First Report of Postharvest Blue Mold decay caused by Penicillium expansum on Lemon (Citrus limon) fruit in China.

Lemon (Citrus limon) is one of the most important commercial (both dried and fresh) citrus fruits in China. In the spring of 2019, postharvest blue mold decay was observed at an incidence of 3-5% on lemon fruit at the local markets in Beijing, China. Fruit lesions were circular, brown, soft, and watery, and rapidly expanded at 25°C. To isolate the causal organism, small pieces (2 mm3) were cut from the lesions, surface-sterilized for 1 min in 1.5% NaOCl, rinsed three times with sterilized water, dried with sterile filter paper, placed onto potato dextrose agar (PDA) medium, and incubated at 25°C for 6 days. Eight morphologically similar single-colony fungal isolates were recovered from six lemon fruit. Colony surfaces were bluish-green on the upper surface and cream to yellow-brown one the reverse. Hyphae on colony margins were entirely subsurface and cream in color. Mycelium was highly branched, septate, and colorless, and conidiophores were 250 to 450 × 3.0 to 4.0 µm in size. Stipe of conidiophores were smooth-walled, bearing terminal penicilli, typically terverticillate or less commonly birverticillate, rami occurring singly, 16 to 23 × 3.0 to 4.0 µm, metulae in 3 to 6, measuring 12 to 15 × 3.0 to 4.0 µm. Phialides were ampulliform to almost cylindrical, in verticils of 5 to 8, measuring 8 to 11 × 2.5 to 3.2 µm with collula. Conidia were smooth-walled, ellipsoidal, measuring 3.0 to 3.5 × 2.5 to 3.0 µm. According to morphological characteristics, the fungus was identified as Penicillium expansum (Visagie et al. 2014). For molecular identification, genomic DNA of eight fungal isolates was extracted, regions of the beta-tubulin (TUB), and calmodulin (CAL) genes and ITS region, were amplified using Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 primers respectively. Obtained sequences of all isolates were identical to sequences of the representative isolate YC-IK12, which was submitted in the GenBank. BLAST results of YC-IK12 sequences (ITS; MT856700: TUB; MT856958: CAL; MT856959) showed 98 to 100% similarity with P. expansum accessions (NR-077154, LN896428, JX141581). For pathogenicity tests, 10 µl of conidial suspension (10 × 105 conidia/ml) from seven-day-old YC-IK12 culture was inoculated using a sterilized needle into the surface of each five asymptomatic disinfected lemons. As a control, three lemons were inoculated using sterile distilled water. All inoculated lemons were placed in plastic containers and incubated at 25°C for 7 days. Decay lesions, identical to the original observations, developed on all inoculated lemons, while control lemons remained asymptomatic. Fungus re-isolated from the inoculated lemon was identified as P. expansum on the basis morphology and Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 sequences. Previously, Penicillium spp. including P. expansum have been reported as post-harvest pathogens on various Citrus spp. (Louw & Korsten 2015). However, P. digitatum has been reported on lemons and P. expansum has been reported on stored Kiwifruit (Actinidia arguta), Malus, and Pyrus species in China (Tai, 1979; Wang et al. 2015). To our knowledge, this is the first report of blue mold caused by P. expansum on lemons in China. References Louw, J. P., Korsten, L. 2015. Plant Dis. 99:21-30. Tai, F.L. 1979. Sylloge Fungorum Sinicorum. Sci. Press, Acad. Sin., Peking, 1527 pages. 8097 Visagie, C.M. et al. 2014. Studies. Mycol.78: 343. Wang, C. W. et al. 2015. Plant Dis. 99:1037.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Cuphea hyssopifolia (Lythraceae) in Mainland China.

Cuphea hyssopifolia (Mexican heather) is a popular evergreen perennial shrub used for ornamental and medicinal purposes. Due to its high ornamental value, it is often used as a ground cover in parks and gardens in China. During February and March 2019 & 2020, powdery mildew was observed on C. hyssopifolia in the districts of Minhou and Jinshan of Fuzhou, China. Disease incidence was 70% but of low severity with only a few older leaves showing yellowing and wilting. Sparse irregular patches of white superficial powdery mildew observed on both sides of mature and young leaves. The powdery mildew fungal appressoria that occurred on epigenous hyphae, were indistinct to nipple-shaped, hyaline, and smooth. Conidiophores were erect, smooth, 80 to 210 × 10 to 12 µm, and produced two to eight crenate-shaped conidia in chains. Foot-cells of conidiophores were straight, cylindric, and 30 to 65 × 10 to12 µm. Conidia were hyaline, smooth, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 16 to 20 µm with distinct fibrosin bodies. Germ tubes were simple to forked and produced from the lateral position of the germinating conidia. No chasmothecia were observed on the surface of infected leaves. Based on the morphology of the imperfect state, the powdery mildew fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). To confirm fungal identification, total DNA was extracted (Mukhtar et al., 2018) directly from epiphytic mycelia on infected leaves collected from both districts. Internal transcribed spacer (ITS) regions and the partial large subunit (LSU) rDNA were amplified using primers ITS1/ITS4 and LSU1/LSU2 (Scholin et al. 1994, White et al. 1990), respectively. The sequences were deposited in GenBank (ITS: MW692364, MW692365; LSU: MW699924, MW699925). The ITS and LSU sequences were 99 to 100 % identical to those of P. xanthii in GenBank, (ITS: MT568609, MT472035, MT250855, and AB462800; LSU: AB936276, JX896687, AB936277, and AB936274). Koch's postulates were completed by gently pressing diseased leaves onto leaves of five healthy potted C. hyssopifolia plants that were held in a greenhouse at 24 to 30°C without humidity control. Five non-inoculated plants served as controls. Inoculated plants developed symptoms after 6 to 10 days, whereas the controls remained symptomless. The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Previously, Podosphaera sp. has been reported on C. rosea in the United Kingdom (Beales & Cook 2008) and P. xanthii on C. hyssopifolia in Taiwan (Yeh et al. 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on C. hyssopifolia in mainland China. Our field observations suggest that the P. xanthii infections would be a potential threat to the health of C. hyssopifolia in China. References: Beales, P. A., and Cook, R. T. A. 2008. Plant Pathol. 57:778. Braun, U., Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Yeh, Y. W., et al. 2021. Trop. Plant Pathol. 46:44.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Bidens pilosa in China.

Bidens pilosa L., (spanish needle), is a wild, flowering plant of Asteraceae, that is grown in gardens, fields, roadsides, and riverbanks in Fuzhou, China. It is also used in traditional folk medicine for a broad range of ailments in China. In March 2019 and 2020, hundreds of B. pilosa growing along the roadsides, and gardens in the districts of Minhou and Jinshan were observed to be severely affected by a powdery mildew with approximately 80% disease incidence. Symptoms appeared as circular to irregular small white, powdery patches, typically on the adaxial sides of leaves and progressed to coalescent colonies on the leaves. As the disease developed, the infected leaves became wilted and senesced. Mycelia on leaves were superficial and solitary appressoria were slightly to distinctly nipple-shaped. Conidiophores were erect, 120 to 230 × 10 to 12 µm, and produced two to five conidia in chains with a sinuate outline. Foot-cells were erect, cylindrical, and 60 to 110 µm long. Conidia were hyaline, ellipsoid to barrel-shaped, 26 to 40 × 18 to 24 µm, and devoid of distinct fibrosin bodies. Germ tubes were long and produced at the perihilar position of the conidia. No chasmothecia were observed. Morphological characteristics overlapped with Golovinomyces ambrosiae, G. cichoracearum, and G. spadiceus (Braun and Cook 2012) on hosts within the Asteraceae tribe Heliantheae (Takamatsu et al. 2013). For molecular identification, ITS and IGS regions as well as partial LSU of two representative collections (MJU-IM019- MJU-IM020), were amplified using ITS1/ITS4, IGS-12a/ NS1R and LSU1/LSU2 primers (Carbone & Kohn, 1999; Scholin et al. 1994; White et al. 1990), respectively. The resulting sequences were deposited in GenBank (ITS: MW965777, MW965778; LSU: MW965787, MW965788; IGS: MW981256, MW981257). A BLAST search revealed 99 to 100 % sequence similarity to G. ambrosiae sequences (KX987303, AB769421, AB077689, AB769426, AB077643, and AB769425). Phylogenetic analysis of ITS, LSU and IGS also grouped obtained sequences within the G. ambrosiae complex (Qiu et al. 2020). Pathogenicity was confirmed through inoculation by gently pressing infected leaves onto leaves of five healthy, potted, young B. pilosa plants, while five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25 ± 2°C. Inoculated plants developed symptoms after 7 to 10 days, whereas the control plants remained symptomless. The morphology of the resulting fungus on inoculated plants was identical to that originally observed on diseased plants. Podosphaera spp., have been reported on B. pilosa (Farr & Rossman 2021) from North America, Africa, and Asia. To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on B. pilosa in China and Asia. Wild populations of B. pilosa may be the primary source of powdery mildew inoculum for commercial Asteraceae members and may warrant consideration in the control of this disease. References: Braun, U., and Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, The Netherlands. Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 18 April 2021. Qiu, P. L., et al. 2020. BMC Microbiol. 20:1. Scholin, C. A., et al. 1994. J. Phycol. 30:999. Takamatsu, S., et al. 2013. Mycologia 105:1135. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Sigesbeckia orientalis in China.

Sigesbeckia orientalis L., (St Paul's wort) is an annually grown natural herb of Asteraceae with a long therapeutic history for a wide range of inflammation-related diseases in China (Zhong et al. 2019). In June 2020, typical symptoms of powdery mildew were observed on 30% of wild S. orientalis plants grown along the roadsides and gardens in Minjiang University, Fuzhou, China. Circular to irregular white powdery fungal colonies were observed on both surfaces of the leaves and young stems, causing necrosis and premature senescence. Fungal hyphae were epigenous, flexuous to straight, branched, and septate. Appressoria on the hyphae were nipple-shaped or nearly absent. Conidiophores were straight, 30 to 210× 8 to 12 µm, and produced 3 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindrical, 45 to 75 ×10 to 12 µm, followed by 1 to 2 shorter cells. Conidia were hyaline, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 18 to 23 µm with distinct fibrosin bodies. Germ tubes were produced from a lateral position on the conidia. Chasmothecia were not observed on the infected leaves. Based on anamorph characteristics, fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). For molecular identification, total genomic DNA was extracted (Mukhtar et al. 2018) from fungal colonies on infected leaves of five collections separately. For each DNA sample, the part of LSU and ITS regions were amplified using primers LSU1/LSU2 and ITS1/ITS4 (Scholin et al. 1994; White et al. 1990), respectively. A BLAST search revealed 100 % sequences similarity with P. xanthii sequences reported on Ageratum conyzoides (KY274485), Eclipta prostrata (MT260063), Euphorbia hirta (KY388505), Sonchus asper (MN134013), and Verbena bonariensis (AB462804). Representative sequences (ITS: MZ613309; LSU: MZ614707) of an isolate were deposited in GenBank. The phylogenetic analysis also grouped the obtain sequences into P. xanthii clade. Pathogenicity was confirmed by gently pressing the infected leaves onto young leaves of five healthy one-month-old S. orientalis plants, while three non-inoculated plants were used as controls. All plants were maintained in a greenhouse at 25 ± 2°C. After, seven days, white powdery colonies were observed on inoculated plants, whereas controls remained mildew-free. On inoculated leaves, the fungus was morphologically and molecularly identical to the fungus on the original specimens. P. xanthii has been reported as a significant damaging pathogen on a wide range of plants in China (Farr and Rossman 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. orientalis in China as well as worldwide. S. orientalis is one of the most important commercial Chinese medicinal herbs and the occurrence of powdery mildew is a threat to its production, quality, and marketability. References: Braun, U., and Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 9 October 2021. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Zhong, Z., et al., 2019. Chin. Med. (U. K.) 14, 1-12. 10.1186/s13020-019-0260-y.

Biodegradation of endocrine disruptor Bisphenol A by Pseudomonas putida strain YC-AE1 isolated from polluted soil, Guangdong, China.

BACKGROUND: Bisphenol A is an important organic chemical as an intermediate, final and inert ingredient in manufacturing of many important products like polycarbonate plastics, epoxy resins, flame retardants, food-drink packaging coating, and other. BPA is an endocrine disruptor compound that mimics the function of estrogen causing damage to reproductive organs. Bacterial degradation has been consider as a cost effective and eco-friendly method for BPA degradation compared with physical and chemical methods. This study aimed to isolate and identify bacterial strain capable to degrade and tolerate high concentrations of this pollutant, studying the factors affecting the degradation process and study the degradation mechanism of this strain. RESULTS: YC-AE1 is a Gram negative bacterial strain isolated from soil and identified as Pseudomonas putida by 16S rRNA gene sequence and BIOLOG identification system. This strain found to have a high capacity to degrade the endocrine disruptor Bisphenol A (BPA). Response surface methodology using central composite design was used to statistically optimize the environmental factors during BPA degradation and the results obtained by significant model were 7.2, 30 °C and 2.5% for optimum initial pH, temperature and inoculum size, respectively. Prolonged incubation period with low NaCl concentration improve the biodegradation of BPA. Analysis of variance (ANOVA) showed high coefficient of determination, R2 and Adj-R2 which were 0.9979 and 0.9935, respectively. Substrate analysis found that, strain YC-AE1 could degrade a wide variety of bisphenol A-related pollutants such as bisphenol B, bisphenol F, bisphenol S, Dibutyl phthalate, Diethylhexyl phthalate and Diethyl phthalate in varying proportion. Pseudomonas putida YC-AE1 showed high ability to degrade a wide range of BPA concentrations (0.5-1000 mg l- 1) with completely degradation for 500 mg l- 1 within 72 h. Metabolic intermediates detected in this study by HPLC-MS were identified as 4,4-dihydroxy-alpha-methylstilbene, p-hydroxybenzaldeyde, p-hydroxyacetophenone, 4-hydroxyphenylacetate, 4-hydroxyphenacyl alcohol, 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl) propanoate. CONCLUSIONS: This study reports Pseudomonas putida YC-AE1 as BPA biodegrader with high performance in degradation and tolerance to high BPA concentration. It exhibited strong degradation capacity and prominent adaptability towards a wide range of environmental conditions. Moreover, it degrades BPA in a short time via two different degradation pathways.

First Report of Pseudopestalotiopsis theae Causing Leaf Spot of Date Palm (Phoenix dactylifera) in China.

Date palm (Phoenix dactylifera L.) is a popular landscape tree in Fujian province, in South China. In November 2018 and June 2019, a leaf spot disease was observed on date palm in Fuzhou city. A survey of date palm plants grown in four different locations revealed that the disease incidence was almost 20%. The spots were brown with a yellow margin, 1 to 20 mm in diameter, and oval to irregular. In later stages, the spots gradually expanded and coalesced, became dry and died. For isolation, small pieces (0.5 cm2) were cut from leaf spots obtained from seven trees and disinfested with 70% alcohol. Leaf pieces were then placed onto 2% potato dextrose agar (PDA) and incubated at 25±2°C for 3 to 4 days. One fungus was consistently isolated from fifteen leaves. Fungal colonies were white with undulating margins and a light cream on the reverse side. Black globose to oblate conidiomata were irregularly distributed throughout ten-day-old colonies. The conidiogenous cells were septate, colorless, smooth-walled, straight to slightly curved, ampulliform or subcylindrical, and 6.0 to 13.5 × 1.3 to 3.0 µm [(n=50); x̄ ± SD = 9.5 ± 2× 2 ± 0.5µm]. Conidia were fusiform and five-celled with constrictions at the septa, measuring 18.5 to 31.5 × 5.0 to 7.5 µm [(n=50); x̄ ± SD = 25.5 ± 2 × 6.5± 0.2µm]. The three median cells were light to dark brown and the two end cells were colorless. Apical cells had 2 to 4 appendages ranging from 10.2 to 22.5 µm long. Basal cells had one appendage ranging from 3.5 to 5.5 µm long. The internal transcribed spacer (ITS) region of the ribosomal DNA and translation elongation factor 1-alpha (TEF1-α) gene of fungus were amplified using primers ITS1/ITS4 and EF1728F/EF1986R, respectively. Amplified products (ITS: MN294700 and TEF1-α: MN970514) showed 99% sequence identity to Pestalotiopsis sp., and Pseudoestalotiopsis theae sequences in GenBank. A comparison of MRC12 sequences with the type culture sequences (ITS: JQ683727 and TEF1-a: JQ683743) also showed high similarity, where ITS sequences exhibited only a three-nucleotide difference at the start of the sequences. No differences, however, were found between the TEF1-α sequences. On the basis of morphology and molecular characteristics, the fungus was identified as Ps. theae (Sawada) Maharachch., K.D. Hyde & Crous Steyaert (Maharachchikumbura et al. 2014). To confirm pathogenicity, five disinfested leaves on three healthy five-year-old date palm plants in a nursery (average temperature 26°C), were punctured 3 to 5 times with a sterilized needle, and then 10 to 15 mL conidial suspension (105 conidia/mL in sterilized distilled water) was sprayed over punctured areas of the leaves. For the control treatment, punctured leaves were sprayed with sterilized distilled water. All inoculated leaves plus the control were covered with plastic bags. After 10 days, brown leaf spots similar in appearance to those observed in the field appeared on all wounded leaves, and Ps. theae was successfully re-isolated; the control leaves remained asymptomatic. Previously, Ps. theae was reported on oil palm (Elaeis guineensis Jacq.) from Sierra Leone and Thailand (Turner, 1971; Suwannarach et al. 2013). To our knowledge, this is the first report of Ps. theae on date palm in China. This report expands the host range Ps. theae to date palm and underscores the potential threat of an emerging leaf spot pathogen on Phoenix species. References Maharachchikumbura, K.D., et al. 2014. Stud. Mycol. 79: 121-186. Suwannarach, N., et al. 2013. J. Gen. Plant Pathol. 79: 277-279. Turner, P.D. 1971. Phytopathol. 14: 1-58.

First Report of Cladosporium Blossom Blight Caused by Cladosporium cladosporioides on Calliandra haematocephala in China.

Calliandra haematocephala Hassk., commonly called red powder puff, is widely cultivated as an ornamental plant in Taiwan, Hainan, Guangdong and Fujian in China (CAS, 1988). The flowers are dark crimson with conspicuous stamens, which give them the appearance of powder-puffs. Blossom blight on C. haematocephala was first observed in early January 2019 on plants grown on the university campus as well as in parks in Fuzhou city, with nearly 80% of flowers on individual plants infected. At various locations in the city, disease incidence was 100%. Symptoms appeared as grayish green fungal growth on the stamens with the entire flower eventually turning black and covered with masses of fungal spores. Fifteen single spore isolates obtained from nine necrotic stamen samples were purified and cultured on Potato dextrose agar (PDA) plates at 24 ℃.The resultant fungal colonies were olivaceous-green to olivaceous-brown and had a velvet-like appearance. Conidiophores were smooth-walled, solitary, non-nodulose, and measuring 40 to 340 × 3 to 4 µm (n=50). Ramoconidia were cylindrical-oblong or slightly curved with 0 to 3 septa, and measuring 10 to 25 × 3 to 4 µm (n=50). Conidia were smooth-walled and prolifically produced in long chains. Terminal conidia were aseptate, subglobose, ovoid to limoniform, measuring 3 to 6 × 2 to 2.5 µm (n=50). Intercalary conidia were elliptical to limoniform or subcylindrical, aseptate, measuring 5 to 12 × 2.5 to 3 µm (n=50). On the basis of its morphology, the causal organism was identified as Cladosporium cladosporioides (Bensch et al. 2010). For molecular identification, pure cultures of five single-spore isolates were used for DNA extraction. A fragment in the ITS regions of the fungal rDNA, the ACT and the TEF1-α, was amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, and EF1728 F/EF1-986R. The DNA sequences obtained from all five isolates were identical. The resulting ITS (MK720012) and ACT (MN013164), and TEFl-α (MK752020) sequences from a representative isolate MRCIM19 were 98-100% identical to the C. cladosporioides accessions (ITS: MH863979, MG228421; ACT: HM148509, JF499878, HM148532; TEFl-α: JF499872). To test pathogenicity, a spore suspension (1×105 conidia/mL) was prepared from a seven- day- old culture of isolate MRCIM19 and 10 mL of the suspension was sprayed onto six flowers on each of three C. haematocephala plants. Sterile distilled water was sprayed onto three flowers of two plants as control. The inoculated flowers were covered with plastic bags which were removed two days post inoculation. Disease symptoms were recorded on each flower at 10 days post inoculation. Based on the morpho-molecular characters, the re-isolated fungus from the inoculated flowers was C. cladosporioides. This fungus was previously reported to cause blossom blight in strawberry in the USA and Korea (Gubler et al. 1999; Nam et al. 2015). Although it has been reported from many plants (Zhang 2003) in China, this is the first report of C. cladosporioides on C. haematocephala worldwide. References Bensch, K. et al. 2010. Stud Mycol. 67:1-94. Chinese Academy of Sciences (CAS), 1988. Flora Republicae Popularis Sinicae Editorial Committee, Beijing Sci. Press., 39: 38. Gubler, W. D. et al. 1999. Plant Dis. 83:400. Nam, M. H. et al. 2015. Microbiol. 43: 354-359. Zhang Z., Ed. 2003. Flora fungorum sinicorum, Vol. 14. Cladosporium, Fusicladium, Pyricularia. Beijing Science Press. 297.

Identification and characterization of a meta-cleavage product hydrolase involved in biphenyl degradation from Arthrobacter sp. YC-RL1.

Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants (POPs) widely existing in the environment. Arthrobacter sp. YC-RL1 is a biphenyl-degrading bacterium that shows metabolic versatility towards aromatic compounds. A 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate (HOPDA) hydrolase (BphD) gene involved in the biodegradation of biphenyl was cloned from strain YC-RL1 and heterologously expressed in Escherichia coli BL21 (DE3). The recombinant BphDYC-RL1 was purified and characterized. BphDYC-RL1 showed the highest activity at 45 °C and pH 7. It was stable under a wide range of temperature (20-50 °C). The enzyme had a Km value of 0.14 mM, Kcat of 11.61 s-1, and Vmax of 0.027 U/mg. Temperature dependence catalysis exhibited a biphasic Arrhenius Plot with a transition at 20 °C. BphDYC-RL1 was inactivated by SDS, Tween 20, Tween 80, Trition X-100, DTT, CHAPS, NBS, PMSF, and DEPC, but insensitive to EDTA. Site-directed mutagenesis of the active-site residues revealed that the catalytic triad residues (Ser115, His275, and Asp247) of BphDYC-RL1 were necessary for its activity. The investigation of BphDYC-RL1 not only provides new potential enzyme resource for the biodegradation of biphenyl but also helps deepen our understanding on the catalytic process and mechanism.

In silico genome analysis reveals the metabolic versatility and biotechnology potential of a halotorelant phthalic acid esters degrading Gordonia alkanivorans strain YC-RL2.

Members of genus Gordonia are known to degrade various xenobitics and produce secondary metabolites. The genome of a halotorelant phthalic acid ester (PAEs) degrading actinobacterium Gordonia alkanivorans strain YC-RL2 was sequenced using Biosciences RS II platform and Single Molecular Real-Time (SMRT) technology. The reads were assembled de novo by hierarchical genome assembly process (HGAP) algorithm version 2. Genes were annotated by NCBI Prokaryotic Genome Annotation Pipeline. The generated genome sequence was 4,979,656 bp with an average G+C content of 67.45%. Calculation of ANI confirmed previous classification that strain YC-RL2 is G. alkanivorans. The sequences were searched against KEGG and COG databases; 3132 CDSs were assigned to COG families and 1808 CDSs were predicted to be involved in 111 pathways. 95 of the KEGG annotated genes were predicted to be involved in the degradation of xenobiotics. A phthalate degradation operon could not be identified in the genome indicating that strain YC-RL2 possesses a novel way of phthalate degradation. A total of 203 and 22 CDSs were annotated as esterase/hydrolase and dioxygenase genes respectively. A total of 53 biosynthetic gene clusters (BGCs) were predicted by antiSMASH (antibiotics & Secondary Metabolite Analysis Shell) bacterial version 4.0. The genome also contained putative genes for heavy metal metabolism. The strain could tolerate 1 mM of Cd2+, Co2+, Cu2+, Ni2+, Zn2+, Mn2+ and Pb2+ ions. These results show that strain YC-RL2 has a great potential to degrade various xenobiotics in different environments and will provide a rich genetic resource for further biotechnological and remediation studies.

Characterization and 16S metagenomic analysis of organophosphorus flame retardants degrading consortia.

Three bacterial consortia, named YC-SY1, YC-BJ1 and YC-GZ1, were enriched from different areas of China. Bacterial consortia YC-SY1, YC-BJ1 and YC-GZ1 could efficiently degrade triphenyl phosphate (TPhP) (100 mg/L) by approximately 79.4%, 99.8% and 99.6%, tricresyl phosphate (TCrP) by 90.6%, 91.9% and 96.3%, respectively, within 4 days. And they could retain high degrading efficiency under a broad range of temperature (15-40 ℃), pH (6.0-10.0) and salinity (0-4%). A total of 10 bacterial isolates were selected and investigated their degradation capacity. Among these isolates, two were significantly superior to the others. Strain Rhodococcus sp. YC-JH2 could utilize TPhP (50 mg/L) as sole carbon source for growth with 37.36% degradation within 7 days. Strain Sphingopyxis sp. YC-JH3 could efficiently degrade 96.2% of TPhP (50 mg/L) within 7 days, except that no cell growth was observed. Combined with 16S diversity analysis, our results suggest that the effective components of three bacterial consortia responsible for TPhP and TCrP degradation were almost the same, that is, bacteria capable of degrading TPhP and TCrP are limited, in this study, the most efficient component is Sphingopyxis. This study provides abundant microorganism sources for research on organophosphorus flame retardants (OPFRs) metabolism and bioremediation towards OPFRs-contaminated environments.

Cometabolic biodegradation of quizalofop-p-ethyl by Methylobacterium populi YC-XJ1 and identification of QPEH1 esterase

BACKGROUND: Quizalofop-p-ethyl (QPE), a unitary R configuration aromatic oxyphenoxypropionic acid ester (AOPP) herbicide, was widely used and had led to detrimental environmental effects. For finding the QPEdegrading bacteria and promoting the biodegradation of QPE, a series of studies were carried out. RESULTS: A QPE-degrading bacterial strain YC-XJ1 was isolated from desert soil and identified as Methylobacterium populi, which could degrade QPE with methanol by cometabolism. Ninety-seven percent of QPE (50 mg/L) could be degraded within 72 h under optimum biodegradation condition of 35°C and pH 8.0. The maximum degradation rate of QPE was 1.4 mg/L/h, and the strain YC-XJ1 exhibited some certain salinity tolerance. Two novel metabolites, 2-hydroxy-6-chloroquinoxaline and quinoxaline, were found by high-performance liquid chromatography/mass spectroscopy analysis. The metabolic pathway of QPE was predicted. The catalytic efficiency of strain YC-XJ1 toward different AOPPs herbicides in descending order was as follows: haloxyfop-pmethyl ≈ diclofop-methyl ≈ fluazifop-p-butyl N clodinafop-propargyl N cyhalofop-butyl N quizalofop-p-ethyl N fenoxaprop-p-ethyl N propaquizafop N quizalofop-p-tefuryl. The genome of strain YC-XJ1 was sequenced using a combination of PacBio RS II and Illumina platforms. According to the annotation result, one α/ß hydrolase gene was selected and named qpeh1, for which QPE-degrading function has obtained validation. Based on the phylogenetic analysis and multiple sequence alignment with other QPE-degrading esterases reported previously, the QPEH1 was clustered with esterase family V. CONCLUSION: M. populi YC-XJ1 could degrade QPE with a novel pathway, and the qpeh1 gene was identified as one of QPE-degrading esterase gene.