Pyrethrum Plants Research Articles

Paraphoma Crown Rot of Pyrethrum (Tanacetum cinerariifolium).

Pyrethrum (Tanacetum cinerariifolium) is commercially cultivated for the extraction of natural pyrethrin insecticides from the oil glands inside seed. Yield decline has caused significant yield losses in Tasmania during the last decade. A new pathogen of pyrethrum causing crown rot and reduced growth of the plants in yield decline affected fields of northern Tasmania was isolated from necrotic crown tissue and described as Paraphoma vinacea. Multigene phylogenetic identification of the pathogen also revealed that P. vinacea was a new species different from other Paraphoma type strains. Glasshouse pathogenicity experiments showed that P. vinacea significantly reduced belowground and total biomass of pyrethrum plants 2 months after inoculation. Dull-tan to reddish-brown discoloration of the cortical and subcortical crown tissue was observed in 100% of the infected plants. P. vinacea infected 75% of the plants inoculated with root dip and soil drench inoculation techniques in an inoculation optimization experiment. P. vinacea, the causal agent of Paraphoma crown rot disease, represents an important pathogen that will negatively impact the commercial cultivation of pyrethrum in Tasmania.

Development of a multiplex PCR diagnostic assay for the detection of Stagonosporopsis species associated with ray blight of Asteraceae

Ray blight, a destructive disease of Asteraceae worldwide, is caused by three morphologically similar but phylogenetically distinct species; Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti. Stagonosporopsis chrysanthemi has been reported as a specific pathogen of chrysanthemum while S. inoxydabilis has been found associated with various Asteraceae. Stagonosporopsis tanaceti has only been reported in Australia, causing substantial crop loss on pyrethrum. All three species were shown to infect and cause disease on in vitro grown pyrethrum plants, hence, S. chrysanthemi and S. inoxydabilis may pose a significant biosecurity threat to the Australian pyrethrum industry. All these Stagonosporopsis species are also Level 2 quarantine pathogens in Europe. Rapid and accurate detection and differentiation of these species is a priority for ray blight management in Australia and in Europe. Accordingly, three species-specific PCR-based assays, targeted to the intergenic spacer of the nuclear ribosomal DNA, were developed. The specificity of each assay was confirmed against 21 Stagonosporopsis spp. as well as 14 pathogenic and saprophytic fungal species commonly found in association with pyrethrum in Australia. The primers were highly sensitive and specific to the target species, detecting down to 4 fg of genomic DNA. These primers were further used in a multiplex PCR to differentiate the presence of the three Stagonosporopsis spp. based on variable sized amplicons in a single reaction.

Histopathology of S. tanaceti infection in pyrethrum leaf lamina

The infection process and life cycle of S. tanaceti in leaf lamina of pyrethrum plants was investigated using histopathology. Conidia attached firmly to the leaf surface before the infection hyphae penetrated directly into the epidermal cells of the leaf without forming appressoria. The maximum germination of conidia on leaf surface was 85 % at 54 HAI. Infection hyphae infected the epidermal and palisade parenchyma cells through the middle lamella. Brown lesions on the leaf were a result of infected necrotic epidermal cells. Extensive colonization through both intra- and intercellular hyphae along with pycnidia formation caused enormous damage to the infected cells at 12 DAI. Unlike the quadruple stain, both single and dual stains had very limited ability to visualise infection structures. These results have provided a better understanding of the physical interaction between the pathogen and the pyrethrum leaf tissues and will help to elucidate the complete disease cycle of S. tanaceti on pyrethrum plant.

Micropropagation of Anacyclus pyrethrum and chemical profiling of the regenerated plants for pellitorine, the active principle

The paper reports the development of a protocol for large-scale micropropagation of Anacyclus pyrethrum, an invaluable medicinal plant, and the presence of pellitorine, the active principle, in leaves and roots of regenerated plants. Three explants—cotyledonary nodes, hypocotyls and cotyledons—were compared for their shoot development/differentiation ability on Murashige and Skoog’s (MS, Physiol Plant 15:473–497, 1962) medium supplemented with 1–10 µM of 6-benzylaminopurine (BAP) or kinetin (Kn). The best response (8.88 ± 0.28 shoots per explant) was elicited from the cotyledonary nodal explants on MS + 2.5 µM Kn. The in vitro regenerated shoots continued to multiply on being sub-cultured on the same medium. Elongated shoots were cultured on MS medium augmented with different concentrations (0.2–15 µM) of 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for induction of roots. The media augmented with 5 or 10 µM IAA or 5 µM NAA were the most effective for the initiation and proliferation of roots. The plantlets were successfully transferred to the following three potting mixtures: garden soil, vermiculite and garden soil (1:1), vermiculite, garden soil and perlite (1:1:1). Among these, the maximum number (75 %) of the transferred plants survived in the garden soil. The high performance liquid chromatographic analyses of roots and leaves of the regenerated plants as well as those from the native habitat revealed the presence of pellitorine in all, with the content being higher in the roots than the corresponding leaves. The developed protocol would be of use for production of A. pyrethrum plants throughout the year, and thus could become a perennial source of the herb and its active principle.

Elemental Composition and Chemometric Characterization of Pyrethrum Plant Materials and Insecticidal Flower Extracts

The elemental composition of native and cultivated pyrethrum plants (Tanacetum cinerariifolium) and their insecticidal extracts obtained from dried flower heads was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The accuracy of the analytical procedure which included pressurized digestion of plant samples in HNO3/H2O2 was tested by certified reference materials. Acceptable recoveries (90–110%) for the majority of elements were obtained measured in a simultaneous multi-element mode. The total pyrethrins content was determined by HPLC and it was 2.09% for cultivated plants and 1.58% for native species. The concentrations of 18 elements and pyrethrin content were subjected to statistical multivariate analysis which showed different patterns among plant parts. The roots were characterized by a combination of loadings of iron, aluminum, nickel, chromium, strontium, and barium, while the stems, leaves, and flowers showed more contributions from sodium, potassium, calcium, magnesium, phosphorus, sulfur, manganese, and copper. A higher loading of pyrethrins was the main characteristic of dried flower extracts. It was also observed that microelements such as copper, aluminum, and iron were more readily transferred into natural insecticide extracts.

Interactions between waterlogging and ray blight in pyrethrum

The effects of waterlogging, alone and combined with ray blight disease (caused by Stagonosporopsis tanaceti), on pyrethrum (Tanacetum cinerariifolium) plant growth were quantified in glasshouse trials. Six pyrethrum cultivars were initially studied for their response to 6 days of waterlogging and their recovery from waterlogging during 26 days post-waterlogging. Waterlogging caused substantial root death and leaf wilting and accelerated senescence in all cultivars. Root growth was 80% more reduced than shoot growth. Cultivar ‘F’ showed significantly higher root porosity and growth following waterlogging than other cultivars. In contrast, cv. ‘C’ had the greatest growth reduction from waterlogging and poor root-system recovery after waterlogging. Plants of cvv. C and F inoculated with S. tanaceti and then waterlogged were more significantly affected than were those exposed to waterlogging only. For both cultivars, shoot growth under the combined treatment, relative to initial growth, recovered up to 25%, but root growth suffered irreversible damage. The combined treatment decreased the number of stems by 39% compared with waterlogging alone after the post-waterlogging period. In conclusion, pyrethrum cultivars showed differential reactions to waterlogging; but growth in all cultivars was seriously affected by a combination of waterlogging and infection by ray blight.

First Report of Boeremia Blight Caused by Boeremia exigua var. exigua on Pyrethrum in Australia.

Pyrethrum (Tanacetum cinerariifolium) is produced for extraction of insecticidal compounds from the flower achenes. In 2004 and 2006, isolations from necrotic lesions on stems and leaves in three fields in northern Tasmania, Australia yielded four unidentified fungal isolates. Leaf lesions were medium brown and circular (2 to 4 mm in diameter) or irregular in shape (2 to 5 mm long). Stem lesions were irregular, necrotic spots, 5 to 15 mm below the flower peduncle, medium brown, 2 to 4 mm long, and 1 to 2 mm wide. Isolations were conducted on water agar following surface sterilization. Isolates were identified by colony characteristics and the presence of metabolite 'E' (1). On oatmeal agar (OA), colonies had irregular margins, were greenish olivaceous-to-olivaceous gray with sparse, white, floccose, aerial mycelia. On malt extract agar (MEA), cultures were variable in color with olivaceous black centers with soft, dense, aerial mycelia. Conidia were hyaline, ellipsoidal to oblong, mainly aseptate, but occasionally 1-septate with dimensions ranging from 2.5 to 7.5 × 1.8 to 3.8 μm (length/width ratio = 1.7 to 2.1). All isolates had moderate reactions to the NaOH test for metabolite 'E'. DNA was extracted from all four isolates with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA). For identification, the internal transcribed spacer region (ITS1, 5.8s, and ITS2) and part of the translation elongation factor (TEF) region were amplified and sequenced. Primers ITS1 and ITS4 (2) were used for the ITS region and primers EFCF1 (5'-AGTGCGGTGGTATCGACAAG) and EFCF6 (3'-CATGTCACGGACGGCGAAAC) were used for the TEF. Amplicons were sequenced in both directions and consensus sequences assembled. The ITS sequence was 100% identical to Boeremia exigua var. exigua (GenBank Accession No. GU237715). Base pairs 413 to 1,214 of the TEF sequence from the pyrethrum isolates matched base pairs 1 to 802 (799 of 802 identities) of B. exigua var. exigua (GenBank Accession No. GU349080). All isolates were confirmed as B. exigua var. exigua using morphology and sequencing. Pathogenicity tests were conducted three times in separate glasshouse trials for two of the four isolates. For each isolate, conidial suspensions in water (3 ml/plant) from MEA, adjusted to 5 × 105/ml were applied with Tween 20 (1 drop per 100 ml of water) to 8-week-old pyrethrum plants (five pots per isolate with four plants per pot) using a hand-held spray bottle. Twenty plants were sprayed with water and Tween 20 as nontreated controls. Plants were covered with plastic bags for 48 h after inoculation and examined for symptoms after 15 days at 20°C. Disease incidence (number of symptomatic leaves affected per total number of leaves) of the inoculated plants varied from 7.5 to 9.4%. Noninoculated plants did not develop symptoms. Isolations resulted in cultures morphologically identical on MEA and OA to those inoculated. To our knowledge, this is the first report of B. exigua var. exigua causing disease in pyrethrum. Cultures were deposited in the New South Wales Department of Agriculture collection (DAR79101 to 79104) and TEF and ITS sequences for DAR79101 in GenBank (Accession Nos. JF925328 and JF925329, respectively). Boeremia blight is likely to contribute to the fungal disease complex causing reductions in green leaf area in Australian pyrethrum production.

Comparative Brine Shrimp Toxicity of <i>Withenia somnifera</i> and <i>Chrysanthemum cinerariaefolium</i> Extracts and <i>In Vitro</i> Antimicrobial Activity of <i>W. Somnifera</i>

This study reports on the bioactivity and in vitro antimicrobial action of methanol extract of Withania somnifera using in vitro cultures of Staphylococcus aureus, Escherichia coli, Candida albicans, Salmonella typhimurium and Pseudomonas aeruginosa . The extract of W. somniferum inhibited the growth of S. aureus and C. albicans but not E. coli, S. typhimurium and P. aeruginosa. The diameters of the zones of inhibitions of microbial growth ranged from 14 mm to 16 mm. The results of antimicrobial testing of W. somnifera extract indicated that it has antimicrobial properties, which support its continued use as a herbal remedy. Comparative toxicity of extracts of W. somnifera and Chrysananthemum cinerariaefolium (pyrethrum plant) was done using the brine shrimp lethality test at serial dilutions of 1000 μg/ml, 100 μg/ ml and 10 μg/ml. The median lethal concentration (LC50) for each extract was determined. Both plant extracts were found to be toxic to brine shrimp with LC50 of 110.3 μg/ml and 106.6 μg/ml for W. somnifera and C. cinerarieafolium respectively. Kenya Veterinarian Vol. 30 (2) 2006: pp. 53-56

Effect of ethrel, chlormequat chloride and paclobutrazol on growth and pyrethrins accumulation in Chrysanthemum cinerariaefolium Vis.

Pyrethrins and flower yield of pyrethrum (Chrysanthemum cinerariaefolium Viz.) plants were determined after application of ethrel, chlormequat chloride and paclobutrazol. Ethrel at 50, 100, 250 and 500 mg l−l produced a significant positive effect on pyrethrins level, decreased plant height, while 50 and 100 mg l−l significantly increased fresh and dry flower yield. Chlormequat chloride at 1000 and 2000 mg l−l and paclobutrazol (80 and 160 mg l−l) increased pyrethrins level, single flower weight and decreased plant height and flower yield. 14C-acetate incorporation studies further substantiated positive effect of growth retardants on pyrethrins biosynthesis. The effect of growth retardants on pyrethrins seems to be mediated through its effect on biosynthesis.

Pathogenicity of fungi commonly isolated from foliar disease in Tasmanian pyrethrum crops

The pathogenicity of Alternaria alternata, A. tenuissima, Stemphylium botryosum, Cladosporium cladosporioides and Ulocladium atrum commonly associated with diseased foliage of pyrethrum (Tanacetum cineariifolium) was characterised by inoculating glasshouse-grown pyrethrum plants in three replicated experiments. In the first experiment only one cultivar was used, whilst in the remaining two experiments the susceptibility of cultivars was evaluated. Inoculation with A. alternata, C. cladosporioides and U. atrum failed to induce disease in any cultivar. Inoculation with A. tenuissima resulted in the development of necrotic spots which coalesced to cover large areas of the leaves in all cultivars, whereas foliage of those inoculated with S. botryosum developed necrotic halos surrounded by a pink/brown margin. No significant differences were detected in disease severity following inoculation with A. tenuissima or S. botryosum between the four cultivars commonly grown in Tasmania. However, some significant differences were noted between cultivars in the number of lesions per leaf following inoculation with S. botryosum.

Susceptibility of pyrethrum and opium poppy to tomato spotted wilt tospovirus infection

Artificial glasshouse challenges of a range of commercial cultivars and breeding lines of opium poppy and pyrethrum with a selection of tomato spotted wilt tospovirus isolates were carried out. Infection of both pyrethrum and poppy was shown. In pyrethrum, evidence for erratic virus distribution, limited systemic infection or reduced virus titre in some tissues was shown. Neither species showed symptoms of infection nor was any effect on plant growth observed. Testing of a small number of field pyrethrum plants indicated natural infection may occur.

The life history of chrysanthemum thrips, Thrips nigropilosus Uzel (Thripidae: Thysanoptera) and an evaluation of the effects of temperature on its life cycle

The biology of Thrips nigropilosus Uzel was studied in a laboratory. The life history was examined at 20°C as well as the effect of temperatures on its life cycle at 12, 20, 25 and 30°C with fixed 70% r.h. The thrips pass through egg, two larval instars, prepupae and pupae to become adults. The development of these pre-adults occur on the pyrethrum plants except that prepupal and pupal stages occur in the soil. The emerged adults display sexual dimorphism, with lower ratio of male to female (2:11) and adult longevity range of 1–21 days. The thrips development duration decreased with increasing temperatures. When expressed as rates, it was found out that faster development occurred for the second larval and pupal instars than other stages with an increase in temperature. The optimum temperature for the thrips development was 20°C, mortality being higher at other temperatures.

A large-scale application of tissue culture: The mass propagation of pyrethrum clones in Ecuador

Developments in plant tissue culture techniques have raised expectations regarding the possibility of their practical applications on a commercial scale. However, serious doubts have been voiced regarding the economics and technical feasibility of a scale-up of the tissue culture techniques for plant multiplication at levels of thousands of plants per day. Indeed, it has been stated that most probably the same targets can be achieved generally, through conventional vegetative propagation, and that the tissue culture techniques belong mainly into the research laboratory. The first large-scale, commercial application on in vitro cultures for the mass propagation of clones of pyrethrum plants in reported here. Considerable economic advantages have been found in this application of tissue culture as compared to conventional means of vegetative propagation only. The current rate of plant propagation in this operation is of the order of 2000 plants per day at the tissue culture level which, combined with one last step of conventional propagation has given a net over-all propagation rate of nearly 1,000,000 plants per month. It is concluded that for this particular application, the scale-up of tissue culture techniques has been proven technically possible, economically feasible and is indeed being considered as the key to continued success of the commercial pyrethrum production of Ecuador.

Biosynthesis of the pyrethrins.

RECENT work1 has indicated that mevalonic acid (I) is an intermediate in the biosynthesis of terpenoid compounds. We now wish to report some experiments which indicate that this acid is also involved in the biosynthesis of the ‘pyrethrins’, the insecticidal constituents of the pyrethrum plant, Chrysanthemum cinerariaefolium. The ‘pyrethrins’ are a mixture of four closely related esters2 derived from two acids, chrysanthemum monocarboxylic acid (II, R = —Me) and pyrethric acid (II, R = —COOMe), and two keto-alcohols, pyrethrolone (III, R = —CH = CH2) and cinerolone (III, R = —CH3). It is customary to refer to the mixture of the two esters derived from chrysanthemum monocarboxylic acid and the keto-alcohols as ‘pyrethrin I’ and to that from pyrethric acid as ‘pyrethrin II’. The two acids have carbon skeletons which can be built up from two isoprene units, and thus might be expected to be derived from mevalonic acid. The two keto-alcohols cannot be so regarded, but it is of interest to note that the methylpentenone ring could be formed in theory by the cyclization of mevalonic acid. We have now shown that this theoretically attractive route is not employed by the plant.

PYRETHRUM CULTIVATION IN ENGLAND, 1925‐37

Small plot and field experiments on the cultivation of the insecticidal pyrethrum plant in England during 1925‐37 showed that the plant grew well and produced satisfactory yields of flowers of high pyrethrin content: costings of the field trials, however, showed that the cultivation of the plant was uneconomic. Individual plants grown from seed differed considerably in growth habit and in the pyrethrin content of their flowers.

Plants for special uses

Dependence of the United States upon foreign sources of Belladonna, Henbane, Stramonium, Digitalis, Ergot, Pyrethrum, Rotenone and numerous other plant products has stimulated attempts to produce the necessary crops domestically.

THE MANURIAL REQUIREMENTS OE PYRETHRUM (CHRYSANTHEMUM CINERARIAEFOLIUM TREV.)

SummaryA small field experiment upon the manurial requirements of the insecticidal pyrethrum plant, grown upon sandy soil of low fertility, is described. Lime had the effect of producing slight, but not significant, increases each year in the yield of flowers and their content of the pyrethrins, and decreased the percentages of plant failures in the fourth and fifth years of the experiment. There was a significant depression in the yield of flowers in the year after the single application of double dressings of the manures, but no effect in later years. The yearly application of moderate dressings of manures gave significant increases in the yield of flowers in the second and fifth years, and significant increases in the pyrethrin I content of the flowers in the fourth and fifth years of the experiment.

PYRETHRUM SENSITIZATION COMPLICATING HAY FEVER

To the Editor.— Commercial Pyrethrum is widely used for its toxic effects in various types of insecticides. The flowers of certain species of pyrethrum plants, of the genus Chrysanthemum, are used for the preparation of the substance, and closed flowers, which contain much pollen, apparently are the most satisfactory. For some years it has been known that dermatitis and allergic manifestations in the respiratory tract may occur as the result of exposure to this toxic material. Ramirez (Pyrethrum, J. Allergy 1 :149 [Jan.] 1930) reported two cases, one of sensitiveness to grass and the other to ragweed pollen, in which sensitization rhinitis occurred after the regular pollen seasons had terminated. These patients were found to be sensitive to pyrethrum and had been exposed to insecticides containing it. Feinberg (Pyrethrum Sensitization,The Journal, May 12, 1934, p. 1557) called attention to the frequency of sensitization to pyrethrum in the ragweed pollen

Pyrethrum Flowers: A Quantitative Study Of Their Development

Summary. An account is given of the examination of the flowers of pyrethrum plants (C. cinerariafoolium) grown upon a bed in Harpenden. The plants were divided into blocks and randomised, the flowers being harvested from a dozen plants each week over a period of 81/2 weeks, the flower heads ranged from the small bud stage in the first week to the overblown stage in the last week. The categories into which the flowers were divided to indicate maturity are defined and illustrated. The yield in numbers and weight of heads per plant, the diameters of the receptacles and the content of pyrethrin I and II were determined. There was a considerable amount of variation in all these factors in the flowers from different plants. A statistical analysis showed in this experiment: That there was no significant variation in the numbers of the flowers with time, but that position of the plant in the bed had a significant effect. That the time of harvesting had a significant effect upon the content of the pyrethrins whether taken separately or together and whether expressed in percentages, parts per flower head or parts per plant. The effect due to‐ position was only definitely significant in the case of pyrethrin I when expressed in parts per flower head. That there was on the different dates a significant correlation between the contents of pyrethrin I and II expressed in parts per flower head or plant. That there was, for the blocks of material examined, a significant correlation between the contents of pyrethrin I and II, whether expressed in percentages, parts‐per head or parts per plant, but that owing to the heterogeneity of the material the correlation was only significant for individual plants when the pyrethrias were expressed in parts per plant. The data in this experiment indicate that for the material examined, there is a quantitative development of the active principles in the flower heads from the small bud stage up to the time of maturity of the flowers, which more than keeps pace on the whole with the increase in weight of the flowers. Thus the content of pyrethrins, both relatively and absolutely, rises to a maximum at the maturity of the flowers. The mean percentage content of pyrethrins fell after pollination and the fading of the flowers; this corresponds with the rapid increase in weight of the heads on the formation of seed. There would thus appear to be a loss, which might be serious, both in percentage content of active principles and in yield of flowers if harvested before being fully open. There seems to be no useful purpose served in leaving the flowers to the overblown condition. I wish to express my great indebtedness to Dr R. A. Fisher and Dr J. Wishart for carrying out the greater part of the statistica.1 analyses incorporated in this paper and for much helpful advice. I am also indebted to Mr J. T. Martin for checking many of the analyses by an alternative method.