Isolation and Electrochemical Analysis of a Facultative Anaerobic Electrogenic Strain Klebsiella sp. SQ-1.

Electricigens decompose organic matter and convert stored chemical energy into electrical energy through extracellular electron transfer. They are significant biocatalysts for microbial fuel cells with practical applications in green energy generation, effluent treatment, and bioremediation. A facultative anaerobic electrogenic strain SQ-1 is isolated from sludge in a biotechnology factory. The strain SQ-1 is a close relative of Klebsiella variicola. Multilayered biofilms form on the surface of a carbon electrode after the isolated bacteria are inoculated into a microbial fuel cell device. This strain produces high current densities of 625 µA cm-2 by using acetate as the carbon source in a three-electrode configuration. The electricity generation performance is also analyzed in a dual-chamber microbial fuel cell. It reaches a maximum power density of 560 mW m-2 when the corresponding output voltage is 0.59 V. The facultative strain SQ-1 utilizes hydrous ferric oxide as an electron acceptor to perform extracellular electricigenic respiration in anaerobic conditions. Since facultative strains possess better properties than anaerobic strains, Klebsiella sp. SQ-1 may be a promising exoelectrogenic strain for applications in microbial electrochemistry.

The Complete Genome Sequence Resource of Rhizospheric Soil-Derived Bacillus velezensis Yao, with Biocontrol Potential Against Fusarium solani-Induced Pepper Root Rot.

The pepper rhizospheric soil-derived Bacillus velezensis Yao from the Shangqiu region of the Henan province in China possesses antagonistic activity against Fusarium solani, which causes pepper root rot. In this report, we introduced the entire genomic sequence of B. velezensis Yao, which is 3,951,864 bp long, with 46.61% G+C content, and 4,097 genes. Using antiSMASH analysis, we predicted 12 gene clusters that encode for secondary antimicrobial metabolites and multiple genes that regulate plant bacterial interactions. The B. velezensis Yao genome data may be a valuable resource as this strain may serve as an effective biocontrol agent against pepper root rot.

Chrysanthemum morifolium ß-carotene hydroxylase overexpression promotes Arabidopsis thaliana tolerance to high light stress.

The ß-carotene hydroxylase gene (BCH) regulates zeaxanthin production in response to high light levels ro protect Chrysanthemum morifolium plants against light-induced damage. In this study, the Chrysanthemum morifolium CmBCH1 and CmBCH2 genes were cloned and their functional importance was assessed by overexpressing them in Arabidopsis thaliana. These transgenic plants were evaluated for gene-related changes in phenotypic characteristics, photosynthetic activity, fluorescence properties, carotenoid biosynthesis, aboveground/belowground biomass, pigment content, and the expression of light-regulated genes under conditions of high light stress relative to wild-type (WT) plants. When exposed to high light stress, WT A. thaliana leaves turned yellow and the overall biomass was reduced compared to that of the transgenic plants. WT plants exposed to high light stress also exhibited significant reductions in the net photosynthetic rate, stomatal conductance, Fv/Fm, qP, and ETR, whereas these changes were not observed in the transgenic CmBCH1 and CmBCH2 plants. Lutein and zaxanthin levels were significantly increased in the transgenic CmBCH1 and CmBCH2 lines, with progressive induction with prolonged light exposure, whereas no significant changes were observed in light-exposed WT plants. The transgenic plants also expressed higher levels of most carotenoid biosynthesis pathway genes, including phytoene synthase (AtPSY), phytoene desaturase (AtPDS), lycopene-ß-cyclase (AtLYCB), and ζ-carotene desaturase (AtZDS). The elongated hypocotyl 5 (HY5) and succinate dehydrogenase (SDH) genes were significantly induced following exposure to high light conditions for 12h, whereas phytochrome-interacting factor 7 (PIF7) was significantly downregulated in these plants.

nifH gene expression and diversity in geothermal springs of Tengchong, China.

Terrestrial hot springs have been suggested to harbor diverse diazotrophic lineages by using DNA-based nifH gene phylogenetic analysis. However, only a small amount of diazotrophs were ever confirmed to perform nitrogen fixation. In order to explore the compositions of active diazotrophic populations in hot springs, the in situ expression and diversity of nifH and 16S rRNA genes were investigated in the sediments of hot springs (pH 4.3-9.1; temperature 34-84°C) in Tengchong, China, by using high-throughput sequencing. The results showed that active diazotrophs were diverse in the studied Tengchong hot springs. The main active diazotrophs in high-temperature hot springs were affiliated with Aquificae, while those in low-temperature hot springs belonged to Cyanobacteria and Nitrospirae. Such dominance of Aquificae and Nitrospirae of diazotrophs has not been reported in other ecosystems. This suggests that hot springs may harbor unique active diazotrophs in comparison with other type of ecosystems. Furthermore, there were significant differences in the phylogenetic lineages of diazotrophs between hot springs of Tengchong and other regions, indicating that diazotrophs have geographical distribution patterns. Statistical analysis suggests that the expression and distribution of nifH gene were influenced by temperature and concentrations of ammonia and sulfur seem in Tengchong hot springs. These findings avail us to understand element cycling mediated by diazotrophs in hot spring ecosystems.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Glandularia tenera in China.

Glandularia tenera (syn. Verbena tenera) is an herbaceous perennial ornamental plant used in gardens as an edging plant with beautiful white, red, or purple flowers. In autumn 2020 and 2021, severe powdery mildew infection was observed on G. tenera cultivar Xianghe in Renming Botanical garden in Shangqiu, Henan province, China (34.4568° N, 115.6640° E). Approximately 80% of leaves on each plant were symptomatic, and about 90% of the plants were infected. Powdery mildew colonies appeared as white spots on the adaxial surface of the leaves and stems of the plants in the initial infection stage. Later, mycelial growth was amphigenous, thick, forming irregular white patches, and effused to cover the whole leaf surface. Finally, leaves turned yellow and senescence occurred. Samples of symptomatic leaves were stained with trypan blue and examined under a Leica DM2500 microscope. Microscopic observations showed that conidia on infected leaves were hyaline and ellipsoid to oval with fibrosin bodies, measured 25 to 37 × 14 to 23 µm with a length/width ratio of 1.4 to 2.0. Conidiophores were unbranched, straight, 80 to 210× 10 to 14 µm in size, and produced two to five immature conidia in chains. Foot cells of conidiophores were cylindrical with slight constrictions at basal septa, and followed by one to three short cells. Fungal hyphae were septate, branched, and flexuous to straight and 4 to 7µm wide with indistinct to slightly nipple-shaped appressoria. Chasmothecia were not observed. These morphological characteristics were identical with the previous description of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012). To confirm the identification, the sequence of ITS1-5.8s-ITS2 region of rDNA for the isolate SQVT was amplified from conidia collected from infected leaves with universal primers ITS1 and ITS4, sequenced and analyzed using the BLASTn search of GenBank. Amplicon was 565 bp (OM293967) and showed 99.82% similarity with sequence of P. xanthii from Eclipta prostrate (MT260063) in China (Xu et al. 2020), from Youngia denticulate (AB040351) in Japan (Hirata et al. 2000), and 99.65% with sequence of P. xanthii from V. brasiliensis in Korea (Cho et al. 2014). The domains D1 and D2 of the 28S rDNA for the isolate SQVTPX-1 was amplified with primer NL1/NL4. Amplicon was 613 bp (ON259308) and showed 100% similarity with sequence of P. xanthii from V. brasiliensis (AB936277) (Meeboon and Takamatsu, 2015). Pathogenicity tests were conducted by gently pressing the infected leaves onto leaves of five healthy G. tenera cultivar Xianghe plants. Five non-inoculated plants served as controls. Plants were maintained in a greenhouse at 25 ± 2°C. Eight days after inoculation, symptoms similar to those observed under natural conditions developed on the inoculated leaves of G. tenera plants, whereas the control plants remained symptomless. The fungus on inoculated leaves was morphologically identical to that first observed in the field. P. xanthii is a cosmopolitan powdery mildew fungus, parasitic on numerous plant species, especially Cucurbitaceae and Compositae plants. The pathogen has been reported infecting V. bonariensis (Hong et al. 2021), V. × hybrida in China (Zhuang 2005), and V. brasiliensis in Korea (Cho et al. 2014). Interestingly, G. tenera plants infected by P. xanthii were adjacent with V. × hybrida plants infected by P. xanthii in Renming Botanical garden. Incidence of P. xanthii on G. tenera add information on pathogen's host range and help us develop comprehensive survey and effective management of the disease. To our knowledge, this is the first report of P. xanthii on G. tenera in China (Farr and Rossman 2021). Braun, U., and Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series No. 11. CBS, Utrecht, the Netherlands. Cho, S. E., et al. 2014. Plant Dis. 98: 1159. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases, Syst. Mycol. Microbiol. Lab., ARS, USDA. Hong, Q. Q., et al. 2021. Plant Dis. 105: 3297. Hirata, T., et al. 2000. Can. J. Bot. 78: 1521-1530. Meeboon, J. and Takamatsu, S. 2015. Mycoscience . 56: 243-251. Xu, D. D., et al. 2020. Plant Dis. 104: 3263. Zhuang, W. Y. 2005. Fungi of northwestern China. Mycotaxon, Ltd., Ithaca, NY.

First report of powdery mildew caused by Erysiphe euonymicola on wintercreeper (Euonymus fortunei) in China.

Wintercreeper (Euonymus fortunei) is an evergreen shrub, a semi climbing plants with very strong vitality. It can tolerate a broad range of environmental conditions varying from full sun to deep shade. So, wintercreeper is a good groundcover plants and tree species of vertical greening. It is widely used in urban greening in China. In May 2021, severe powdery mildew signs were found on wintercreeper plant in a garden of Luohe cities of Henan province, China. Approximately 40% of leaves on a plant were symptomatic, and about 60% of the plants were infected. Powdery mildew colonies appeared as white spots on the upper surface of the leaf and stem of the plants in the initial stage. Later, mycelial growth was amphigenous, thick, forming irregular white patches, effused to cover the entire leaf surface. At last, leaves turned yellow and senescence. One representative voucher specimen was deposited at the herbarium of Shangqiu Normal University (SQNU), Shangqiu, China, under the accession number of EF02. Conidiophores arising terminally from the mother cell, mostly central, erect, straight, 40.7 to 67.7µm (average 49.1µm) 6.1 to 8.5µm (average 7.5µm) (n=30) composed of 3 to 4 cells and produced conidia singly. Conidia were obovoid-ellipsoid, apex rounded, base subtruncate, ends truncate or subtruncate, long 26.1 to 36.4µm (average 30.7µm) and width 9.9 to 16.0µm (average 11.8µm) (n=30). No chasmothecia were observed. The morphological characteristics and measurements were consistent with those of Erysiphe euonymicola (Braun and Cook 2012). The internal transcribed spacer regions of EF02 were amplified using primers ITS1/ITS4, and sequences directly. The obtained sequence of 648 bp was deposited in GenBank (accession no. OM304857). The isolate (EF02) was 99.83% similarity with E. euonymicola on E. fortunei var. radicans (KM361621 from Korea) (Lee et al. 2015), 99.23% with E. euonymicola on E. fortunei (MT510003 from USA), 99.23% with E. euonymicola on E. japonicus (LC270841and LC270834 from Azerbaijan, AB250228 from Japan ). The domains D1 and D2 of the 28S rDNA obtained 619 bp sequences from the powdery mildew of isolate EF02 with primer NL1/NL4, deposited in GenBank (OM302187). A NCBI BLAST search of the EF02 isolate showed 100% similarity with 28S rDNA sequence of E. euonymicola on E. japonicus (AB250230 from Japan) (Limkaisang et al. 2006), and 28S phylogenetic tree analysis EF02 isolate is located in the same branch as E. euonymicola. The pathogenicity was confirmed by gently pressing the infected leaves onto five healthy plants. Five uninoculated plants served as controls. Ten inoculated and non-inoculated plants were placed in different growth chambers with 14-h photoperiod at 22±2°C and 60% of relative humidity. After 11 to 13 days, powdery mildew colonies developed on inoculated plants. Non-inoculated control plants did not show powdery mildew symptoms. The fungus on inoculated leaves was morphologically identical to that first observed in the field. Although E. euonymi-japonici (synonym E. euonymicola) has been recorded on E. japonicus in China (Li et al. 2011), this is the first report of powdery mildew caused by E. euonymicola on E. fortunei in China. It could become a threat to the widespread planting of wintercreeper, Similar report has been in Korea (Lee, C. K., et al. 2015).

Biological Control of Verticillium Wilt and Growth Promotion in Tomato by Rhizospheric Soil-Derived Bacillus amyloliquefaciens Oj-2.16.

Verticillium wilt disease caused by Verticillium dahliae seriously affects tomato quality and yield. In this work, strain Oj-2.16 was isolated from rhizosphere soil of the medicinal plant Ophiopogon japonicas and identified as Bacillus amyloliquefaciens on the basis of morphological, physiological, and biochemical characteristics and 16S rDNA sequencing. Strain Oj-2.16 exhibited a high inhibition rate against V. dahliae, and the hyphae inhibited by Oj-2.16 were found to be destroyed on scanning electron microscopy. Lipopeptide and dipeptide genes were detected in the Oj-2.16 genome by PCR amplification involved in surfactin, iturin, fengycin, and bacilysin biosynthesis. In pot experiments, the biocontrol efficacy of strain Oj-2.16 against Verticillium wilt in tomato was 89.26%, which was slightly higher than the efficacy of the chemical fungicide carbendazim. Strain Oj-2.16 can produce indole acetic acid, siderophores, assimilate various carbon sources, and significantly promoted the growth of tomato seedlings by increasing plant height, root length, stem width, fresh weight, and dry weight by 44.44%, 122.22%, 80.19%, 57.65%, 64.00%, respectively. Furthermore, defense-related antioxidant CAT, SOD, POD, and PAL enzyme activities significantly increased and MDA contents significantly decreased in tomato seedlings treated with strain Oj-2.16 upon inoculation of V. dahliae compared with the pathogen-inoculated control. In summary, we concluded that B. amyloliquefaciens Oj-2.16 could be used as a promising candidate for the biocontrol of Verticillium wilt and as plant growth stimulator of tomato.

Mini Review: Advances in 2-Haloacid Dehalogenases.

The 2-haloacid dehalogenases (EC 3.8.1.X) are industrially important enzymes that catalyze the cleavage of carbon-halogen bonds in 2-haloalkanoic acids, releasing halogen ions and producing corresponding 2-hydroxyl acids. These enzymes are of particular interest in environmental remediation and environmentally friendly synthesis of optically pure chiral compounds due to their ability to degrade a wide range of halogenated compounds with astonishing efficiency for enantiomer resolution. The 2-haloacid dehalogenases have been extensively studied with regard to their biochemical characterization, protein crystal structures, and catalytic mechanisms. This paper comprehensively reviews the source of isolation, classification, protein structures, reaction mechanisms, biochemical properties, and application of 2-haloacid dehalogenases; current trends and avenues for further development have also been included.

Comparison of the transcriptomic responses of two Chrysanthemum morifolium cultivars to low light.

BACKGROUND: Low light is a primary regulator of chrysanthemum growth. Our aim was to analyse the different transcriptomic responses of two Chrysanthemum morifolium cultivars to low light. METHODS AND RESULTS: We conducted a transcriptomic analysis of leaf samples from the 'Nannonggongfen' and 'Nannongxuefeng' chrysanthemum cultivars following a 5-day exposure to optimal light (70%, control [CK]) or low-light (20%, LL) conditions. Gene Ontology (GO) classification of upregulated genes revealed these genes to be associated with 11 cellular components, 9 molecular functions, and 15 biological processes, with the majority being localized to the chloroplast, highlighting the role of chloroplast proteins as regulators of shading tolerance. Downregulated genes were associated with 11 cellular components, 8 molecular functions, and 16 biological processes. Heat map analyses suggested that basic helix-loop-helix domain genes and elongation factors were markedly downregulated in 'Nannongxuefeng' leaves, consistent with the maintenance of normal stem length, whereas no comparable changes were observed in 'Nanonggongfen' leaves. Subsequent qPCR analyses revealed that phytochrome-interacting factors and dormancy-associated genes were significantly upregulated under LL conditions relative to CK conditions, while succinate dehydrogenase 1, elongated hypocotyls 5, and auxin-responsive gene of were significantly downregulated under LL conditions. CONCLUSIONS: These findings suggest that LL plants were significantly lower than those of the CK plants. Low-light tolerant chrysanthemum cultivars may maintain reduced indole-3-acetic acid (IAA) and elongation factor expression as a means of preventing the onset of shade-avoidance symptoms.

First report of chili pepper fruit rot caused by Fusarium incarnatum in China.

Pepper (Capsicum annuum L.), with annual production over 1 million tons, is ranked the first vegetable crop in Hainan Province, China. In December 2018, fruit rot of chili pepper , with yield loss of up to 15%, was found in 10 fields (12 hm2) in Yacheng (18°N, 109°E), Hainan Province, China. Water-soaked and soft lesions developed on fruit, with white to light gray fungal mycelium present inside. The diseased fruit turned soft and decayed at the later stages. Diseased tissue was cut into 12 pieces of 0.5×0.5 cm, surface-disinfected with 2% sodium hypochlorite for 2 min, followed by 70% ethanol for 30 s, rinsed with sterile distilled water five times, and plated onto potato dextrose agar (PDA). After growing on PDA for 2 to 3 days at 28°C in an incubator without light, 10 pure culture isolates were obtained. All isolates had abundant dense white aerial mycelia that became beige with age. The macroconidia were slightly curved with four to seven septa, 29.51 to 42.15 × 4.29 to 6.22 µm. Spindle-shaped mesoconidia with three to four septa were abundantly produced, 20.34 to 24.54 × 4.58 to to 11.70 × 2.35 to 3.20 µm. Chlamydospores were absent. Based on the morphological characteristics, the fungus was tentatively identified as Fusarium incarnatum (Leslie and Summerell 2006). An isolate SQHP-01 was chosen for molecular identification and pathogenicity test. Two DNA fragments of the isolate, the internal transcribed spacer (ITS) and translation elongation factor genes (EF-1α) were amplified for sequencing. BLAST analysis showed that sequences of ITS (GenBank acc. no. MN317371) and EF-1α (acc. No. MN928788) had 99.61 to 100% identity with those of known F. incarnatum (MN480497 and KF993969). Phylogenetic analysis was conducted using neighbor-joining algorithm based on ITS and EF-1a genes separately, and the isolate was well clustered with F. incarnatum both with 100% bootstrap support. Pathogenicity test of the isolate were carried out twice on five healthy chili pepper fruit. After surface-disinfection, fruit were wounded at three different points and 20 µl of conidial suspension (106 conidia/ml) were deposited on each wound. Unwounded inoculation was conducted by spreading 100 µl of the suspension on each fruit surface including the pedicel and calyx. The fruit spread with sterile distilled water represented the negative control. All fruit treatments were placed on the moist sterile cotton in moist chambers at 25°C with 16 h light and 8 h darkness. After 4 to 6 days, water-soaked necrotic lesions appeared on the wounded fruit, the symptoms identical to those observed in the field. Water-soaked necrotic lesions developed on the pedicel and calyx of unwounded fruit. No symptoms were observed on the control fruit. The morphology and sequences of re-isolated fungal isolates from the tested peppers were the same as the original isolate. To our knowledge, this is the first report of F. incarnatum (synonym of F. semitectum) causing fruit rot on chili pepper in China. F. incarnatum has been reported to cause root rot of greenhouse pepper in China (Li et al. 2018), fruit rot of bell pepper in Trinidad (Ramdial et al. 2016) and Pakistan (Tariq et al. 2018). Effective control strategies need to be developed to prevent the economic losses caused by the disease in chili pepper.

CRISPR/Cas9 edited HSFA6a and HSFA6b of Arabidopsis thaliana offers ABA and osmotic stress insensitivity by modulation of ROS homeostasis.

The role of Heat Shock Transcription Factor 6 (HSFA6a & HSFA6b) in response to abiotic stresses such as ABA, drought, salinity, drought, and osmotic stress is individually well established. Unfortunately, the functional redundancy between the HSFA6a and HSFA6b as well as the consequences of simultaneous editing of both in response to aforementioned stresses remains elusive. Therefore, this study was designed with the aim of addressing whether there is any functional redundancy between HSFA6a and HSFA6b as well as to decipher their role in abiotic stresses tolerance in Arabidopsis thaliana, by using the CRISPR-Cas9. We have generated the single (hsfa6a and hsfa6b) as well as double mutants (hsfa6a/hsfa6b-1 and hsfa6a/hsfa6b-2) of HSFA6a and HSFA6b with higher frequencies of deletion, insertion, and substitution. The phenotypic characterization of generated double and single mutants under abiotic stresses such as ABA, mannitol, and NaCl identified double mutants more tolerant to subjected abiotic stresses than those of their single mutants. It warrants mentioning that we have identified that HSFA6a and HSFA6b also involved in other major ABA responses, including ABA-inhibited seed germination, stomatal movement, and water loss. In addition to the above, the simultaneous editing of HSFA6a and HSFA6b lead to a reduced ROS accumulation, accompanied by increased expression of much abiotic stress and ABA-responsive genes, including involved in regulation of ROS level. In conclusion, these results suggest that HSFA6a and HSFA6b may offer abiotic stress tolerance by regulating the ROS homeostasis in plants.

First Report of Powdery Mildew Caused by Podosphaera fusca on Helianthus tuberosus in China.

Helianthus tuberosus L. (Jerusalem artichoke) is an herbaceous perennial plant in the Asteraceae that is native to North America but introduced to China in the 17th century. The tubers of H. tuberosus are used as a vegetable, for the pharmaceutical production of inulin and as a source of ethanol biofuel, several B vitamins and minerals。From June to September 2009, severe powdery mildew infection was observed on H. tuberosus in a vegetable garden at Shangqiu Normal University, Shangqiu, China. Approximately 60% of leaves on individual plants were symptomatic, and almost 70% of the plants were infected. Initially, discrete powdery mildew colonies appeared on the upper surface of the leaves and on stems of the plants. Later, mycelial growth was amphigenous, thick, forming irregular white patches, covering the whole leaf surface. Eventually, leaves turned yellow, withered, and abscissed. Microscopic observations showed that conidia on infected leaves were ellipsoid-ovoid to barrel-shaped, with distinct fibrosin bodies visible in their cytoplasm, measuring 28 to 38 × 15 to 22 µm (n = 40). Conidiophores were unbranched, straight, 80 to 210 × 8 to 14 µm (n = 40) in size, and produced two to six immature conidia in chains with intercellular diaphragms. Foot cells of conidiophores were cylindrical and 45 to 65 µm × 8 to 14 µm (n = 40), with slight constrictions at basal septa, and followed by one to three short cells. Fungal hyphae were septate, branched, and flexuous to straight and 5 to 8 µm wide with indistinct to slightly nipple-shaped appressoria. These structures are typical of the genus Podosphaera, although chasmothecia were not observed. The sequence of ITS1-5.8s-ITS2 region of rDNA were amplified from conidia collected from infected leaves with universal primers ITS1F and ITS4 (White et al. 1990), sequenced and analyzed using the BLASTn search of GenBank. Amplicons were 603 bp (GQ927254) and exhibited 99.83% sequence identity with sequence of P. fusca from Cucurbita pepo (KJ698669) in Italy (Pirondi et al. 2015). The fungal species was identified as P. fusca (synonym P. xanthii ) by morphological characteristics and molecular analysis (Braun and Takamatsu 2000; Braun and Cook 2012). Pathogenicity tests were conducted by gently pressing the infected leaves onto leaves of six healthy H. tuberosus plants while six noninoculated plants served as controls. Plants were maintained in a greenhouse at 25 ± 2°C. Eight days after inoculation, symptoms similar to those observed under natural conditions developed on the inoculated leaves of H. tuberosus plants, whereas the control plants remained symptomless. The fungus on inoculated leaves was morphologically identical to that first observed in the field. P. fusca parasitizes a large number of asteraceous species including Euryops pectinatus (Saenz et al. 2000), Coreopsis lanceolata (Garibaldi et al. 2007), Cosmos caudatus (Siddiqui et al. 2011), Herba eupatorii (Ding et al. 2013) and etc. Powdery mildew caused by P. fusca has been reported on H. tuberosus in Russia (Farr and Rossman 2019). Golovinomyces ambrosiae was previously recorded on H. tuberosus in China (Huang et al. 2017; Radisek et al. 2018). This is the first report to our knowledge of powdery mildew P. fusca on H. tuberosus in China. It could cause significant yield losses and become a threat to production of H. tuberosus .

[Recent progress in 2-haloacid dehalogenases].

2-Haloacid dehalogenases (EC 3.8.1.X) catalyze the hydrolytic dehalogenation of 2-haloacids, releasing halogen ions and producing corresponding 2-hydroxyacids. The enzymes not only degrade xenobiotic halogenated pollutants, but also show wide substrate profile and astonishing efficiency for enantiomer resolution, making them valuable in environmental protection and the green synthesis of optically pure chiral compounds. A variety of 2-haloacid dehalogenases have been biochemically characterized so far. Further studies have been made in protein crystal structures and catalytic mechanisms. Here, we review the recent progresses of 2-haloacid dehalogenases in their source, protein structures, reaction mechanisms, catalytic properties and application. We also suggest further research directions for 2-haloacid dehalogenase.

ShORR-1, a Novel Tomato Gene, Confers Enhanced Host Resistance to Oidium neolycopersici.

A previous complementary cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis examined responses to the powdery mildew pathogen Oidium neolycopersici (On) of the resistant cultivar Solanum habrochiates G1.1560, carrying the Ol-1 resistance gene, and susceptible cultivar S. lycopersicum Moneymaker (MM). Among other findings, a differentially expressed transcript-derived fragment (DE-TDF) (M14E72-213) was upregulated in near isogenic line (NIL)-Ol-1, but absent in MM. This DE-TDF showed high homology to a gene of unknown function, which we named ShORR-1 (Solanum habrochaites Oidium Resistance Required-1). However, MM homolog of ShORR-1 (named ShORR-1-M) was still found with 95.26% nucleic acid sequence similarity to ShORR-1 from G1.1560 (named ShORR-1-G); this was because the cut sites of restriction enzymes in the previous complementary cDNA-AFLP analysis was absent in ShORR-1-M and differs at 13 amino acids from ShORR-1-G. Transient expression in onion epidermal cells showed that ShORR-1 is a membrane-localized protein. Virus-induced gene silencing (VIGS) of ShORR-1-G in G1.1560 plants increased susceptibility to On. Furthermore, overexpressing of ShORR-1-G conferred MM with resistance to On, involving extensive hydrogen peroxide accumulation and formation of abnormal haustoria. Knockdown of ShORR-1-M in MM did not affect its susceptibility to On, while overexpressing of ShORR-1-M enhanced MM's susceptibility to On. We also found that changes in transcript levels of six well-known hormone signaling and defense-related genes are involved in ShORR-1-G-mediated resistance to On. The results indicate that ShORR-1-M and ShORR-1-G have antagonistic effects in tomato responses to On, and that ShORR-1 is essential for Ol-1-mediated resistance in tomato.

Bioaccumulation and chemical forms of cadmium, copper and lead in aquatic plants

The cadmium(Cd), copper(Cu) and lead(Pb) accumulation, as well as their relative content of different chemical forms in Sagittaria sagittifolia L. and Potamogeton crispus L. were determined. The results showed that both the plants had the ability to accumulate large amounts of Cd, Cu and Pb, and they absorbed metals in dose-dependent manners. The roots of S. sagittifolia appeared more sensitive to Cd and Pb than the leaves of P. crispus. The potential of Cu uptake by these two plant tissues was similar. Under the same concentration, the uptake of Cu for both the plants was higher than Pb and Cd, while that of Pb was lowest. The Cd, Cu and Pb existed with various forms in the plants. Cd and Pb were mainly in the NaCl extractable form in S. sagittifolia and P. crispus. The HAc and ethanol extractable Cu were the main forms in the root, whereas the ethanol extractable form was the dominant chemical form in the caulis and bulb of the S. sagittifolia L.

RNase HII from Chlamydia pneumoniae discriminates mismatches incorporation into DNA-rN1-DNA/DNA duplexes.

It was reported that RNase HII from Chlamydia pneumoniae (CpRNase HII) had RNase H activity on RNA/DNA duplex. We have analyzed the cleavage specificity of CpRNase HII on DNA-rN1-DNA/DNA duplex (rN1, one ribonucleotide). Various mismatches were introduced into the DNA-rN1-DNA/DNA duplexes at or around the ribonucleotide. The mismatches of duplexes resulted in slower cleavage rates compared to the matched duplexes. Furthermore, a greater reduction in cleavage activity was observed for the mismatches located at or adjacent to the ribonucleotide. The mismatches at the same position of DNA-rN1-DNA/DNA duplexes have different impact on the cleavage rates of CpRNase HII depending on the types of mismatches. These findings may offer further insights into the physical binding and catalytic properties of CpRNase HII-substrate interaction.

Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39.

Chlamydophila pneumoniae AR39 contains two different ORFs (CP0654 and CP0782) encoding ribonuclease H (RNase H) homologues, Cpn-RNase HII and Cpn-RNase HIII. Sequence alignments show that the two homologues both contain the conserved motifs of type 2 RNase H, and Cpn-RNase HII has the conserved active-site motif (DEDD) of RNase HII. Cpn-RNase HIII also contains a unique active-site motif (DEDE), common to other RNase HIIIs. Complementation assays indicated that Cpn-RNase HII can complement both Escherichia coli RNase HII and RNase HI, but Cpn-RNase HIII can only complement the latter. In vitro enzyme activity experiments showed that neither Cpn-RNase HII nor Cpn-RNase HIII is thermostable and their optimum pH values were 9.0 and 10.0, respectively. Cpn-RNase HII cleaves a 12 bp RNA-DNA substrate at multiple sites, but Cpn-RNase HIII at only one site. When a 35 bp DNA-RNA-DNA/DNA chimeric substrate was used, cleavage was only observed with Cpn-RNase HII. These results indicate that the RNase H combination of C. pneumoniae AR39 is not simple substitution of E. coli RNase H, perhaps representing a more primordial type. This is believed to be the first in vivo functional study of Chlamydophila RNase Hs and the results should contribute to the analysis of RNase Hs of other parasite species.

Correlation between antifungal agent phenazine-1-carboxylic acid and pyoluteorin biosynthesis in Pseudomonas sp. M18.

Biosynthesis and secretion of two different types of antifungal compound [phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) in Pseudomonas sp. M18] contribute to its suppression of soil-borne root pathogens. To better understand the correlation between two antifungal agents in secondary metabolism, a DNA fragment covering partial pltC and pltD coding sequences was obtained by screening the genomic library of Pseudomonas sp. M18. A mutant, M18T, was then constructed by insertion of the aacC1 gene cassette (encoding gentamycin resistance). With the same methods, one PCA biosynthetic gene cluster was insertionally inactivated and a mutant M18Z1 was created. The mutant strain M18T produces no Plt and the same amount of PCA in comparison with the wild-type strain M18. The mutant M18Z1, however, produces less PCA but more Plt than the wild-type strain M18. According to the documented data on strain M18, it is suggested that production of PCA is not influenced by Plt yield, but Plt biosynthesis is influenced by an alteration of PCA production.

Autoinduction of RpoS biosynthesis in the biocontrol strain Pseudomonas sp. M18.

The rpoS gene from Pseudomonas sp. M18, which encodes predicted protein (an alternative sigma factor s, sigma(S), or sigma(38)) with 99.5% sequence identity with RpoS from Pseudomonas aeruginosa PAO1, was first cloned. In order to investigate the mechanism of rpoS expression, an rpoS null mutant, named M18S, was constructed with insertion of aacC1 cassette bearing a gentamycin resistance gene. With introduction of a plasmid containing an rpoS'-'lacZ translational fusion (pMERS) to wild-type strain M18 or M18S, it was first found that beta-galactosidase activity expressed in strain M18S (pMERS) decreased to fourfold of that expressed in the strain M18 (pMERS). When strain M18S (pMERS) was introduced with another plasmid pBBS containing the wild-type rpoS gene, its beta-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of beta-galactosidase from a chromosomal fusion of the promoter of the wild-type rpoS gene with lacZ (rpoS-lacZ) was enhanced fivefold in the presence of a plasmid with the wild-type rpoS gene. With these findings, it is suggested that RpoS sigma factor may be involved in autoinducing its own gene expression in Pseudomonas sp. M18.

[Effects of sigma38 subunit deletion of RNA polymerase on antibiotics biosynthesis in pseudomonas].

With the designed primers, PCR was carried out using the genomic DNA of Pseudomonas sp. M18 as a template and a 378bp DNA fragment of the rpoS gene was amplified. Then, a 3. 1kb EcoR I -Xho I fragment containing the rpoS gene and its flanking sequence was obtained by screening the genomic DNA library of Pseudomonas sp. M18.A sigma38-subunit-deficient mutant M18S was constructed with insertional gentamycin gene cassette. In PPM medium, the mutant M18S produced 20.4 microg/mL of PCA and 75 microg/mL of Plt. In KMB medium, the mutant M18S produced no PCA and 185.6 microg/mL of Pit. It is obvious that the deficiency of sigma38 subunit in the mutant M18S leads less or no PCA production and much more Plt production than those in the wild type strain M18. PCA and Plt production were restored to the levels in wild type strain after complementation with rpoS gene in trans in strain M18S. Moreover, beta-galactosidase activities of the translational fusions phzA'-'lacZ and pltA'-'lacZ in strain M18S confirmed the effects of sigma38 subunit on PCA and Plt biosynthetic operons. With these results, it is suggested that sigma38 subunit gives a differential impacts on PCA and Plt biosynthesis, i. e, PCA production is positively regulated but Plt production is negatively influenced by sigma38 subunit in Pseudomonas sp. M18.