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"Bioactive natural products often occur as a plant of a family of related molecules so that it is possible to isolate a number of homologous and obtain structure-activity information" (Raaman, 2006). Plants are known to be a very rich source of bioactive organic chemicals and more than 400,000 secondary metabolites may be present in the plant kingdom (Swain, 1977). Natural products, which often have an ecological role in regulating the interactions between plants, microorganisms, insects and animals (Hanson, 2003) can be defensive substances, antifeedants, attractants and pheromones.
Plutella Xylostella (L.) (=P. manulipennis (Curt))
Common name - Diamondback moth
Family - Yponomeutidae (Plutellidae) (Chand, 1995)
Hosts (main) - Brassicae of all species
(Alternative) - A wide range of wild and cultivated Crucifereae (Dennis, 1983)
Diamondback moth is possibly of European origin but has become rather cosmopolitan, and is now found all over the Americas and in Europe, Southern Asia, Australia, and New Zealand. It was first observed in North America in 1854, in Illinois which was spread quickly. In North America, everywhere where cabbage is grown, diamondback moth is now recorded (John, 2001). Yadav et al., (1983) concluded that 25-26°C was almost appropriate for development of moth in his study about the effect of temperature on growth of Plutella Xylostella.
Life cycle of Diamondback moth
According to John (2001), the total development time, which depends on weather conditions (range about 17-51 days), from the egg to pupal stage averages 25-30 days. Annually, there are 4-6 generations (Harcourt, 1957). As with most moths, the Diamondback moth is active mostly at dusk and at night for mating and laying eggs (Pluke et al., 1999).
Diamondback moth eggs are oval and flattened, and measure 0.44 mm long and 0.26 mm wide. They are yellow or pale green in color, and are deposited separately or in small groups of 2-8 eggs in depression on the surface of the leaves, or sometime on other parts of the plants (John, 2001). Incubation takes about 3-8 days (Dennis, 1983).
Diamondback moth has four instars. Average and range of growth time for a larva is about 4.5 (3-7), 4(2-7), 4(2-8), and 5(2-10) days respectively. Throughout their development, larvae stay quite small and active (John, 2001). The lepidopteran larva wriggle violently if disturbed and often drop off the leaf, remaining suspended from it by a silk thread (Dennis, 1983). During the first instars, the larvae are colourless, but thereafter they become green (John, 2001).
Pupation occurs in a loose silk cocoon which is usually formed on the lower or outer leaves. The yellowish pupa is 7-9 mm long. The period of the cocoon averages about 8.5 days (range 5-15 days), but during this time the insect is in the pre-pupal rather than pupal stage (John, 2001).
The adult is a small, slender, grayish-brown moth with distinct antennae. It is about 6 mm long, and marked with a broad cream or light-brown band along the back. Moths usually mate at dusk, immediately after coming out from the cocoon. Flight and oviposition take place from dusk to midnight, and the adults can be found feeding at blossoms on nectar. Moth males and females live about 12 and 16 days respectively, and female deposit eggs for about 10 days (John, 2001).
Hosts and distribution of Diamondback moth in Mauritius
In Mauritius, Dunhawoor and Abeeluck (1997) stated that the insect was first identified in 1945 and has now turned into one of the most destructive pests. In Mauritius, almost all the possible methods and pest control technologies have been investigated to manage this serious pest (Facknath, 1997). Until 1980, this pest was controlled effectively by synthetic insecticides (Seewooruthun et al., 1984).
Damaged caused by Diamondback moth
John (2001) reported that diamondback moth was long considered a relatively insignificant insect. During the larval stage, there is most damage caused. Though the larvae are very small, they can be numerous; thus, resulting in complete removal of foliar tissue except for the leaf veins (John, 2001). The seedlings are particularly spoiled, which may disturb head formation in cabbage, broccoli, and cauliflower. According to Dennis (1983), severe attacks sometimes occur, particularly in hot dry weather.
Control of diamondback moth
Head and Savinelli (2008) stated that the major problem is that these pests have developed resistance to many conventional insecticides. Parasitic wasps are known to be the natural enemies of diamondback moth larvae (Cranshaw, 1998). Starting a plantation with DBM-free plants will provide the greatest income for a farmer (Pluke, 1999).
At University of Mauritus
Pathogens (Bacillus thuringiensis)
Intercropping and use of trap crops
Combinations of above
Botanicals and cultural
At Ministry of Agriculture
Synthetic pesticides and growth regulators
Table 1: Research Strategies presently being explored for the control of Plutella Xylostella.
Plant species (Pitaya)
Almost unknown fifteen years ago, pitahaya (different spellings are used: Pitahaya, pitaya, pitajaya, pitajuia or pitalla) nowadyas occupies a growing niche in Europe's exotic fruit market (Mizrahi et al., 1997) as well as in the domestic markets of producer countries, such as Vietnam (N'Guyen, 1996), Colombia (Mizrahi et al., 1997), Mexico (De Dios, 2004), Costa Rica and Nicaragua (Daubresse, 1999). Currently, pitahaya has only a few species usually found on the market: yellow pitayaha [S. megalanthus (Schum.) Britt & Rose] and red pitayaha (Hylocereus spp. Britt & Rose) (Fabrice et al., 2006). There are 16 species of Hylocereus whose ornamental value is because of the beauty of their large flowers (15-25cm) (Fabrice et al., 2006). Most Hylocereus species originate mainly from Latin America (probably from Mexico and Colombia), with others possibly from the West Indies (Britton and Rose, 1963). Hylocereus varieties can adapt to different types of well-drained soil (Barbeau, 1990).
Pitaya plant worldwide
Pitaya is known to be the one of the most beautiful and widespread members of the family Cactacea (Julia, 1987). It is usually cultivated and naturalized throughout tropical American lowlands, the West Indies, the Bahamas, Bermuda, southern Florida and the tropics of the Old World.
The plant is a climbing cactus known as 'cactus torue' and 'raquette tortue' (MSIRI, 2006) in Mauritius which is widespread as a Hylocereus undatus clone (Govinden, 2007).Pitaya grows wild in dry zone (MSIRI, 2006). During the French colonization, it has been introduced in Mauritius to feed giant land tortoises (Govinden, 2007). As the clone Hylocereus undatus do not produce fruits normally, it must be crossed pollinated with a genetically- different clone set of fruits (MSIRI, 2006). Now, subhumid zone is found to be appropriate for cultivation of Pitaya (MSIRI, 2006). Pitaya can be grown on all soil types from sand to heavy clays (Govinden, 2006). This plant does not need any pest control apart from snails during the first year and it produces its first fruit within a year of plantation (Govinden, 2006).
Hylocereus undatus is a climbing plant which does not have leaves as other cacti (Govinden, 2007). The role of the leaves is given by the triangular-shaped, segmented and green succulent stems (Govinden, 2007). Pitayas have easily small detached spines (Pimienta-Barrtos and Nobel, 1994), branches, flowers and fruits (Govinden, 2007). The plant has areoles spaced at 3 to 4 cm and each areole has 2 or 3 spines in it (Govinden, 2007). They have magnificient white flowers that are night blooming and very fragrant (Morton, 1987) which are 29 cm long (Fabrice et al., 2006). The fruit is non-spiny (Morton, 1987) and is around 15-22 cm long (Fabrice, 2006). They are a red fruit with edible white flesh and tiny black seeds (Govinden, 2007).
Metabolites in plants
Plants synthesize a vast range of organic compounds that are usually classified as primary and secondary metabolites although the precise limits between the two groups can in some instances be somewhat blurred (Alan and Hiroshi, 2006). Plants have many natural enemies, thus they have to evolve mechanism to protect them.
Primary metabolites are compounds that have important roles associated with photosynthesis, respiration, and growth and development (Alan and Hiroshi, 2006) which are present throughout the life cycle. They are simple compounds with direct connections to the metabolic pathway of the microorganisms (Schügerl, 1994). Consequently, close relationships exist between growth rate and their production rate (Schügerl, 1994). Most primary metabolites exert their biological effect inside the cell or organism that is responsible for their production (Hanson, 2003).
Based on their biosynthesis origins, plant secondary metabolites can be divided into three main groups: (i) flavonoids and allied phenolic and polyphenolic compounds, (ii) terpenoids and (iii) nitrogen-containing alkaloids and sulphur-containing compounds (Alan and Hiroshi, 2006). In most plants, synthesis and accumulation of secondary metabolites is controlled in space and time (Wink, 2010). Secondary metabolites are complex metabolites (Schügerl, 1994).the end products play no obvious role in the economy of the organism. Specific secondary metabolites may attract insects to particular plants in order to lay their eggs (Hanson, 2003). The biosynthesis of Secondary metabolites has no direct connection to the cell metabolism (Schügerl, 1994). Other role of the secondary metabolites is to act as defence such as pigmentation or support.
Role of secondary metabolites
Whatever the initial reason for their evolution, secondary metabolites are now an essential part of the armamentaria used by plants in the battle to survive and propagate, to the extent where the expenditure of energy, photosynthate and nutrients for their production can be 'cost effective' for that (Chadwick, 1992)).
-root nodule bacteris
-Growth of seedlings
Figure 1: Ecological and physiological functions of plant secondary metabolites (Wink, 2010)
Substances in plants which affect insect behavior
Brian (1977) stated that during all stages of their life-cycles, plants are exposed to many potentially parasitic micro-organisms. The idea that plants produce defensive chemical substances after infection was expressed by a number of research workers in the first half of this century, but the conception was formalized by Müller and Börger (1941) (Brian, 1977). Alkaloids, sequiterpenes, flavonoids, limonoids, phenols, coumarins and stilbenes of plant origin possess toxic, antifeedant, and growth regulating effects against a wide range of insect pest (Hassanali and Lwande, 1989). Highly elaborate chemical defences have been eveolved by plants against attack and these have provided a rich source of biologically actuve compounds which may be used as novel crop-protecting agents (Chadwick and ÄŒhulÄphÅn, 1990).
Antifeedants, known as the defense substances of higher plants against insects, protect the plants against pests. Insect antifeedants have been studied as a part of our efforts to defend our crops against pest insects (Kenji, 2010).
A dominant role is played by attractant in many vital aspects of insect behavior (Jacobson, 1965). The activity of insects in seeking food, the opposite sex, or a place to lay their eggs are governed by attractants.
In addition to the attractive odors, plants also produce volatile chemicals that function as repellents (from a distance) or feeding inhibitors (at close range) which form part of the plant defense mechanism against insect attack (Hill, 2008). Repellents are known to have tremendous practical importance (Garson and Winkie, 1968). Terpenes, tannins and various alkaloids are seemed to be the major chemical repellents in plants (Hill, 2008). Insect repellents are considered to be insecticides in a broad sense (Troy, 2006). The plant must have the ability to produce an adaptive balance in which it accumulates enough pepellents to defend itself but not enough to poison itself (Whittaker, 1971).
Sondheimer and Simeone (1970) have stated that recently, great potential for insect control has been shown by insect hormones. The occurrence of insect moulting hormones provide a fascinating insight into the way plants may be evolved in order to protect themselves from insect predation (Hardorne, 1998).recording have been made for plant analogues of insect moulting hormones in plants and it has a wide distribution. These substances may cause sterility and death, as they have the ability to upset normal metamorphosis.
Defence mechanisms of plant against insect pests
Plants have developed over time a number of tactics that allow them to resist attack that are being attacked by insects. One or more of several different kinds of resistance mechanisms can cause resistance to animal pests (Painter, 1951). Separation, purification and identification of many different constituents present in plants need to be done by different methods (Harborne, 1988).
Extraction is a method whereby the desired constituents of plant are removed using a solvent. Organically soluble compounds are extracted with solvents (Raaman, 2006). There are several methods used for preparing extracts, including organic solvent extraction, supercritical gas extraction and steam distillation (Raaman, 2006).
Organic solvent extraction
Organic solvent extraction is one process of separating desired substances from plant material. Both fresh plant and dried plants can be used for extraction (Harborne, 1988). In this method, the plants are first crushed and then carefully mixed with a solvent such as hexane, benzene, methanol or ethanol inside a closed container. Raaman (2006) stated that the selection of solvent depends on several factors as well as the characteristics of the constituents being extracted, cost and environment issues. The success of the extraction with alcohol is directly related to the amount chlorophyll is removed into solvent (Raaman, 2006). The extract obtained is clarified by filtration, which is concentrated in vacuo in rotary evaporator (Raaman, 2006). Fungal growth is prevented by addition of a trace of toluene to the concentrated extracts and they are stored in refrigerator. As the solvent is dissolved in the desired substances of the plant, it is called "miscella". It is then separated from the plant material. There are a number of techniques for solvent extraction, which consist of maceration, percolation and countercurrent extraction (Raaman, 2006).
This technique involves soaking and agitating the solvent and plant material together (Raaman, 2006). The solvent is then drained off (Ramaan, 2006). Remaining miscella is removed from the plant material through pressing (Ramaan, 2006).
Biochemically active natural groups
Tannins are divided into two groups: hydrosable and condensed tannins. Hydrosable tannins are astringent which have the ability to tan hide. Condensed tannins are related to flavonoids as they are polymers of phenolic compounds.
Heterosides are organic compounds found in plants. They make up the active components of many plants.
Terpenoids are generally lipid-soluble which are located in the cytoplasm of the plant cell (Harborne, 1998). They are the largest group of secondary compounds (Schoonhoven et al., 2005). They are of importance in plant growth, metabolism or ecology.
Steroid is a group formed from triterpenoids which occur mainly as glycosides.
Anthraquinones are known to be the largest group of naturally occurring quinine substances. They are used as laxative.
Alkaloids include the largest single class of secondary plant metabolites. The functions of alkaloids in plants are still largely vague, although individual substances have been reported as growth regulators or as insect repellents or attractants (Harborne, 1998). Most alkaloids are physiologically active compounds having a range of toxic effects on animals (Margaret and Michael, 1998). Nicotine is a very respective example of the potency of the insecticidal behavior of alkaloids and was used extensively until its neurotoxic effects not only on insects but also on human, bird and mammals were discovered (Regnault-Rogen and Philogène, 2008). Many alkaloids have neuroactive properties and work together with the receptors at nerve endings (Hanson, 2003). Alkaloids are thought to play a defensive role in the plant against herbivores and pathogens as secondary metabolites (Crozier and Ashihara, 2006). They are known to block the ion channel, slow down enzymes or interfere neurotransmission. Alkaloids can also produce hallucinations, failure of coordination, convulsions and death.
The flavonoids are all structurally derived from the parent compound flavones (Harborne, 1998). Flavonoids, which are generally water-soluble compounds, can be extracted with 70% ethanol. Flavonoids are phenolic and hence modify in color when treated with base or with ammonia (Harborne, 1998). They are there in all vascular plants. Flavonols appear to be significant in regulating control of growth in the pea plant (Galston, 1969) and their adverse effects on insect feeding (Isman and Duffey, 1981) have indicated that they may be natural resistance factors.
Coumarin is an aromatic which work as pesticides in plants. These plants have sedative properties and they also increase the resistance of capillary walls.
Phenols are a wide variety of secondary compounds that include glycosides, tannins, coumarins, quinines and flavonoids (Facknath and Lalljee, 2000). Phenolic just tastes awful which block enzyme activity, slows growth and interfere with digestion.
Saponins are glycosides of both triterpenes and sterols which have been detected in over seventy families of plants (Hostettmann and Marston, 1995). They are known to be widely distributed in the plant kingdom (Hostettmann and Marston, 1995. Saponins have foaming characteristics.
MATERIALS AND METHODS
For this project, the morphogenetic effects of hylocereus undatus (Pitaya) were studied on the insect pests DBM. The biological process that causes the insects' organism to develop its shape, where cell growth and differentiation is controlled, is known as the morphogentic effect. Together with testing if Pitaya has bioactive compounds that can be used to control DBM, two other activities were undertaken: the rearing of DBM larvae which was carried out at in the laboratory and the growing of cabbage.
Round bottom flask, measuring cylinders, petri dishes, beakers, funnel, filter paper (Whatmann, 18.5 cm diameter, pore size 42), aluminum foil, test tubes, conical flask.
Solvents used: Methanol, ethanol, dichloromethane, hexane. These solvents were used for extraction of metabolites.
Sulphuric acid was used for washing glasswares, round bottom flask, beakers and test tubes.
Rotary evaporator was used to concentrate the extracts.
Test insect: Diamondback moth
Diamondback moth, also known as Plutella xylostella, was used as insect test for this project.
Classification of diamondback moth
Kingdom: Animalia (Animals)
Phylum: Arthropoda (Arthropods)
Class: Insecta (Insects)
Order: Lepidoptera (Butterflies and Moths)
No Taxon: (Moths)
Species: xylostella (Diamondback moth)
Plutella xylostella (Linnaeus) (Lepidoptera: Yponomuetidae) commonly called Diamondback moth (DBM) is believed to have originated in the Mediterranean area, where most of the cruciferous crop plants have originated (Mukerji, 2004).
Test organism: Hyloceureus Undatus
The test material whose pesticidal properties were evaluated is Hylocereus Undatus.
Classification of Hylocerus Undatus
Hulocereus undatus was collected at Barkly Experiment Station. Immediately after collection, the specimen was brought in the laboratory where it was watered n kept at room temperature.
Rearing of diamondback moth
Larvae Plutella xylostella were captured from cabbage plants and brought to laboratory to be reared under conditions at room temperature in wooden cages (75 x 50 x 50 cm) covered with fine nylon mesh. The lepidopteran larvae were fed on clean leaves of cabbage. The adults were fed on 5% honey solution which was soaked in a cotton swab and untreated cabbage leaves were placed in small bottles filled with water in the cage for oviposition.
The eggs that were laid were transferred to another cage to allow it to hatch into larva and feed on untreated leaves. After pupation of the larvae, they were transferred together with their leaves on moist filter paper and were allowed to emerge into adults. Consequently, the adults were breed to increase population.
Growing of cabbage
Classification of cabbage
SPECIES: Oleracea (var. capitata)
COMMON NAME: cabbage
Cultivation of cabbage
Demarcation of land
(10 x 10 m)
Planting distance was done and holes were dug
Holes were filled with manure and then convered with a layer of soil
Seedlings were transplanted and watered
Seedlings were covered by putting branches with leaves next to them to serve as shading
After month, top dressing was carried out
Figure 2: Cultivation procedure
Method of extraction
Maceration is the process of extracting metabolites with the solvent with several days shaking or stirring at room temperature. Ideally, fresh plant tissues should be used for phytochemical analysis (Harborne, 1988). Solvent chosen depends upon the characteristics of the secondary metabolites in the plant. Boiling ethanol is a good all-purpose solvent for preliminary extraction (Harborne, 1988).
After addition of each fresh solvent, the mixture should be allowed to macerate overnight. The solvent is filtered and concentrated by a rotary evaporator. Anhydrous magnesium sulphate is added to the filtrate to remove the water added in it.
Method of separation
Hylocereus Undatus (crushed)
Extraction with MEOH (70%) for 3 days
Extraction with ethanol (70%) overnight
Extraction with DCM (80%) overnight
Extraction with hexane (80%) overnight
Filtrate is concentrated individually in rotary evaporator
Figure 3: Extraction and portioning procedure
Phytochemical screening of the extracts
Test for Tannins
50 mg of extract was dissolved in 5 ml of alcohol and few fragments of magnesium powder and concentrated Hydrochloric acid (drop wise) were added. The presence of tannins is revealed by the formation of any pink to crimson colour.
Test for Heterosides
10 cm3 of 50% sulphuric acid was added to 1 cm3 of the extract in a test tube. The mixture was heated in a boiling tube water bath for 15minutes. 10 cm3 of Fehling's solution was added and the mixture was boiled and observed for a brick red precipitate.
Test for Terpenoids
2ml of chloroform was added to the extract followed by 3ml of concentrated sulphuric acid to form a layer. A reddish brown colouration is indicated as positive.
Test for Steroids
3 drops of acetic anhydride and 1 drop of concentrated sulphuric acid were added to 1ml of extract. The presence of steroids was detected by a colour change from deep green turning to brown/dark brown.
Test for Antraquinones
4 ml of hexane was shaken with 2 ml of extract. The upper lipophilic layer was seperated and treated ith 4 ml of diluted ammonia. The presence of anthraquinones is indicated by a colour change of violet to pink in the lower layer.
Test for Alkaloids
50 mg of extract was stirred in a few ml of dilute Hcl acid and then filtered. The filtrate was tested with Mayer's test and Wagner's test.
Wagner's testÂ : A few drops of Wagner's Reagent were added by the side of the test tube to a few ml of filtrate. The presence of alkaloids was confirmed by a reddish-brown colour.
Wagner's reagent- 1.27 g of Iodine and 2.0 g of potassium iodide were dissolved in 5 ml of water and made up to 100 ml with distilled water.
Mayer's testÂ : A drop or two of Mayer's reagent was added to a few ml of filtrate. The test is indicated as positive by a white/creamy precipitate.
Mayer's reagent- 1.358 g of mercuric acid was dissolved in 60 ml of water and 5.0 g of potassium iodided was dissolved in 10 ml of water. The two solution were mixed and made up to 100 ml with water.
Test for Flavoinods
2 ml of concentrated HCL and 0.2 g of magnesium powder were added to 2 ml of extract. The test is indicated as positive by a colouration change to red/red-orange.
Test for Coumarins
Some concetrated ammonia is added to the extract. A smear of solution is placed on a microscope slide and viewed under long wave (366nm) UV light. The presence of coumarins is indicated by a green fluorescence.
Test for Phenols
A few drops of iron (III) chloride were added to 1 ml of the etxract. The presence of phenolic compound was confirmed by a brownish colouration.
Test for Saponins
50 mg of extract was diluted with distilled water and made up to 20 ml. The suspension was shaken in a graduated cylinder for 15 minutes. The presence of saponins was indicated by a two cm layer of foam.
HCL + magenisum powder
Iron (III) chloride
Results of chemical tests on extracts of Hylocereus Undatus.
-Â : indicates negative test
+ : indicates trace of test
++ : indicates positive test