Objective To isolate and purify cytotoxin isoforms from Naja atra and further appraise its properties. Methods Cytotoxins were purified from Naja atra venom by successive separation on a SP|Sephadex C|25 column and a Sephasil Peptide C-{18} column. The purity of cytotoxins was appraised by SDS|polyacrylamide gel electrophoresis(Tris|Tricine system). N|terminal amino acid sequences of cytotoxins were determined by Edman degradation. Results Ion|exchange chromatography of lyophilized crude venom yielded 14 fractions. By reverse phase high performance liquid chromatography, four cytotoxins were purified from fractions Ⅹ~ⅩⅢ which have cytotoxic activities. The total yield of cytotoxin was 32 48%. SDS|PAGE and N|terminal sequencing analysis of them showed that molecular were 7 28, 7 33, 7 24 and 7 38 kD orderly. Conclusion Four cytotoxin isoforms were obtained from Naja atra venom. It is proved that the method of a successive cation|exchange and RP|HPLC is efficient in cytotoxin purification.
Postharvest diseases in lily plants are prevalent during storage and transportation, leading to potentially catastrophic economic losses for the lily industry. Specifically, bulb rot has been observed in Lanzhou lily (Lilium davidii var. unicolor) during cold storage in Beijing, China. In this study, fungal isolates were obtained from decayed bulbs using a conventional fungal separation method, and these isolates were confirmed to be the causative agent of lily bulb rot, according to Koch postulates. A representative isolate, LZ-3-10, was selected for further identification. Based on morphological features and internal transcribed spacer sequencing results, the LZ-3-10 isolate was identified as Rhizopus arrhizus. Subsequently, an endophytic bacterial strain exhibiting robust antagonistic ability, Bacillus siamensis B55, was screened from the roots of lily plants. Evaluation of its biocontrol ability revealed that strain B55 could effectively protect L. davidii var. unicolor bulbs from infection by LZ-3-10, demonstrating a biocontrol efficacy of 51.2% and significantly reducing the severity of lily Rhizopus rot. In summary, this study identifies R. arrhizus as the cause of postharvest bulb rot in L. davidii var. unicolor and, for the first time, showcases the biocontrol activity of the endophytic bacterial strain B. siamensis B55 against the isolated pathogenic fungus. These findings not only provide insights into lily bulb rot but also highlight the potential of B. siamensis B55 as a biocontrol agent for managing this disease during postharvest storage.
The present study was aimed at isolating endophytic fungi from the Asian culinary and medicinal plant Lilium davidii and analyzing its antifungal and plant growth-promoting effects. In this study, the fungal endophyte Acremonium sp. Ld-03 was isolated from the bulbs of L. davidii and identified through morphological and molecular analysis. The molecular and morphological analysis confirmed the endophytic fungal strain as Acremonium sp. Ld-03. Antifungal effects of Ld-03 were observed against Fusarium oxysporum, Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi. The highest growth inhibition, i.e., , was observed for B. dothidea followed by , , and for B. cinerea, F. fujikuroi, and F. oxysporum, respectively. Analysis of the ethyl acetate fraction through UHPLC-LTQ-IT-MS/MS revealed putative secondary metabolites which included xanthurenic acid, valyl aspartic acid, gancidin W, peptides, and cyclic dipeptides such as valylarginine, cyclo-[L-(4-hydroxy-Pro)-L-leu], cyclo(Pro-Phe), and (3S,6S)-3-benzyl-6-(4-hydroxybenzyl)piperazine-2,5-dione. Other metabolites included (S)-3-(4-hydroxyphenyl)-2-((S)-pyrrolidine-2-carboxamido)propanoic acid, dibutyl phthalate (DBP), 9-octadecenamide, D-erythro-C18-Sphingosine, N-palmitoyl sphinganine, and hydroxypalmitoyl sphinganine. The strain Ld-03 showed indole acetic acid (IAA) production with or without the application of exogenous tryptophan. The IAA ranged from μg ml-1 to μg ml-1 under different tryptophan concentrations. The strain was able to produce siderophore, and its production was significantly decreased with increasing Fe(III) citrate concentrations in the medium. The endophytic fungal strain also showed production of organic acids and phosphate solubilization activity. Plant growth-promoting effects of the strain were evaluated on in vitro seedling growth of Allium tuberosum. Application of 40% culture dilution resulted in a significant increase in root and shoot length, i.e., mm and mm, respectively, compared to nontreated control plants. The fungal endophyte Ld-03 demonstrated the potential of conferring disease resistance and plant growth promotion. Therefore, we conclude that the isolated Acremonium sp. Ld-03 should be further investigated before utilization as a biocontrol agent and plant growth stimulator.
A plant growth-promoting and antifungal endophytic bacteria designated as Ld-08 isolated from the bulbs of Lilium davidii was identified as Pseudomonas aeruginosa based on phenotypic, microscopic, and 16S rRNA gene sequence analysis. Ld-08 exhibited antifungal effects against Fusarium oxysporum, Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi. Ld-08 showed the highest growth inhibition, i.e., 83.82±4.76% against B. dothidea followed by 74.12±3.87%, 67.56±3.35%, and 63.67±3.39% against F. fujikuroi, B. cinerea, and F. oxysporum, respectively. The ethyl acetate fraction of Ld-08 revealed the presence of several bioactive secondary metabolites. Prominent compounds were quinolones; 3,9-dimethoxypterocarpan; cascaroside B; dehydroabietylamine; epiandrosterone; nocodazole; oxolinic acid; pyochelin; rhodotulic acid; 9,12-octadecadienoic acid; di-peptides; tri-peptides; ursodiol, and venlafaxine. The strain Ld-08 showed organic acids, ACC deaminase, phosphate solubilization, IAA, and siderophore. The sterilized bulbs of a Lilium variety, inoculated with Ld-08, were further studied for plant growth-promoting traits. The inoculated plants showed improved growth than the control plants. Importantly, some growth parameters such as plant height, leaf length, bulb weight, and root length were significantly (P ≤0.05) increased in the inoculated plants than in the control un-inoculated plants. Further investigations are required to explore the potential of this strain to be used as a plant growth-promoting and biocontrol agent in sustainable agriculture.
The genus Fritillaria comprises approximately 130 perennial herbaceous species. In the Pharmacopoeia of the People's Republic of China, the bulbs of 11 Fritillaria species are used in Chinese herbal medicines. However, the traditional methods of morphological classification cannot accurately identify closely related species of Fritillaria. Previous studies have attempted to identify these species with universal molecular markers, but insufficient phylogenetic signal was available. In this study, the complete chloroplast genomes of eight Fritillaria species were compared. The length of the eight Fritillaria chloroplast genomes ranges from 151,009 bp to 152,224 bp. A total of 136 SSR loci were identified, including 124 polymorphic SSR loci. For large repeat sequences, 108 repeat loci and four types of repeats were observed. Ten highly variable regions were identified as potential molecular markers. These SSRs, large repeat sequences and highly variable regions provide important information for the development of genetic markers and DNA fingerprints. Phylogenetic analyses showed that the topological structures of all data sets (except the IR regions) were in complete agreement and well resolved. Overall, this study provides comprehensive chloroplast genomic resources, which will be valuable for future studies of evolution and species identification in Fritillaria.
Abstract Trichome is a specialized structure found on the surface of the plant with important function in survival against abiotic and biotic stress. It is also an important economic trait in crop breeding. Extensive research has investigated the foliar trichome in model plants (Arabidopsis and tomato). However, the developmental mechanism of tepal trichome remains elusive. Lilium pumilum is an edible ornamental bulb and a good breeding parent possessing cold and salt-alkali resistance. Here, we found a natural mutant of Lilium pumilum grown on a highland whose tepals are covered by trichomes. Our data indicate that trichomes of this mutant are multicellular and branchless. Notably, stomata are also developed on the tepal of the mutant as well, suggesting there may be a correlated between trichome and stomata regulation. Furthermore, we isolated 27 differentially expressed genes (DEGs) by comparing the transcriptome profiling between the natural mutant and the wild type. These twenty-seven genes belong to four groups: epidermal cell cycle and division, trichome morphogenesis, stress response, and transcription factors. Quantitative real-time PCR in Lilium pumilum (natural mutant and the wild type) and other lily species ( Lilium leichtlinii var. maximowiczii / trichome; Lilium davidii var. willmottiael , trichomeless) confirmed the validation of RNA-seq data and identified several trichome-related genes.
Abstract Background Lilium is an important ornamental bulb, possesses medicinal properties, and is also edible. Species within the Lilium genus share very similar morphology and macroscopic characteristics, thus they cannot be easily and clearly distinguished from one another. To date, no efficient species-specific markers have been developed for classifying wild lily species, which poses an issue with further characterizing its medicinal properties. Results To develop a simple and reliable identification system for Lilium , 45 representative species from 6 sections were used to develop a DNA barcoding system, which was based on DNA sequence polymorphisms. In this study, we assessed five commonly used DNA barcode candidates ( ITS , rbc L , ycf1b , mat K and psbA-trnH ) and five novel barcode candidates obtained from highly variable chloroplast genomic regions ( trnL-trnF , trnS-trnG , trnF-ndhJ , trnP-psaJ-rpI33 and psbB-psbH ). We showed that a set of three novel DNA barcodes ( ITS + trnP-psaJ-rpI33 + psbB-psbH ) could be efficiently used as a genetic marker to distinguish between lily species, as assessed by methods including DNAsp, BI and ML tree, and Pair Wise Group (PWG). Conclusions A rapid and reliable DNA barcoding method was developed for all 45 wild Lilium species by using ITS , trnP-psaJ-rpI33 , and psbB-psbH as DNA barcoding markers. The method can be used in the classification of wild Lilium species, especially endangered species, and also provides an effective method for selective lily breeding.
Abstract Dynamic miRNA detection using the qRT-PCR technique requires appropriate reference genes to ensure data reliability. Previous studies have screened plant internal reference genes during embryonic development and various stress treatment, involving relatively few tissues and organs. There is no relevant miRNA study in Lilium henryi Baker and limited research on the optimal miRNA reference genes in lilies. Twelve genes were selected as candidate reference genes whose expression stability was analyzed in petals at different developmental stages and other tissues using various algorithms, such as geNorm, Normfinder, Bestkeeper, and Delta CT. The results revealed that the optimal combination of reference genes in the Lilium henryi Baker petals at different developmental stages was osa-miR166m and osa-miR166a-3p, while that in the different tissues of Lilium henryi Baker was osa-miR166g-3p and osa-miR166a-3p.Four important genes related to growth and development regulation, namely, osa-miR156a, osa-miR395b, osa-miR396a-3p, and osa-miR396a-5p, were selected for validation. The findings of the present study would contribute to future investigations on the miRNA expression and the related functions in Lilium henryi Baker while providing important references for the normalization of the miRNA expression in other varieties of lily.
Lilium is a large genus that includes approximately 110 species distributed throughout cold and temperate regions of the Northern Hemisphere. The species-level phylogeny of Lilium remains unclear; previous studies have found universal markers but insufficient phylogenetic signals. In this study, we present the use of complete chloroplast genomes to explore the phylogeny of this genus. We sequenced nine Lilium chloroplast genomes and retrieved seven published chloroplast genomes for comparative and phylogenetic analyses. The genomes ranged from 151,655 bp to 153,235 bp in length and had a typical quadripartite structure with a conserved genome arrangement and moderate divergence. A comparison of sixteen Lilium chloroplast genomes revealed ten mutation hotspots. Single nucleotide polymorphisms (SNPs) for any two Lilium chloroplast genomes ranged from 8 to 1,178 and provided robust data for phylogeny. Except for some of the shortest internodes, phylogenetic relationships of the Lilium species inferred from the chloroplast genome obtained high support, indicating that chloroplast genome data will be useful to help resolve the deeper branches of phylogeny.
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