Pyrethrum Research Articles

Understanding pyrethrin biosynthesis: toward and beyond natural pesticide overproduction.

Pyrethrins are natural insecticides biosynthesised by Asteraceae plants, such as Tanacetum cinerariifolium and have a long history, dating back to ancient times. Pyrethrins are often used as low-persistence and safe insecticides to control household, horticultural, and agricultural insect pests. Despite its long history of use, pyrethrin biosynthesis remains a mystery, presenting a significant opportunity to improve yields and meet the growing demand for organic agriculture. To achieve this, both genetic modification and non-genetic methods, such as chemical activation and priming, are indispensable. Plants use pyrethrins as a defence against herbivores, but pyrethrin biosynthesis pathways are shared with plant hormones and signal molecules. Hence, the insight that pyrethrins may play broader roles than those traditionally expected is invaluable to advance the basic and applied sciences of pyrethrins.

Nocturnal burst emissions of germacrene D from the open disk florets of pyrethrum flowers induce moths to oviposit on a nonhost and improve pollination success.

Recent studies show that nocturnal pollinators may be more important to ecosystem function and food production than is currently appreciated. Here, we describe an agricultural field study of pyrethrum (Tanacetum cinerariifolium) flower pollination. Pyrethrum is genetically self-incompatible and thus is reliant on pollinators for seed set. Our pollinator exclusion experiment showed that nocturnal insects, particularly moths, significantly contribute to seed set and quality. We discovered that the most abundant floral volatile, the sesquiterpene (-)-germacrene D (GD), is key in attracting the noctuid moths Peridroma saucia and Helicoverpa armigera. Germacrene D synthase (GDS) gene expression regulates the specific GD production and accumulation in flowers, which, in contrast to related species, lose the habit of closing at night. We did observe that female moths also oviposited on pyrethrum leaves and flower peduncles, but found that only a small fraction of those eggs hatched. Larvae were severely stunted in development, most likely due to the presence of pyrethrin defense compounds. This example of exploitative mutualism, which blocks the reproductive success of the moth pollinator and depends on nocturnal interactions, is placed into an ecological context to explain why it may have developed.

Extraction of Pyrethrins from Chrysanthemum cinerariifolium petals, its application as a Bioinsecticide and study of its effects on Rice Weevil Sitophilus oryzae

Extraction of Pyrethrins from Chrysanthemum cinerariifolium petals, its application as a Bioinsecticide and study of its effects on Rice Weevil Sitophilus oryzae

Local adaptation to the native environment affects pyrethrin variability in Dalmatian pyrethrum populations.

The insecticidal compound pyrethrin is synthesized in Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch.Bip.; Asteraceae), a plant species endemic to the eastern Mediterranean. Pyrethrin is a mixture of six compounds, pyrethrin I and II, cinerin I and II, and jasmolin I and II. For this study we sampled 15 natural Dalmatian pyrethrum populations covering the entire natural distribution range of the species; Croatian coastal regions and the islands, inland Bosnia and Herzegovina and Montenegro. The plants were grown in a field experiment under uniform growing conditions to exclude a short-term response to environmental factors and instead observe variation in pyrethrin content and composition among and within populations due to genetic adaptation to the native environment. The drivers of local adaptation were explored by examining the role of bioclimatic factors as a cause of population differentiation. Pyrethrins were extracted by ultrasound-assisted extraction, and the extracts were analyzed by HPLC-UV-DAD. The populations differed significantly in the content and composition of pyrethrins. The highest levels of total pyrethrins (1.27% flower DW), were found in population P14 Budva and the significantly highest levels of pyrethrin I in population P14 Vranjske Njive, Podgorica (66.47% of total pyrethrin). Based on bioclimatic conditions of the sampling sites, populations were grouped into five bioclimatic groups (A, B, C, D, and E), which showed qualitative and quantitative variability in pyrethrin content. The most abundant bioclimatic group was bioclimatic group E, which was characterized by the highest average values for pyrethrin I (53.87% of total pyrethrin), total pyrethrin content (1.06% flower DW) and the ratio of pyrethrin I and II (1.85). The correlation analysis between the pyrethrin compounds and some of the bioclimatic variables (e. g., BIO03 Isothermality and BIO04 Temperature seasonality) showed their significant contribution in explaining the variation of pyrethrins in T. cinerariifolium. The differences in pyrethrin content and composition may be partly due to genetic adaptation to the ecological conditions of the native environment. The obtained data would enable the selection of source populations for breeding programs aimed at producing cultivars with desirable biochemical properties and adaptation to different bioclimatic conditions.

Aphid alarm pheromone mimicry in transgenic Chrysanthemum morifolium: insights into the potential of (E)-β-farnesene for aphid resistance.

(E)-β-Farnesene (EBF) serves as the primary component of the alarm pheromone used by most aphid pest species. Pyrethrum (Tanacetum cinerariifolium) exhibits tissue-specific regulation of EBF accumulation and release, effectively mimicking the aphid alarm signal, deterring aphid attacks while attracting aphid predators. However, cultivated chrysanthemum (Chrysanthemum morifolium), a popular and economically significant flower, is highly vulnerable to aphid infestations. In this study, we investigated the high expression of the pyrethrum EBF synthase (TcEbFS) gene promoter in the flower head and stem, particularly in the parenchyma cells. Subsequently, we introduced the TcEbFS gene, under the control of its native promoter, into cultivated chrysanthemum. This genetic modification led to increased EBF accumulation in the flower stem and young flower bud, which are the most susceptible tissues to aphid attacks. Analysis revealed that aphids feeding on transgenic chrysanthemum exhibited prolonged probing times and extended salivation durations during the phloem phase, indicating that EBF in the cortex cells hindered their host-location behavior. Interestingly, the heightened emission of EBF was only observed in transgenic chrysanthemum flowers after mechanical damage. Furthermore, we explored the potential of this transgenic chrysanthemum for aphid resistance by comparing the spatial distribution and storage of terpene volatiles in different organs and tissues of pyrethrum and chrysanthemum. This study provides valuable insights into future trials aiming for a more accurate replication of alarm pheromone release in plants. It highlights the complexities of utilizing EBF for aphid resistance in cultivated chrysanthemum and calls for further investigations to enhance our understanding of this defense mechanism.

Establishment of an efficient genetic transformation system for Tanacetum cinerariifolium

The Dalmatian Daisy Tanacetum cinerariifolium is an Asteraceae plant species that produces the natural insecticide “pyrethrum”, which is effective against mosquito disease vectors and household pests. To enhance the content of pyrethrum in flowers, a more detailed understanding of the mechanisms underlying pyrethrum biosynthesis is needed. Even though gene transformation and genome editing techniques are vital for investigating pyrethrin biosynthesis, limited information is available on the transformation of T. cinerariifolium. Furthermore, each seedling possesses a distinct genotype with large variations by self-incompatibility. We herein employed T. cinerariifolium line #14 with weak self-incompatibility to establish a protocol of efficient regeneration from leaf segments and transformation. Leaf segments formed calli on 1/2 Murashige and Skoog’s basal medium (MS) with naphthalene acetic acid 1 mg L−1 and 6-benzylaminopurine (BAP) 2 mg L−1, regenerated shoots from calli on 1/2 MS with BAP 0.5 mg L−1 and GA3 0.2 mg L−1, and elongated shoot stems on 1/2 MS with indole-3-butyric acid 0.5 mg L−1 and BAP 0.5 mg L−1. To establish genetic transformation, Rhizobium radiobacter strain EHA105 with the highest infectivity and the mas1'-2' bidirectional promoter with the highest expression of the nptII resistance gene were used, and the antibiotic G418 was added to medium at a concentration of 10 to 20 mg L−1 to select transformed cells. Using established regeneration techniques, we successfully obtained transformants that highly expressed the transgene gusA. This technique will be useful for creating genetically modified T. cinerariifolium, particularly for elucidating the mechanism of pyrethrin biosynthesis toward the creation of pyrethrin-rich traits.

Exploring the co-operativity of secretory structures for defense and pollination in flowering plants.

In flowers multiple secretory systems cooperate to deliver specialized metabolites to support specific roles in defence and pollination. The collective roles of cell types, enzymes, and transporters are discussed. The interplay between reproductive strategies and defense mechanisms in flowering plants has long been recognized, with trade-offs between investment in defense and reproduction predicted. Glandular trichomes and secretory cavities or ducts, which are epidermal and internal structures, play a pivotal role in the secretion, accumulation, and transport of specialized secondary metabolites, and contribute significantly to defense and pollination. Recent investigations have revealed an intricate connection between these two structures, whereby specialized volatile and non-volatile metabolites are exchanged, collectively shaping their respective ecological functions. However, a comprehensive understanding of this profound integration remains largely elusive. In this review, we explore the secretory systems and associated secondary metabolism primarily in Asteraceous species to propose potential shared mechanisms facilitating the directional translocation of these metabolites to diverse destinations. We summarize recent advances in our understanding of the cooperativity between epidermal and internal secretory structures in the biosynthesis, secretion, accumulation, and emission of terpenes, providing specific well-documented examples from pyrethrum (Tanacetum cinerariifolium). Pyrethrum is renowned for its natural pyrethrin insecticides, which accumulate in the flower head, and more recently, for emitting an aphid alarm pheromone. These examples highlight the diverse specializations of secondary metabolism in pyrethrum and raise intriguing questions regarding the regulation of production and translocation of these compounds within and between its various epidermal and internal secretory systems, spanning multiple tissues, to serve distinct ecological purposes. By discussing the cooperative nature of secretory structures in flowering plants, this review sheds light on the intricate mechanisms underlying the ecological roles of terpenes in defense and pollination.

Evolution of the Genetic Structure of the Didymella tanaceti Population During Development of Succinate Dehydrogenase Inhibitor Resistance.

Emergence of pathogens with decreased sensitivity to succinate dehydrogenase inhibitor fungicides is a global agronomical issue. Analysis of Didymella tanaceti isolates (n = 173), which cause tan spot of pyrethrum (Tanacetum cinerariifolium), collected prior to (2004 to 2005) and after (2009, 2010, 2012, and 2014) the commercial implementation of boscalid in Tasmanian pyrethrum fields identified that insensitivity developed over time and has become widespread. To evaluate temporal change, isolates were characterized for frequency of mutations in the succinate dehydrogenase (Sdh) B, C, and D subunits associated with boscalid resistance, mating type, and SSR genotype. All isolates from 2004 and 2005 exhibited wild-type (WT) Sdh alleles. Seven known Sdh substitutions were identified in isolates collected from 2009 to 2014. In 2009, 60.7% had Sdh substitutions associated with boscalid resistance in D. tanaceti. The frequency of WT isolates decreased over time, with no WT isolates identified in 2014. The frequency of the SdhB-H277Y genotype increased from 10.7 to 77.8% between 2009 and 2014. Genotypic evidence suggested that a shift in the population structure occurred between 2005 and 2009, with decreases in gene diversity (uh; 0.51 to 0.34), genotypic evenness (E5; 0.96 to 0.67), genotypic diversity (G; 9.3 to 6.8), and allele frequencies. No evidence was obtained to support the rapid spread of Sdh genotypes by clonal expansion of the population. Thus, insensitivity to boscalid has developed and become widespread within a diverse population within 4 years of usage. These results suggest that D. tanaceti can disperse insensitivity through repeated frequent mutation, sexual recombination, or a combination of both.

Investigation Of The Biosynthesis Pathway That Generates cis-Jasmone.

Researchers have established that (+)-7-iso-jasmonic acid ((+)-7-iso-JA) is an intermediate in the production of cis-jasmone (CJ); however, the biosynthetic pathway of CJ has not been fully described. Previous reports stated that CJ, a substructure of pyrethrin II produced by pyrethrum (Tanacetum cinerariifolium), is not biosynthesized through this biosynthetic pathway. To clarify the ambiguity, stable isotope-labelled jasmonates were synthesized, and compounds were applied to apple mint (Mentha suaveolens) via air propagation. The results showed that cis-jasmone is not generated from intermediate (+)-7-iso-JA, and (+)-7-iso-JA is not produced from 3,7-dideydro-JA (3,7-ddh-JA); however, 3,7-didehydro-JA and 4,5-didehydro-7-iso-JA were converted into CJ and JA, respectively.

Pyrethrins Profiles and Contents in Seedlings, in Vitro Cultures, and Acclimatized Plants of Tanacetum cinerariifolium Clones (Asteraceae)

Tanacetum cinerariifolium (Trevir.) Sch. Bip., Asteraceae, known as pyrethrum, is a perennial herb endemic to the eastern Adriatic coast. It is cultivated worldwide for its valuable secondary metabolites: pyrethrins, which are natural insecticides. Pyrethrins are monoterpene esters of chrysanthemum acid (Pyrethrin-I, Cinerin-I, Jasmolin-I) or pyrethric acid (Pyrethrin-II, Cinerin-II, Jasmolin-II) with lethal or knock-down insecticidal properties, respectively. The market demand of pyrethrin-based products is increasing due to the organic farming requirements. In the present study the content of pyrethrins was determined by GC/MS in n-hexane extracts of shoots of several in vitro clones and in flower heads of outdoor acclimatized plants from the same clones during primary and secondary blooming, as well as in seedlings grown on different soil types. Statistical analyses were done by One-way ANOVA Post Hoc LSD analysis. All pyrethrins were detected in all samples, including a Pyrethrin-I isomer but they differed in content and proportion. As expected, pyrethrins in in vitro shoots and in seedlings were in small amounts. However, in the flower heads of the acclimatized plants, pyrethrin content was comparable to that of the native Dalmatian populations. The main component in all samples was Pyrethrin-I, possessing the strongest insecticidal activity, with the highest content in the flower heads of the primary flowering: 4.87 mg/g with a CV of 24.9%. Statistical differences were noted between clones for Pyrethrin-II and the Pyrethrin-I isomer in June samples, and Pyrethrin-I in September samples (p < 0.05). A wide range of variation was also found in seedlings due to different soil types. Growing conditions should be optimized in terms of sunlight and watering, and further research on pyrethrin content should be carried out in several consecutive years.

Synergistic effect of the combination of Chrysanthemum cinerariifolium (Trev.) and doxorubicin in inhibiting PI3K and Cyclin D in oral squamous cell carcinoma: in vitro study

Background: The most common type of lips and oral cavity cancer is oral squamous cell carcinoma (OSCC). Doxorubicin (DX) is commonly used as a chemotherapy drug, but its use is limited due to risk factors and drug resistance. Chrysanthemum cinerariifolium (Trev.) (CC) has potential as an anticancer agent. Combining the plant extract and chemotherapy drug might prevent OSCC proliferation by inhibiting PI3K and cyclin D protein. Therefore, the present study aimed to determine the synergistic effect of the combination of C. cinerariifolium (Trev.) and doxorubicin in inhibiting PI3K and Cyclin D protein. Methods: Human oral squamous carcinoma cell lines SCC-9 were used in this study. A cytotoxicity assay was performed to obtain the IC50 value of CC ethanol extract and DX on the SCC-9 cell line. Synergism evaluation of the combination CC and DX was analyzed using CompuSyn software. ELISA and the immunofluorescent assay were performed to determine the level of PI3K and cyclin D in the SCC-9 cell line after being treated with IC50 value of CC, IC50 value of DX and three combinations of CC and DX [7/8 IC50 CC + 1/8 IC50 DX (dose 1), 6/8 IC50 CC + 2/8 IC50 DX (dose 2), and 4/8 IC50 CC + 4/8 IC50 DX (dose 3). Results: CC stem ethanol extract and DX inhibited the proliferation of SCC-9 cell lines with the IC50 value of 133.4 µg/mL and 288.3 nM, respectively. The combination of CC and DX at dose 2 (6/8 IC50 CC + 2/8 IC50 DX) exhibited a high decrease in PI3K and cyclin D expression. Conclusions: The combination of C. cinerariifolium and doxorubicin synergistically declined OSCC proliferation by inhibiting PI3K and cyclin D expression.

Confirmation of Itersonilia perplexans infecting pyrethrum (Tanacetum cinerariifolium) in Australia.

Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.) is grown to extract pyrethrins which are active ingredients for insecticides (Greenhill 2007). The Australian pyrethrum industry supplies over 50% of the world market. Surveys of Tasmanian crops in spring 2013, detected the presence of a fungus putatively identified as Itersonilia perplexans Derx. on foliage in 54 of 86 surveyed fields (Hay et al. 2015). This fungus was associated with necrotic leaf tips often spreading to encompass whole leaves. However, pathogenicity to pyrethrum was not confirmed. To isolate, tissue was excised from foliar lesions, surface sterilised using 0.4% NaClO, placed onto 2% water agar and incubated at 20°C for 5 days. Colonies were pure-cultured by hyphal-tip transfer onto potato-dextrose agar. Eleven isolates were cultured onto yeast mold agar (YMA) for 14 days at 15°C in the dark (Horita and Yasuoka 2002). Colonies were slow growing (1.9 to 2.3 mm/day) white to buff on both surfaces, with a darker center visible on lower surfaces. Mycelia were straight and hyaline with clamp connections at the septa. Squares transferred from the edge of YMA colonies onto microscope slides produced ballistoconidia that were aseptate, granular and lunate, kidney or lemon-shaped after 24 h. Ballistoconidia lengths and widths (n = 50/isolate) ranged from 14.6 to 20.4 µm and 10.0 to 13.6 µm. Chlamydospores were not observed. These observations were consistent with descriptions of I. perplexans (Koike and Tjosvold 2001; Liu et al. 2015). All 11 isolates were sequenced across the internal transcribed spacer (ITS) region of rDNA (ITS; primers V9G/ITS4; de Hoog and van den Ende 1998; White et al. 1990), and large (LSU; primers LROR/LR7; Rehner and Samuels 1995), and small (SSU; NS1/NS4; White et al. 1990) subunits of rDNA (Genbank accession nos. KU563626 to KU563658). The ITS (673 bp), SSU (1,047 bp) and LSU (1,318 bp) differed by 3, 1 and 0 bp, respectively, across isolates. Maximum parsimony and maximum likelihood analyses of a concatenated 3 loci alignment with Cystofilobasidiales representatives (Liu et al. 2015) placed all isolates and the I. perplexans ex-neotype strain CBS 363.85 within a single monophyletic clade with 100% bootstrap support. Two representative isolates are stored at the Plant Pathology Herbarium (accession nos. BRIP 57986 and 57987). Leaves of 46-day-old pyrethrum plants (n = 45), generated from surface sterilised seed, were inoculated with a 1.5 × 105 ballistoconidia/ml suspension (equal mix of eight isolates) and maintained between 10 and 22°C under a 12-h photoperiod for 14 days. Brown necrotic leaf tips, consistent with reported field symptoms were observed on 71% of plants and I. perplexans was recovered from 69% of symptomatic plants. For flower inoculations, pyrethrum plants were removed from fields as vegetative plants in spring and maintained in a greenhouse set at 20:14°C and 14:10 h day:night. Open flowers (10 per plant) were dipped into a 1.2 × 104 ballistoconidia/ml suspension mix of three isolates. Brown withered ray florets were observed on 10/12 plants 18 days post-inoculation, matching those described in petal blight of chrysanthemum (McRitchie et al. 1973). I. perplexans was re-isolated from 11/12 inoculated plants and 1 control plant (of 12) which exhibited the same symptoms. In both experiments, I. perplexans was identified based on its distinctive morphology. This confirms the pathogenicity of I. perplexans to both pyrethrum leaves and flowers.

TcWRKY75 participates in pyrethrin biosynthesis by positively regulating the expression of TcCHS, TcAOC, and TcGLIP in Tanacetum cinerariifolium

Pyrethrin, a combination of six esters, is a well-known kind of natural insecticide extracted primarily from the flowers of Tanacetum cinerariifolium. Yet the regulatory network of the pyrethrin biosynthetic pathway remains unclear. In this study TcWRKY75 was cloned. This gene exhibited an expression pattern similar to that of some genes in the pyrethrin biosynthesis pathway at different stages of flower development. Transient overexpression of TcWRKY75 in T. cinerariifolium significantly increased its pyrethrin content; conversely, transient silencing of TcWRKY75 decreased its pyrethrin content. Further, in the TcWRKY75-overexpressing transgenic hairy-root lines, the expression levels of TcCHS and TcAOC were both significantly upregulated. Follow-up investigation revealed that the protein encoded by TcWRKY75 positively regulated the expression of TcCHS, TcGLIP, and TcAOC by directly binding to the W-box in their promoters. These results demonstrate that TcWRKY75 functions as a positive regulator in the pyrethrin biosynthesis pathway.

Combined heat and water stress leads to local xylem failure and tissue damage in pyrethrum flowers.

Flowers are critical for angiosperm reproduction and the production of food, fibre and pharmaceuticals, yet for unknown reasons they appear particularly sensitive to combined heat and drought stress. A possible explanation for this may be the co-occurrence of leaky cuticles in flower petals and a vascular system that has a low capacity to supply water and is prone to failure under water stress. These characteristics may render reproductive structures more susceptible than leaves to run-away cavitation - an uncontrolled feedback cycle between rising water stress and declining water transport efficiency that can rapidly lead to lethal tissue desiccation. We provide modelling and empirical evidence to demonstrate that flower damage in the perennial crop pyrethrum (Tanacetum cinerariifolium), in the form of irreversible desiccation, corresponds with run-away cavitation in the flowering stem after a combination of heat and water stress. We show that tissue damage is linked to greater evaporative demand during high temperatures rather than direct thermal stress. High floral transpiration dramatically reduced the soil water deficit at which run-away cavitation was triggered in pyrethrum flowering stems. Identifying run-away cavitation as a mechanism leading to heat damage and reproductive losses in pyrethrum provides different avenues for process-based modelling to understand the impact of climate change on cultivated and natural plant systems. This framework allows future investigation of the relative susceptibility of diverse plant species to reproductive failure under hot and dry conditions.

Two New Cytotoxic Sesquiterpene-Amino Acid Conjugates and a Coumarin-Glucoside from Crossostephium chinense.

The Asteraceae family is a promising source of bioactive compounds, such as the famous Asteraceae plants Tanacetum cinerariifolium (pyrethrin) and Artemisia annua (artemisinin). As a result of our series of phytochemical studies of the subtropical plants, two novel sesquiterpenes, named crossoseamines A and B in this study (1 and 2, respectively), one undescribed coumarin-glucoside (3), and eighteen known compounds (4-21) were isolated from the aerial part of Crossostephium chinense (Asteraceae). The structures of isolated compounds were elucidated by spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, HMBC, and NOESY), IR spectrum, circular dichroism spectrum (CD), and high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). All isolated compounds were evaluated for their cytotoxic activities against Leishmania major, Plasmodium falciparum, Trypanosoma brucei (gambiense and rhodesiense), and human lung cancer cell line A549 because of the high demand for the discovery of new drug leads to overcome the present side effects and emerging drug-resistant strains. As a result, the new compounds (1 and 2) showed significant activities against A549 (IC50, 1: 3.3 ± 0.3; 2: 12.3 ± 1.0 μg/mL), L. major (IC50, 1: 6.9 ± 0.6; 2: 24.9 ± 2.2 μg/mL), and P. falciparum (IC50, 1: 12.1 ± 1.1; 2: 15.6 ± 1.2 μg/mL).

In vitro clonal propagation of Tanacetum cinerariifolium and establishment of an ex situ collection of selected clones

Dalmatian pyrethrum Tanacetum cinerariifolium (Trevir.) Sch. Bip. (Asteraceae) is a perennial herb endemic to the eastern coast of the Adriatic Sea. The species is widely cultivated in many countries for its bioactive compounds pyrethrins, which are used as natural insecticides. Plants derived from seeds vary greatly in pyrethrin content; therefore, the vegetative propagation of high-quality individuals is very important for the establishment of agricultural pyrethrum crops. The present study deals with rapid in vitro multiplication of pyrethrum, ex vitro adaptation of selected clones and creation of an ex situ collection, as a first step towards introducing the species into agriculture in Bulgaria. Seeds from a private ex situ collection in Bulgaria and from a natural Croatian population were used as initial material for in vitro cultures initiation. Basal MS medium (Murashige and Skoog 1962) or MS supplemented with different concentrations of kinetin and indole-3-butyric acid were used for seed germination and multiplication of one-seed derived clones by consecutive subcultivations. The propagation effectiveness was evaluated as a number of new plants obtained per initial shoot. Considerable losses were noticed due to both endophytic contaminations and necrosis, especially on media supplemented with plant growth regulators. These problems were overcome by medium optimization: adding an antibiotic and modifying the medium to increase the calcium concentration using CaCO3. In the best medium variant (basal MS + 200 mg/L Medaxone + 75 mg/L Ca) no more infected plants were observed, and the percentage of necrotic plants decreased threefold, which resulted in formation of 38.06±10.11 new plants per initial shoot for a period of 7 months. Three hundred and sixty plants were ex vitro adapted in a phytotron (88% surviving rate), then 16 plants from 4 selected clones were transferred to the ex situ collection and bloomed twice from the very first growing season (June and September). The number of the flower heads increased in the second year of field cultivation and an average of 328±138 capitula per plant were counted for the best clone. The first trials to establish a pilot plantation of pyrethrum are promising.

Analysis of the Metabolite Compound of the Ethanol Extract of Chrysanthemum cinerariifolium Stem and Activity for inhibition of Oral Squamous Cell Carcinoma (OSCC) in silico study

Analysis of the Metabolite Compound of the Ethanol Extract of Chrysanthemum cinerariifolium Stem and Activity for inhibition of Oral Squamous Cell Carcinoma (OSCC) in silico study

TcbHLH14 a Jasmonate Associated MYC2-like Transcription Factor Positively Regulates Pyrethrin Biosynthesis in Tanacetum cinerariifolium.

Natural pyrethrins have high application value, and are widely used as a green pesticide in crop pest prevention and control. Pyrethrins are mainly extracted from the flower heads of Tanacetum cinerariifolium; however, the natural content is low. Therefore, it is essential to understand the regulatory mechanisms underlying the synthesis of pyrethrins through identification of key transcription factors. We identified a gene encoding a MYC2-like transcription factor named TcbHLH14 from T. cinerariifolium transcriptome, which is induced by methyl jasmonate. In the present study, we evaluated the regulatory effects and mechanisms of TcbHLH14 using expression analysis, a yeast one-hybrid assay, electrophoretic mobility shift assay, and overexpression/virus-induced gene silencing experiments. We found that TcbHLH14 can directly bind to the cis-elements of the pyrethrins synthesis genes TcAOC and TcGLIP to activate their expression. The transient overexpression of TcbHLH14 enhanced expression of the TcAOC and TcGLIP genes. Conversely, transient silencing of TcbHLH14 downregulated the expression of TcAOC and TcGLIP and reduced the content of pyrethrins. In summary, these results indicate that the potential application of TcbHLH14 in improving the germplasm resources and provide a new insight into the regulatory network of pyrethrins biosynthesis of T. cinerariifolium to further inform the development of engineering strategies for increasing pyrethrins contents.

Chrysanthemum cinerariifolium (Trev.) Vis leaves extract reduces the level of Macrophages, Interleukin-17, and Keratinization in Oral squamous cell carcinoma rat model

Chrysanthemum cinerariifolium (Trev.) Vis. leaves have potential anticancer activity and are expected to inhibit oral squamous cell carcinoma (OSCC) progression. The present study aimed to determine the effect of ethanol extract of C. cinerariifolium leaves on the number of macrophages, IL-17 levels, and degree of keratinization in the OSCC rat model. Thirty male Sprague Dawley rats (weighing 80-160g, 2-3 months old) were randomly divided into five groups (n=6): (1) negative control (healthy rat, untreated); (2) positive control (OSCC rat); (3-5) dose 1, 2 and 3 (OSCC rat received 50, 100 and 200mg/kg BW of C. cinerariifolium leaves, respectively). The tongues of the rats were prepared for Hematoxylin-Eosin (HE) staining and immunohistochemistry. The number of macrophages was determined under a light microscope with 400x magnification. The level of IL-17 was examined using an ELISA assay. The degree of keratinization was calculated using the ImageJ software. All doses of C. cinerariifolium leaves extract could reduce the number of macrophages in OSCC rats. The levels of IL-17 in OSCC rats were decreased after being treated with 100 and 200mg/kg BW of C. cinerariifolium leaves extract. The high dose of C. cinerariifolium leaves extract (200mg/kg BW) reduced the degree of keratinization in the OSCC rat model. The study revealed that C. cinerariifolium leaves extract could inhibit the progression of OSCC by reducing macrophage, IL-17 and keratinization.

Globisporangium and Pythium Species Associated with Yield Decline of Pyrethrum (Tanacetum cinerariifolium) in Australia

Pyrethrum (Tanacetum cinerariifolium) cultivation in Australia, which accounts for the majority of global production of natural insecticidal pyrethrins, is affected by a persistent yield decline which in part is caused by a complex of pathogens. Globisporangium and Pythium species were isolated from crown and roots of pyrethrum plants showing stunting and brown discoloration of crown tissue, and from soil adjacent to diseased plants from yield-decline-affected sites in Tasmania and Victoria, Australia. Ten known Globisporangium species (Globisporangium attrantheridium, G. erinaceum, G. intermedium, G. irregulare, G. macrosporum, G. recalcitrans, G. rostratifingens, G. sylvaticum, G. terrestris and G. ultimum var. ultimum), two new Globisporangium species (Globisporangium capense sp. nov. and Globisporangium commune sp. nov.) and three Pythium species (Pythium diclinum/lutarium, P. tracheiphilum and P. vanterpoolii) were identified through morphological studies and multigene phylogenetic analyses using ITS and Cox1 sequences. Globisporangium ultimum var. ultimum, G. sylvaticum, G. commune sp. nov. and G. irregulare were most abundant. Globisporangium attrantheridium, G. macrosporum and G. terrestris were reported for the first time in Australia. Seven Globisporangium species were pathogenic on both pyrethrum seeds (in vitro assays) and seedlings (glasshouse bioassays), while two Globisporangium species and three Pythium species only caused significant symptoms on pyrethrum seeds. Globisporangium irregulare and G. ultimum var. ultimum were the most aggressive species, causing pyrethrum seed rot, seedling damping-off and significant plant biomass reduction. This is the first report of Globisporangium and Pythium species causing disease in pyrethrum globally and suggests that oomycete species in the family Pythiaceae may have an important role in the yield decline of pyrethrum in Australia.