceus. MATERIALS AND METHODS: Hericenone C, D, F were isolated from H. ernaceus by open column chromatography and identified on the basis of spectroscopic analyses including (1)H NMR, (13)C NMR and MS. The amounts of hericenone C, D, and F in Hericium strains were determined by HPLC/UV analysis. In order to investigate the anti-inflammatory effect of Hericium strains extracts, RAW 264.7 cells were treated with 200µg/mL of Hericium strains extracts for 48h. Cell growth was assessed by MTT assay. RESULTS: Phytochemical constituents were isolated from H. ernaceus by open column chromatography. Their structures were elucidated as hericenones C, D, and F on the basis of spectroscopic analyses including (1)H NMR, (13)C NMR and MS. The amounts of hericenones C, D, and F in Hericium strains were determined by HPLC/UV analysis. Hericenones C, D, and F contents were highest in Norugungdenglee-2 (8.289±0.593mg/g), KFRI-1453 (4.657±0.462mg/g), and KFRI-1093 (5.408±0.420mg/g) strains, respectively. All Hericium strains extracts tested inhibited the lipopolysaccharide- and interferon-γ-induced inflammatory activity of RAW264.7 cells. The strain KFRI-1093 about 39.6% reduced NO generation with compared to control. CONCLUSION: We believe that the anti-inflammatory effect of KFRI-1093 was due to hericenone F content. Our results contribute towards validation of the traditional use, natural drugs and health supplements. And also, the developed simple, accurate and rapid LC method can be used determinate the content of hericenones from other Hericium strains.

Oxalic Acid from Lentinula edodes Culture Filtrate: Antimicrobial Activity on Phytopathogenic Bacteria and Qualitative and Quantitative Analyses.

The culture filtrate of Lentinula edodes shows potent antimicrobial activity against the plant pathogenic bacteria Ralstonia solanacearum. Bioassay-guided fractionation was conducted using Diaion HP-20 column chromatography, and the insoluble active compound was not adsorbed on the resin. Further fractionation by high-performance liquid chromatography (HPLC) suggested that the active compounds were organic acids. Nine organic acids were detected in the culture filtrate of L. edodes; oxalic acid was the major component and exhibited antibacterial activity against nine different phytopathogenic bacteria. Quantitative analysis by HPLC revealed that the content of oxalic acid was higher in the water extract from spent mushroom substrate than in liquid culture. This suggests that the water extract of spent L. edodes substrate is an eco-friendly control agent for plant diseases.

Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato.

Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding ß-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction.

Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.

Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model.

Cloning and Characterization of a Novel Laccase Gene, fvlac7, Based on the Genomic Sequence of Flammulina velutipes.

Laccases (EC 1.10.3.2) are copper-containing polyphenol oxidases found in white-rot fungi. Here, we report the cloning and analysis of the nucleotide sequence of a new laccase gene, fvlac7, based on the genomic sequence of Flammulina velutipes. A primer set was designed from the putative mRNA that was aligned to the genomic DNA of F. velutipes. A cDNA fragment approximately 1.6-kb long was then amplified by reverse transcriptase-PCR using total RNA, which was subsequently cloned and sequenced. The cDNA sequence of fvlac7 was then compared to that of the genomic DNA, and 16 introns were found in the genomic DNA sequence. The fvlac7 protein, which consists of 538 amino acids, showed only 42~51% identity with 12 different mushroom species containing two laccases of F. velutipes, suggesting the fvlac7 is a novel laccase gene. The first 25 amino acids of Fvlac7 correspond to a predicted signal sequence, four copper-binding sites, and four N-glycosylation sites. Fvlac7 cDNA was heterologously overexpressed in an Escherichia coli system with an approximate expected molecular weight of 60 kDa.

Efficient Recovery of Lignocellulolytic Enzymes of Spent Mushroom Compost from Oyster Mushrooms, Pleurotus spp., and Potential Use in Dye Decolorization.

This study was conducted in order to perform efficient extraction of lignocellulolytic enzymes amylase (EC 3.2.1.1), cellulase (EC 3.2.1.4), laccase (EC 1.10.3.2), and xylanase (EC 3.2.1.8) from spent mushroom compost (SMC) of Pleurotus ostreatus, P. eryngii, and P. cornucopiae. Optimal enzyme recovery was achieved when SMCs were extracted with 50 mM sodium citrate (pH 4.5) buffer at 4℃ for 2 hr. Amylase, cellulase, and xylanase activities showed high values in extracts from P. ostreatus SMC, with 2.97 U/g, 1.67 U/g, and 91.56 U/g, respectively, whereas laccase activity and filter paper degradation ability were highest in extracts from P. eryngii SMC, with values of 9.01 U/g and 0.21 U/g, respectively. Enzymatic activities varied according to the SMCs released from different mushroom farms. The synthetic dyes remazol brilliant blue R and Congo red were decolorized completely by the SMC extract of P. eryngii within 120 min, and the decolorization ability of the extract was comparable to that of 0.3 U of commercial laccase. In addition, laccase activity of the SMC extract from P. eryngii was compared to that of commercial enzymes or its industrial application in decolorization.

Electrophoretic karyotyping and construction of a bacterial artificial chromosome library of the winter mushroom Flammulina velutipes.

An electrophoretic karyotype of Korean Flammulina velutipes was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The monokaryotic progenies (4019-20 and 4019-18) and a dikaryotic strain (4019-2018) had 6-8 chromosomes, with sizes ranging from 1.6 to 5.8 megabase (Mb) pairs. Among the 3 strains that were examined, strain 4019-20 resolved into at least 7 chromosomes, with a total genome size of approximately 26.7Mb. We selected several chromosome-specific genes from the cDNA library of F. velutipes using Southern hybridization analysis. In order to determine the whole genome sequence, we constructed a bacterial artificial chromosome (BAC) library of the 4019-20 strain. The BAC library comprised 3840 clones, and the insert size ranged from 60 to 228kb, with an average size of 136kb.

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes.

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/µg of DNA in 1 × 10(7) protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

Phellinins A1 and A2, new styrylpyrones from the culture broth of Phellinus sp. KACC93057P: I. Fermentation, taxonomy, isolation and biological properties.

Novel styrylpyrones, phellinins A1 and A2, were isolated together with known styrylpyrone compounds, hispidin and 1,1-distyrylpyrylethan, from the cultured broth of Phellinus sp. KACC93057P. These compounds were purified by solvent partition, Sephadex LH-20 column chromatography, C(18)-solid phase extraction and finally by reversed-phase (ODS) TLC. To identify the phellinin producer Phellinus sp. KACC93057P, the ribosomal DNA (rDNA) internal transcribed space regions containing 5.8 rDNA were sequenced and compared with those of the known Phellinus isolates. Phellinus sp. KACC93057P was 94.8% identical to P. baumii and P. linteus, all of which did not produce phellinins A1 and A2. These compounds significantly scavenged free radicals such as 1,1-diphenyl-2-picrylhydrazyl, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and superoxide.

Virulence analysis and gene expression profiling of the pigment-deficient mutant of Xanthomonas oryzae pathovar oryzae.

Xanthomonas oryzae pathovar oryzae (Xoo) causes bacterial blight disease in rice (Oryza sativa L.). For a study of function, we constructed a random insertion mutant library of Xoo using a Tn5 transposon and isolated the mutant strain (M11; aroK::Tn5) that had extremely low pigment production. In addition, M11 had decreased virulence against the susceptible rice cultivar IR24. Thermal asymmetric interlaced-PCR and sequence analysis of M11 revealed that the transposon was inserted into the aroK gene (which encodes a shikimate kinase). To investigate the expression patterns of the pigment- and virulence-deficient mutant, DNA microarray analysis was performed. In addition, reverse transcriptase-PCR was performed to confirm the expression levels of several genes, including the aro genes of the aroK mutant. Our findings reveal that several crucial genes for virulence, including cellulase and hypersensitive response and pathogenicity (hrp) genes, were regulated by mutations in the aroK gene.

Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains.

An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5x10(2) and 1x10(3)cfu/ml and the detection sensitivity was increased to as few as 2cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls.

Expression, crystallization and preliminary X-ray crystallographic analysis of XometC, a cystathionine gamma-lyase-like protein from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice (Oryza sativa L.), one of the most devastating diseases of rice in most rice-growing countries. XometC, a cystathionine gamma-lyase (CGL) like protein that is an antibacterial drug-target protein against Xoo, was cloned, expressed, purified and crystallized. CGL catalyzes the second step in the reverse-transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine. Crystals of two different shapes, plate-shaped and pyramid-shaped, diffracted to 2.9 and 3.2 A resolution and belonged to the primitive orthogonal space group P2(1)2(1)2(1) and the tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = 73.0, b = 144.9, c = 152.3 A and a = b = 78.2, c = 300.7 A, respectively. For the P2(1)2(1)2(1) crystals, three or four monomers exist in the asymmetric unit with a corresponding V(M) of 3.02 or 2.26 A(3) Da(-1) and a solvent content of 59.3 or 45.7%. For the P4(1) (or P4(3)) crystals, four or five monomers exist in the asymmetric unit with a corresponding V(M) of 2.59 or 2.09 A(3) Da(-1) and a solvent content of 52.5 or 40.6%.

Functional analysis of pilQ gene in Xanthomonas oryzae pv. oryzae, bacterial blight pathogen of rice.

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease in rice. A virulence-attenuated mutant strain HNU89K9 of X. oryzae pv. oryzae (KACC10331), with a transposon insertion in the pilQ gene was used for this study. The pilQ was involved in the gene cluster pilMNOPQ of the Xoo genome. Growth rate of the pilQ mutant was similar to that of wild-type. At level of amino acids, PilQ of Xoo showed that a high sequence identities more than 94% and 70% to Xanthomonas species and to Xyllela fastidiosa, respectively but a low sequence homology less than 30% to other bacterial species. The twitching motility forming a marginal fringe on PSA media was observed on colony of the wild-type strain KACC10331, but not in mutant HNU89K9. Wild-type Xoo cells formed a biofilm on the surface of the PVC plastic test tube, while the mutant strain HNU89K9 did not form a biofilm. The results suggest that the pilQ gene of X. oryzae pv. oryzae plays a critical role in pathogenicity, twitching motility, and biofilm formation.

Overexpression of the pineapple fruit bromelain gene (BAA) in transgenic Chinese cabbage (Brassica rapa) results in enhanced resistance to bacterial soft rot

Bromelain is a crude protein extract obtained from pineapple stems, which comprises a variety of proteolytic enzymes. It exhibits potential therapeutic activities against trauma, inflammation, autoimmune diseases and malignant disorders. In this study, we cloned BAA1 (the gene encoding fruit bromelain) into a plant expression vector that was then used to transform Brassica rapa and overexpress BAA1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. We demonstrate that constitutive overexpression of BAA1 in B. rapa confers enhanced resistance to the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum. These results suggest that it could be utilized for protecting plants from attack by bacterial pathogens.

Analysis of virulence and growth of a purine auxotrophic mutant of Xanthomonas oryzae pathovar oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice. A random insertional mutant library of Xoo KACC10331 was constructed using a Tn5-derived transposon, and the virulence of the mutants against the susceptible rice cultivar IR24 was assayed. After the virulence assay, the M793 (purD::Tn5) mutant that had reduced virulence against the rice plants was isolated. Thermal asymmetric interlaced-PCR and sequence analysis revealed that the transposon was inserted into the purD gene (encodes a phosphoribosylamine-glycine ligase) of the M793 mutant. The reverse transcriptase-PCR assay revealed that the mutation of the purD gene did not affect the expression of other purine biosynthesis genes. However, the M793 mutant required exogenous purines and thiamine for growth in minimal media. These results indicate that the purD gene plays a crucial role in the growth and virulence of Xoo.

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences.

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice.

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.