Leaf Spot caused by Alternaria alternata on Macleaya cordata in China.

Macleaya cordata is a perennial herb that belongs to the Papaveraceae and is typically prescribed as a traditional antibacterial medicine in China (Kosina et al. 2010). The extract from M. cordata has been widely used in the manufacturing of natural growth promoters as an alternative to antibiotic growth promoters in the livestock industry (Liu et al. 2017), and the products are marketed in 70 countries such as Germany, China, etc (Ikezawa et al. 2009). During the summer of 2019, symptoms of leaf spot were observed on M. cordata (cv. HNXN-001) in two commercial fields (approximately 1, 300 m2 and 2, 100 m2) of Xinning county, Shaoyang City, Hunan Province, China, where approximately 2 to 3% of the plants were affected. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to leaf blight. Six symptomatic basal leaf sections from six plants from two fields were surface disinfested in 0.5% NaClO for 1 min, then 75% ethanol for 20 s, rinsed in sterile water three times, air dried, and placed onto potato dextrose agar (PDA), one dish for samples from a single leaf. Plates were incubated at 26°C in darkness. Nine strains with similar morphological characters were isolated, and one representative isolate ( BLH-YB-08) was used for morphological and molecular characterization. The colonies on PDA were grayish-green with white round margins. Conidia were typically obclavate to obpyriform, brown to dark brown, and 12.0 to 35.0 × 6.0 to 15.0 µm, and with 1 to 5 transverse septa and 0 to 2 longitudinal septa (n=50). Isolates were identified as Alternaria sp. on the basis of mycelial characteristics, color, and conidial morphology. To confirm identity of the pathogen, DNA was extracted from isolate BLH-YB-08 with the DNAsecure Plant Kit (TIANGEN, Biotech, China). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), actin (ACT), 28S nrDNA (LSU), 18S nuclear ribosomal DNA (SSU), histone 3 (HIS3), internal transcribed spacer (ITS) region of ribosomal DNA, and translation elongation factor 1-α (TEF) genes ( Berbee et al. 1999; Carbone and Kohn. 1999; Glass and Donaldson. 1995; White et al. 1990.) were amplified and sequenced. Sequences were deposited into the GenBank database. They were 100% sequence identity of GAPDH (OQ224996) with A. alternata strain AA2-8 (MH65578; 578/578bp), 100% sequence identity of RPB2 (OQ190460) with A. alternata strain SAX-WN-30-2 ( MK605877; 933/933bp), 100% sequence identity of ACT (OQ923292) with A. alternata strain FCBP0352 (OL830257; 939/939 bp), 100% sequence identity of LSU (OQ891167) with A. alternata XL14 (MG839509 ; 908/908 bp), 100% sequence identity of SSU (OQ139544) with A. alternata strain BJ19.4.1(OM736063; 1,067/1,067 bp), 100% sequence identity of HIS3 (MT454856) with A. alternata YJ-CYC-HC2 (OQ116440 ; 442/442 bp), 100% sequence identity of ITS (MT212225) with A. alternata CS-1-3 (OQ947366; 543/543bp), and 100% sequence identity of TEF (OQ190461) with A. alternata strain YZU 221185 (OQ512730; 252/252 bp). To test pathogenicity, the isolate BLH-YB-08 was cultured on PDA for 7 days to prepare conidial suspensions and the spore concentration adjusted to a final concentration of 1×106 spores/ml. The leaves of five potted 45-day-old M. cordata (cv. HNXN-001) plants were sprayed with conidial suspensions, and five control potted plants were wiped with 75% alcohol and washed five times with sterile distilled water. They were then sprayed with sterile distilled water. Plants were placed in a greenhouse at 25 to 30°C with 90% relative humidity. Pathogenicity tests were conducted twice. Fifteen days after inoculation, lesions were found on inoculated leaves, and the symptoms were the same as those in the field, whereas the controls were healthy. A fungus was consistently isolated from the inoculated leaves and identified as A. alternata by DNA sequencing of the GAPDH, ITS, and HIS3 genes, fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on M. cordata caused by A. alternata in China. Understanding its etiology may help to control this fungal pathogen, thus reducing economic losses. Funding: Hunan Provincial Natural Science Foundation General Project (2023JJ30341) Hunan Provincial Natural Science Foundation Youth Fund (2023JJ40367) Seed Industry Innovation Project of Hunan Provincial Science and Technology Department Special project for the construction of Chinese herbal medicine industry technology system in Hunan Province "Xiangjiuwei" Industrial Cluster Project of the Ministry of Agriculture and Rural Affairs.

Effects of codon optimization, N-terminal truncation and gene dose on the heterologous expression of berberine bridge enzyme.

Morphine, sanguinarine and chelerythrine are benzylisoquinoline alkaloids (BIAs), and these compounds possess strong biological activities. (S)-scoulerine is a commonly shared precursor of these compounds, and berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the synthesis of (S)-scoulerine. We isolated the BBE gene from Macleaya cordata (McBBE) and used CEN.PK2-1C as a chassis strain. We compared the catalytic efficiency of five codon-optimized McBBE genes in Saccharomyces cerevisiae and finally obtained a yeast strain (YH03) that exhibited a 58-fold increase in yield (1.12 mg/L). Then, we truncated the N-terminus of McBBE by 8 and 22 amino acids and found that with the increase in the number of N-terminal truncated amino acids, the production of (S)-scoulerine gradually decreased. Additionally, we used CRISPR-Cas9 to integrate the McBBE gene at the delta site of the S. cerevisiae genome to achieve stable genetic inheritance and found that the yield of (S)-scoulerine was not significantly increased in the integrated strain. In conclusion, our work achieved high-efficiency expression of McBBE in S. cerevisiae, explored the influence of N-terminal truncation on the yield of (S)-scoulerine, and obtained a genetically stable S. cerevisiae strain with high McBBE expression. This study provides a reference for further complex metabolic engineering optimization and lays a foundation for the efficient biosynthesis of BIAs.

Plant-microbe hybrid synthesis provides new insights for the efficient use of Macleaya cordata.

Sanguinarine and chelerytrine have antibacterial and anti-inflammatory effects and is the main active ingredients of growth promoters in animals. Currently, Sanguinarine and chelerytrine were extracted from the capsules of the medicinal plant Macleaya cordata. However, the biomass of M. cordata nonmedicinal parts (leaves) accounted for a large proportion and contained a rich presentation of protopine and allocryptopine which are the precursor compounds of sanguinarine and chelerytrine. The aim of this study was to develop a new method for producing sanguinarine and chelerytrine through yeast transformation of protopine and allocryptopine in M. cordata leaves. First, we isolated different genes from Papaver somniferum (PsP6H, PsCPR, PsDBOX), Eschscholtzia californica (EcP6H), Cucumis sativus (CuCPR), Arabidopsis thaliana (AtCPR) and M. cordata (Mc11229, Mc11218, Mc6408, Mc6407, Mc19967, Mc13802). Additionally, some of the gene sequences were codon optimized. Then, we transformed these genes into yeast cells to compare the catalytic efficiency. Second, we used the most efficient strains to biotransform the leaves of M. cordata. Finally, we obtained 85.415 ± 11.887 ng mL-1 sanguinarine and 4.288 ± 1.395 ng mL-1 chelerytrine, which was more than 2-3 times the content in leaves of M. cordata. Overall, we using the nonmedicinal parts of M. cordata and successfully obtained sanguinarine and chelerytrine by the plant-microbial hybrid synthesis method.

Functional analysis of a lily SHORT VEGETATIVE PHASE ortholog in flowering transition and floral development.

Lilium is a commercially important genus of bulbous flowers, investigating the flowering molecular mechanisms is important for flowering regulation of lily. MADS-box SHORT VEGETATIVE PHASE (SVP) orthologs are involved in the flowering transition and floral organ differentiation in many plants. In this study, we identified an SVP ortholog from L. × formolongi (LfSVP), which was closely related to Arabidopsis SVP according to phylogenetic analysis. Tissue-specific expression patterns indicated that LfSVP expression levels peaked in the leaves and showed low expression levels in flowering tepals. Stage-dependent expression patterns of LfSVP showed high transcription level in the flowering induction stage under different photoperiods and exhibited transcription peak in the floral budding development stage under long days. Overexpressed LfSVP led to delayed flowering and floral organ defects in Arabidopsis independent of photoperiod. Tobacco rattle virus -induced gene silencing of LfSVP caused a strongly earlier flowering time and floral organ defects of L. × formolongi. Moreover, LfSVP can interact with L. × formolongi APETALA1 (AP1) in both yeast and tobacco cells, and the two may interact to regulate floral organ differentiation. In conclusion, LfSVP is a flowering repressor and may be involved in the regulation of floral organ differentiation. This study will be helpful for the molecular breeding of short-life-period and rich floral patterns lily varieties.

Fermented traditional Chinese medicine alters the intestinal microbiota composition of broiler chickens.

For a long time, phytogenic resources have been widely used as substitutes for antibiotics in livestock production. However, few studies have examined the relationship between the intestinal microbiota and fermented herbal medicines. Here, 252 Arbor Acres broiler plus broiler chickens were randomly assigned to a control group, which was fed a basal diet; an unfermented healthy chicken powder group (JJS) fed a basal diet containing 20 g/kg JJS; or one of 5 fermented JJS groups, which were fed a basal diet containing 20 g/kg JJS, fermented with by 5 different bacterial strains for 42 days. The growth performances of the different groups were measured and the changes in the intestinal microbiota were analyzed. The body weight gain in the Bacillus subtilis group (Bs) was the highest among the 6 groups, while the feed conversion ratio (FCR) was best with Z. rouxii fermentation. The result indicated that products of JJS fermentation products of JJS by B. subtilis and Z. rouxii had important effects on chicken growth performance. The foregut and hindgut microbial communities of Bs, Zr, the control group and the JJS group, were collected for 16S rDNA sequencing. The results showed that JJS and its fermentation products mainly acted on the foregut and had little effect on the hindgut, and Z. rouxii fermentation products can increased the diversity in the foregut microbial community. In addition, the relative abundance of Bifidobacterium in the foregut of the Z. rouxii group was significantly increased, which may be an important factor in promoting growth.

The chromosome-level Hemerocallis citrina Borani genome provides new insights into the rutin biosynthesis and the lack of colchicine.

Hemerocallis citrina Borani (huang hua cai in Chinese) is an important horticultural crop whose flower buds are widely consumed as a delicious vegetable in Asia. Here we assembled a high-quality reference genome of H. citrina using single-molecule sequencing and Hi-C technologies. The genome assembly was 3.77 Gb and consisted of 3183 contigs with a contig N50 of 2.09 Mb, which were further clustered into 11 pseudochromosomes. A larger portion (3.25 Gb or 86.20%) was annotated as a repetitive content and 54,295 protein-coding genes were annotated in the genome. Genome evolution analysis showed that H. citrina experienced a recent whole-genome duplication (WGD) event at ~15.73 million years ago (Mya), which was the main factor leading to many multiple copies of orthologous genes. We used this reference genome to predict 20 genes involved in the rutin biosynthesis pathway. Moreover, our metabolomics data revealed neither colchicine nor its precursors in H. citrina, challenging the long-standing belief that this alkaloid causes poisoning by the plant. The results of our disruptive research are further substantiated by our genomic finding that H. citrina does not contain any genes involved in colchicine biosynthesis. The high-quality genome lays a solid foundation for genetic research and molecular breeding of H. citrina.