REVIEW ARTICLE 73

Morphological and Biochemical Diversity of Dalmatian Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.)

Martina GRDIŠA ( ) Klaudija CAROVIĆ-STANKO Ivan KOLAK Zlatko ŠATOVIĆ

Summary Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.) is a perennial herbaceous plant belonging to the Asteraceae family. It is endemic to the East coast of the Adriatic Sea and its natural habitat extends from Italy to northern Albania and up in the mountainous regions of Croatia, Bosnia and Herzegovina and Montenegro. Pyrethrum fl owers yield an important insecticide, the pyrethrin. Pyrethrin is mainly concentrated in oil glands on the surface of the seed inside the tightly packed fl ower head, but they can also be found in the other plant parts, however in much lower concentrations. Th e pyrethrin exist as a combination of six insecticide active ingredients: pyrethrin I, cinerin I, jasmolin I, pyrethrin II, cinerin II and jasmolin II, with pyrethrin I and pyrethrin II present in higher concentrations. Pyrethrin is non-toxic to mammals and other worm-blooded animals, it is unstable in light, oxygen, water and at elevated temperatures and therefore highly biodegradable. Due to the fact it is environmentally safe it is leading insecticide in organic farming systems. Th e most scientifi c work concerning Dalmatian pyrethrum was focused on its morphological and biochemical traits that are relevant in breeding. Breeding programmes are primarily focused on increasing the yield of pyrethrin per unit area. Relative to dry fl ower weight, fl ower heads contain the majority of the pyrethrin. Croatian wild populations contain approximately 0.60 to 0.79 %, while clones in breeding programmes of Australia and Kenya contain up to 3.0 % of pyrethrin.

Key words pyrethrin; pest control; natural insecticides; insect repellent; organic farming

University of Zagreb, Faculty of Agriculture, Department of Seed Science and Technology, Svetošimunska cesta 25, 10000 Zagreb, Croatia e-mail: [email protected] Received: December 12, 2008 | Accepted: February 20, 2009

Agriculturae Conspectus Scientifi cus | Vol. 74 (2009) No. 2 (73-80) 74 Martina GRDIŠA, Klaudija CAROVIĆ-STANKO, Ivan KOLAK, Zlatko ŠATOVIĆ

Introduction Pyrethrum achilleifolium M.Bieb. subsp. achilleifolium, Pyrethrin has highly unusual insecticidal activity, with the Pyrethrum achilleifolium M.Bieb. ability to control or repel numerous insect species, with little 2. Tanacetum annuum L., syn.: Chrysanthemum annuum or no adverse environmental impact (Casida and Quistad, (L.) Hayek 1995). Its insecticidal properties have been known for cen- 3. Tanacetum bipinnatum (L.) Sch.Bip., syn.: Pyrethrum turies. It has a quick knock-down eff ect on a wide range of bipinnatum (L.) Willd, Chrysanthemum bipinnatum L. insect species (Bhat and Menary, 1984), causing paralysis 4. Tanacetum cinerariifolium (Trevir.) Sch.Bip., syn.: within a few minutes. It acts as contact poison and aff ects the Pyrethrum cinerariifolium Trevir., Chrysanthemum cin- central nervous system of insects, by blocking their nervous erariifolium (Trevir.) Vis. functions. Th e insect’s nervous impulses fail, causing them 5. Tanacetum corymbosum (L.) Sch.Bip., syn.: Pyrethrum to die. In low concentrations, pyrethrin acts as a repellent, subcorymbosum (Sch.Bip.) Schur, Pyrethrum corymb- causing the insect to fl ee the source of the chemicals. It is iferum (L.) Schrank, Chrysanthemum corymbosum L. non-toxic to humans and worm-blooded animals. In mam- 6. Tanacetum corymbosum (L.) Sch.Bip. subsp. co- mals, pyrethrin is readily broken down into inactive forms rymbosum, syn.: Leucanthemum corymbosum (L.) and passes from the body quickly. Additional advantages of Gren. & Godr., Pyrethrum corymbosum (L.) Scop., pyrethrin are twofold: it is not persistent as it breaks down Chrysanthemum corymbosum L. subsp. corymbosum readily in sunlight and evolved insect resistance has not been 7. Tanacetum corymbosum (L.) Sch.Bip. subsp. clusii recorded so far. (Fisch. ex Rchb.) Heywood, syn.: Tanacetum subcorym- Dried and grounded fl owers have been traditionally used bosum (Schur) Sch.Bip., Chrysanthemum subcorym- in Croatian agriculture. Th ey were also used as the remedy bosum Schur., Pyrethrum clusii Fisch. ex Rchb. against fl eas and lice, and burned to repel the fl ying insects. 8. Tanacetum funkii Sch.Bip. ex Willk. Dalmatian pyrethrum was cultivated in Croatia from 9. Tanacetum macrophyllum (Waldst. & Kit.) Sch.Bip., the end of 19th century. From 1930 the production gradually syn.: Chrysanthemum macrophyllum Waldst. & Kit., decreased, especially during the World War II. Commercial Pyrethrum macrophyllum (Waldst. & Kit.) Willd. use of DDT (Dichloro-Diphenyl-Trichloroethane) chemical 10. Tanacetum microphyllum DC. insecticides ended the production of Dalmatian pyrethrum 11. Tanacetum millefolium (L.) Tzvelev, syn.: Pyrethrum in Croatia. Nevertheless, production expanded to the other millefoliatum (L.) Willd., Tanacetum kittaryanum parts of the world. Nowadays, it is cultivated in more than (C.A.Mey.) Tzvelev, Chrysanthemum millefoliatum L. 10 countries: Kenya, Australia (mainly north-west coast of Tasmania), Tanzania, Rwanda, Ecuador, France, Chile etc. 12. Tanacetum mucronulatum (Hoff manns. & Link) Heywood, syn.: Pyrethrum mucronulatum Hoff manns. According to the Article 16(3) (c) of European Union & Link, Chrysanthemum mucronulatum (Hoff manns. & Regulation (EC) No. 834/2007 on Organic Production and Link) Cout. Labeling of Organic Products, use of pyrethrin extracts as a 13. Tanacetum odessanum (Klokov) Tzvelev plant protection products is permitted in organic farming. Th ey are considered as environmentally friendly insecticides 14. Tanacetum paczoskii (Zefi r.) Tzvelev and widely used in agriculture, horticulture, store products 15. Tanacetum parthenifolium (Willd.) Sch.Bip. syn.: (Silcox and Roth, 1995), households (Kennedy and Hamilton, Pyrethrum parthenifolium Willd. 1995) and for the control of animal pests (Gerberg, 1995). 16. Tanacetum parthenium (L.) Sch.Bip., syn.: Leucanthemum parthenium (L.) Gren. & Godr., Taxonomy and distribution Pyrethrum parthenium (L.) Sm., Chrysanthemum Dalmatian pyrethrum (Tanacetum cinerariifolium /Trev./ parthenium (L.) Bernh., non (Lam.) Gaterau Schultz Bip.) is herbaceous perennial herb, belonging to the 17. Tanacetum santolina M.Winkl. Asteraceae (Compositae) family. Th e Asteraceae family is one 18. Tanacetum sclerophyllum (Krasch.) Tzvelev of the largest angiosperm family and comprises around 1300 19. Tanacetum uralense (Krasch.) Tzvelev genera and 25 000 species (Bremer, 1994). Genus Tanacetum 20. Tanacetum vahlii DC. is distributed in Europe, temperate Asia, North Africa, and 21. Tanacetum willkommii Sch.Bip. North America. Some species have been introduced into 22. Tanacetum vulgare L. syn.: Chrysanthemum au- South Africa, South America, Australia and New Zealand. diberti (DC.) P.Fourn., Chrysanthemum tanacetum Th e genus contains 152 species, including those earlier con- Karsch, non Vis., Chrysanthemum vulgare (L.) Bernh., sidered to belong to the separate genus Pyrethrum Zinn. non (Lam.) Gaterau, Pyrethrum vulgare (L.) Boiss., (Bremer and Humphries, 1993). Th e following species can Tanacetum audibertii (Req.) DC. be found in Europe (Tutin et al., 1964-1980): In Croatia, the following species can be found: Dalmatian 1. Tanacetum achilleifolium (M.Bieb.) Sch.Bip., syn.: pyrethrum (Tanacetum cinerariifolium/Trevir./ Sch. Bip.), Pyrethrum achilleifolium M.Bieb. subsp. wisockianum common tansy (Tanacetum vulgare L.), tansy daisy (Tanacetum Pacz, Chrysanthemum achilleifolium (M.Bieb.) Prodán, macrophyllum /Waldst. & Kit./ Sch. Bip.), feverfew (Tanacetum

Agric. conspec. sci. Vol. 74 (2009) No. 2 Morphological and Biochemical Diversity of Dalmatian Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.) 75

parthenium /L./ Sch. Bip.) and corymbfl ower tansy (Tanacetum it was not profi table, due to the fact that the price of imported corymbosum /L./ Sch. Bip.) (Nikolić et al., 2009). pyrethrum extract was much lower than production costs. Wild populations of Dalmatian pyrethrum in Croatia are Nowadays, Kenya is a major source of pyrethrum, ac- distributed on the Eastern sub- Mediterranean dry grasslands counting for more than 70 % of the global supply. Other larger and abundant populations can be found in southern parts producers of pyrethrum are Australia (Tasmania), Tanzania of Istria (Premantura), Kvarner islands (Krk, Cres, Lošinj), and Rwanda. Th e pyrethrum extracts are mainly exported to mountains Velebit and Biokovo, and along Dalmatian coast- USA, Europe and Asian countries. According to FAOSTAT al region and its islands (Brač, Hvar, Biševo, Vis, Korčula, in 2003, 2004 and 2005 Kenya produced 8000 tons of dried Lastovo, Mljet) (unpublished data). pyrethrum fl owers on the production area of 9000 hectares, while Tanzania produced 2500 tons on the production area of History 13500 hectares. In the same period, Rwanda produced 1000 Th e history of pyrethrum usually starts with the mention of tons of dried pyrethrum fl owers on the production area of identifi ed species Chrysanthemum cinerariaefolium Vis. which 2200 hectares. On average, 3 to 4 kg of fresh fl owers yield 1 was fi rst recorded in Dalmatia (Visiani, 1847). Pharmacist kg of dried fl owers. Around 250 kg of dried fl owers are pro- Antun Drobac (1810-1882) from Dubrovnik, Croatia was the duced per hectare during the fi rst year, increasing to 1000- fi rst to prove its insecticidal activity (Bakarić, 2005). Around 1200 kg per hectare in the second and third year. Aft er the 1854 Dalmatian pyrethrum was introduced into cultivation third year yield declines. along Dalmatian coast. It was a very profi table production for local producers and it infl uenced the agricultural develop- Morphology ment of Dalmatian coast. Ground pyrethrum fl owers could Dalmatian pyrethrum is a perennial herb, 30 to 100 cm be found for sale by the name ‘Dalmatian Insect Powder’ in high. Th e shoots branch a few times before terminating into a most European pharmacies. From 1885 dried fl owers were white, daisy-like fl ower heads. Th e infl orescence is composed also exported to the USA. Th e primary use of dried pyre- from two types of sessile fl orets. Th e white petalled ray fl o- thrum fl owers was the control of mosquitoes and body lice rets are placed on the margin and the yellow disc-fl orets are on humans and animals (Glynne-Jones, 2001). Pyrethrum in the centre, and they are placed on a slightly convex recep- products were available for use even though the chemistry of tacle (Brewer, 1968). Disc fl orets have both male and female active ingredients was unknown. In 1924, German chemist organs, while the ray fl orets are female (Bhat, 1995). On a single Herman Staudinger (1881-1965) and Lavoslav Ružička (1887- fl ower head, fl orets are arranged in a form of dense spiral. Th e 1976), a Croatian scientist, winner of the 1939 Nobel Prize in ray fl orets have a strap shaped corolla with three teeth at the Chemistry, identifi ed chemical structure of the active pyrethrin end. Th e base of corolla is tubular and envelopes a bi-lobed ingredients, pyrethrin I and pyrethrin II (Coomber, 1948). cylindrical style which is placed in the center of the fl oret on Based on these discoveries it was possible to proceed with the top of the ovary. Ovary is pentagonal and green calyx is the synthesis of pyrethroids. Pyrethroids are manufactured attached to it. A single erect ovule stands in the ovary. Th e chemicals that are very similar in structure to pyrethrin, but yellow corolla of the disc fl orets is also tubular. Disc fl orets are oft en more toxic to insects, as well as to mammals, and are bisexual. Stamens are inserted on short stalks at the base last longer in the environment. Chemical structure of cinerin of the inner surface of the corolla. At anthesis, the bi-lobed I and II was identifi ed in 1945, while the chemical structure style grows through a tube formed by fi ve anthers which of jasmolin I and II was identifi ed in 1966. are united along the edges. Green calyx is also attached to a ‘Golden years’ of pyrethrum production in Dalmatia were pentagonal ovary. Pollen grains are tri-nucleate. Th e fl owers from 1910 until 1930. In that time, phylloxera (Daktulosphaira open approximately one month aft er the fi rst buds become vitifoliae Fitch) destroyed many vineyards and peasants began visible. Th e fl owering rhythm encourages crosspollination. cultivating Dalmatian pyrethrum in abandoned vineyards. Fruits of pyrethrum are pale brown, cylindrical or semicu- Pyrethrum production saved many families from starvation neiform achenes, with 5 to 7 ridges and glabrous and slightly and emigration. From 1930 to the beginning of the World lustrous surface (Bojnanský and Fargašová, 2007). Th e active War II production rapidly decreased and discovery of syn- ingredient of pyrethrum, the pyrethrin, is mainly located in thetic pesticide DDT signifi cantly reduced pyrethrum pro- tiny oil glands on the surface of the achenes. duction. In 1928, it was introduced to the British Colony of Kenya. Captain Gilbert Walker was the fi rst settler to grow Pollination pyrethrum commercially in Kenya. He imported some seed Th e insects, mostly Hymenoptera , carry out the pollination from Dalmatia and planted it on his farm in the highland in pyrethrum. In order to collect pollen and nectar insects near Nakuru (Chandler, 1948). During the World War II visit the infl orescence. Th eir path follows the development of Kenya produced large amounts of this natural insecticide to the fl ower, from margin to the center, and through that proc- delouse allied armies (Mocatta, 2003). By the 1945 it became ess, they deposit foreign pollen on the styles of fully opened the world’s main producer and it still holds the leading po- fl owers. A discharge of the ripe pollen takes place under in- sition. From 1964 to 1966 some attempts have been made to fl uence of the elongation process of the style. As soon as the revitalize pyrethrum production in Dalmatia, unfortunately, pollen ripens, the style grows through the tube formed by

Agric. conspec. sci. Vol. 74 (2009) No. 2 76 Martina GRDIŠA, Klaudija CAROVIĆ-STANKO, Ivan KOLAK, Zlatko ŠATOVIĆ

the united edges of the anthers. Th e pollen reaches the re- Other important breeding objectives have been the de- ceptive surfaces of the style with the help of the long hairs velopment of nonlodging cultivars resistant to economically on the top of the lobes. Few hours aft er pollen discharge the important pests and diseases, diff ering in fl ower maturity and style unfolds and is ready to accept foreign pollen. Th e ger- adaptable to diff erent environmental conditions. According to mination percentage of the pollen decreases relatively rap- Bhat (1995), the most important traits in pyrethrum breeding idly aft er anthesis (the period during which a fl ower is fully are pyrethrin yield and quality, morphological traits (such as open and functional). Since the individual fl oret discharges bush size, fl ower shape and size and plant height), resistance the ripe pollen before it unfolds the receptive surfaces of the to lodging and synchronous fl owering. style, the limited life of the pollen aft er anthesis reduces the Pyrethrin yield and quality are determined by dry fl ower chance for own pollen to germinate on styles of the same fl oret. yield per unit area, pyrethrin content in the dry matter of the Th erefore the germination percentage of the own pollen by fl owers and the ratio of pyrethrin I to pyrethrin II. the time the style becomes receptive can be ignored. Th is is Dry fl ower yield depends on fresh fl ower yield and mois- a well known mechanism to avoid self-pollination (Bremer, ture content and it must be expressed on moisture free bases. 1968). Another incompatibility system also operates with Fresh fl ower yield depends on the weight and the number even higher effi ciency- sporophytically determined system. of fl owers. It is believed that genotypes with higher fl ower (Brewer, 1974; Parlevliet, 1975; Bhat, 1995). number per plant produce high fl ower yield. Flower weight Within sporophytic incompatibility, the reaction of the is determined as dry or fresh weight of 100 fl owers. Th e sam- pollen is linked to a trinucleate pollen grains and inhibition of ples must be collected at the same maturity and dried at the germination or early tube growth. In Dalmatian pyrethrum, same nondegrading temperature (50 ºC) (Ngugi and Ikadu, self-fertilization is prevented by inhibition of germination of 1990). Th e ratio between pyrethrin I and pyrethrin II deter- the trinucleate pollen on the stigma. Despite the above de- mines the quality of pyrethrum extract (Morris et al., 2006). scribed mechanism, the self-fertilization has been also re- Bush size, fl ower shape and size and plant height are mor- ported (Brewer, 1968). phological traits important for the design and development Genetics of the appropriate harvesting equipment. Bush size shows a strong interaction with environmental factors such as tem- Th e basic chromosome number in pyrethrum is 2n=2x=18 perature, soil type and moisture. Larger bushes usually yield (MacDonald, 1995), but triploids (2n=3x=27) (MacDonald, more fl owers. 1995; Ottaro, 1977) and tetraploids (2n=4x=36) (MacDonald, Flower shape and size are usually determined by the di- 1995) have been also recognized. To produce a triploid plant ameter of the disc fl orets. Larger fl owers are more attractive, with 27 chromosomes, the tetraploids must be produced fi rst but because of their mass, they might cause lodging in weaker and then crossed with a diploid parent. In pyrethrum, tetra- plants. Another disadvantage of large fl owers is that they take ploids have been produced by treating the seed with 0.2 % col- longer to dry and therefore increase production costs. chicine for fi ve days. Vegetative buds can also be treated with colchicine to double the chromosome number (Bhat, 1995). Plant height is measured as the length of the central fl owering shoots from the ground to the level of the fl owers. Ottaro (1977) reported close relationship between trip- Tall and weak plants with heavy fl owers tend to lodge which loidy and an increase in the expression of some morphologi- is undesirable characteristic. Lodging is determined as an cal traits. Th e aim of study was to determine morphological angle at which the fl owering shoots stand in the relation to characteristics that could be used for simple identifi cation of the ground level (Bhat, 1995). Other diff erent classifi cations diploid and triploid pyrethrum plants. Reliable distinguish- concerning this trait have also been described. For example, ing traits were: fl ower diameter, number of pollen germpo- depending upon whether the growth was dense (stems grew res and number of stomata per unit area of the leaf. Flower erect), open (stems inclined to spread) or very open (stems weight, plant height and length of stomata were found to be lay on the ground), Chamberlain and Clark (1947) used the less reliable traits. Th e results showed that triploid plants had terms “compact”, “open” or “prostrate”. Scales of 1 to 10 and signifi cantly larger and heavier fl owers than diploids. Th e tri- 1 to 5 have also been used for scoring this trait, with 1 being ploids were also signifi cantly higher than diploid plants but the most susceptible to lodging (Parlevliet and Contant, 1970; it was not found to be a reliable method for separating dip- Parlevliet, 1974). Number of fl owers per stem along with fl ower loids from triploids, due to overlapping observed in the dis- size infl uences resistance to lodging. Resistance to lodging is tribution of the two groups, unlike germpore count as well required for easy picking as well as for mechanized harvest as number of stomata per unit area of the leaf. (Parlevliet, 1974). Harvest of pyrethrum fl owers is determined Breeding by their maturity. Some authors consider fl owers mature when ½ to ¾ disc fl orets are open (Bhat, 1995). Th e most scientifi c work concerning Dalmatian pyrethrum has been focused on those morphological and biochemical Th e relationships between morphological and chemical traits that have been relevant in breeding, with the aim of in- traits have been studied in order to facilitate selection, by creasing the yield of pyrethrin per unit area (Parlevliet, 1969, enabling identifi cation of genotypes with higher pyrethrin 1974, 1975; Bhat and Menary, 1986; Bhat, 1995). content without performing chemical analysis.

Agric. conspec. sci. Vol. 74 (2009) No. 2 Morphological and Biochemical Diversity of Dalmatian Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.) 77

Pandita and Bhat (1986) have conducted a study on 100 yield were not related and therefore total fl ower yield and lodg- randomly selected pyrethrum plants with the aim of fi nding ing were also unrelated. Another explanation of conclusion out the correlation of pyrethrin content with fl oral traits. Th e made by Parlevliet is the fact that in Kenya pyrethrum fl owers results of the study showed that the pyrethrin content was are harvested every 2-3 weeks at their maturity and therefore signifi cantly positively correlated with the width of the disc the weight of the fl owers on the stem is reduced (Bhat, 1995). fl oret. Th ey concluded that it is possible that more pyrethrin Parent-off spring correlations were found to be highly in the ovary induces a swelling of fl orets and therefore in- signifi cant for number of fl owers per plant, fl ower weight, crease the fl ower width. pyrethrin content and yield, signifying the highly heritable Th e investigations to determine the nature and magni- nature of these traits (Singh et al., 1988). tude of genotypic and phenotypic relations between various traits determining the pyrethrin yield have been conducted. Chemistry Parlevliet (1974) has determined that quantity of pyrethrin Pyrethrin is mainly concentrated in the fl ower heads, harvested per hectare depends on fresh fl ower yield and dry namely 93.7 % of pyrethrin is accumulated in achenes, and matter content of the fl owers. Fresh fl ower yield is correlated minor quantities in disc fl orets (2.0 %), ray fl orets (2.6 %), with the fl ower size, fresh weight of 100 fl owers, number of and receptacles (2.6 %). Th e term pyrethrin is referred to the fl owers per stem and number of stems during the growing six insecticide active ingredients: pyrethrin I, cinerin I, jas- period. Th is investigation resulted with the conclusion that molin I, pyrethrin II, cinerin II and jasmolin II (Figure 1). fl ower size is negatively correlated with dry matter content of Pyrethrin I, cinerin I and jasmolin I are closely related in- the fl owers and the fl ower yield, while the pyrethrin content secticidal esters of chrysanthemic acid, while pyrethrin II, had no correlation with fl ower yield and fl ower size. However, cinerin II and jasmolin II are insecticidal esters of pyrethric Parlevliet did not present the data concerning the magnitude acid. Th e three chrysanthemic acid esters are commonly re- or the nature of these relationships. ferred as pyrethrins I, and pyrethric acid esters as pyrethrins II Bhat and Menary (1986) have also tested genotypic and (Essig and Zao, 2001b). Among these compounds pyrethrin I phenotypic correlations in pyrethrum. Tested parameters and pyrethrin II are the most predominant and active (Casida of yield were pyrethrin content in dry matter of the fl owers, and Quistad, 1995). Pyrethrin I alone is toxic, acting in min- dry fl ower yield, number of fl owers per plant and 100 fl ow- utes, while pyrethrin II has a high knock-down eff ect. Insects ers dry weight. Th e results have shown that pyrethrin yield easily metabolize pyrethrin II and recover in a few hours. was correlated to four traits in following order: fl ower yield, Nevertheless, combination of pyrethrin I and pyrethrin II pyrethrin content, number of fl owers per plant and 100 fl ow- has an outstanding eff ect on wide range of insect species ers weight. Th e results also showed that fl ower yield depends (Winney, 1979). Th e relative amount of each ingredient de- more on the number of fl owers per plant than on 100 fl owers pends on the particular plant type, geographical source and dry weight (Pandita and Bhat, 1984; Bhat and Menary, 1986; time of harvest (Glynne-Jones A., 2001). Ratio of pyrethrin Singh et al., 1987). Th ere was also a negative correlation be- I to pyrethrin II determines the quality of the pyrethrin ex- tween the number of fl owers per plant and 100 fl owers dry tract. Th e insecticidal activity increases with the increased weight. Pyrethrin content did not show signifi cant correla- ratio, which varies between 0.9-1.3 (Maciver, 1995). As the tion with any character other than fl owers yield (Bhat and fl owers approach maturity, a decrease in pyrethrin I/II ratio Menary, 1986). has been reported. (Pattenden, 1970). According to Singh et al. (1987), traits signifi cantly corre- Gnadinger and Corl (1930) conducted a study to deter- lated with fl ower yield were bush diameter and plant height. mine the relation between maturity of the fl owers and py- Bush diameter is positively correlated with number of fl ow- rethrin content. Th e results showed that before the buds were ers per plant and plant height. Between fl ower size (diameter) formed, the roots, leaves and stems did not contain any py- and fl ower yield (Pandita and Bhat, 1986; Singh et al., 1988) rethrin. As the buds formed and developed, their pyrethrin as well as between fl ower size (diameter) and number of fl ow- content gradually increased. Th e last and the most mature ers per plant negative correlation has been observed. Negative samples contained four times the percentage of pyrethrin correlation between fl ower yield and pyrethrin content was found in the unexpanded buds. Th e results indicated that reported by Kroll (1958). the pyrethrin content depends on the maturity of the plant Th e relationship between fl ower yield and lodging is also as well as on the development of the fl owers. important in pyrethrum breeding. Parlevliet (1974) has ob- Head (1966) conducted a study to determine the appro- served that lodging was highly aff ected by fl ower size and the priate time for harvesting the crop. In his investigation, sam- number of fl owers per stem. He also reported minimal rela- ples of fl ower heads were taken at diff erent stages of maturity, tionship between lodging and total fl ower yield. According ranging from closed bud stage to full fl ower maturity (fl ower to Bhat (1995), these results are opposite to the observations heads dry and seed suitable for collection). Th e results have in the fi eld, where it was shown that higher fl ower yield re- shown that fl owers had maximum pyrethrin content when sulted with more lodging of the plants. Explanation of the 3-4 rows of disc fl orets were open and that the pyrethrin yield conclusion made by Parlevliet is the fact that it was based on did not signifi cantly increase beyond this stage. a presumption that the number of fl owers per stem and fl ower

Agric. conspec. sci. Vol. 74 (2009) No. 2 78 Martina GRDIŠA, Klaudija CAROVIĆ-STANKO, Ivan KOLAK, Zlatko ŠATOVIĆ

Figure 1. Chemical structure of pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I and jasmolin II.

In Dalmatia, the fl ower heads were traditionally picked were accompanied by the loss of pyrethrin. Th e loss of py- around June 13th (St. Anthony’s Day). According to Bakarić rethrin depended upon the air temperature and the duration (2005), fl owers should be picked third or the fourth day of over drying. aft er the blooming starts, i.e. when half of disc fl orets open. Because of the diffi culties concerning the separation of Pyrethrin content varies from 0.91 to 1.30 % of the dry individual pyrethrin component and because of the lack of fl ower weight (Kolak et al., 1999; Casida and Quistad, 1995). absolute standards for each component the content of pyre- According to Bakarić (2005), wild Croatian populations of thrum extract is usually analyzed for total pyrethrin, or total Dalmatian pyrethrum contain 0.60-0.79 % of pyrethrin. pyrethrins I and total pyrethrins II. Many methods of py- Parlevliet et al. (1979) analyzed twenty wild populations of rethrin extraction have been tested and used. Th e main ob- Dalmatian pyrethrum for pyrethrin content. Wild popula- jective of the extraction process is to obtain a light colored tions were collected in Dalmatia and fi eld trials were set on product, with a high recovery of pyrethrin active ingredients two locations in Kenya. Th e pyrethrin content ranged from (Kiriamiti et al. 2003). Th e most commonly used methods 0.75 to 1.04 % (Bhat, 1995). Morris et al. (2005) reported for of extraction in industry and laboratories are Soxhlet (SOX) the Tasmanian commercial varieties to contain approximate- (Otterbach and Wenclawiak, 1999), Ultrasonic (USE) (Kasaj ly 1.80 to 2.50 % of pyrethrin. According to Kiriamiti et al. et al., 1999) and recently the Supercritical Fluid Extraction (2003) in most cases pyrethrin content in the fl owers ranges (SFE) (Pan et al., 1995; Otterbach and Wenclawiack, 1999; from 0.50 to 2.0% of the dry fl ower weight, while Pandita Della Porta and Reverchon, 2002). It is of great importance and Sharma (1990) reported for the pyrethrin content in the to use suitable solvent for a particular extraction method. Dalmatian pyrethrum, bred and grown in India, to vary from Many solvents have been used in pyrethrin extraction: pro- 0.90 to 1.50 % of dried fl ower weight. In breeding programs panol, ethanol, methanol (EU project, 2002), n-hexane (Pan of Australia, Kenya and the USA today, it is common to fi nd et al., 1995, Kasaj et al., 1999, Kiriamiti et al., 2003), petro- clones with pyrethrin content of 3.0 % and more (Casida and lether (Della Porta and Reverchon, 2002) and carbon dioxide Quistad, 1995). (Kiriamiti et al., 2003). Th ree diff erent extraction methods Many investigations concerning the eff ect of drying air (USE, SOX and SFE) were compared for the extraction of temperature and drying time on pyrethrin content have been pyrethrin. For USE the samples were extracted with 10 ml conducted. Gnadinger (1936) reported that artifi cial drying at isopropanol, the SOX extraction was performed with 50 ml 50 °C does not aff ect pyrethrin content, but at 60 °C a slight isopropanol in a micro-Soxhlet apparatus. Th e extraction loss has been observed and the loss increased at higher tem- conditions for SFE were a pressure of 30 MPa and tempera- peratures. Th e same results were obtained by Beckley (1952) ture of 100 ºC. To control the expansion of carbon dioxide, a and Ngugi and Ikadu (1990). Githinji (1973) conducted a series heated (50 ºC) regulated restrictor was used. Separation and of drying experiments at 60 ° C, 80 °C, 100 °C, 120 °C, 140°C determination of the samples was performed by Super Critical and 160 °C, using a thin-layer of fl owers. Th e best results were Fluid Chromatography (SFC- FID). Th e results have shown obtained while drying at 80 °C, with regular turning of the that the total amount of recovered pyrethrin in the solvents fl owers, to avoid over drying of the fl owers at the bottom of is very similar in all three extractions. Extraction time in the layer. Shorter drying periods using higher temperatures USE and SOX is slightly higher (60 min) than in SFE extrac-

Agric. conspec. sci. Vol. 74 (2009) No. 2 Morphological and Biochemical Diversity of Dalmatian Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.) 79 tion (20 min) (Otterbach and Wenclawiak, 1999). Wynn et References al. (1995) described a preparative Supercritical carbon diox- Bakarić P. (2005). Buhač - prirodni insekticid. Gospodarski list 17: ide extraction process from pyrethrum fl owers at 40 ºC and 41-45 80 bars. Th e extraction effi ciencies of supercritical carbon B e ckley V.A. (1952). Pyrethrum drying. Pyrethrum Post 1 (3): 9-11 dioxide were much better than those of hexane. During the Bhat B.K. (1995). Breeding Methodologies Applicable to Pyrethrum: extraction process, the most effi cient extraction period was Pyrethrum Flowers Production, Chemistry, Toxicology and the fi rst three hours of the experiment. Kiriamiti et al. (2003) Uses. Oxford University Press, New York Bhat B.K., Menary R.C. (1984). Pyrethrum production in Australia: compared extraction of pyrethrin using supercritical carbon Its Past and Present Potential. Th e Journal of the Australian dioxide as a solvent with hexan Soxhlet extraction. Th e ob- Institute of agricultural Science 494: 189-192 tained extracts were very similar, whereas the only diff erence Bhat B.K., Menary R.C. (1986). Genotypic and Phenotypic was in ratio of pyrethrin I to pyrethrin II, which was lower correlation in Pyrethrum (Chrysanthemum cinerariaefolium with supercritical carbon dioxide. Vis) and their implication in selection. Pyrethrum Post 16 (2): 61-65 Many separation techniques have been compared Bojnanský V., Fargašová A. (2007). Atlas of Seeds and Fruits of and reported: Reversed-Phase High-Performance Liquid Central and East-European Flora: Th e Carpathian Mountains Chromatography (RP-HPLC) (Pan et al, 1995, Wang et al., Region. Springer, Netherlands 1997, Kasaj et al.,1999; Morris, 2006), Normal-Phase High- Bremer K. (1994). Asteraceae: Cladistics and Classifi cation. Timber Performance Liquid Chromatography (NP-HPLC) (Essig Press, Portland and Zao, 2001a), Gas Chromatography (GC) (Nguyen et al., Bremer K., Humphries C.J. (1993) Bull. Nat. Hist. Mus. Lond. (Bot.) 23, p. 71Brewer J.G. (1968). Flowering and seed setting in 1998) and some others. Due to thermal instability of py- pyrethrum (Chrysanthemum cinerariaefolium Vis.). Pyrethrum rethrin, HPLC methods are preferred over GC methods for Post 9(4): 18-21 analyzing pyrethrin (Wang et al., 1997). Kasaj et al. (1999) Brewer J.G. (1974). Incompatibility relationship in pyrethrum have developed fast and precise RP-HPLC method for the (Chrysanthemum cinerariaefolium Vis.). Euphytica 23: 45-47 quantifi cation of total and individual amounts of the py- Casida J.E., Quistad G.B. (1995). Pyrethrum fl owers: Production, rethrin from pyrethrum extract and fl owers by internal and Chemistry, Toxicology, and Uses. Oxford University Press, New York. external standardization. In the development of this method, Chamberlain E.E., Clark P.J. (1947). Investigation on growing the individual pyrethrins were isolated from the extract by pyrethrum in New Zealand II. Selection of clones: their preparative NP-HPLC. NP-HPLC is an analytical method- variation in habit of growth, susceptibility to root-rot, yield ology, which provides the basis for quantitative analysis of and pyrethrins content of fl ower. New Zealand J Sci and Tech each component in pyrethrum extract. It is usually used for 29:215-222 the isolation of the individual compound while RP-HPLC is Chandler S.E. (1948). Th e Origin and Early History of the used for their quantitative determination (Kasaj et al., 1999). Production of Pyrethrum in Kenya. Pyrethrum Post 1 (1): 10-13 Coomber H.E. (1948). Th e chemical evaluation of Pyrethrum According to Wang et al. (1997), the advantage of RP-HPLC fl owers. Pyrethrum Post 1 (1): 16-19 over NP-HPLC is very low level of interferences in the chro- Della Porta G., Reverchon E. (2002). Supercritical Fluids Extraction matography. and Fractionation of Pyrethrins from Pyrethrums. In: Symposium Proceedings of 4th International Symposium on Conclusion high pressure process technology and chemical engineering. Insecticidal effi ciency of pyrethrin has been known for cen- Venice, Italy. turies and the powder prepared from dried fl owers has been Essig K., Zhao Z. (2001a). Method Development and Validation of a High-Performance Liquid Chromatographic Method for used in traditional Croatian farming systems and household Pyrethrum Extract. J Chromatogr Sci 39 (4): 473-480 (8) pest control. With the increase of production and consump- Essig K., Zhao Z. J. (2001b). Preparation and characterization of a tion of healthy products as well as an increased resistance of Pyrethrum extract standard. LC/GC 19(7): 722-730 pests to synthetic pesticides and more strict environmental EU project (FAIR Programme, Agriculture and Fisheries) No legislation, interest in pyrethrin has been expanding con- 1436 “Improvement of effi ciency and reduction of application tinuously in recent years (Kolak, et al., 1999). Th eir very low rates of preferable naturally grown biocides by complexing with gamma-cyclodextrin”, 1999-2002, Available: http://www. toxicity to man and animals are ecological benefi ts which nf-2000.org/secure/Fair, 23.06.2004 have led to increased production of this natural insecticide Gerberg E.J. (1995). Pyrethrum for control of pests of medical and world-wide (Kasaj et al., 1999). veterinary importance. In: Casida, J.E. and Quistad, G.B. (eds). Formulations containing natural pyrethrin are widely Pyrethrum fl owers: Production, chemistry, toxicology,and uses. Oxford University Press, New York, pp. 302 – 310 used as household insecticides, for the control of pest in ag- Githinji P.M. (1973). Th e eff ects of drying air temperature and riculture as well as for the prevention of infestations during drying time on pyrethrins content of pyrethrum fl owers. storage. For the past 130 years of using pyrethrin on humans Pyrethrum Post 12(2): 77-82 or near humans, the only complaints were due to the appear- Glynne-Jones A. (2001). Pyrethrum, Pesticide Outlook- ance of the skin allergies (Glynne-Jones, 2001). Biopesticides. October, 195-198 Gnadinger C.B., Corl C. S. (1930). Studies on pyrethrum fl owers II: Th e relation between maturity and pyrethrin content. J Am Chem Soc 52 (2) 680–684

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