Lippia multiflora leaf extract to control the influence of Aspergillus niger

Sorghum, (Sorghum bicolor(L.) Moench)  belongs to the grass family Graminece.  It is a cultivated tropical cereal grass.  It is generally, although not universally considered to have first been domesticated in North Africa. It is an important crop in West Africa. In fresh forms, immature pods and seeds are used as vegetables while Snack and main dishes are prepared from the dried grain. All the plant parts used for food are nutritious, providing carbohydrate, vitamin and minerals. FAO (2000), estimates that 2.3 million tons of sorghum dry grains produced world wide. Nigeria produces 1.8 Million tons of these, making it the world largest producer followed by Niger. (540000) and Mail (9000). The total area grown by sorghum was  9.5millilon hectare,  about 90 hecter of these in West Africa. (FAO 2000). It is quantitatively the world’s 5^th largest most important cereal grain after wheat, maize, rice and barely. There are many varieties and are classified into four groups ;( Trojanhn, 1996).

Grain Sorghum: grown especially for their rounded starchy seeds. The grain serves as a substitute for corn in feeding animals. Some grain sorghums grow as  much as 15 feet high.  Farmers feed  the seed to livestock or make the entire plant into silage.  

Grass Sorghum: grown  for  green  feed  and  hay  but  can  be  found  in  Kansas  fields as weeds. Two  types  that  grow  in  Kansa  are  Sudan  and  Johnson  grass.  Sudan  is  an  annual  sorghum  grown  for  feed  and  hay  that  grows  quickly  and  can  reach 10 feet in height. Johnson  grass  is  a  perennial  that  grows  as  a  weed  in  the  US and Kansas.

Sweet Sorghum: also known as sorgos have sweet juicy stems. They are grown  especially for the production of sorghum syrup. Syrup is made by pressing the  juice  out  of  the  stems  with  rollers  and  boiling  it  down  to  the  proper  thickness. Animal  feed  and  silage  can  also  be  made  from  sweet  sorghum.

Broomcorn Sorghum : is  a  kind  of  sorghum  grown  for  the  brush  or  the  branches of the seed cluster.  Fibers from broomcorn are used to make brooms. The  main  use  of  sorghum  is  for  livestock  feeds  and  as  food  for  human.  The nutritive  value  is  similar   to  corn  though  have  more  protein  and  fat  than  corn  but  lower in vitamin A.  (Kapoor L.D., 1989).  The chemical composition of Sorghum correspond with most edible cereals (Trojanhn,1996.The include; carbohydrates, protein, water, food fiber, ash, fat, phosphorous, calcium and iron. The seed also contains small amount of Vitamin A, Folic  acid, Ascorbic acid, Thiamine, Riboflavin, Niacin.(Cross,1977 and Tondy, 1980). The use of grains provide an inexpensive source of carbohydrate in diet. The  major  sorghum  diseases  in  West  Africa  (Nigeria)  were  primarily  fungal  diseases. Pathogens associated with sorghum seeds have been reported to  be limiting production   of crops in Nigeria and world wide. (Lambo J.O., 1978). Heavy  infection  of  sorghum  by  Smut,  caused  by  Sporangium,  Rust,  caused  by Puccunia  purpure, and other agents like Fusarium  moniliforme, Claviceps sorghi, Bipolaris sorghicola, Acremonium strictum,   caused spoilage of  seeds  and  seedlings,   pre  and  post  emergence  mortality  ( Sofowora  et.al., 1985 ). Fungi  as  poisonous  agent  of  mycotoxin  has  been  appreciated  for  many  years,  but  in  recent  times,  the  scope  and  magnitude  of  the  losses  that  they  cause  have become obvious.  Poisonous symbiotic and parasitic fungi causes loss in the yield, quality,  nutritional  value  and  viability  of  foods  and   feed  stuffs  especially  cereal crops.  Fungi and the secondary metabolites (mycotoxins) they produce, constitute health  hazards  to  animals  and  man,  following  consumption  of  contaminated grains.  Due  to  high  growth  of  fungi  on  feeds  and  the  mycotoxins  the  produce, many  diseases   in  animal  are  known  to  be  of  fungi  origin  (Gbodi, et al,  1988).    Fungi  effects  on  sorghum  have  been  well  managed  and  effectively  controlled  with  various antifungicidal substances and chemicals which chemical effects have shown great  disadvantage  to  animal  in  mammalian  toxicity,  development  of  resistance  strains,  increase  in  cost  and  have  high  toxic  residues  due  to  their  metabolic  composition which is heavily concentrated. These  bioactive  fungicides  are  not  environment  friendly  and  could  lead  to  further  degenerating  diseases  even  so,  they  are  of  high  cost  beyond  the  local  farmers, which  gives  rise  to  alternative  source  of  antifungal  for  seed  dressing and  preservation  to  control  or  even  eradicate  fungicidal  activities  which  reduces  sorghum  production  and  its  nutritive  values  and  in  turn  causes  diseases  due  to  secondary  metabolites (mycotoxins) they  produce.  The aim of this research work then involves;

The study evaluates antifungal activities of bioactive fungi agents from Lippia  multiflora (L) modenke. In view of these reasons, the survey of mycotoxigenic fungi contaminating Sorghum in Minna, Niger State would be of great importance with respect to public health, agriculture and economic growth of Nigeria, a populous nation with increasing demands for food and fund that are now limited. The study will also increase knowledge in the area of mycotoxicology as little has been done in this field in Niger State.

Sorghum is annual plant belonging to the family Graminecea. It is an important crop in West Africa. In fresh form, the young leaves, immature pods and peas are used as vegetable, while snacks and main dishes are prepared from dried grains. All the plant parts that are used for food are nutritious, providing proteins, vitamins and minerals. FAO (2007) estimates that 3.3 million tons of sorghum dried grains were produced world wide. Nigeriaproduces 2.1 million, making it the world largest producers.

 

Chemical composition of sorghum include; carbohydrates, proteins, water, crude fiber, Ash, phosphorous, calcium and iron and these corresponds with most edible crops (Toetzee, 1995). The seed also contains small amount of B-carotenes equivalent, thiamine, riboflavin, vitamin A, niacin, folic acid and ascorbic acid ( Kay 1979).

Fungi are nucleated, spore-bearing achlorophyllous organism that reproduce with both asexually and sexually, and whose usually filamentous, branched somatic structures are typically surrounded by cells containing cellulose or chitin or both (Alexopulous, 1962).

The Deuteromycetous fungi is the most important terrestrial fungal group that concerns the mycotoxicologist. These fungal can asexually produce small dry spores that are readily distributed into the atmosphere by the slightest physical disturbance, making them ubiquitous. This feature promotes their ability to contaminate great varieties of field crops stored agricultural products, and a host of other materials.

They are filamentous fungi (mould) and many are able to produce a wide range of secondary metabolites. Some of these metabolites are pigments, some have antibiotic properties and same metabolites are toxic to plants and animals. The three genera, Aspergillus, Penicillum, and Fusarium are considered to be the most significant toxigenic moulds at the present time (Smith and Moss, 1985). Other toxigenic Deuteromycetes species other than Aspergilli ,Penicillia and Fusaria include  Alternaria alteranata, Pithomyces chartarium, Trichithecium rouseum, Rhizoctonia legumini cola, stachybotry atra, Myrothecium roridum and phomopsis lephtrostromiformis

            A range of pathogens, primarily fungi, damage the Sorghum plant which include the following:

Cereal grain, stored grain and processed foods of both plant and animal origin harbour large number of fungi under conditions. Those fungi include parasitic species that are host specific, saprophytic species which even tend to be selective in their choice of living substrate and environment. Fungi are major cause of spoilage in stored grain and seeds, and rank second only to insect as a case of deterioration and loss (Miller and Trenholn, 1994).

In 1974, Christensen, classified the fungi identified from cereal into three groups namely: field, storage and advanced decay fungi. Field fungi invade developing and mature seed before harvesting and include species of Alternaria fusarium, Helminthosporium, Cladosporium, chaetomium and curvularia in order of predominance (Javis, 1971). All field fungi required high speed moisture content of between 20-25% water content to grow and so are referred to as hydrophilic fungi (Lillehoj, 1973).

The storage fungi that invade grains after harvesting in storage consist of the few yeast (Christensen, 1974, Javis1971). This group also known as mesophytic storage fungi that has the following as their representative species. Aspergillus flavus, A. fumigatus, A terreus ,paecilomyce Variotti ,Penicilium guarantiogriseum, P. citrinum  and P.viridicatum (Lillehoj, 1973). The major factor influencing the development of this group of fungi are moisture content of the stored grain temperature, storage period, degree of earlier invasion, before arrival at storage site, amount of foreign material and the activities of insects and mites (Miller and Trenholen, 1994).

Advanced decay fungi require the same general moisture range as field fungi but rarely develop on seed in the field and consist of the genera, Fusarium and chaetomium. This fungi grow after considerable damage from other micro organism has occurred. (Javis, 1971 and Lillethoj 1973).

The occurrence of mycotoxins in foods and feeds depends on factors such as geographical locations, season and the conditions under which a particular crop is grown, harvested processed and stored. Production of mycotoxins can occur in the field before harvest, or post harvest during storage, processing, or feeding. Several factors that influencing the degree of fungal growth include.

The pathogenic nature of certain species of fungi to plant has been observed virtually since the beginning of agriculture. These plant pathogens can produce metabolites that show toxic effects when they are ingested. Several examples in recent history exemplify this property. In 1960, turkey X disease killed 100,000Turkeys, 14,000 ducklings and thousands of partridge and pheasant poultry inEngland.

In the mid 1930s and late 1970s there were outbreaks of a sickness in horses called equine leukoencephalomalacia in the united state (Hasseltine and Mehlhman; 1978) and alimentary toxic aleukia has been responsible for the distress and death of thousands of people since it was first recorded in the 19^th century (Moss and smith 1985).

Mycotoxins are metabolites that are produced by fungi growing on cereals, nuts, soybeans and several other crops including fruit. The Turkey X disease outbreak in Englandwas traced to contaminated peanuts from Braziland led to the discovery of aflatoxin produced primarily by Aspergillusniger and A. flavus. Equine leukoncephalomalacia is doubtless caused by toxins form Fusariummoniliforme and alimentary toxic aleukia was thought to be caused by the trichothecenes mycotoxins. Since mycotoxins producing fungi grow on some of the staple foods of both humans and animals, both populations are affected by them. Also, products such as eggs, milk, dairy products, and meat can be contaminated through the ingestion of feed containing mycotoxins (Moss and Smith 1985).

Toxigenic Aspergillus species are now recognised to be a major agricultural problem. The extensive research carried out over the past two decades has revealed a large number of toxic Aspergillus secondary metabolites. There are at least 24 species of Aspergillusthat have been associated with a large number of secondary metabolites of varied concentration (Marasas et al; 1984). The majority of Aspergillus toxin has been discovered in the last decade and represents an amazing variety of biosynthetic origins. The genus Aspergillus is a versatile biosynthetic apparatus capable of producing secondary metabolites by all of the major known routes for secondary metabolite formation (Ap Simon et al; 1991).

Aflatoxin are family of extremely toxic, mutagenic and carcinogenic compounds produced by Aspeigillus niger and A parasiticus (Deiner et al; 1987) Aflatoxin contaminations of sorghum, corn peanuts, tree nuts, cotton seed and other commodities are constituting world wide problem. Toxigenic A. nigerisolates produce aflatoxin B1 and B2 and toxigenic A parasiticus isolate produce aflatoxin B1, B2, Gland G2 (cotty et al; 1994). A. niger is the predominant fungus in aflatoxin contaminating Sorghum and cotton seed while A. parasiticus is probably more common in peanuts (Davis and Diener; 1983). A. niger and  A. parasiticus are temperature tolerant and can be selectively isolated on a high salt culture medium incubated at 37⁰C.

Before the 1970s, most of the aflatoxin Sorghum were general believed to originate after harvest. Improperly stored Sorghum can and does become contaminated with aflatoxin (Lillehoj and Fenuel, 1975). However, after aflatoxin was identified in Sorghum before harvest, it has become clear that most of the aflatoxin problem in Sorghum originates in the field.

Zearalenone and zearalenol are estrogenic metabolites of several species of Fusarium chemically, zearalenone (ZEN) is a resorcylic acid lactone which does not have actual toxicity. Fusarium graminearum is the major ZEN-producing fungus of the Fusarium species that causes corn ear and stalk rots, but other species of Fusarium produce ZEN, as well as other mycotoxins. (Christense et al; 1988). Zearalenone has been reported to occur in Sorghum, other grains, and silage in many areas of the world. (Hagler et al; 1989). ZEN is also found in wheat, barley, oats corn, sesame seed, hay and silages condition exacerbating ZEN accumulations in corn include weather which holds moistures contents at 22 – 25% or delayed harvest (Abbas et al; 1988).

Trichothecenes are a family of 200-300 related compounds that apparently exert their toxicity through protein synthesis inhibition at the ribosomal level. Several species of Fusarium and related genera produce trichothences. T-2 toxin, diace toxyscripenol (DAS), and deoxynivalenol (DON), are commonly found in agricultural commodities (Desjardins et al; 1993).

The toxic effects of trichothecenes include gastrointestinal effects such as vomiting, diarrhoea and bowel inflammation. Anaemia, leukopenia, skin irritation, feed refusal and abortion are also common. The trichothecenes, as a group, are immune suppressive (Sharma, 1993). T-2 toxin (T-2) is produced primarily by F. sporotrichioides and F.poae, but is also produced by other species of Fusarium (Maracas et al; 1984). T-2 is often found in barley, wheat, safflower, reduced gain low milk production, reproductive failure, gastrointestinal haemorrhage and increased mortality occur when cattle consume rations contaminated with these trichothecenes.

Fumonisin B1 was first isolated in South Africa where Fusarium moniliforme has long associated with animal problems (Gelderblom et al; 1985). Fumonisin has been shown to cause leucoencephalomalacia in horses (Marasas, et al; 1985), pulmonary edema in swine (Harrison et al; 1990). F. verticilloides, a species that is almost ubiquitous in Sorghum, and F. poliferatum are the main species producing high yields of Fumonisms B1, B2 and B3 (FB1, FB2 and FB3) are fumonisin in fungal cultures or found in naturally contaminated corn samples (Cawood et al; 1991).  F. verticiloides  and  F. proliferatum  are recently receiving increasing alterations in scientific literatures because they have been implicated in a number of animal diseases which involves a massive liquefaction of the cerebral hemisphere of the brain with neurological manifestation such as abnormal movement, aimless circling, lameness, etc (Morasa, 1995).

Ochratoxins A (OTA) is produced by species of Penicillium and Aspergillus, and is a causative agents of kidney disease in pigs that has been referred to as mycotoxins porcine nephropathy (Krogh, 1979). OTA can reduce weight gains and performance in swine (cook et al; 1986). Other symptoms include diarrhoea, increased water consumption, diuresis and dehydration.

Patulin is produced by Penicillium Aspergillus, and Byssochlamy  and may be found in silage (Dutton, 1984). Patulin has been incriminated as a possible toxin in Europe andNew Zealand (Lacey, 1991).

Mycotoxins contamination of food and feed grains is a serious economic problem for grain producers. The reason there is a market effect is that some of mycotoxins adversely affect animal and human health. The fungi that produce mycotoxins in grains commonly Fusarium Aspergillus and Penicillium species mycotoxins play a global role in human and animal health; some of their effect are well documented. Examples of major Fusarium mycotoxins with known biological activity include fumonisin with known biological activity include fumonisms (neurotoxity hepatotoxicity, cardiotoxicity, carcinogenesis), T-2 toxin (haemorrhage, enteritis immune suppression) deoxynivalenol (decrease weight gain), zearalenone (estrogenism) and fusarochromanone (Bone deformation many of the other known mycotoxins are acutely toxic or have sub acute effect on crop production economic impact of mycotoxins is difficult to determine, primarily  because of the sub clinical effect on crops.

The economic and health risk as with mycotoxins are presently poorly defined because mycotoxin contamination all segments production, marketing and utilization of grains in the Midwest region of the united state, losses due to fusarum head blight in wheat as the accompanying production of vomitoixin can be devastating as evidence by the severe outbreaks in south Dokota, Minnesota. The effects include crop production with the inability to use the crop for human (milling and baking) and animal food. However, different routes and levels of mycotoxin exposure may affect the out come of the toxicity. These variables must be defined so that reliable toxicity information can be documented.

Plant extracts have played significant role in the inhibition of seed borne pathogen and in the improvement of seed quality and field emergency of plant seeds. The development of non toxic, safe and effective biodegradable alternative to synthetic fungicides has in recent year, led to global at screening various plant for bioactivity against plant pathogenic organisms (Onifade, 2000, Yorinori 1994). However, it is estimated that about 10% of the over 250,000 different plant species in the world today have been examined chemically for antimicrobial activity (Earn worth, 1990).

The continued use of natural plant products is particularly important in countries likeNigeriawhere synthetic fungicides are not readily available, farmers are poorly equipped to handle them and their use is uneconomical. Fungi are more devastating in tropical and subtropical regions in the world. InNigeria, the pathogenic is of major concern to vegetable growers in the northern savannah beets, which potentially rank among the world’s best vegetable production zone (Yorinori, 1994).

Fungi toxic activity of vernolepin and vernodalin isolated from vernonia amydalina Del (Maboul et al, 1997). They found that Aspergillus niger and Candida albicans were sensitive to both pigments. Two new flavones 4;6,7- trihydioxy 3,’5-dimethyl flavones and 5,’5-dihydroxy-3,’4,’8-reimethoxy flavones was isolated from Artemisia giraldii these two new flavones also showed antitoxic activity against Aspergillus flavus, Trichodering viride (Zherg et al, 1990). Essential oils from Azadirachla indica and Morinda lucida were found to inhibit  the growth of toxigenic A. flavus  and significantly reduced aflatoxin synthesis in inoculated maize grains (Bankole, 1997).

The efficacy of plant material in controlling mycotoxigenic moulds, there has not been any concerted effort of a large scale trial of these plants on the farmers’ field. Udoh et al; (2000) was of the view that caution must be exercised in using plant material to control mycotoxins, because some of these materials are natural media for A. flavus Khaya senegalenisi bark to protect maize against insect increased the risk of aflatoxin development and that even the farmers were aware of low efficiency of the indigenous products, most of the plants being screened for ability to control storage fungi are traditional medicine (Hell et al; 2000).

It is a fact that natural products from plants remain a vastly under utilised resources for the discovery of novel antimicrobial compounds, yet we live in a world where most pathogens can be controlled by these natural products. The majority of higher plant species are yet to be explored as potential sources of antimicrobial agents. The use of botanicals in disease management has been going on for a very long time in traditional practices.

Lippia is a member of the bitter leaf family Meliacea. It is drought-tolerant species that grows well in the tropical and sub-tropical regions with. Semi-arid and humid climates (Taylor, 1984). Lippia contains a group of compounds called ‘triterpenes’ more specifically ‘limonoids’ the part of the plant are used in treatment of malaria, a portion of the leaves and stem bark are drunk while in some cases the stem bark is also used to treat fevers by inhalation or hydrotherapy. This plant has shown to possess a steam volatile, only constituent in trace amounts, which showed only one component common to leaf, stem and root when examined chromatographically (Sofowora, 1978). The tree has been known for its insect antifeedant properties, Numbin and nimbidin have treatment of a variety of human ailments particularly against disease of bacterial and fungal origin. In the world of human medicine, it has been used as fungicide, antibacterial, and antiviral agents, for dermatological infections dental treatments, chaga’s disease, malaria, pain relief and fever reduction and birth control. In field of veterinary medicine, it is used in controlling insects, bacteria and intestinal worms (Murty et al; 1978).

Researchers have found out that herb, spices and seed out of the Lippia tree have fungicidal activity and could also inhibit mycotoxin formation. (Ibrahim, 1987) studied the storage of yam tubers and showed that yams treated with Lippia bark water extract, Lippia bark slurry and Lippia leaves have been stored for six months. The study further showed that rot in yams treated with bark extract were delayed for three months. Over the last some years, Lippia has been shown to possess active principles in its leaves, bark and fruit and the can be exploited for their medicinal, biological and biocides properties Olabiyi (1992), found out that Lippia  leaf extract has some nematode properties.

Lippia leaves are widely used to cure a number of human and animal diseases. The physio-chemical properties of Lippia leaves  are also used to manufacture a number of drugs and medicines. They have been traditionally used to give bath to patients suffering from measles or chicken pox.

Lippia leaves are generally gathered only from organic trees, this is so because it ensures the protection of natural elements and reduction of contamination by environment/synthetic toxin. Lippia  leaves can be taken as;

Lippia leaf extract has a fruit like smell and contains essential fatty acids; this extract finds large scale personal and industrial application. They are used in a number of pesticides and insecticides, high potency extracts are used to manufacture personal product like facial creams, skin creams, cleansers and oral care products.

Although human faces health risk stemming from contamination of grains with other naturally occurring substances, mycotoxins are unique in that they are produced naturally on grains. Their presence is usually associated with uncontrollable factor such as climatic conditions. Mycotoxins are produced by certain fungi (e.g. Aspergillus spp, Penicillin spp and Fusarium spp) that grow on grain and feed ingredients such as Sorghum, maize, wheat, barley, peanuts.

Research results support the contention that generally soft endosperm sorghum is more susceptible to seed pest than harder endosperm in types and that the biggest problem with the dense genotypes is endosperm softness (Groiler, 1994) despite insecticide application to reduce core earworm these insect caused major cob damage in predisposition of plants to seed mould damage by providing entry point and vectoring moulds if a substrate is spoiled by the growth of mould, that is caused above all by the production of mycotoxins (Flepporly et al 1989).

Mycotoxins occurrence is not restricted to under developed countries. Aflatoxin in sorghum in the South Eastern United States is the known mycotoxins that are more likely to occur at higher concentration in the-tropical or sub tropical developing countries of the world, the bulk of mycotoxin research in Latin America has been conducted on sorghum and specifically on aflatoxin although other toxins such as zearalenone, T-2 toxin, deoxynivalenol, penicillic acid Kojic acid and Ochratoxins have been detected in sorghum.

Aflatoxin is the major food borne caranogenic, hepatoxins of great public health importance in Africaand indeed in the world. They are produced by Aspergillus niger, A. flavus, A. parasiticn  and A. nomius  on cereal, nuts, dried fruits and even water (Patterson et al; 1979) which when consumed elecits in mah and animal causing damage of the lever and kidney intestinal haemorrhages and death of affected organisms (Smith and Moss, 1985).

Ochratoxins A (OTA) is a mycotoxin produced by different species of Aspergillus and Penicillium, though it was first isolated from cultures of Aspergillus ochreaceus (Van dermerwe et al; 1965) it is found as natural contaminants in many foodstuffs such as dried fruits, corn etc.

Zearalenone is produced mainly by Fusariumgraminearum and related species. It is found principally in maize and wheat zearalenone and its derivatives produce estrogenic effects in various animal species (infertility, vulvae oedema, vaginal prolapsed and mammary hypotrophy in females and fertilization of males in Puerto Rico, zearalenone was found in the blood of children with precocious sexual development (Saenz de Rodriguez, 1984) contaminated food. Zearalenone was also found together with other fusarium mycotoxin in “Scabby grain toxicosis”  (Luo, 1988).

Trichothecenes are mycotoxin produced mostly by members of the Fusarum genus. The most frequent contaminants are deoxynivalenol (DON) known as vomitoxin, nivalenol (NIV), diacetoxyscirpenol (DAS), while T-2 toxin in rarer (WHO, 1990). Common manifestations of trichothenes toxicity are depression of immune responses, nausea, and sometimes vomiting. The first recognised trichothecenes mycotoxicoses was alimentary toxin a leukia in theUSSR in 1932 with mortality rate of 60% (Gajduslek, 1953).

Fumonisms discovered in South Africa in 1988 (Marasas, 1995), and produced by F. Verticiliodes and  F. proliferatum are recently receiving increasing attention in scientific literature  because they have been implicated in a number of animal diseases such as levcoencephalomalacia in equines, which involves a massive  liquefactions of the cerebral hemisphere of the brain (Marasas, 1995). Fumonism has also been identified as a carcinogen in humans, in addition to adversely affecting brain, liver and lung function. It can cause hepatotoxicity and rephrotoicity in many animals (Howard et al, 2001).

Due to the presence of fungi and mycotoxin in sorghum grains, the different methods of prevention could be used such as the use of pesticides on grains, avoidance of physical damage to the grain, inhibition of biosynthetic pathway all seem or observed before harvest (pre harvest prevention). Post harvest detoxification methods include the use of physical methods like temperature, moisture content, time, irradiation and chemical method application of acids, oxidizing agents etc (Okoye, 1992).

Plant leaves of Lippia multiflora was collected from healthy plants, rinsed and air- dried. Pestle and mortar was used to crush the dried leaves and further blended into homogenous powered using and electric blender (National MX391N) to enhance penetration of extracting solvent (ethanol) and to facilitate the release of active ingredient. The powdered sample was stored in well labeled and clean container until used.

The cold decortion / extraction method was used to obtain the crude ethanolic extract of the powdered leave. One thousand five hundred grams of the powdered leave was weighed into a Bochuner flask and five hundred ml (500ml) of 95% ethanol (CH3CH2OH) was used to immerse it. This was left sealed with a foil for 4 days after which filtration followed. Two hundred ml (200ml) of the 95% ethanol was used on the residue to ensure proper extraction of the active constituent of the leave.

The reflux apparatus ( Heidulph rotary evaporator) was used for the concentration of the crude extract. The ethanolic crude extract was poured into the round bottom flask. The set was placed on a heating mantle until it became volatilized. The process continued for about 1hour 20 minutes to ensure complete concentration of the extract. A water bath was used to achieve absolute concentration of the extract. The extract was transferred to a beaker and refrigerated for further chemical test and uses.

Sorghum grain samples were obtained from three markets ( Tunga,Mobileand Bosso) inMinna,Nigerstate during the raining season between August and September 2007. Nine market samples were collected from three different strategic points from each market. The samples were made up of white and red varieties, each in polythene bags. They were appropriately labeled and stored in the refrigerator at 20C until used.

Sabrose Dextrose Agar (SDA) medium was used. In preparing it, 62 grams of the agar was     weighed  and was poured 1liter flat bottom flask. 1liter of distilled water was used to dissolve it and 0.5grams of Chloramphenicol was added to inhibit bacteria growth and subsequent contamination. The solution was boiled for 30 minutes to ensure proper dissolution/mixing. It was sterilized by auto cleaving at 121C for 15 minutes. It was allowed to cool t a reasonable extent, poured into petric dishes, which was then allowed to solidify.

Inoculation was carried out after solidification of the medium. Sorghum grains ( about 5-6) grains were inoculated on the solidified medium. Prior to the inoculation, the inoculums were washed with 5.25% Sodium hypo chloride (NaOCl) and distilled water to sterilize and make prevent bacteria growth. Inoculated mediums were incubated in the inoculation hood for four days, for fungi growth and isolation according to the method described by Smith et al (1985) and Barkai-Golan et al (1999).

Each fungi cultures were aseptically placed on a sterile slide using a forcep. The fungi on the slide was stained with dye (Lacto phenol blue) and viewed under X10 and X40 objective lens of the microscope. Identification of each fungi growth was ascertained using a fungi catalogue.

The pure culture of different isolates were aseptically sub cultured in Sabrose Dextrose Agar slants and incubated at room temperature,(27^oC) until appreciable fungi growth were observed. These were kept as stock culture in the refrigerator for further analysis and the two most common colonies of fungi species; Aspergillus niger and Aspergillus flavus collected from the stock culture were used for the study.

Mycotoxins (fungi secondary metabolites) production was obtained using Maize grains which is the perfect and most suitable substrate for mycotoxins production. To 500grams of the maize weighed into eight different Buchuner flask, 200 ml of distilled water was added and mixed thoroughly and left overnight for moisture equilibration ( Gbodi, 1986). Sterilization of the maize was achieved using an auto cleave for 20 minutes at 15pi and 121^oC. The sample was left to cool after which the old pure culture of the Aspergillus species grown on the SDA in slant tubes of seven days were inoculated into each maize samples under sterilized condition to prevent contamination. The set up was left in the inoculation hood for 21 days for massive growth of fungi hence synthesis of secondary metabolites.

Extraction of the mycotoxins was with dichloromethane (CH2CL2) 84.93g/mol and 1molar phosphoric  acid according to the method described by Gbodi,(1986). 500 ml to 50 ml respectively of the solvent and the acid was poured into the Buchuner flasks of the maize samples grown with large biomass of fungi for homogeneity. It was left to stand for 30minutes after which, blended with a blender ( National MX391N ) and filtered through fast fluted filter paper. The extract was concentrated in a beaker on a heating water bath at 55^oC. As the solvent distilled off, the residue was left in the beaker and kept in the fridge at 25^oC till used for toxicity testing on Sorghum.

Soil sample was obtained from the field and was sterilized. Sterilization was by massive heating under high temperature using fire wood for 1hr 30 minute. The soil was left standing to cool till the next day after which it was bagged for 5kg per bag and the treated seeds were planted and germination was for four days.

The treatments are as follows:

 Treatments

Table 4.1, and 4.2 shows the list and occurrence of fungi isolated from sorghum grains sample collected from different market places in Minna market, Nigerstate. Ninety seven fungi isolates were cultured and identified from the sorghum samples. The fungal genera identified contaminating the selected isolates sorghum in Minna, in order of decreasing predominance were Aspergillus, Fusarium, Neurospora, , Rhizopus, Nigrospora and Mucor (Table 4.1) while the percentage incidence (Table 4.2 and Fig 4.1) of the major fungi species contaminating sorghum in Minna are Aspergillus species ( 58.76%), Fusarium species (27.84%), Neurospora species (5.15%), Rhizopus species(4.12%),  Nigrospora species (2.06%) and Mucor (2.02%).

Table 4.3 and Fig. 4.2, Fig. 4.3 and Fig. 4.4 shows the effects of the various individual treatments of the seed samples in terms of root and shoot lengths, seedling vigor and weights of dry root and shoot after four and eight days of germination.  All the treatments gave varying levels of germinability and vigor when compared with the control. Remarkable levels of 80%, 65% and 60% was recorded with treatments with Lippia extract.

Seeds treated with A. niger, its metabolite and synergetic treatment of the fungi and its metabolite and with metabolite of Fusarium recorded the low, lower and lowest germinability and vigor respectively both in root and shoot length.  

Table 4.1 Incidence of fungi in sorghum seeds samples collected from three markets in Minna. 

 

Table 4.2: Percentage occurrence of the incidence of fungi species in sorghum seed samples collected from three markets in Minna.

Table 4.3: Effects of Aspergillus niger, its metabolite and Lippia leaf extract on the germination and seedling vigor indices of sorghum after four and eight days   

 

Microorganisms especially fungi are known to be the major cause of market and field losses of crop (Okoli et al, 1898, and Onifade, 2000). Sorghum seeds were collected market in Minna, Niger state. Different fungal genera such as (Aspegillus, FusariumNeurospora, Nigrospora, Rhizopus and Mucor) were found to contaminate sorghum. Many of these families of fungi have also been shown to cause spoilage to sorghum in other parts of the world and other grains as have been reported by Brandyopadyay et al, (2005), Taylor et al, (2000) and Leslie, (1992). From the results of fungi identification obtained, the flora of sorghum was dominated by Aspergillus niger / species. This corroborate the work Collinson et al., (1999).  

The most important field fungi of sorghum in Africaand worldwide are Aspergillus species. The main groups of Aspergillus toxins commonly found is aflatoxin it is known to produce over 100 secondary metabolites that can adversely affect human and animal health (Visconti, 2001). The production of mycotoxin such as aflatoxin, fumonism etc were reported by Scott, (1994). The toxins are in human (Peraica et al., 1999 and Bankole and Adebayo, 2003) and animal (Gbodi and Nwude, 1988) maladies. Of the major concern is the presence of aflatoxin B1 in our foods which has been isolated from sorghum, soybean oil, kernel, garri, yam flour, ginger, cowpea, maize, millet, rice, cottonseed and groundnut and melon seed crops from northern and southern parts of the country. In a paper by Obidoa and Guguani (1990) presented at a workshop. Aspergillus flavus and parasiticus were reported to have been found to grow and produce Aflatoxin.

Fusarium was isolated from sorghum, wheat, soybeans, flour, cowpea, maize from northern and southern part of the country. These Fusarium spp namely F. graminearium, F. culmoun F. ceredes, F. equiseti, F. nivales, F. sporotrichiodes. F. oxysporum, F. verticilliode, F. gibbogum and F. avenaceum produces zearalenone, anon-sterodial estrogenic mycotoxin. Zearalenone, Fumonisin and trichothecen are the metabolite of fusarium spp and the importance of these fusariotoxins are also well documented.

The co-occurrence of toxigenic fungi in some sample was common in this study. The natural combination of fungi in the same crop could be synergetic or antagonistic in the host.

In this investigation, the leaves extract of Lippia multiflora demonstrated and confirmed its antifungi and fungitoxic potency against Aspergillus niger and its metabolite and the synergetic metabolite of Aspergillus niger and Fusarium. . Many plants especially spices have been used severally in the preservation of plants and animal products and in the treatment of various human (Hirt et al., 1995 and Pamploma, 2001) and plant diseases (Onifade, 2000 and Onifade 2000). The Lippia leaf extract exhibited its fungicidal action through inhibition of growth of the fungi ( Nair and Arora, 1996) and  stimulation of  the percentage  germinability and seedling vigor including root and shoot lengths of the sorghum seed sample. The existence of fungi toxic material has been reported in some West African plants including Lippp the root length, shoot length and also increase in the number of seedling emergence even with fungi contamination. Therefore, Lippia multiflora has shown to be fungistatic on seed borne fung of sorghum and the higher the concentration of the extract the less growth of organis under study.

From the findings of this research work, natural plant like Lippia multiflora  extracts could therefore be used in agriculture to enhance and protect economic important crops. The leaf  may be of commercial value in the protection of food and feeds against fungi contamination. This could reduce the incidence of human exposure to mycotoxins. The fact that the extract have inhibitory and stimulating effect on growth of Aspergillus niger, it metabolite, their utility as a biocontrol agent  should not be an oversight. It is recommended that further studies should be done on the fungicidal effects of the leaf extract and also the possibilities of producing them in commercial quantities. 

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