Flavonoids are important plant natural products with variable structures and bioactivities. All known plant flavonoids are generated under the catalysis of a type III polyketide synthase (PKS) followed by a chalcone isomerase (CHI) and a flavone synthase (FNS). In this study, the biosynthetic gene cluster of chlorflavonin, a fungal flavonoid with acetolactate synthase inhibitory activity, was discovered using a self‐resistance‐gene‐directed strategy. A novel flavonoid biosynthetic pathway in fungi was revealed. A core nonribosomal peptide synthetase‐polyketide synthase (NRPS‐PKS) is responsible for the generation of the key precursor chalcone. Then, a new type of CHI catalyzes the conversion of a chalcone into a flavanone by a histidine‐mediated oxa‐Michael addition mechanism. Finally, the desaturation of flavanone to flavone is catalyzed by a new type of FNS, a flavin mononucleotide (FMN)‐dependent oxidoreductase.
Broussonetia papyrifera (paper mulberry) is a deciduous tree with a number of uses and is native to northeastern Asia. Because of its fast-growing nature and high tolerance to dust, smoke, and high temperatures, paper mulberry is regarded as an important and economically-valuable component of a biologically diverse community and is used extensively in several areas including medicine, animal husbandry, paper making, weaving, afforestation and light industry (Mei et al. 2016). From June to August of 2019, symptoms on paper mulberry trees were observed in Shiniushan village, Sanhua town, Xishui County, Hubei province of China. Typical symptoms on leaves included small, angular, brown spots surrounded by yellow haloes. These spots coalesced into necrotic areas. The incidence was around 30%, which threatened the survival and reduced the yield of paper mulberry. In order to identify the causal pathogenic organism, leaf samples from 10 different infected trees were collected every two weeks and isolations made over three months. Several circular, flat, granulated colonies with entire margins were isolated on King’s B medium (KB). The biochemical and physiological characteristics of thirty typical strains were tested and listed as following: gram negative, aerobic, rod shaped, and non-fluorescent on King’s B medium; positive for carbohydrate utilization (sucrose, glucose, fructose and arabinose), levan production, hypersensitive on tobacco, potato and tomato; and negative for oxidase, arginine dehydrolase, tyrosinase and urease activity, gelatin liquefaction, and reduction of nitrate. Psa pathovar-specific primers PsaF1/PsaR2 (280bp product ) identified two representative strains as Psa (Rees-George et al. 2010). BLAST analysis further confirmed that the 16S rDNA region amplified by primers 27F/1492R (NCBI accession nos. MT472100 and MT472101) shared 99.84% and 99.77% identity with the Psa type strain ICMP 18884 (CP011972) respectively (Weisburg et al. 1991). For ten typical strains, pathogenicity was confirmed by spraying a bacterial suspension (108 cfu/mL) onto fifty one-year seedlings of B. papyrifera, five seedlings repetitions for each strain. Symptoms of infection similar to those observed initially in the field were detected within 7 days after incubation at 25°C with 80–85% humidity. No symptoms were observed on control plants. The pathogen was re-isolated from symptomatic leaves and re-identified as Psa by morphological characteristics and sequencing. To our knowledge, this is the first report of Psa causing bacterial leaf spot disease on B. papyrifera, China. Psa has been reported as a pathogen causing bacterial canker of kiwifruit worldwide, resulting in severe economic losses to kiwifruit growers (McCann & Li, 2017). As a host of Psa, B. papyrifera may be a source of inoculum for nearby kiwifruit orchards, and consequently effective control measures should be taken to manage this disease. Funding: This study was supported by the National Natural Science Foundation of China (31701974; 31901980), Science and technology program funded by Wuhan Science and Technology Bureau (2018020401011307). References: Mei et al. 2016. Eur J Plant Pathol. 145: 203. McCann & Li et al. 2017. Genome Biol Evol. 9: 932. Rees-George et al. 2010. Plant Pathol. 59: 453 Weisburg et al. 1991. J Bacteriol. 173: 697.
Akebia trifoliata (Thunberg) Koidzumi (three-leaf akebia), a climbing deciduous woody plant, grows wild in mountains of China and Japan. It has long been prized for its delicious sweet taste and medicinal value (Lu et al., 2019). Few pests and diseases reportedly affect this plant (Ye et al., 2013), but with more commercial planting of A. trifoliata in China, symptoms of anthracnose on leaves and fruits have increased. Between December 2018 and May 2019, typical anthracnose symptoms were first observed on A. trifoliata grown in Wuhan, China, with an incidence up to 15%. Diseased leaves exhibited irregular gray-brown spots with dark brown edges, and dark brown undersides, substantially affecting photosynthesis and growth. As disease progressed, white mycelium appeared on stems causing stem rot and fruit drop. Several round or needle-shaped dark brown spots formed on fruit peel, coalescing into irregular, slightly sunken blotches. Under high humidity, the whole fruit turned brown and the spots were covered by white mycelia, greatly affecting the fruits’ ornamental quality. To isolate the pathogen, 5-mm2 pieces of symptomatic tissue from 10 infected leaves and fruits were surface-disinfected for 90 s in 1% sodium hypochlorite then 30 s in 75% ethanol, rinsed twice with sterile water, then incubated on potato dextrose agar (PDA, Oxoid) at 25°C under 12 h light/dark photoperiod. Pure cultures were obtained from hyphal tips of each colony. Initially, colonies produced white mycelia, turning gray after 5 days. The isolates produced abundant hyaline, single celled, straight and cylindrical conidia, with mean size 10.35 to 15.58 × 3.46 to 5.69 μm. Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides (Cannon et al. 2012). Genomic DNA of three isolates was extracted for PCR amplification of the internal transcribed spacer (ITS) region, and β-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Weir et al. 2012). BLAST search identified all sequences (GenBank accession nos. MT451846 to MT451848 for ITS, MT573957 to MT573959 for TUB2, and MT573960 to MT573962 for GAPDH) as 100% matches to C. gloeosporioides (Penz.) Penz. & Sacc. CBS 112999 strain (JQ005152 for ITS, JQ005587 for TUB2, JQ005239 for GAPDH) (Damm et al. 2009). Identification was confirmed by maximum likelihood phylogenetic analysis using MEGA7 . To evaluate pathogenicity, isolates were inoculated onto one side of 10 wounded healthy leaves of 1-year-old pot-grown A. trifoliata plants and 10 nearly mature fruits, with 10 μl of conidial suspension (106/ml) and colonized PDA pieces (5 mm diam.) from 7-day-old cultures of the fungus in Petri dishes; control sides received 10 μl sterile distilled water and sterile agar pieces. The test was performed twice. After incubation at 25°C, 70% humidity under 12 h fluorescent illumination/12 h dark for 5 days, similar spots were observed on all inoculated leaves and fruits. Controls remained asymptomatic. The re-isolated pathogen was identified as C. gloeosporioides by biological characteristics and sequencing analysis, indicating that C. gloeosporioides was a causal agent of anthracnose of A. trifoliata. Anthracnose caused by C. acutatum has been reported on A. trifoliata in Japan (Kobayshi et al. 2004). To our knowledge, this is the first report of C. gloeosporioides found on Akebia species. The new disease primarily reduces the quality and yield of A. trifoliata. Effective measures should be taken to manage this disease. Funding: This study was supported by the National Natural Science Foundation of China (31701974; 31901980), Science and technology program funded by Wuhan Science and Technology Bureau (2018020401011307). References: Lu, W.L., et al. 2019. J. Ethnopharmacol. 234:204. Ye, Y.F., et al. 2013. Plant Dis. 97:1659. Kobayshi, Y., et al. 2004. J. Gen. Plant Pathol. 70:295. Cannon, P.F., et al. 2012. Stud. Mycol. 73:181. Weir, B.S., et al. 2012. Stud. Mycol. 73:115. Damm, U., et al. 2009. Fungal Divers. 39:45.
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