CA2712463A1 - Environmentally friendly plant protection agents - Google Patents

Abstract

The invention relates to the use of compositions comprising herbal preparations for the treatment and prevention of plant diseases caused by pests such as nematodes, fungi and insects. Said compositions comprise a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala,Myristica fragrans,Syzygium aromaticumand Rhazya stricta.
Also provided are methods for the treatment or prevention of disease in plants which comprise applying said compositions; seeds, plant growth media, food wrapping and pesticide formulations comprising said compositions; and methods forthe prevention of termite damage to buildings which comprise applying said compositions to buildings or to building materials.

Description

ENVIRONMENTALLY FRIENDLY PLANT PROTECTION AGENTS

The invention relates to compositions for use in the treatment and prevention of disease in plants. In particular, the invention relates to the use of compositions comprising herbal preparations for the treatment and prevention of plant diseases caused by soilborne pests such as nematodes, fungi and insects. Said compositions comprise effective plant-derived anti-nematode, antifungal and anti-insect agents with minimal harmful effect to the environment.
Soilborne pests can cause substantial crop damage and economic losses. This is particularly true in intensive agriculture where crops are planted in the same place year after year, allowing pest populations to accumulate in the soil. Soilborne pests include nematodes, fungi, bacteria, viruses and insects.
Plant parasitic nematodes are small worm-shaped invertebrates, ranging in length from 0.5-10mm. Nematodes (derived from the Greek word for thread) are active, flexible, elongate, organisms that live on moist surfaces or in liquid environments, including films of water within soil and moist tissues within other organisms. While only 20,000 species of nematode have been identified, it is estimated that 40,000 to 10 million actually exist. Some species of nematodes have evolved to be very successful parasites of both plants and animals and are responsible for significant economic losses in agriculture and livestock and for morbidity and mortality in humans (Whitehead, 1998). They affect agricultural land worldwide and are some of the most damaging pests of tropical, sub-tropical and temperate agriculture. They can cause serious yield losses in crops such as citrus, potatoes, banana, rice, pineapple, coffee, peanuts, sugar cane, and tobacco. Nematodes damage crops by direct feeding, transmitting viruses and facilitating bacterial and fungal infections.
Nematode parasites of plants can inhabit all parts of plants, including roots, developing flower buds, leaves, and stems. Plant parasites are classified on the basis of their feeding habits into the broad categories:
migratory ectoparasites, migratory endoparasites, and sedentary endoparasites. Sedentary endoparasites, which include the root knot nematodes (Meloidogyne) and cyst nematodes (Globodera and Heterodera) induce feeding sites and establish long-term infections within roots that are often very damaging to crops (Whitehead, supra).
As nematodes often do not provide clear symptoms, their economic effect tends to be underestimated by growers, however annual losses to world agriculture are estimated to be US$100 billion (Sasser and Freckman, 1987).
This is based on an estimated average 12% annual loss spread across all major crops. For example, it is estimated that nematodes cause soybean losses of approximately $3.2 billion annually worldwide (Barker et al., 1994).
Several factors make the need for safe and effective nematode controls urgent. Continuing population growth, famines, and environmental degradation have heightened concern for the sustainability of agriculture, and new government regulations may prevent or severely restrict the use of many available agricultural anthelmintic agents. Nematodes are a significant problem in the agriculture of developing, as well as developed, countries.
The most economically damaging plant parasitic nematode genera belong to the family Heterderidae of the order Tylenchida, and include the cyst nematodes (genera Heterodera and Globodera) and the root-knot nematodes (genus Meloidogyne). The soybean cyst nematode (H. glycines) and potato cyst nematodes (G. pallida and G. rostochiensis) are important examples. Root-knot nematodes infect thousands of different plant species including vegetables, fruits, and row crops in poor countries.
There are very small arrays of chemicals available to control nematodes (Becker, 1999). Nevertheless, the application of chemical nematicides remains the major means of nematode control. In general, chemical nematicides are highly toxic compounds known to cause substantial environmental damage and are increasingly restricted in the amounts and locations in which then can be used. For example, the soil fumigant methyl bromide, which has been used effectively to reduce nematode infestations in a variety of specialty crops, is regulated under the U.N. Montreal Protocol as an ozone-depleting substance and is scheduled for elimination in 2005 in the US (Carter et al., 2005). It is expected that strawberry and other commodity crop industries will be significantly impacted if a suitable replacement for methyl bromide is not found. Similarly, broad-spectrum nematicides such as Telone (various formulations of 1,3-dichloropropene) have significant restrictions on their use because of toxicological concerns (Carter et al., 2005).
In order to kill nematodes in soil, most nematicides are toxic at low levels and are water soluble in order to move down to where the nematodes are. Many of the effective nematicides used in the past have been withdrawn from the market during the last 25 years for environmental and health reasons until only a handful remain. When using any nematicide the product label must be strictly adhered to in order to minimize human and environmental health impacts and to avoid liability. Table 1 is a list of nematicides labelled for use on golf course turf (Crow, 2005) and illustrates the restrictions on the use of these products necessitated by their environmental toxicity.

Table 1. Nematicides for use on golf course turf in Florida (Crow, 2005).
:Product Legal sites, application notes Rates ......... ......... ......... ......... ......... ......... .........
......... ......... ...... ......... ........
ÃNemacur Restricted use Pesticide. Golf courses and sod 2.3 lb/1000 1OG farms only, do not use on lawns or public areas:sq ft, or Mother than golf courses. Irrigate immediately after: 100 lb/acre application with at least 1/2 inch of water; do not:
allow puddling or run-off to occur. Do not treat:
newly-seeded areas until plants have developed secondary root systems. Do not apply a total of:
more than 200 lb/acre/year. Do not apply to more:
than 10 acres per golf course per day; wait 3 days before treating any additional area. After May 31, 2005, do not apply to hydrologic soil group A soils:
that are excessively drained and predominately:
sand or loamy sand such as soils in the suborder:

Product Legal sites, application notes Rates psamments with shallow water tables (less than 50 feet deep).
.........
.Nemacur Restricted use Pesticide. Golf courses and sod ::9.7 fl Ã3 farms only; do not use on lawns or public areasoz/1000 sq other than golf courses. Apply in a minimum of 0.5ft, or 3.3 gallon of water per 1000 sq ft (approx. 20 gallons gal/acre per acre). Irrigate immediately after treatment with a minimum of 1/2 inch of water; do not allow:
puddling or run-off to occur. Do not treat newly:
seeded areas until plants have developed secondary root systems. Do not use more than twice per year. Do not apply to more than 10 acres per golf course per day; wait 3 days before treating any additional area. After May 31, 2005, do not:
apply to hydrologic soil group A soils that are excessively drained and predominately sand or:
loamy sand such as soils in the suborder psamments with shallow water tables (less than 50 feet deep).

Curfew For use on golf courses and athletic fields. Must be 5 gal/acre Soil applied by an approved custom applicator. Do not:
Fumigant apply within 100 ft. of buildings or wells. 24 hour::
reentry restriction. Cannot be used on areas with Karst geology.

?Turfcure For use on fairways, driving ranges and roughs. 5 gal/acre 376 Must be applied by an approved custom applicator.
Do not apply within 50 ft. of buildings. 48 hour re entry restriction.

The information in Table 1 is not a substitute for the product label.
Always follow directions on the product label when applying any pesticide.
The above-mentioned products all have a "Danger" label. This is a concern in an industry such as the golf course industry where there is a high 5 degree of visibility and human involvement.
In the wider agricultural setting, current nematode control strategies often combine crop rotation, nematicide application and the use of resistant varieties within an integrated pest management system. However these conventional approaches are becoming increasingly unsatisfactory. Crop rotation is of limited value for controlling nematodes with a wide host range and is impractical for specialist growers. Modern intensive farming methods rely heavily on the use of chemical control, but, as mentioned above, this is causing concern as nematicides are amongst the most toxic and environmentally damaging of all crop protection agents. A number of nematicides have been withdrawn [e.g. see the Montreal Agreement: (United Nations Environment Programme, Ozone Secretariat, Montreal Protocol -Celebrating 20 years of progress in 2007: http://ozone.uneg.org/) and the use of methyl bromide (United Nations Environment Programme, Ozone Secretariat: http://ozone.unep.org/teap/Reports/MBTOC/index.shtmi)] and many countries have proposed a reduction in use of the remaining chemicals.
With the reduction in the choice of chemical control agents, resistant plants are becoming increasingly important in the battle to reduce crop losses to plant parasitic nematodes. Resistant cultivars of several crop plants have been successfully developed by conventional plant breeding methods, however, this approach has limited value for a variety of reasons (Roberts, 1992). The main problems centre on the specificity of the resistance which is often restricted to particular nematode species or even races and the difficulties often encountered when trying to transfer the desired resistance trait to commercial plant varieties.
As a result of these limitations, researchers are seeking to apply modern molecular techniques to provide effective and durable forms of nematode control. Transgenic nematode resistance and the cloning of natural resistance genes (Jung and Wyss, 1999; Cai et al., 1997; Milligan et al., 1998) have been investigated, which may allow their introduction into related species and/or elite commercial crop lines whilst the design of novel, engineered transgenic plant defences may provide alternative forms of effective and durable nematode control.
A further avenue of research in the quest for alternative anti-nematode agents is the investigation of plant, e.g. herb, species that exhibit high levels of nematode resistance with a view to developing a plant-derived nematicide.
Some plant species are known to be highly resistant to nematodes. The best documented of these include marigolds (Tagetes spp.), rattlebox (Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.), castor bean (Ricinus communis), margosa (Azardiracta indica), and many members of the family Asteraceae (family Compositae) (Hackney & Dickerson, 1975). In the case of the Asteraceae, the photodynamic compound alpha-terthienyl has been shown to account for the strong nematicidal activity of the roots. Castor beans are ploughed under as a green manure before a seed crop is set. However, a significant drawback of the castor plant is that the seed contains toxic compounds, such as ricin, that can kill humans, pets and livestock and is also highly allergenic. In many cases, however, the active principle(s) for plant nematicidal activity has not been discovered and it remains difficult to derive commercially successful nematicidal products from these resistant plants.
US patent application no. 20040127362 discloses that cloves and clove oil are included in the US Environmental Protection Agency's list of minimum risk pesticides. The application relates to a nematicide comprising an aqueous formulation of one or more oils in combination with an inert ingredient such as molasses and/or cheese.
Certain soil-dwelling fungi, such as species of Fusarium, Verticillium and Phytphthora, attack plant roots or the base of stems, causing diseases in the plants and reducing crop yields. Fungal diseases remain a principal limitation to increased agricultural production of food. Fungal species such as Fusarium, Rhizoctonia, Pythium, Phytophthora, etc. continue to stifle development efforts in many countries and account for billions of dollars of damage annually. Thus, protection of crop plants from fungal pathogens remains a primary preoccupation of agricultural scientists.
For example, Cucurbitaceae plants are infected worldwide by species of soilborne fungi such as Pythium sp., Rhizoctonia sp., Fusarium sp., and Phytophthora sp. Most of these fungi remain in the soil for several seasons, then, when suitable conditions such as high temperature and humidity occur, the fungi will develop and infect plants. This is a particular problem in greenhouses where temperatures of 28-35 C and humidity of 60-75%
typically occur. The main fungi causing damping-off of cucumber seedlings at high frequency are Pythium sp. and, to a lesser extent, Rhizoctonia sp. These two fungi can cause seedling wilt in approximately 10-15% of a cucumber crop. Another fungus, Fusarium sp, infects the plants mid season causing wilt in the more mature plants.
Measures to control fungal diseases frequently involve targeting pathogen propagules in soil with fungicide drenches to reduce initial inoculum.
Commercially available pesticides include: Rhizolex 50WP, Vongared, Tichigaren 30L, Tatto C SC, Terlai 67% WP, Rridomil 5G, Fungarid, Rhizolex, Ridomil, Tachigareen. Chemical fungicides remain the first line of defence against fungal pests despite serious environmental concerns over their use and abuse.
There are growing concerns over the residual effect of the widespread use of these chemicals on the environment and on human health. Some chemicals have been reported as being capable of causing birth defects in test animals when administered at doses as low as 30 mg/kg (Bulletin of Environmental Contamination Toxicology, 54:363-369, 1995;
httg:,'/www.chem-tox.com/pesticides!/). Mice given the fungicide maneb showed clear signs of Parkinson's, a progressive and incurable brain illness (Parkinson's Disease Linked to Pesticide Combination: http://www.chem-tox.com/ esticides/). For this reason, and because of safety and environmental problems, many synthetic agricultural agents have been or are being targeted for removal from the market. Hence, disease control options using pesticides are being lost, which creates a need to find alternative ways to control fungal pathogens.
One of the alternative ways to control fungal diseases is through the development of new crop varieties with enhanced resistance to fungal diseases and effort in this area continues, both by traditional plant breeding and using techniques of genetic engineering. Nevertheless, resistance, whether natural or engineered, will not provide a complete solution and there is a continuing interest in developing novel anti-fungal agents with improved properties.
One avenue of investigation is the screening of plant-derived products for anti-fungal activity. Bowers and Locke (2000 and 2004) investigated the effect of botanical extracts on Fusarium oxysporum and Phytophthora nicotianae in soil and in the control of Fusarium wilt and Phytophthora blight in the greenhouse. They reported that pepper/mustard, cassia, and clove oil extracts reduced the pathogen population and suppressed disease development compared with untreated infested soil. Clove oil has also been proposed for use in the management of post harvest fungal diseases of banana (Ranasinghe et al. 2002).
Certain insects are also significant pests. Insects that feed on crop plants cause reduced yields. Plants that have been damaged by insect feeding may be more susceptible to infection; the insect may also act as a vector for plant pathogens such as bacteria and viruses. Protection of crop plants from insect pests including Lepidoptera and Coleoptera is therefore an important goal of agricultural research.
Termites are a group of social insects usually classified as belonging to the order Isoptera. Termites mostly feed on dead plant material, e.g. wood, leaf litter, and about 10% of the estimated 4,000 species are economically significant as pests that can cause severe losses in crops or plantation forests or serious structural damage to buildings (e.g. Yamano, 2000 and Mulrooney et al., 2007). Although chemicals have been traditionally used for termite control, the extensive use of chemicals causes environmental hazards and there is a risk of termites developing a resistance to these chemicals (Zoberi, 1995). Furthermore, these pesticides have been banned or withdrawn from the market in an increasing number of countries from the late eighties and the nineties for human health and environmental reasons. The move away from hazardous chemical agents for the control of termites has been further accelerated in recent years by efforts by the United Nations Environment Program (UNEP) and Food and Agriculture Organization (FAO) to eliminate globally the production and use of certain persistent organic pollutants which include the organochlorine pesticides (UNEP/FAO/Global IPM Facility 2000).
As a consequence of these developments, the focus in termite management has shifted increasingly to alternative methods for dealing with termite problems. Lenz (2005) reviewed the options for biological control of termites and found there to be a limited biological activity for sustainable control of termites (Sosa-Gomez et al. 1996; Castrillo et al., 2005; Lenz 2005;
Yanagawa et al 2008). The use of microbial pathogens is proposed as a solution for certain termite problems, but may not help with others because available pathogens may be effective against certain species and less so against others (Lenz 2005).
There remains an urgent need for new, effective, target-specific, environmentally safe compositions and methods for controlling plant pests and diseases such as those caused by plant parasitic nematodes, by fungal plant pathogens and by insect pests.
The present inventors investigated the activity of a number herbs i.e.
plant species used for flavouring and/or herbal medicine, against nematode, fungal and insect pests of plants.
The present invention arises from the inventors' finding that simple, ground preparations of certain herbs exhibit a marked pesticidal effect when tested against nematode, fungal and insect plant pests.
Pimpinella anisum fruit (aniseed) is used for flavouring baked foods such as cakes, biscuits and confectionery, as well as rye breads. It is also used in much the same way as fennel to flavour fish, poultry, soups and root vegetable dishes. Numerous alcoholic drinks and cordials are flavoured with aniseed, such as French pastis, Greek ouzo, Turkish raki, Arab arrak etc. Anise has long been known for its carminative properties. It is chewed after meals in parts of Europe, the Middle East and India to aid digestion and freshen breath. A few seeds taken with water are recommended for hiccups. It is a mild expectorant, often used in mixtures and lozenges for dry coughs. Antiseptic, antispasmodic 5 and soporific effects are attributed to the herb: it is used to relieve toothache and as a treatment for sleeplessness. Extracts of Pimpinella anisum seed are reported to have antioxidant capacity (Ilhami et al. 2003). Its essential oil is used to treat lice and scabies and, mixed with other ingredients, against insects.
The essential oil is reported to have antifungal (Shukla and Tripathi 1987) and 10 antiviral (Shukla et al. 1989) activities suggesting its use in the treatment of colds and `flu. The principal component of anise oil is anethole, a precursor that can eventually produce 2,5-dimethoxybenzaldehyde which is used in the clandestine synthesis of psychedelic drugs (Waumans et al. 2004).
Peganum harmala L. (Syrian rue) is a wild-growing flowering plant belonging to the Zygophylaceae family and is found abundantly in Middle East and North Africa. From ancient times, it has been claimed to be an important medicinal plant. The brown triangular-conical seeds of P. harmala are known to possess hypothermic and hallucinogenic properties (Lamchouri et al., 1999;
Kuhn and Winston 2000). It has been used traditionally as an emmenagogue and an abortifacient agent in the Middle East and North Africa (Fleming).
There are several reports in the literature indicating a great variety of pharmacological activities for P. harmala L (Abdel-Fattah et al., 1997). It has also been known to interact with a2-Adrenoceptor subtypes (Saleem et al., 2001) and have hallucination potency and be effective in the treatment of dermatosis (Saad and Rifaie 1980), hypothermic (Abdel-Fattah et al., 1995) and cancer (Adams 1983) with anti-nociceptive activity (Monsef et al., 2004).
The seeds are used to remove moisture and heat from the body, as a purifying medicine, as an aphrodisiac and are also regarded as an emmenagogue, diuretic and vomitive. Juice of the seed and leaves also used in various prescriptions including remedies for insanity and epilepsy, baldness and haemorrhoids (Lebling 2002).

The nutmegs Myristica are a genus of evergreen trees indigenous to tropical Southeast Asia and Australasia. They are important for two spices derived from the fruit, nutmeg and mace. Nutmeg is the seed kernel inside the fruit and mace is the lacy covering (aril) on the kernel. Powdered or grated nutmeg is found variously in savoury and sweet dishes in Asian, Middle Eastern and European cuisines. The essential oil is used as a natural food flavouring in baked goods, syrups (e.g. Coca Cola), beverages, sweets etc. Nutmeg has aromatic, stimulant, narcotic, carminative, astringent, aphrodisiac, hypolipidemic, antithrombotic, anti-platelet aggregation, antifungal, antidysenteric, anti-inflammatory activities (Nadkarni 1988). It is used as a remedy for stomach ache, rheumatism and vomiting in pregnancy (Nadkarni 1988), and is an important source of natural antioxidants (Juki et al. 2006).
The essential oil, obtained by the steam distillation of ground nutmeg, is also used in the cosmetic and pharmaceutical industries for instance in toothpaste and as the major ingredient in some cough syrups. In traditional medicine nutmeg and nutmeg oil were used for illnesses related to the nervous and digestive systems. Myristicin and elemicin are believed to be the chemical constituents responsible for the subtle hallucinogenic properties of nutmeg oil. Other known chemical ingredients of the oil are a-pinene, sabinene, y-terpinene and safrole.
Syzygium aromaticum (syn. Eugenia aromaticum, Eugenia caryophyllata, Eugenia caryophyllus or Caryophyllus aromaticus) is a tropical tree, the immature, unopened flower buds of which provide, when dried, the spice known as cloves. Cloves are a popular spice in North African and Middle Eastern cuisine, used whole or ground, and are also used in European dishes, both savoury and sweet. Cloves contain 15 to 20% essential oil which is mostly Eugenol, the principal flavour-giving volatile oil which is a very strong antiseptic. Clove oil is a strong stimulant and carminative and is used to treat nausea, indigestion and dyspepsia. It is used to treat toothache. It has been suggested as a treatment for rheumatism, arthritis and mouth sores and also as a mosquito repellent. Clove oil is highly irritating to the skin, unless well diluted.

Rhazya stricta is an evergreen dwarf shrub and a traditional medicinal herb once considered effective in treating venereal diseases. The dried leaves were smoked in a pipe, sometimes mixed with other kinds of leaves, to cure syphilis. Today, the herb is still sometimes smoked, but as a treatment for rheumatism. The plant is considered somewhat toxic and is avoided by livestock although it is not regarded as a serious threat. The herb has been used in small quantities to relieve upset stomachs (Lebling 2002). The roots, leaves and branches of Rhazya stricta were traditionally used for tooth diseases, diabetes, constipation and intestinal diseases (Ahmad et al. 2006).
Rhazya stricta is rich in alkaloids thought to have anticancer properties (Gilani et al. 2006). It should be noted that the vernacular name "harmal" is in use both for Rhazya stricta (family Apocynaceae) and Peganum harmala (family Zygophyllaceae).
The inventors' work disclosed herein shows that preparations of P.
anisum, P. harmala, M. fragrans, S. aromaticum and R. stricta each show effective pesticidal activity individually. Additionally, the inventors have observed synergistic effects with combinations of two or more herbs. For example, each of P. anisum and M. fragrans is individually effective against nematodes, but the combination of P. anisum, P. harmala and M. fragrans gives a synergistic increase in nematicidal activity, providing particularly effective nematicidal activity against all stages of root-knot nemotodes, especially egg masses (Example 3).
Accordingly, the invention provides the use of a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta for the treatment or prevention of disease in plants. In the context of the present invention, "disease" includes both infections caused by plant pathogens such as nematodes and fungi, and adverse health effects resulting from infestation by pests such as insects.
Herbal preparations according to the invention exhibit "nematicidal" or "anti-nematode" activity. These terms are used interchangeably herein and encompass not only actual killing of nematodes, but also arresting or retarding their normal growth and development such that the host plant is better able to survive or tolerate the nematode infection.
Herbal preparations according to the invention also show antifungal activity. The terms "fungicidal" and "antifungal" have equivalent meaning with respect to fungi and fungal infection as the above terms "nematicidal" and "anti-nematode" have with respect to nematodes.
The term "pesticidal" as used herein refers to the property of killing, or arresting or retarding the growth of, plant pests including, but not limited to, disease-causing pathogens such as nematodes and fungi and pests such as insects which adversely affect plant health by feeding on them.
Preferably, the pesticidal composition of the invention comprises a preparation or extract of three or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta, more preferably four or more plants selected from the aforesaid group or all five plants.
A preferred composition comprises a preparation or extract of Pimpinella anisum or Myristica fragrans as preparations of these herbs have been found to be particularly effective against nematodes. More preferably, the composition further comprises a preparation or extract of each of Pimpinella anisum, Peganum harmala and Myristica fragrans, as this combination shows a synergistic effect providing effective nematicidal activity against all stages of root-knot nematodes, including egg masses.
A composition comprising a preparation or extract of each of Pimpinella anisum, Peganum harmala, Myristica fragrans, and Syzygium aromaticum has been found to have a broad spectrum pesticide activity including activity against soilborne nematodes and fungi and against insects.
Because the pesticidal preparations of the invention are of plant origin, said plants being herbs already consumed by man, there is a low risk of toxicity to man or the environment from the use of said preparations as pesticides (plant protection agents).
Conveniently, the preparation is powdered dried plant material e.g. the dry seed or kernel, or fruit or part thereof (e.g. aril), or bud, or flower, which has been ground to a powder. Suitable and readily available dry herb materials include: fruit of P. anisum (anise "seed" or aniseed), seeds of P.
harmala, seeds (nutmeg) or arils (mace) of M. fragrans dried flower buds (sometimes called pods) of S. aromaticum (cloves) and seeds of Rhazia stricta. The foregoing substances are readily obtainable owing to their culinary use as spices. The plant material is ground to a powder to allow easy dispersal of the preparation and promote diffusion of the nematicidal agent.
The above mentioned plant parts have been identified by the inventors as having profound pesticidal effects, however, other plant parts may be used in the plant protection composition of the invention. The observed nematicidal, fungicidal and insecticidal effects will be brought about by one or more active agents produced by the respective plant. These active agents may be found in other parts of the plant to those specifically mentioned above. Any plant part containing the active agent(s) is a candidate material for preparing a pesticide composition according to the invention. Plant parts containing the active agent(s) may be determined empirically by measuring pest killing or inhibitory activity in a test assay such as those described herein.
Alternatively, once the active agent(s) has been identified, the concentration of the active molecule(s) in different tissues or organs of the plant can be measured directly. For practical purposes, preferred plant parts are those which accumulate the active ingredient in relatively large amounts. Ideally, the plant part is relatively easy to harvest and handle, such as a seed or fruit.
However, other parts such as leaves, roots, stems or flowers or parts thereof may be suitable provided that the active agent(s) is present in sufficient quantities to give the desired nematicidal effect.
It is, in addition, conceivable that the pesticide properties of the plants described herein could be exploited to protect crop plants from disease, e.g.
nematode or fungal infection or insect infestation, by co-cultivating the herb plant with the crop plant as a form of biological pest control, i.e. growing the herb near or within the crop to deter pests.
The invention further provides a method for the treatment or prevention of disease in plants which comprises applying to said plants a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta.
In the nematode assay system described below, the dried herb (flower 5 bud, seed, fruit etc.) is ground or milled to a powder which is mixed with a homogenized suspension of nematode-infected roots in water. Accordingly, the anti-nematode composition is applied to the nematode-infected tissue as an aqueous solution/suspension (solution as regards water-soluble components of the herb preparation; suspension as regards components that 10 are insoluble in water). For practical application to treat or prevent disease, caused by e.g. nematode or fungal infection or insect infestation, in an agricultural crop in a field or glasshouse, the preparation can similarly be applied to the field (prior to sowing or planting) or the crop (after sowing or planting) as an aqueous solution/suspension by spraying or watering. In case 15 suspended particles of the preparation cause blockage of the spraying or watering apparatus, the preparation could be filtered prior to application, although it would first be necessary to test that removal of the insoluble fraction did not compromise the efficacy of the composition. Alternatively, the preparation can be applied to the field or the crop as a dry powder and subsequently watered-in. The powder can be formed into granules, pellets or cakes for ease of handling and application. For pot-grown plants, the dry powder can be mixed with the soil, compost or other growing medium prior to use. In a hydroponic system, the powder can be mixed with the aqueous nutrient medium. Plant pots, especially degradable pots such as those made from peat or fibre which allow the plant to be planted out without disturbing the roots, can be impregnated with the pesticide (e.g. anti-nematode, antifungal or insecticidal) composition. Seeds may be conveniently treated, e.g. dusted or sprayed, with the pesticide composition prior to storage or planting to prevent infection of the germinating seed. Crops can even be protected from infection post-harvest by employing containers impregnated with the pesticide composition for storage and transport. Similarly, vegetable (i.e. plant) foodstuffs can be protected from spoilage by storing/transporting them in containers or wrappings impregnated with the pesticide composition.
The invention accordingly further provides:
seeds coated or impregnated with a composition according to the invention;
a plant growth medium such as soil, peat, or hydroponic nutrient solution, or a degradable container such as a plug or pot, comprising a composition according to the invention;
a container or wrapping, such as a box, carton or film, for vegetable foodstuff, which is coated or impregnated with a composition according to the invention.
The invention still further provides a composition according to the invention (i.e. a composition as described herein) formulated for use as a pesticide, i.e. a pesticide formulation comprising a composition according to the invention and one or more vehicles, carriers, binders or excipients.
Pesticide (e.g. anti-nematode, antifungal and insecticide) compositions comprising ground herbs as described herein are simple to make and, therefore, relatively inexpensive. Furthermore, the anti-nematode, antifungal and insecticidal activity demonstrated by the inventors opens the possibility to protect a crop against nematode and fungal infection and insect attack with the application of a single composition, providing savings in time and cost.
The fact that the herbal compositions of the invention have activity against these disparate groups of pests also suggests that they may have a mode of action that will also be active against further classes of pests such as bacteria and viruses.
As an alternative to using the powdered herb directly in the pesticide composition, an extraction, e.g. with alcohol, water or organic solvent, or decoction of the powdered herb may be performed and the product of said process used in the pesticide composition.
It may be desirable to purify the pesticide fraction of the herb preparation to a lesser or greater extent, or even to isolate the pesticide factor, prior to use in a pesticide composition.

As illustrated by the examples, compositions according to the invention are effective in the treatment and/or prevention of plant diseases caused by a wide range of plant pathogens/pests including nematodes, fungi and insects.
The pesticide compositions of the invention can be applied to any plant susceptible to infection by pests. The invention is most useful in the protection of crop plants in which infection by pests, e.g. nematodes, fungi or insects, causes significant losses in yield and, hence, significant economic losses.
In the specialty crop markets, economic hardship resulting from nematode infection is highest in high value vegetables and fruits such as strawberries and bananas. In the high-acreage crops, nematode damage is greatest in soybeans and cotton. Non-limiting examples of crops to which the anti-nematode compositions of the invention can be applied include:
strawberries, bananas, soybeans, cotton, potato, pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals and amenity (e.g. golf course) turf grasses.
As regards fungal infection/disease, the greatest benefit is again likely to be gained in the protection of crops in which fungal disease causes significant economic loss through loss of yield, both in high value vegetables and fruits, and in high acreage crops, such as those listed above as well as cucumber and eggplant. Plantation crops such as date palm, oil palm, peach palm, pineapple and sugarcane are also susceptible to fungal disease, as well as tree species such as maple and tulip poplar. Fungicidal compositions of the invention may also be used to protect turfgrass, as well as ornamental plants such as Ficus, Guzmania, Areca and Howea, from fungal pathogens.
The inventors' findings suggest that the compositions described herein could also be used to protect crops susceptible to damage by insect pests, for example termites.
An advantage of the pesticidal compositions of the invention is the broad spectrum of pests against which the compositions are active, allowing crops to be protected against multiple, diverse pests by application of a single composition, providing reduced costs and reduced impact on the environment.

The anti-termite results presented herein indicate that pesticide compositions according to the invention could further be used to treat termite infestations in buildings, or to prevent structural damage to buildings caused by termites by coating (e.g. painting) or impregnating building materials such as wood, cements, gypsum etc. with such a composition.
The wood-eating habits of many termite species can do great damage to unprotected buildings and other wooden structures. However, once termites have entered a building, they also damage other cellulosic materials such as paper, cloth and carpets. Other soft materials such as soft plastics, plaster, rubber, and sealants such as silicon rubber and acrylics may be damaged by the termites which use particles of these materials for construction. Pesticide compositions according to the invention could be used to protect both food (i.e. cellulosic) and non-food building materials from attack by termites, or to treat existing infestations.
Accordingly, in a further embodiment, the invention provides a method for the treatment or prevention of termite damage to a building which comprises applying to said building or part thereof a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta.
Also provided is a method for the construction of buildings resistant to damage by termites, which comprises the steps of: i) treating one or more building materials with a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta; and ii) using said treated building materials in the construction of a building.
The anti-nematode activity of preparations according to the invention is demonstrated in the following examples in a test system using root-knot nematodes (genus Meloidogyne), however, there is no reason to suppose that the anti-nematode activity will be restricted to this genus of nematodes alone.
The anti-nematode preparations of the invention are expected to have utility against any soil-borne nematode pest including, but not limited to, those belonging to the genera Meloidogyne, Heterodera, Globodera, Ditylenchus, Aphelenchoides and Heliotylenchus, and any others mentioned herein.
Similarly, it is expected that the antifungal activity of the herbal preparations described herein will extend beyond the fungal species tested so far (Examples 5 and 6), to include, for example, pathogenic species of Fusarium, Verticillium, Rhizoctonia, Pythium and Phytophthora and other species mentioned herein.
The inventors have found that herbal preparations according to the invention are effective against pests (e.g. nematodes, fungi and insects) at concentrations of 1% (w/v) (i.e. 10g/I) or less. Preferably, preparations according to the invention show pesticidal activity at concentrations of 0.75%, 0.5%, 0.33%, 0.25% (w/v) or less. More preferably, said preparations show pesticidal activity at concentrations as low as 0.2%, 0.15%, 0.1% (w/v) or even less.
The final concentrations of the herbal preparations may be varied independently, i.e. the different herbs may be present in the pesticide composition at different concentrations.
For example, the inventors have found that a higher concentration of P.
anisum, relative to the other herbal ingredients, results in good nematicidal activity. An efficacious anti-nematode composition is produced by mixing 0.4% (w/v) Pimpinella anisum and 0.2% of each of Peganum harmala, Myristica fragrans and Syzygium aromaticum. Similarly, a preparation comprising 0.3% Pimpinella anisum and 0.1 % of each of Peganum harmala, Myristica fragrans and Syzygium aromaticum was found to be effective for controlling termites.
Compositions with a relatively higher concentration of Syzygium aromaticum have performed well in field tests against fungal pathogens, for example 0.1% Pimpinella anisum, 0.25% of each of Peganum harmala, and Myristica fragrans, and 0.4% Syzygium aromaticum.
Advantageously, preparations according to the invention are effective against all stages of the nematode lifecycle i.e. egg masses, consecutive larval stages and adults. It is particularly important that the preparations are effective against the egg masses which persist in the soil and the free-living second stage larvae that invade the roots of the host plant.
Preferred features of different embodiments of the invention are as to each other mutatis mutandis.
5 The invention is illustrated by way of the following non-limiting examples which make reference to the following figures:-Figure 1 is a photograph showing different formulations of clove anti-nematode agent according to Example 1.
Figure 2 shows chromatographic analysis of chloroform extract of M.
10 fragrans (nutmeg) at (from left to right) room temperature, 150 C and 200 C.
Figure 3 shows chromatographic analysis of chloroform extract of S.
aromaticum (clove) at (from left to right) room temperature, 1500C and 200 C.
Figure 4 shows microscopic examination of egg masses and juveniles of Meloidogyne incognita infected Ficus benjamina roots (a) non-treated with 15 herbal agent (control) (b) treated with herbal agent.
Figure 5 shows photographs of the root system of root-knot nematode-infected Ficus benjamina: (a) control; (b) after treatment with herbal agent composed from 0.3% Pimpinella anisum and 0.1% of each Peganum harmala, Myristica fragrans and Syzygium aromaticum; and (c) after chemical 20 treatment with Nemamort agent.
Figure 6 shows representative microscopic examinations of egg masses and juveniles of Helicotylenchus sp. infected on ornamental plants Guzmani, Areca and Howea roots: (a) before treatment with herbal agent (control) and (b) after treatment with herbal agent.
Figures 7 and 8 show symptoms of disease caused by soil-borne fungal pathogens in untreated turfgrass.
Figures 9 and 10 show healthy growth and colour in turfgrass treated with fungicidal herbal agent.
Figure 11 shows cultures of soil-borne Fusarium sp. and Pythium sp.
on PDA isolated from non-treated turfgrass. Petri dishes were incubated at 28 C for 7 days.

Figure 12 shows plots of alfalfa (a) before treatment, and (b) after treatment with herbal agent composed from percentage solution (w/v) of 0.2%
Pimpinella anisum and 0.15% of each Peganum harmala and Myristica fragrans and 0.1% Syzygium aromaticum. The treatment applied 10gm/L or 30gm/m2 as drench and repeated twice within 30 days.
Figure 13 is a photograph showing F1 hybrid cucumbers Miracle (Brusima) grown in plastic greenhouses with sand base: (A) treated with herbal mixture - the plants look very healthy absence of soil-borne fungi; (B) treated with NIPROTTM - the soil-borne fungi symptoms appeared on the plants.
Figure 14 shows photographs of F1 hybrid cucumbers Miracle (Brusima) grown in plastic greenhouses with sand base: (A) treated with herbal fungicidal mixture as described in Example 11 - the plants look very healthy without appearance of soil-borne fungi symptoms and yielded with high number of fruits compared to (B) and (C) control plants.
Figure 15 shows photographs of F1 hybrid cucumbers Miracle (Brusima) grown in plastic greenhouses with sand base: (A) treated by herbal fungicidal mixture as described in Example 11 - the plants are taller than (B) control plants.
Figure 16 shows photographs of F1 hybrid cucumbers Miracle (Brusima) grown in plastic greenhouses with sand base: (A) treated by herbal fungicidal mixture as described in Example 11; (B) control plants grown in FYM enriched soil. Fewer dead plants are observed in the plants treated with the herbal mixture.
Figure 17 shows photographs of F1 hybrid cucumbers Miracle (Brusima) grown in plastic greenhouses with sand base (A) treated by herbal fungicidal mixture as described in Example 11; (B) control plants grown in FYM enriched soil. A significantly greater yield of fruit is observed in the plants treated with the herbal mixture.
Figure 18 shows photographs of date palm trees infected with Thielaviopsis paradoxa. (A) date palm trees treated by 1% w/v herbal fungicidal aqueous solution of mixture 0.1% Pimpinella anisum and 0.5% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum applied once every 15 days for 1 month; (B) control date palm tree received no treatment and clearly shows symptoms of black scorch and bending head diseases in the heart and leaves.

Clove formulation:
Dried clove pods were purchased from Dubai local herbal market. The cloves were cleaned from dust by air-drying for 15 min in metallic strainers.
The clean pods were powdered in a grinder. 1 kg of cleaned clove pods powder was placed in plastic container and 450m1 tap water was added and promptly mixed well until a dough was formed. The clove pod dough was passed through 0.4cm diameter hole of mince machine to form granules. The granules were collected in a stainless steel tray and placed in an oven to dry at 45 C for 2 days with two times daily rotation. Various clove formulations were prepared, as shown in Figure 1.

Crop under investigation:
Okra or tomato No. of samples under treatments:
1) Clove 0 gm/I (control) 2) Clove 5 gm/l 3) Clove 7.5 gm/I
4) Clove 10 gm/I
5) A commercial recommended chemical nematocide used as another control sample.

Treatment intervals:
One treatment per every 2 weeks No. of replicates:
1. For pot experiment are five pots per treatment, and five plants per pot.
2. For field experiment are 5 lines per treatment, and 50 plants per line Nematode used for investigation:
Root-knot nematode is to be use for artificial infection.
Pots experiment:
Experiments performed with pot-grown plants are probably the most common in plant research. The need to perform controlled experiments often replaces observations on plants grown in their native environment by bringing plants for closer observation in the controlled laboratory. Plants are then grown in an artificial environment in various containers with various concentrations (w/v) of herbal anti-nematode and placed greenhouses. To avoid error variance an optimal growth conditions will be implemented.

Field experiment:
This method is experimentally examining an intervention of the herbal anti-nematode and the viability of nematode in the open fields. Field experiments will be compared with pot experiment groups.
Two fields experiment will be carried-out as follows:
Under the Authority of Ministry of Environment and Water in Al Himraniya, Agriculture station, Ras-Al-khaima.
Al Dhaid area.
Results The results will confirm that the clove anti-nematode agent is an environmentally safe, target-specific product for controlling plant parasitic nematodes in agricultural fields.

MATERIALS AND METHODS
Preparation a homogenized suspension of root-knot nematode Infected roots of squash with root-knot nematode were crushed with 200 ml tap water to prepare a homogenized suspension of root-knot nematode. Twenty millilitre samples of the homogenized suspension of root-knot nematode were placed in 50 ml glass beakers and number of egg masses, second stage larvae and other stages of root-knot nematode were checked microscopically and recorded. On average each 20 ml of the homogenized suspension root-knot nematode contained approximately 50-60 of the second stage larvae.

Herbal preparation Dried clove pods Syzygium aromaticum, Peganum harmala seed and Myristica fragrans seed were purchased from Dubai local herbal market. All herbal material was cleaned from dust by air-dried for 15 min in metallic strainers and powdered individually in a grinder.

Anti-nematode assays Different concentrations of 0.33% (w/v) and 0.5% (w/v) were prepared in 20 ml sample of homogenized suspension of root-knot nematode and 20 ml control sample (herbal material not added) (Table 2). After incubation for 24h at room temperature the numbers of egg masses, second stage larvae and other stages of root-knot nematode were checked microscopically and recorded for each sample.

RESULTS
The results are set out in Table 2 indicates that each aqueous solutions of 0.3% of clove pods Syzygium aromaticum, Peganum harmala seed and Myristica fragrans seed powder has effective nematocidal activity alone in comparisons to untreated control sample. The combination of 0.5% of each Peganum harmala and Myristica fragrans or 0.3% of each Cloves, Peganum harmala and Myristica fragrans profound novel nematocidal synergy has observed against egg masses and other stages in particular the second stage of root-knot nematode (Table 2).
In this regard a profound synergy was observed with 0.5% of each 5 Cloves and Peganum harmala in which egg masses, second stage and other stages larvaes were abolished (Table 2). However, clove pods presence or absence from the combination of Peganum harmala and Myristica fragrans appears has no significant effect in which egg masses and other stages were absence and only 1.23% and 1.8% and of second stages were observed, 10 respectively (Table 2).
The aqueous solutions 0.33 % of Peganum harmala seed appear has specific anti-egg masses nematodes activity in which they were absence from the sample under treatment. This phenomenon has also seen again in the combination of Peganum harmala seed with each clove pods and Myristica 15 fragrans seed or both (Table 2). The role Peganum harmala seed in interaction with both clove pods and Myristica fragrans seed needs further investigation.

Table 2. The survival of egg masses, second stage larvae and other stages of 20 Root-knot nematode in aqueous solutions of different herbal compositions.
Percentage of Egg masses % of Alive Larvae aqueous solutions of herbal materials Second stage Other stages***
Control (tap water) Transparent* 100% 100% Available 0.33% S. aromaticum Black** 4% Absence "Cloves"
0.33% P. harmala Absence 6% 20%
0.33% M. fragrans Black 4.4% 10%
0.33% Cloves, Absence 1.23% Absence 0.33% P. harmala and 0.33% M. fragrans 0.5% P. harmala and Absence 1.8% Absence 0.5% M. fragrans 0.5% Cloves and Absence 0% Absence 0.5% P. harmala 0.5% Cloves and Black 6.3% 5%
0.5% M. fragrans *Transparent means that larvae inside still alive and will hatch.
**Black means damage and will not hatch.
***Third, fourth & adult stages larvae are normally not moving.
Note: Similar results were obtained by placing infected roots of squash with Root-knot nematode in 1000 ml glass beakers containing 500m1 of different concentration of herbal aqueous solution of 0.33% and 0.5% and 500 ml tap water (control sample). All samples were left standing at 25 C and after 48 h the numbers of egg masses, second stage larvae and other stages of root-knot nematode from infected squash roots were checked microscopically and recorded for each sample as above.

CONCLUSION
Although clove oil has previously been proposed for use in a nematicide and in a fungicide (US 20040127362; Bowers et al, supra, Ramasinghe supra), it has not previously been reported that a simple preparation of ground, dried cloves is effective for the treatment or prevention of disease in plants. Neither Peganum harmala or Myristica fragrans has been investigated previously in the literatures as anti-nematodes.
It appears that each of cloves, Peganum harmala and Myristica fragrans has now been shown as effective protectants against plant nematode. The nematicide activity of Peganum harmala and Myristica fragrans will be further studied in the field.
Nemato-toxic compounds of the Peganum harmala and Myristica fragrans plant will be investigated. We are assuming that the modes of action of these compounds are complex, and a number of mechanisms in relation to nematode management are yet to be fully explored.

MATERIALS AND METHODS
Preparation a homogenized suspension of root knot nematode Infected roots of squash with root knot nematodes were crushed with 500 ml tap water to prepare a homogenized suspension of root knot nematodes. One hundred millilitre samples of the homogenized suspension of root knot nematode were placed in 750 ml plastic beakers and the number of egg masses, second stage larvae and other stages of root knot nematodes were checked microscopically and recorded. In average each 100 ml of the homogenized suspension root knot nematodes contained approximately 2500-3000 of the second stage larvae.
Herbal preparation Dried clove pods Syzygium aromaticum, Pimpinella anisum L. seed, Peganum harmala seed and Myristica fragrans seed were purchased from a local Dubai herbal market. All herbal materials were cleaned from dust by being air-dried for 15 min in metallic strainers and were then powdered individually in a grinder.

Anti-nematode assays Different concentrations of herbal materials, 0.1% (w/v), 0.2% (w/v) and 0.15% (w/v), were prepared in 750 ml sample of homogenized suspension of root knot nematodes, along with, a 100 ml control sample (herbal material not added) (Table 3). After incubation for 12 h at room temperature the sample suspension were passed through a 53 pm sieve, and the material remaining on the sieve were washed with tap water and resuspended in 100 ml then the numbers of egg masses, second stage larvae and other stages of root knot nematodes were checked microscopically and recorded for each sample.

RESULTS
The results, as set out in Table 3, indicate that each aqueous solution of 0.1 % of clove pods of Syzygium aromaticum, 0.2% of Pimpinella anisum seed, 0.15% of Peganum harmala seed and 0.15% Myristica fragrans seed powder has effective nematocidal activity alone in comparison to untreated control sample. The combination of 0.15% of both Peganum harmala and Myristica fragrans and 0.2% of Pimpinella anisum showed profound and novel nematocidal synergy against egg masses and other stages of root knot nematodes, in particular the egg masses.
However, the presence of 0.1 % clove pods Syzygium aromaticum in the combination of 0.15% of both Peganum harmala and Myristica fragrans and 0.2% of Pimpinella anisum has reduced the nematocidal synergy against egg masses by more than 4.5 fold (Table 3). Though it has shown to be effective against egg masses with the combination of 0.2% Pimpinella anisum seed extract. Further studies to find out the mode of action of the four herbal materials on egg masses are planned.

Table 3. The survival of egg masses, second stage larvae and other stages of root knot nematodes in aqueous solutions of different herbal compositions.
Percentage of aqueous Egg % of Living Larvae solutions of herbal masses materials Second Other stages stage 3rd 4th adult Control (tap water) 550 100% 8 8 17 0.1 % S. aromaticum 600 30-35 21 1 1 0.2% P. anisum 200 10-15 7 7 16 0.15% P. harmala 400 30 3 2 2 0.15% M. fragrans 500 25 17 8 12 0.1 % S. aromaticum and 100 5 7 3 1 0.2% P. anisum Percentage of aqueous Egg % of Living Larvae solutions of herbal masses materials Second Other stages stage 3rd 4th adult 0.1 % S. aromaticum and 400 5 8 5 4 0.15% P. harmala 0.1 % S. aromaticum and 350 20 17 1 3 0.15% M. fragrans 0.1 % S. aromaticum, 450 15-20 5 1 0.00 0.2% P. anisum, 0.15% P. harmala and 0.15% M. fragrans 0.2% P. anisum and 800 5-10 11 2 8 0.15% P. harmala 0.2% P. anisum and 200 7 7 1 4 0.15% M. fragrans 0.2% P. anisum, <100 10-15 9 0.0 1 0.15% P. harmala and 0.15% M. fragrans 0.15% P. harmala and 300 20 12 10 3 0.15% M. fragrans 0.1 % S. aromaticum, 100-150 20 7 0.0 2 0.2% P. anisum and 0.15% P. harmala 0.1 % S. aromaticum, 200 10 10 5 4 0.15 % P. harmala and 0.15%M. fragrans CONCLUSION
The herbal medicines Pimpinella anisum, Peganum harmala and Myristica fragrans have not been investigated previously in the literatures as anti-nematodes. Each of Pimpinella anisum, Peganum harmala and Myristica fragrans has been shown to be effective as protectants against plant nematodes. Use of 0.2% P. anisum in combination with 0.15% each P.
harmala and M. fragrans gives a synergistic increase in anti-nematode activity. These results suggest the use of P. anisum as the main effective material against egg masses and that supplementing this main material with P. harmala and M. fragrans can give an effective, and synergistic, increase in activity. Numerous studies are being implemented in the agriculture fields at the present time.
Nemato-toxic compounds of the Pimpinella anisum, Peganum harmala and Myristica fragrans plant will be investigated. We are assuming 5 that the modes of action of these compounds are complex, and a number of mechanisms in relation to nematode management are yet to be fully explored.

EXAMPLE 4: Chemical Analysis of Syzygium aromaticum (Clove), 10 Myristica fragrans (Nutmeg), Peganum harmala (Harmala) and Pimpinella anisum (Anise) under different grade of heating MATERIALS AND METHODS
Dry seeds of Syzygium aromaticum (Clove), Myristica fragrans 15 (Nutmeg), Peganum harmala (Harmala) and Pimpinella anisum (Anise) were purchased from a local Dubai herbal market. All herbal materials were cleaned from dust by being air-dried for 15 min in metallic strainers and were then powdered individually in a grinder.
The seeds were extracted with chloroform in Soxhlet extractor. The 20 extracts were evaporated under pressure and the dried residue was weighed.
Five grams of dried residue was used from 100g of seeds.
Establishing of general chemical composition of a vegetal material is done by means of qualitative chemical analysis, which uses the selective extraction with solvents and subsequent fractionating for identifying the active 25 ingredients (Ciulei, 1982).
Chromatographic analysis is effectuated for terpenoids using TLC G60 F254 as stationary phase, two solvent systems Toluen - Ethyl acetate (93:7) and Ethyl acetate - Glacial acetic acid - Formaldehyde - Water (100:11:11:27), detected with 5% vanillin in H2SO4 at 105 C (Wagner, 1984).

RESULTS AND DISCUSSION
Results of qualitative measurement of terpenoids are given in the following Table 4:

Sample Temp. treatment Volatile oils Sterols Un-heated +++ +++
Syzygium aromaticum 40 C +++ +++
(Clove) 100 C +++ +++
Un-heated +++ -Myristica fragrans (Nutmeg) 40 C +++ -100 C ++ -Un-heated - -Peganum harmala (Harmala) Un-heated + -Pimpinella anisum (Anise) 40 C + -100 C + -The data show that clove is richer than others in the amount of volatile oils and sterols, then nutmeg but without contains of sterols. Anise present very light positive marks related to volatile oils. Harmala show negative results in both tests.
The results of chromatography analysis are given in the following Table 5:

Sample Treatment Rf Un-heated 0.58, 0.64, 0.86, 0.88 *Clove 40 C 0.58, 0.64, 0.86, 0.88 100 C 0.58, 0.64, 0.86, 0.88 Un-heated 0.52, 0.53, 0. 57, 0.61 *Nutmeg 40 C 0.52, 0.53, 0. 57 1 00 C 0.52, 0.53, 0. 57 Un-heated No spot evidence *+**Harmala 40 C No spot evidence 100 C No spot evidence Un-heated 0.23, 0.24, 0.26 *Anise 40 C 0.23, 0.24, 0.26 100 C 0.23, 0.24, 0.26 *solvent system (Toluen - Ethyl acetate 93:7) **solvent system (Ethyl acetate - Glacial acetic acid - Formaldehyde - Water 100:11:11:27) OBSERVATIONS
1. From phytochemical point of view there are no significant differences in chemical constituents under different grade of temperature from qualitative and quantitative point of view.

2. Clove and Nutmeg present significant amount of active ingredient than Anise.

3. Harmala show no significant in both specific solvent systems used for terpenoids.

Recommendation for further investigation: to treat Clove and Nutmeg with 150 C and 200 C probably make differentiation in active ingredients amount as follows:-Myristica fragrans (Nutmeg) The samples from nutmeg with different grade of temperature (room temperature, 150 C and 200 C), put on chromatographic analysis using TLC
G60 F254 as stationary phase, and Toluen - Ethyl acetate (93:7) as solvent system. Detected with UV254 then by 5% vanillin in H2SO4 at 105 C. The results are shown in Figure 2. The following features are observed:
1. Note the presence of the principle material that is the more contain of "Myristicin" to Rf = 0.75, in three different heat treatment samples.

2. Note the presence of other less quantitative levels in the three samples convergent Rf = 0.59, 0.57, and 0.55, since it with the same colour UV believe that the differences resulted from the beginning of affected material is thermally.
The differences in the Rf value from different experiments is not a concern since we are not working at standard conditions, but the Myristicin is always the first compounds that go out with a supreme Rf and largest content.
Syzygium aromaticum (Clove) The samples from clove with different grade of temperature (room temperature, 150 C and 200 C), put on chromatographic analysis using TLC
G60 F254 as stationary phase, and Toluen- Ethyl acetate (93:7) as solvent system. Detected with UV254 then by 5% vanillin in H2SO4 at 105 C. The results are shown in Figure 3. The following features are observed:
1. Note the presence of the two materials the first is on Rf = 0.96 and is located in three transactions at the same size. The second one is the Eugenol and is on Rf = 0.6 and it is the largest in the three transactions.
2. There are a perfect concordance between spots on UV and spots which result after detection with reagent.
SUMMARY
The chloroform extract of Syzygium aromaticum (Clove), Myristica fragrans (Nutmeg), Peganum harmala (Harmala) and Pimpinella anisum (Anise) did not affected by different grade of heating, 40 C and 100 C. The clove and nutmeg are significantly richer in active ingredients than anise, but harmala extract show no significant. Therefore, probably the treatments of the extracts for higher temperature have an influence on present of active ingredients. To this point it was found that at 150 C to 200 C the principle material in Myristica fragrans (Nutmeg) contain of "Myristicin" to Rf = 0.75, whilst for Syzygium aromaticum (Clove) at the same temperatures were found the presence of the two materials the first is on Rf = 0.96 and is located in three transactions at the same size. The second one is the Eugenol and is on Rf = 0.6 and it is the largest.
The chemical structures of Myristicin and Eugenol are shown below in Table 6:

Myristicin Eugenol Formula C11H1203 C10H1202 Structure Origin Phenol (Allyl-benzene) Phenol (Allyl-guaiacol) Pathway Shikimic acid Shikimic acid Found in Nutmeg Clove + Nutmeg EXAMPLE 5: Effect of aqueous Syzygium aromaticum "clove" pod powder on the population of soilborne fungi on Cucurbitaceae and eggplant plants in greenhouse and open field Ground dried clove pods were tested for antifungal effect in vitro and in the control of soilborne fungi in the greenhouse and field.
Potato Dextrose Agar (PDA) was supplemented with 0.5% and 1 % w/v clove pod powder. Growth of Fusarium oxysporum, Fusarium wilt, Phytophthora blight, Phytophthora nicotianae and Pythium sp. was abolished in PDA supplemented with 1 % clove pod powder compared to control treatments (no clove powder) after incubation for 7 days at 28 C.
Eggplant (var. Black Beauty) and cucumber (var. Queen) plants were grown from seed in pots containing soil previously inoculated with Phytophthora sp. and Pythium sp. The effect of weekly treatment with 0.5%
and 1% w/v clove pod aqueous extract on the number of wilted plants was measured compared to untreated controls. Treatment with 0.5% clove pod aqueous extract was found to significantly reduce disease severity; 1 %
extract had an even greater effect.
Clove pod aqueous extract 0.25%, 0.5% and 1 % w/v was also tested for efficacy against soilborne fungal diseases of eggplant and cucumber in the 5 greenhouse and in open field. Treatments were applied on the date of sowing with two further applications at weekly intervals. The results showed that clove pod aqueous extract in the range of 0.5% to 1 % w/v was able to demolish fungal infection (rot, crown rot and stem) of cucumber var. Queen and eggplant var. Black Beauty in greenhouse and farms in less than 7 days, 10 and is more effective in the management of fungal diseases in situ than commercial chemical products tested (Fungarid 0.1%, Rhizolex 0.15%, Ridomil plus 0.2%, Tachigareen 30% 0.2%).
Clove pod granules prepared according to Example 1 were evaluated for efficacy against soilborne fungi. Treatment of coconut soil with 1% w/v 15 (10g/L) clove pod granules was found to significantly reduce soil populations of Phytophthora sp. and Pythium sp. compared with untreated coconut soil and significantly reduce the frequency of eggplant var. Black Beauty seedlings showing seedling wilt symptoms.
The foregoing results demonstrate that clove pod powder, granules 20 and aqueous extract are an effective antifungal compositions, as well as being environmentally friendly, safe to consumers and inexpensive to farmers.

EXAMPLE 6: The antifungal activity of Pimpinella anisum (Anise), Peganum harmala (harmala), Myristica fragrans (nutmeg) and Syzygium 25 aromaticum (cloves) against selected species of soilborne fungi MATERIALS AND METHODS
Inoculum preparation Fungi used in the present investigation were isolated from damping-off 30 seedlings and wilted plants of cucumber var. Queen from different farms of Northern Emirates (Al-Dhaid and Ras-El Khaima, UAE) The collected diseased materials were thoroughly washed in running tap water and then surface sterilized by dipping in 0.1% mercuric chloride solution for 3 minutes and rinsing twice in sterile water. Such sterilized materials were cut into small pieces (0.5-1 cm in length), dried between sterilized filter papers and then planted on potato dextrose agar (PDA; Difco) medium. Plates were incubated for 3-6 days at 28 C and examined daily for the occurrence of the fungal growth. Purification of the resultant fungal growth was made through the hyphal-tip or single spore isolation techniques (Hildebrand, 1938; Domsch, et.a1.1980) and the pure cultures were maintained on PDA slants. Isolated fungi examined microscopically and identified (Ellis, 1971).
In vitro evaluation of herbal anti-soilborne fungal efficiency Dried Pimpinella anisum (Anise), Peganum harmala (harmala), Myristica fragrans (nutmeg) and Syzygium aromaticum (cloves) pods were cleaned from dust by air-dried. The clean herbal materials were powdered individually in a blender. Then 0.15% w/v of Peganum harmala (harmala) or Myristica fragrans (nutmeg), and 0.2% w/v of Pimpinella anisum (Anise) or Syzygium aromaticum clove pods powder were supplemented in individually or mixed (Table 7) in PDA and autoclaved at 121 C for 20 min. The herbal mixture media were cooled to approximately 45 C mixed well and promptly dispensing to 20 ml in sterile disposable Petri dishes and allow the contents to solidify at room temperature.
Soilborne fungal inoculums of 0.5 X 0.5 cm2 from 7 day cultures on PDA were placed on the center of the PDA supplemented with herbal materials. The Petri dishes were inverted and incubated for 5 days at a 28 C.
In addition, plates of PDA without herbal powders were inoculated as described above with soilborne fungi as a control test. Five replicates were used for each fungus, on PDA with and without herbal powder.
The term "growth" is used in a special sense herein, i.e. to designate the presence and presumed proliferation of viable fungi. Following incubation, fungal growth diameter (cm) was calculated and the colony size was calculated using experimental data by Gorbushina (1997) partially represented in Table 7.

RESULTS
Two fungal species were isolated from fungus-infected cucumber plants and identified as Fusarium sp and Pythium sp.
To investigate the nature of the selected herbal materials for the inhibition of other soilborne diseases for instance soilborne fungi, a series of experiments was performed to evaluate the kinetics of 0.15% Peganum harmala (harmala), 0.15% Myristica fragrans (nutmeg), 0.2% Syzygium aromaticum (cloves) and 0.2% Pimpinella anisum (Anise) supplemented PDA
against growth survival of soilborne Fusarium sp and Pythium sp.
The colony diameter, the size of the zone of nutrition and the input of herbal matter on the PDA to determine the herbal matter available to inhibit the growth for a single fungal colony were determined. This assay measured the rate of fungal inhibition by determination of the colony diameter (mm) which survived exposure to concentration of herbal material-supplemented PDA. Under the conditions used, the mean fungal survival rate of Fusarium sp and Pythium sp which were exposed to PDA without herbal material as a control, as compared to PDA supplemented different concentration of 0.15%
Peganum harmala (harmala), 0.15% Myristica fragrans (nutmeg), 0.2%
Syzygium aromaticum (cloves) and 0.2% Pimpinella anisum (Anise) (Table 7).
Table 7. Colony diameter (mm), mean of five replicates, of two soilborne fungi species on PDA supplemented with different percentage of powdered herbal materials after 5-days growth at 28 C
Treatment: PDA supplemented herbal Colony diameter (mm) : %
materials Fungal survival Fusarium sp Pythium sp..
0.15% Peganum harmala 55 : 68.75% 50 : 66.66%
0.15% Myristica fragrans 70 : 87.5% 65 : 86.66%
0.2% Syzygium aromaticum 55 : 68.75% 55 : 73.3 %
0.2% Pimpinella anisum 80:100% 75:100%
0.15% Peganum harmala + 0.15% Myristica 60:75% 40 : 53.33%
fragrans + 0.2% Pimpinella anisum 0.15% Peganum harmala + 0.15% Myristica 55:68.75% 45 : 60%
fragrans + 0.2% Syzygium aromaticum +
0.2% Pimpinella anisum Control (PDA without herbal materials) 80 : 100% 75 : 100%

The result shows that Peganum harmala has a profound antifungal activity against both soilborne Fusarium sp and Pythium sp, whilst Syzygium aromaticum shows also similar activity against Fusarium sp.
The differences of antifungal activity between samples treated with Peganum harmala and Syzygium aromaticum and those treated with control deprived of herbal materials were significant.
The medicinal herb Rhazya stricta was also tested for pesticide activity, individually and in combination with S. aromaticum, M. fragrans and P.
anisum, against nematodes and fungi and showed similar anti-nematode and antifungal activities to those observed for P. harmala.

EXAMPLE 7: Effect of varying the relative amounts of P. anisum, P.
harmala, M. fragrans and S. aromaticum in herbal mixture on control of root-knot nematode in ornamental plants Different herbal mixture groups were prepared by weighing individually different amounts of dry herbal materials (Pimpinella anisum, Peganum harmala, Myristica fragrans and Syzygium aromaticum) in beakers. Then tap water was added to each group to make up an herbal solution in a final volume of 100 ml. Each herbal solution was then tested on ornamental plants Ficus benjamina infected with root-knot nematodes Meloidogyne incignita.

Table 8. Nematocidal activity of different herbal mixtures on F. benjamina infected with root-knot nematodes.
Herbal Herbal concentration Nematocidal % Nematodes mixture (% w/v) Activity Mortality group 1 Pimpinella anisum 0.01 1 + 10-20 Peganum harmala 0.01 Myristica fragrans 0.01 S z gium aromaticum 0.01 2 Pimpinella anisum 0.03 2+ 20-30 Peganum harmala 0.03 Myristica fragrans 0.03 S z gium aromaticum 0.03 3 Pimpinella anisum 0.05 2+ 20-30 Peganum harmala 0.03 Myristica fragrans 0.03 S z gium aromaticum 0.05 4 Pimpinella anisum 0.1 2+ 20-30 Peganum harmala 0.1 Myristica fragrans 0.05 S z gium aromaticum 0.05 Pimpinella anisum 0.15 3+ 30-50 Peganum harmala 0.1 Myristica fragrans 0.05 Syzygium aromaticum 0.15 6 Pimpinella anisum 0.2 3+ 30-50 Peganum harmala 0.1 Myristica fragrans 0.1 Syzygium aromaticum 0.1 7 Pimpinella anisum 0.2 3+ 30-50 Peganum harmala 0.15 Myristica fragrans 0.1 Syzygium aromaticum 0.15 8 Pimpinella anisum 0.25 4+ 50-75 Peganum harmala 0.1 Myristica fragrans 0.15 Syzygium aromaticum 0.1 9 Pimpinella anisum 0.3 5+ 75-90 Peganum harmala 0.1 Myristica fragrans 0.1 Syzygium aromaticum 0.1 Pimpinella anisum 0.4 6+ 90-100 Peganum harmala 0.2 Myristica fragrans 0.2 Syzygium aromaticum 0.2 11 Pimpinella anisum 1 6+ 90-100 Peganum harmala 0.25 Myristica fragrans 0.25 S z gium aromaticum 0.25 The above results show that the herbal mixture group number 10 gave >_90% nematodes mortality and for this reason it was selected for field experiments and has been further prepared in large proportions in litres as 5 follows:
Weigh individually powdered herbs of 4 Kg of Pimpinella anisum, and 2 Kg of each Peganum harmala, Myristica fragrans and Syzygium aromaticum then mix all together very well. Transfer 10 gm from the powdered herbal dry mixture into a plastic container and 1 litre of tap water added to obtain the 10 final concentration of (w/v %) 0.4% Pimpinella anisum and 0.2% of each of Peganum harmala, Myristica fragrans and Syzygium aromaticum.

EXAMPLE 8: Efficacy of nematocidal herbal agent to control root-knot nematode in ornamental plants This study was conducted to determine the activity of nematocidal herbal agent (consisting of w/v 0.4% Pimpinella anisum and 0.2% of each Peganum harmala, Myristica fragrans and Syzygium aromaticum) performed on ornamental plants Ficus benjamina infected with root-knot nematodes Meloidogyne incognita.

METHODS
1- Treatment of infected root-knot nematode Ficus benjamina Fifty plants of Ficus benjamina infected with root-knot nematode planted in 40 cm diameter potting soil pots were divided into 3 groups and following treatments administered:
i. 10 plants treated with tap water and marked as a control.
ii. 20 plants treated with nematocidal herbal mixture no. 10 (10 gm/L: of 0.4% Pimpinella anisum and 0.2% of each Peganum harmala, Myristica fragrans and Syzygium aromaticum). Every plant/pot was treated by pouring 2 litres of the solution evenly over the soil.
iii. 20 plants treated with chemical anti-nematode Nemamort (1 ml/L) one plant/ pot was treated by pouring 2 litres of the solution evenly over the soil.
Treatments were repeated twice within 30 days and all pots received tap water 3 times per week and pots left for 30 days in which then the roots from each treatment were microscopically inspected visually.

2. Root-knot nematode inspection The root-knot nematode life cycle stages were prepared for inspection microscopically from 50 grams of the infected Ficus benjamina roots, rinsing in running tap water for 2 minutes and then cut into 20mm pieces, placed in blender with 250 ml tap water. 20 ml of the suspension containing nematode were collected through 53 micron sieve and then inspected using a compound microscope at 40 X magnification.

RESULTS AND DISCUSSION
All stages of root-knot nematode Meloidogyne incognita life cycle (egg masses, second, third, fourth stage larvae and adults) were inspected microscopically. The results from control samples shows clearly second stage larvae were alive with transparent egg masses (Fig. 4).
Galls and alive juvenile root-knot nematodes of Meloidogyne incognita were clearly observed on the root system of control Ficus benjamina not treated with any nematocidal agents (Fig. 5(a)). Fewer galls and alive juvenile root-knot nematodes were observed on the root system of Ficus benjamina treated with chemical nematocide (Nemamort) (Fig. 5(c)). By contrast, no root-knot nematode galls were visually observed on the roots of Ficus benjamina treated with nematocidal herbal agent (Fig. 5(b)). Furthermore, approximately 99.2 % of the juvenile were found dead in roots of Ficus benjamina treated with herbal nematocidal agent in comparison to untreated control plants.

Furthermore, same treatments as above were extended to control root-knot nematodes of other parasitic nematodes Ditylenchus sp., Appelenchoides sp. and Helicotylenchus sp. on ornamental plants Guzmania, Areca and Howea and same results were also obtained (Fig. 6).
EXAMPLE 9: Control of turfgrass soil-borne fungi by herbal agent The current example was conducted to determine the fungicidal activity of herbal mixture with the percentage solution (w/v) of 0.1% Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum, based upon its ability to move through the soil and achieve the desired activity against the major root fungi diseases.
INTRODUCTION
Soil-borne turfgrass diseases affect all warm-season grasses and cause major loss of turf quality. They are caused primarily by fungi and there are few examples of true disease resistance in turfgrass. Rhizoctonia diseases of turfgrasses are caused by at least two soilborne fungi, Rhizoctonia solani and R. cerealis. One or both fungi are present in practically all soils throughout the world. Both fungi are composed of a large number of strains or races that attack a wide range of different plants and include most vegetables, flowers, and field crops.
Symptoms of turfgrasses infected by species of Rhizoctonia vary widely and are easily confused with the symptoms of diseases produced by other pathogens. They vary with the specific combinations of turfgrass cultivar, soil and air environmental conditions, and the specific species and strains (or races) of Rhizoctonia. One or more species of Rhizoctonia infect all turfgrasses, causing foliar blights as well as seedling blights.
Higher-cut turf, found on home and industrial lawns, parks, athletic fields, and golf course fairways, diseased patches usually are roughly circular, light brown, matted down, and up to 2 feet in diameter. Both incidence and disease severity are influenced by plant health, site factors like shading and sometimes directly by mowing height. At times environmental factors like shading, water logging or heat stress can cause severe turf injury and stress and are misdiagnosed as diseases.
The patches sometimes develop green centres and resemble the "frogeyes" of summer patch and necrotic ring spot. Diseased patches of grass, however, appear to be sunken.
In light infections of brown patch, the affected turf generally recovers in 2 or 3 weeks. When the attack is severe, however, the crown, rhizomes, stolons, and roots turn brown and rot often killing large areas.
Cultural control for most of these diseases is all about ensuring soil moisture is adequate but not high or excessive. In some soils drainage needs to be improved, while for others, where good drainage is inherent, good irrigation management practices need to be employed.
Many diseases are also facilitated by high rates of applied nitrogen, often because nitrogen facilitates fast growth rates, producing plentiful amounts of young, easily infected tissue. This can be mitigated to some extent by fertilising more frequently with less fertiliser to promote steady growth rates.
There is a range of chemicals registered for the control of diseases in turf, some being registered for use against several diseases.

METHODS
Herbal anti-fungal Powdered herbs were weighed individually: 1 Kg of Pimpinella anisum, and 2.5 Kg of each Peganum harmala and Myristica fragrans and 4 Kg Syzygium aromaticum. All 4 herbs were mixed together very well. Then 30 gm from the herbal dry mixture were placed into a plastic container and 3 litre of tap water was added to obtain the final concentration of herbal antifungal agent (w/v) 0.1% Pimpinella anisum and 0.25 % of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum.

Identification of soil-borne fungi Field soil was collected from around plant roots known to be heavily infested with soil-borne fungi. The standard operating procedure for aerobic plate count was used with the fungal medium the potato dextrose agar (PDA).
Plates were incubated at 28 C and examined daily for 7 days. Pure fungi cultures were selected and identified microscopically and visually.

Treatment of infected area An area of 20m X 20m was divided to 16 blocks 5m X 5m and two treatments were used as follows:
i. Fungicidal herbal agent (10gm/L or 30gm/m2) applied as drench and repeated twice within 30 days.
ii. Tap water as control.

All blocks received tap water 3 times per week. Randomized samples were collected and results recorded.

RESULTS AND DISCUSSION
Identification of the isolated fungi Pure fungi cultures were identified microscopically and visually on PDA
culture. The main fungi identified as follows:
Rhizoctonia solani. The disease most commonly known as "brown patch" and "large patch". Patches of turf usually become light green in color, then yellow, before degenerating into a brown discolored area.
Fusarium spp. It is primarily and is often characterized by an orange/brown color in the patch and patch borders.

Treatment of infected area Untreated, symptoms may show up at any time during the growing season. The grass appears yellowish (chlorotic) and thins out in large, irregular patches 1 foot to more than 20 feet in diameter. The stolons can often be lifted easily from the soil because of the poor root system. Nodes may be discoloured. The yellowish foliage eventually dies and turns brown (Fig.s 7 and 8).
The blocks treated with fungicidal herbal agents became green grass color without any blotches within 30 days (Figs 9 and 10) as compared with 5 untreated area.
Furthermore no fungi growth found after 7 days incubation at 28 C on the plates of PDA from the treated 16 blocks with herbal antifungal agent as compared with from that obtained from untreated control blocks (Fig. 11).
The same experiment extended to control of soil-borne fungi in alfalfa 10 also showed positive results i.e. control of soil-borne fungal pathogens leading to healthier plants (Fig. 12).

EXAMPLE 10: Biological control of soil-borne fungi in cucumber protective cultivation This study was conducted to determine the fungicidal activity of a mixture of herbal materials consisting from percentage solution (w/v) of 0.1%
Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum against soil-borne fungi in green-houses. The results have been compared with treatment of biological agent of the fungal Trichoderma harzianum and Trichoderma viride.

METHOD
Plastic greenhouses A sand base in three plastic greenhouses 8m x 40m were prepared to grow crop of cucumbers hybrid F1 seeds Miracle (Brusima) with 8 lines and 80 plants per line for a period of 3 months. Treated post and high tensile wire trellis systems were installed in houses with eight rows fencing wire was used to support the cucumbers to a height of 7.5 feet. This allowed eight lines of cucumbers with a total of 640 plants in the house. Houses were covered with single layers of 6 mil polyethylene plastic and had existing heating and cooling capability.

Treatments 1. Fungal Trichoderma sp. treatment: Mix 150 gm NIPROTtm of each Trichoderma viride or Trichoderma harzianum with 10 kg of moisture well-decomposed farm yard manure (FYM) and kept for 12 days under a polyethylene cover in shade. The mixture were turned-over every 3 days and after 12 days the mixture equally distributed as 30g/ m2 area then covered with sand and after 2 days cucumber seeds were planted as 40 plants per line.

2. Fungicidal herbal agent: The herbal fungicidal mixture was prepared by weighing powdered herbs in the following amounts and mixing together thoroughly: 1 Kg of Pimpinella anisum, and 2.5 Kg of each Peganum harmala and Myristica fragrans and 4 Kg Syzygium aromaticum. 7 days before planting the seed (pre-plantation) 30 gm from the powdered herbal dry mixture was taken and equally scattered on an area of 1 m2 of sand and mixed thoroughly with a depth of 5 cm and then irrigated with 3 litre of tap water. The herbal fungicidal mixture was reapplied 1 time every 3 weeks for 3 months. The FYM not used with herbal fungicidal mixture.

3. Control: only FYM added to the soil.

All plants in the above three plastic greenhouses were watered from the city water source at pH 8.2 and plant growth was observed for 3 months.
During the indicated period the plants were observed for main and secondary shoot length, biomass, the density of green vegetation, flowering time, fresh weight yield, number of wilted plants, fertilizer deficiency and pH around plant roots.

RESULTS AND DISCUSSION
The crop took about 3 months to complete the growing cycle which in the case of herbal fungicidal mixture the fruit was of good quality and size and yields were fair, the cost of maintaining this crop was inexpensive given yields and time to produce a mature crop. Based on our results, the antifungal herbal formula is a good candidate for greenhouse production due to high yield of production of fruit and health plant (Fig. 13) and is markedly superior to known fungicidal agents (Trichoderma viride or Trichoderma harzianum) conventionally used for soil-borne fungi in agricultural markets as biological control agent (Table 8), whereas, the foot rot caused by Pythium sp and wilt caused by Fusarium oxysporium were cleared from plants treated by herbal fungicide according to the results from the microbiology lab on PDA plate agar culture test.
Table 8. Cucumbers hybrid F1 seeds Miracle (Brusima) grow in plastic greenhouses treated with NIPROTTM (Trichoderma viride or Trichoderma harzianum) and herbal formula composed from (w/v) 0.1% Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4%
Syzygium aromaticum to control soil-borne fungi.
Treatments Rate % Fungal Mortality Yield/plant Foot rot % Wilt Kg Trichoderma 150gm/10kg FYM 99.52 100 1.860 harzianum Trichoderma viride 150gm/10kg FYM 99.04 100 1.736 Herbal agent 1Ogm/L 100 100 1.625 Control ------------- 93.75 97.60 1.060 In the method described above, the herbal fungicidal mixture was applied to the sand bed as a dry powder, mixed and watered in. Similar results were also obtained by first mixing 30 gm of the herbal fungicidal mixture (1 Kg P. anisum, 2.5 Kg each P. harmala and M. fragrans, and 4 Kg S. aromaticum) with 3 litres of water to obtain a final concentration of (w/v) 0.1% P. anisum, 0.25% each of P. harmala and M. fragrans and 0.4% S.
aromaticum; irrigating each 1 m2 of the field with 3 litres of herbal mixture solution; and mixing the herbal solution thoroughly with sand for a depth of 5 cm.

EXAMPLE 11: Control of soil-borne diseases of greenhouse crops METHOD
Plastic greenhouses A sand base in three plastic greenhouses 8m x 40m were prepared to grow crop of cucumbers hybrid F1 seeds Miracle (Brusima) with 8 lines and 80 plants per line for a period of 3 months. Treated post and high tensile wire trellis systems were installed in houses with eight lines fencing wire was used to support the cucumbers to a height of 7.5 feet. This allowed eight rows of cucumbers with a total of 640 plants in each house. Houses were covered with single layers of 6 mil polyethylene plastic and had existing heating and cooling capability.

Treatments 1. FYM was mixed 30g/m2 area mixed with sand and after 2 days cucumber seeds were planted as 80 plants per line to serve as control.

2. Fungicidal herbal agent: The herbal fungicidal mixture of (w/v) 0.1%
Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum applied as a powder by mixing 30gm/m2 of sand 7 days before planted the seed (pre-plantation) and then 1 time every 3 weeks for 3 months without adding the organic and chemical fertilizer.

All plants in the above plastic greenhouses were watered from the city water source at pH 8.2 and plant growth was observed for 3 months. During the indicated period the plants were observed for main and secondary shoot length, biomass, the density of green vegetation, flowering time, fresh weight yield, number of wilted plants, fertilizer deficiency and pH around plant roots.

Identification of soil-borne fungi Field soil was collected from around plant roots known to be heavily infested with soil-borne fungi. The standard operating procedure for aerobic plate count was used with the fungal medium the potato dextrose agar (PDA).
Plates were incubated at 28 C and examined daily for 7 days. Pure fungi cultures were selected and identified microscopically and visually.

RESULTS AND DISCUSSION
During the 3 months of plantation it was observed that the hybrid F1 Miracle (Brusima) cucumber plants grown in greenhouse herbal treated soil were remarkably healthy and yielded a higher number of fruits (Fig.14(a)) than untreated plants (Fig.s 14(b)&(c)) and the results obtained from PDA show that this was due to the marked abolition from soil of the fungi Pythium sp and Fusarium oxysporium (causal agents of foot rot and wilt, respectively) by the herbal treatment.
It was also noticed that the flowering time started in the cucumbers plant grown on treated soil treated by herb 7 days before the same plant grown under the same condition on soil treated by FYM. Furthermore they also exhibited ample clusters of developing fruit earlier (Figs 14a) in which it appears the soil deficient herbal formula has causes a moribund length period for flowering and fruiting. In general, the untreated herbal formula soil exhibited less clusters of developing fruit.
After 35 days the plant length were longer in the average of 120cm in the greenhouse soil enriched with herbal formula, whereas, in the FYM
enriched soil the plant length were in the average of 95 cm (Fig. 15A & B).
The number of dead plant were only 2 observed in the herbal soil treated formula greenhouse (Fig. 16A) in comparison there were 25 plants dead in the in the FYM enriched soil (Fig. 16B).
The fruit yield increased approximately 37% (fresh weight 2.55 kg /
plant) as compared with plants yield grown in the FYM enriched soil (1.85 kg/plant) (Fig. 17A & B).

From the above experiments it appears that herbal mixture of Pimpinella anisum, Peganum harmala, Myristica fragrans and Syzygium aromaticum beside its' fungicidal and nematicide activities and in comparison to FYM it has mediated enhancement on the plant growth very well and 5 yielded with impressive amount of fruits.
On the other hand, the pH value of treated soil with herbal mixture reached to 5.9 (acidic soil) and it was less by 2.34 pH level from that soil treated with FYM pH 8.24 (alkaline soil). It is well known that the pH level influences the availability of nutrients to plants, and the type of plants grown.
10 Some plants like a lot of iron, a micronutrient that becomes less available to plants as soil alkalinity increases. These plants prefer a more acid soil, where iron is freely available otherwise their leaves become yellow between the veins.
All plants need a good balance of the major nutrients (nitrogen, 15 phosphorus and potash), as well as minor or 'micro' nutrients such as magnesium, copper, iron, manganese and many others in minute quantities.
From the chemical analysis, carried-out in Al Dhaid Research Center UAE, apparently the herbal mixture contains all major and minor nutrients with a phenomenal percentage of organic matter 94.68% in comparison to FYM
20 50-55% (Table 9). Accordingly, the herbal mixture works as a soil conditioner to supplement the plants with important nutrients. Beside the pH level of soil treated by herb was around 5.9 which may perfectly influence the availability of nutrients and added value to crops and reduced overall use of fertilizers and pesticides whereas the soil became more healthy and balanced.
Table 9. Chemical analysis of the herbal mixture and farm yard manure (FYM).
Material used Nutrients %/ppm N 2.38%
Herbal mixture: K 1.37%
0.1 % Pimpinella anisum, P 0.31 %
0.25% Peganum Na 0.15%
harmala Fe 145 ppm 0.25% Myristica fragrans Mn 94 pmm 0.4% Syzygium Zn 26 pmm aromaticum Cu 5 pmm NaCl 0.38 %
Organic matter 94.68 %
C/N ratio 23.13 Moisture content 11.19%
pH 5.9 FYM: Animal manure, approximately 70% of K and P 2-3%
treated cow manures S 0.5-0.7%
Ca 1-2%
Mg 0.5-1%
Mn 200 ppm Fe, Cu, Zn, B and Mo 800-1000 ppm NaCl 0.5-1.5%
Total nitrogen (N) 2-3%
Organic matter 50-55%
C/N ratio 1-12 Moisture content 12-25%
pH 8.24 It appears that the herbal mixture presented in the current invention beside it has antifungal and anti-nematodes activities it also has perfect chemical nutrient balances to increase soil fertility for the organic growers to be used in their farms instead of chemical fertilizers and as it has accelerated the production of flowers 7 days with a 37% fruits yield increase over the FYM, therefore, this phenomenon can be used to facilitate of good time management of harvesting of crops and keep them in the market for a long period with less competition and this will have a promising commercial advantages over the other chemical fertilizers which had destroyed soil health and nutrient balances and also have a bad health impact on human and animal.

EXAMPLE 12: Control of date palm black scorch fungus by fungicidal herbal agent This study was conducted to determine the fungicidal activity of a mixture of herbal materials consisting from percentage solution (w/v) of 0.1%
Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum to control black scorch disease affecting date palm trees.

Black scorch disease symptoms Black scorch, also called Medjnoon or Fool's disease, is caused by Ceratocystis paradoxa (Hohn) which is the perfect form of Thielaviopsis paradoxa.
Thielaviopsis paradoxa is an opportunistic secondary pathogen attacking stressed trees growing under conditions of severe drought, hot winds and salinity produced symptoms similar to bunch fading disorder and also causes terminal bud rot, black scorch and bending head diseases on date palms (Karampour and Pejman 2007).
The disease has been reported from many of the economically important crop growing regions with heavy losses under severe condition on date palm trees (Phoenix dactylifera), oil palm (Elaeis guineensis), peach palm (pejibaye, Bactris gasipaes), pineapple, sugarcane (Saccharum officinarum L.) and also found in the eastern United States, causing disease in maple and tulip poplar.
Until now, set-treatment with fungicide is commonly followed for its management. But there is a need to devise the non-chemical methods of control to reduce the adverse effect of toxic chemical on environment particularly date palm trees and other important crops' ecosystem.
In date palm trees the symptoms are usually expressed in four distinct forms: black scorch on the leaves, inflorescence blight, heart or trunk rot and bud rot on palms of all ages. Infections are all characterized by partial to complete necrosis of the tissues. Typical lesions are dark brown to black, hard, carbonaceous, and, as a mass, give the petioles, fruit strands and fruit stalks a scorched, charcoal-like appearance. Decay is most serious when it attacks the terminal bud and heart leading to the death of the palm.
Good sanitation is the first step in the control of black scorch. The affected fronds, leaf bases and inflorescences should be pruned, collected and immediately burned. The pruning cuts and surrounding tissues should be protected by spraying with Bordeaux mixture, lime-sulphur solution, copper sulphate lime mixture, dichlone, thiram or any new copper-based fungicides.
Under a severe attack, affected palms are to be removed and burnt.

MATERIALS AND METHODS
Inoculum preparation The fungi Thielaviopsis paradoxa used in the present investigation was isolated from more than 20 samples of vegetative and generative tissues of affected trees from several areas of Northern Emirates (Al-Dhaid and Ras-El Khaima, UAE). The collected diseased materials were thoroughly washed in running tap water and then surface sterilized by dipping in 0.1% mercuric chloride solution for 3 minutes and rinsing twice in sterile water. Such sterilized materials were cut into small pieces (0.5-1 cm in length), dried between sterilized filter papers and then planted on potato dextrose agar (PDA; Difco) medium. Plates were incubated for 3-7 days at 28 C and examined daily for the occurrence of the fungal growth. Purification of the resultant fungal growth was made through the hyphal-tip or single spore isolation techniques (Hildebrand, 1938; Domsch, et.al. 1980) and the pure cultures were maintained on PDA slants. Isolated fungi examined microscopically and identified (Ellis, 1971).

In vitro evaluation of herbal anti- black scorch fungal efficiency Dried Pimpinella anisum (Anise), Peganum harmala (harmala), Myristica fragrans (nutmeg) and Syzygium aromaticum (cloves) pods were cleaned from dust by air-dry. The clean herbal materials were powdered individually in a blender. Then (w/v) of 0.1% Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum powder were supplemented in mixed in PDA at the following concentration 0.2, 0.5, 0.75, 1 and 2% and autoclaved at 121 C for 20 min.
The herbal mixture media were cooled to approximately 45 C mixed well and promptly dispensing to 20 ml in sterile disposable Petri dishes and allow the contents to solidify at room temperature.

Black scorch fungal inoculums of 0.5 X 0.5 cm2 from 7 day cultures on PDA were placed on the center of the PDA supplemented with herbal materials. The Petri dishes were inverted and incubated for 7 days at a 28 C.
In addition, plates of PDA without herbal powders were inoculated as described above with black scorch fungi as a control test. Five replicates were used on PDA with and without herbal powder. The term "growth" is used in a special sense herein, i.e. to designate the presence and presumed proliferation of viable fungi. Following incubation, fungal growth diameter (cm) was calculated and the colony size was calculated using experimental data by Gorbushina (1997).

Field trial Four infected Thielaviopsis paradoxa date palm trees were treated with an aqueous solution of a herbal mixture prepared according to Example 9, i.e.
1% (w/v) (10 gm/L) of mixed 0.1% Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum by pouring and wetting method in the heart and infected parts of the trees.
Furthermore, two infected Thielaviopsis paradoxa date palm trees were treated by tap water only as control. Treatments replicated two times within one month (once every 15 days), and trees left for observation and after 30 days samples were collected for fungal screening on PDA.

RESULTS
Laboratory experiments The fungal Thielaviopsis paradoxa was isolated from fungus-infected date palm tree tissues such as pedicels, strands and peduncles exclusively and purified and identified based on morphological and growth characters.
To investigate the nature of the selected herbal materials for the inhibition of Thielaviopsis paradoxa fungi, a series of experiments was performed to evaluate the kinetics concentrates concentration of 0.1, 0.25, 0.5, 0.75 and 1 % of a mixture of 0.1 % Pimpinella anisum (Anise) and 0.25%
of each Peganum harmala (harmala), and Myristica fragrans (nutmeg) and 0.4% Syzygium aromaticum (cloves). The colony diameter, the size of the zone of nutrition and the input of herbal matter on the PDA to determine the herbal matter available to inhibit the growth for a single fungal colony were determined. This assay measured the rate of fungal inhibition by 5 determination of the colony diameter (mm) which survived exposure to concentration of herbal material-supplemented PDA. Under the conditions used, the mean fungal survival rate of Thielaviopsis paradoxa which was exposed to PDA without herbal material as a control, as compared to PDA
supplemented different concentration of 0.2, 0.5, 0.75 and 1% of a mixture of 10 0.1% Pimpinella anisum (Anise) and 0.25% of each Peganum harmala (harmala), and Myristica fragrans (nutmeg) and 0.4% Syzygium aromaticum (cloves) (Table 10).

Table 10. Colony diameter (mm), mean of five replicates, of Thielaviopsis 15 paradoxa on PDA supplemented with different percent concentration of powdered mixed herbal materials of 0.1% Pimpinella anisum (Anise) and 0.25% of each Peganum harmala (harmala), and Myristica fragrans (nutmeg) and 0.4% Syzygium aromaticum (cloves) after 7-days growth at 28 C.
% of powdered mixed herbal Colony diameter (mm) : %
materials supplemented PDA Fungal survival Control (PDA without herbal materials) 80 : 100%
0.1 64 : 80%
0.25 44 : 55%
0.5 0:0%
0.75 0:0%
1 0:0%

20 The result shows that concentrations over 0.25% of mixed herbal materials composed from 0.1% Pimpinella anisum (Anise) and 0.25% of each Peganum harmala (harmala), and Myristica fragrans (nutmeg) and 0.4%
Syzygium aromaticum (cloves) have a profound antifungal activity against the black scorch fungus the Thielaviopsis paradoxa.

The differences of antifungal activity between samples treated with the herbal mixture (0.1% Pimpinella anisum, 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum) and those treated with control deprived of herbal materials were significant.
Experimental field From the results presented in Table 10 the concentration of 1% of an aqueous solution of the mixture composed from 0.1% Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum was found to be extremely effective in inhibiting mycelial growth of Thielaviopsis paradoxa on date palm tree from experimental field and which was significantly superior over all other concentrations evaluated.
The date palm trees treated by 1% w/v herbal fungicidal aqueous solution of mixture 0.1% Pimpinella anisum and 0.25% of each Peganum harmala and Myristica fragrans and 0.4% Syzygium aromaticum applied once every 15 days for 1 month the results were remarkably in comparison to untreated trees (control; Fig. 18). The results from PDA show that the herbal fungicidal aqueous solution has disinfected fungal mycelial growth of Thielaviopsis paradoxa from all the parts of the date palm trees as illustrated in Figure 18. Moreover, new growth of healthy leaves shown in heart of the date trees as compared with control untreated trees which symptoms of black scorch and bending head diseases on date palms trees (Fig. 18) that damages both the quality and quantity of date yield.

EXAMPLE 13: Anti-termite activity of herbal preparation This example illustrates the use of herbal preparations as termite control agents.

MATERIALS
Herbal preparation 0.3% Pimpinella anisum and 0.1% of each of Peganum harmala, Myristica fragrans and Syzygium aromaticum The performance of herbs to control termites was evaluated by two methods.

Fifteen square meter of nursery land infested with subterranean termites (800 termites/square meter) were divided equally to 3 quarters.
The first quarter was treated by mixing 1 Kg of powdered herbal preparation with 50 litre potting soil and distributed on the infested area.
The second quarter was treated by a commercial chemical anti-termites compound Dursban 40 C by mixing of 1 litre Dursban 40 C with 50 litre potting soil and distributed on the infested area.
The third quarter was treated with 50 litre of potting soil only as control.
Numbers of dead termites were illustrated and recorded daily for 3 days.
RESULTS
As shown in Table 11, the results indicate that herbal preparation comprised of 0.3% Pimpinella anisum and 0.1 % of each of Peganum harmala, Myristica fragrans and Syzygium aromaticum had the same effect controlling termites after 3 days as chemical treatment by Dursban 40 C.

Table 11. Percentage of dead termites in areas treated by herbal preparation and chemical compound in comparison to control untreated areas Treatment 8 hrs 24 hr 48 hr 72 hr Control untreated 0.00 0.00 0.00 0.00 Herbal preparation 15% 35 % 78 % 100 %
Dursban40C 20% 45% 86% 100%

This experiment was conducted to determine the effectiveness of a herbal preparation against dampwood termite Porotermes adamsoni and drywood termite Incisitermes minor affecting doors and wood in houses at a local area in Dubai. The colonisation of these houses by termites was apparent from the symptoms include holes, light brown cracks and insect colonies (2500 individuals). In addition, hundreds of flying bugs were noted.
Cracks and holes of infected areas were sprayed with an aqueous solution of herbal preparation by mixing 1 Kg of powered herbal preparation with 50 litre of water. Other holes and cracks were sprayed with an aqueous solution of anti-termites compound Dursban 40 C by mixing 1 litre of Dursban 40 C with 50 litre of water.
Colour of cracks and numbers of dead termites were illustrated and recorded daily for 3 days.

RESULTS
According to colour of cracks and percentage of dead termites, the results indicated that chemical compound Dursban 40 C and herbal preparation had the same performance on termites symptoms against dampwood termite Porotermes adamsoni and drywood termite Incisitermes minor affecting doors and wood. Cracks treated with chemical agent and herbal preparation became dark brown to black colour after three days and 100% dead termites were evaluated after three days as compared with untreated cracks.

CONCLUSION
The herbal mixture of the invention is further useful as an effective agent for the control of termites. Effectiveness matching chemical treatment has been demonstrated against a range of termites including subterranean, dampwood and drywood termites.

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Claims (20)

1. Use of a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta for the treatment or prevention of disease in plants.

2. Use according to claim 1, in which the composition comprises a preparation or extract of Pimpinella anisum.

3. Use according to claim 2, in which the composition further comprises a preparation or extract of one or both of Peganum harmala and Myristica fragrans.

4. Use according to claim 3, in which the composition further comprises a preparation or extract of Syzygium aromaticum.

5. Use according to any of claims 1 to 4, wherein the or each plant preparation is powdered dried plant material.

6. Use according to claim 5, wherein the plant material is flower, fruit or seed.

7. Use according to any of claims 1 to 6, in which the plant disease is caused by a nematode infection, a fungal infection or an insect pest.

8. A method for the treatment or prevention of disease in plants which comprises applying to said plants a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta.

9. A method according to claim 8, in which said composition is applied to seeds of the plants in which it is desired to treat or prevent disease, prior to planting said seeds.

10. A method according to claim 8, in which said composition is applied to the plant growth medium prior to planting.

11. A method according to claim 8, in which said composition is applied after planting.

12. A method for protecting vegetable foodstuffs from pests and/or disease which comprises placing said vegetable foodstuff in a container or wrapping which is coated or impregnated with a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta.

13. A method for the treatment or prevention of disease in plants which comprises co-cultivating plants of two or more species selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta with the plants susceptible to disease.

14. A method according to any of claims 8 to 13, in which the disease is caused by a nematode infection, a fungal infection or an insect pest.

15. Seeds coated or impregnated with a composition according to any of claims 1 to 6.

16. A plant growth medium comprising a composition according to any of claims 1 to 6.

17. A container or wrapping for vegetable foodstuff, which is coated or impregnated with a composition according to any of claims 1 to 6.

18. A pesticide formulation comprising a composition according to any of claims 1 to 6 and one or more vehicles, carriers, binders or excipients.

19. A method for the treatment or prevention of termite damage to a building which comprises applying to said building or part thereof a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta.

20. A method for the construction of buildings resistant to damage by termites, which comprises the steps of:
i) treating one or more building materials with a composition comprising a preparation or extract of two or more plants selected from Pimpinella anisum, Peganum harmala, Myristica fragrans, Syzygium aromaticum and Rhazya stricta; and ii) using said treated building materials in the construction of a building.