Journal of Microbiology and Antimicrobials Vol. 4(1), pp. 23-31, January 2012 Available online at http://www.academicjournals.org/JMA DOI: 10.5897/JMA11.106 ISSN 2141-2308 ©2012 Academic Journals

Full Length Research Paper

Evaluation of botanicals as biopesticides on the growth of Fusarium verticillioides causing rot diseases and

fumonisin production of maize

Adejumo T. O.

1Department of Microbiology, Adekunle Ajasin University, P. M. B. 01, Akungba-Akoko, Ondo State, Nigeria.

E-mail: toadejumo@yahoo.com.

Accepted 30 October, 2011

Partially (Pp) and completely purified (Cp) methanolic extracts of leaves of Cassia alata, Cymbopogon citratus (lemongrass), Mangifera indica (mango), Carica papaya (pawpaw), Citrus limon (lemon), fruits of Xylopia aethiopica, seeds of Aframomum melegueta (alligator pepper), Citrus aurantifolia (lime), Garcinia kola (bitter kola), Piper guineense (brown pepper), and rhizome of Zingiber officinale (ginger) were tested for antimicrobial activity using the poisoned medium and disc diffusion assay techniques on maize mycotoxigenic fungus: Fusarium verticillioides. The Pp extracts of P. guineense, G. kola and A. melegueta showed consistent higher growth inhibitions at 2, 5 and 7 days after incubation, while C. papaya and M. indica had the least activities. It was observed that these extracts completely inhibited the mycelial growth of the fungus at 2 days after incubation and produced growth reduction of 82, 80 and 73% respectively. The Cp extracts of P. guineense, G. kola, and A. melegueta also exhibited consistently higher inhibitions at 3, 5, and 7 days after incubation, while C. papaya and M. indica had no inhibitory activity. The three extracts produced reduction of mycelial growth of 76, 54 and 43% respectively. CP extracts of the plant material showed minimum inhibitory activity against F. verticillioides at 10 to 250 mgml

-1 concentration level. The thin layer chromatographic analysis of the plant extracts

showed different fractions, while 4 and 1 fractions of P. guineense and A. melegueta showed bioactivity respectively, which may be the important factor in causing the inhibitory effect. P. guineense, G. kola and A. melegueta showed potential and could be successfully used as environmentally-friendly, cheap, available, effective and sustainable alternative biopesticides. Key words: Alternative pesticides, antifungal, Fusarium verticillioides, maize, medicinal plants.

INTRODUCTION Maize (Zea mays L.) is the 3

rd most important food crop

worldwide, most widely cultivated cereal as a subsistence food crop and most frequently consumed staple crop in all agroecological zones in Nigeria. Twenty per cent of the population consume it at various times in a week (Dixon et al., 2004), as roasted, boiled grain or processed to flour as snacks. It also constitutes a major foodstuff for all classes of livestock. The most commonly isolated Fusarium species on maize was Fusarium verticillioides (Adejumo et al., 2007a, b), which have a direct effect on corn yields by causing plant diseases in infected ears and kernels and sometimes produce mycotoxins like

fumonisins. Fumonisins are believed to cause equine leukoencephalomalacia (ELEM) in horses, pulmonary edema in swine (Harrison et al., 1990; Kellerman et al., 1990), hepatotoxic and carcinogenic to rats (Engelhardt et al., 2006). F. verticillioides has been associated with human esophageal cancer risk in the Transkei region of southern Africa (Marasas et al., 1988) and in China (Li et al., 1980; Yang, 1980).

Maize cultivation in Nigeria is subject to intensive pesticide applications. The excessive use and misuse of chemical pesticides have raised serious concern about health and environmental hazards, and increasingly strict

24 J. Microbiol. Antimicrob. maximum residue limits. These have significant draw-backs including increased cost, handling hazards, and concern about pesticide residues on food (Tzortzakis and Economakis, 2007). It is obvious that recently, there has been a considerable interest in extracts and essential oils from aromatic plants with antimicrobial activities for controlling pathogens and/or toxin producing microorga-nisms in foods (Reddy et al., 1998; Soliman and Badeaa, 2002; Valero and Salmeron, 2003). The advantages of the use of natural plant protectants with pesticidal activity include low mammalian toxicity, less environmental effects and wide public acceptance. They are inexpen-sive, nontoxic and easy biodegradability (Adegoke and Odesola, 1996). The aim of this investigation was to evaluate plant products being used as medicinal plants in Nigeria for safe, cheap and sustainable alternative pesticides. MATERIALS AND METHODS

Collection of plant material and fungus

Eleven commonly and traditionally useful medicinal plants for human ailments and crop storage in Nigeria were used. Their selection was based on their availability. The fresh leaves of Cassia alata, Cymbopogon citratus (lemon grass), Mangifera indica (mango), Carica papaya (pawpaw), Citrus limon (lemon), fruits of Xylopia aethiopica, seeds of Aframomum melegueta (alligator pepper), Citrus aurantifolia (lime), Garcinia kola (bitter kola), Piper guineen-ses (brown pepper), and rhizome of Zingiber officinale (ginger) were collected from Ibadan, Oyo State (7°26’N Longitude 3°54’E), Nigeria.

They were authenticated in the Department of Plant Science and Biotechnology, Adekunle Ajasin University, Akungba-Akoko, Nigeria. The characteristics of the medicinal plants- botanical name and family, common name and part used, uses and constituents are presented in Table 1.

The leaves and rhizome were washed 3 times in running water and once with sterile distilled water. The materials were air-dried on sterile filter paper, and stored in airtight container and kept in the desiccator.

Culture of F. verticillioides 62264 was collected from the German Collection Centre for Microorganisms- Deutsche Sammlong von Mikroorganismen Zell undefelkulturen (DSMZ) GmbH Hnhoffenstraβe 7B, 38124 Bransweig, Germany. It was maintained on potato dextrose agar (PDA) and kept inside the refrigerator until further tests.

Solvent extraction

The dried plant materials were pulverized to powdered form with Warring blender (BRAUN blender and IKA-Universalmuhle M20 Gebr. Liebach, Bielefeld 14). A known quantity of each of the samples was put into the thimble and extracted with methanol in Soxhlet extractor. The extracts were concentrated in the rotary evaporator (Büchi Rotavapor R-134 with Büchi waterbath B-481) into completely purified and partially purified forms (1/3 of its original volume), the pH value was determined using Fisherbrand Hydrus 300 and the material was kept in McCartney bottles at 5°C until further tests. Preparation of inoculum Conidial suspensions of F. verticillioides were prepared from 7

to 10 day-old cultures that were maintained on PDA. The spore density was standardized to a known final concentration (1.0 x 10

6 spores/ml) using the haemocytometer and Olympus

microscope with attached camera according to the modified methods of Strange et al. (2002). Antifungal activity The poisoned food technique (Grover and Moore, 1962; Nene and Thapilyal, 2000) was used to study the antifungal activities. A known concentration (mg mL

-1) of each extract was incorporated into

plates before the PDA was poured and allowed to solidify. A 10-day-old test fungus was placed on the solidified agar, placed in inverted position and enclosed in sealed plastic bags. The plates were allowed to incubate at 28°C for up to 7 days. Nystatin (0.1%) was used as positive control (Mahesh and Satish, 2008). The antifungal activity was assessed by measuring the radial growth of F. verticillioides for different extracts daily after 24 h of incubation until the 7

th day. The percentage growth reduction (GR) of each of the

extracts at 7 days of incubation was calculated by subtracting the radial growth of the fungus with the extracts (E) from that of the control (C), and later divided by the radial growth of the control (C):

GR (%) = (C – E)/C x 100

Determination of minimum inhibitory concentration (MIC) The filter paper disc diffusion (Taylor et al., 1995; Prescott and Harley, 1996) was used for MIC of the completely purified extracts. Tween 80 (0.005% v/v) was used as an emulsifying agent. Paper discs (6 mm) were impregnated with known concentration ((10, 50, 100, 250, 500 and 1000 mg mL

-1) of each of the extracts and were

placed on the already seeded plates with 1.0 x106 spores/ml of F.

verticillioides. The MIC value of the extracts was determined by measuring the diameters of zone of inhibition around the disc after 24 h until the 3

rd day (Sengupta et al., 2008). After the incubation as

outlined, plates were examined for inhibition zones at the minimum concentration level.

Thin layer chromatographic separation of active constituents and antimicrobial tests

The mixture of the active compounds of the best seven extracts (P. guineense, Z. officinale, A. melegueta, Citrus limon, X. aethiopica, Cassia alata and G. kola) that showed high potential from their antifungal effect was separated into different fractions. Methods of Sengupta et al. (2008) was used. The samples were centrifuged for 10 min at 13.100 X g in an Eppendorf centrifuge 5451D at ambient temperature. The supernatants were dissolved in absolute methanol and 15 µL extract was applied as spot on TLC plate coated with silica gel “PLC Silica gel 60 2mm” Merck KGaA, 64271 Darmstadt, Germany. Mobile phase: Acetic acid ethyl ester/n-Hexane (1:1, v/v) acetic acid-ethyl ester and hexane 1:1 were used to separate the probable active principles (Sadasivam and Manickam, 1996; Wagner and Bladt, 1996). Plates were placed in a horizontal glass chamber, developed and the solvents were evaporated with a fan and the resolved components visualized by irradiation with a 340 nm lamp. Horizontal bands were marked with a pencil and scraped off from the plates, and collected in 10 ml glass tubes, flushed with Helium. The tubes were closed with screwed caps and stored at -20°C in the refrigerator. The Rf ranged values of each fraction of P. guineense and A. melegueta samples were calculated and the scraped material from the TLC plates was tested against the test organism by the disc diffusion method.

Adejumo 25

Table 1. Characteristics of the medicinal plants.

S/No. Botanical name and Family

Common name and part used

Uses Constituents References

1 Piper guineense Schum. and Thonn. Black Piperaceae

Brown pepper (seed)

Preparations are drunk as a purge, for thickening of soup (condiment), native medicine for sores and stomach pains, intestinal illnesses. Powder and oils for protecting cowpea against seed beetle and maize against Sitophilus zeamais

Piperine, amide alkaloid: wisanine and okolasine.

Okogun et al., 1977; Oji, et al., 1992; Abila et al., 1993.

2 Zingiber officinale Roscoe

Zingiberaceae

Ginger (rhizome)

Rheumatic and respiratory diseases, loss of appetite, vomiting, nausea and convulsion in children

1,8-cineole, neral and geranial in 5.48:1:2.13.

Ali et al., 2008; Ukeh et al., 2009

3 Aframomum melegueta Schumm

Zingiberaceae

Alligator pepper (seed)

As a spice for flavouring curries, cakes, bread and for other culinary purposes 2-heptanol, 2-heptyl acetate & linalool in 1:6:3.

Ukeh et al., 2009; Purseglove et al., 1981.

4 Citrus limon

Rutaceae

Lemon (leaf) For desserts, flavours are used in beverage, confectionary,cookies. monoterpenes, and limonene

Dongmo et al., 2009.

5 Xylopia aethiopica (Dunal) A.Rich

Annonaceae

Guinea/ Ethiopian pepper (fruit)

In foods (spice), cough remedy, relieve flatulence, a post-partum tonic, stomach ache, bronchitis, biliousness, dysentery and antiplasmodial

Terpenoids: α-copaene, γ-cadinene, δ-adinene.

Iwu, 1993; Boyom, 2003.

6 Cassia alata L.

Fabeceae

Ringworm bush (leaf)

Decoctions- leaf, flower, bark and wood for skin diseases: eczema, pruritis, itching and constipation. Flowers- bronchitis and asthma.

Chrysophanol, a metabolic product of chrysarobin

Viallaroya and Bernal-Sangos, 1976

7 Garcinia kola Heckel

Gultiferae

Bitter kola (seed)

Generally eaten as a stimulant, hops substitute, cough suppresant, anti-tumor, an aphrodisiac.

Polyisoprenyl benzophenone (Kolanone)

Madubunyi, 1995; Ogu and Agu, 1995.

Statistical analysis Statistical analysis was carried out on the collected data using Statistix 8.1 Analytical Software, 2003. Tukey HSD All-Pairwise Comparisons Tests at 5% was used to compare the means.

RESULTS

The results of both the poisoned medium and disc

diffusion assay techniques for partially purified (pp) and completely purified (cp) extracts of A. melegueta, C. limon, X. aethiopica, C. alata, G. kola, P. guineense, Z. officinale, C. papaya, M. indica, C. citratus, and C. aurantifolia showed that they possess antifungal activities against F. verticillioides (Tables 2 and 3). The pp extracts of P. guineense, G. kola and A. melegueta showed consistently higher inhibitions at 2, 5 and 7 days after incubation, while C. papaya and M. indica

had the least activities. It was observed that the extracts of P. guineense, G. kola, and A.

melegueta completely inhibited the mycelial growth of the fungus at 2 days after incubation, and produced a reduction in radial growth of 82, 80 and 73% respectively (Table 2, Plate 1). The cp extracts of P. guineense, G. kola and A. melegueta also exhibited consistent higher inhibitions at 3, 5, and 7 days after incubation, while those of C. papaya and M. indica had no

26 J. Microbiol. Antimicrob.

Table 2. Growth of Fusarium verticillioides on partially purified methanolic extracts of medicinal plants.

Extract

Diameter of growth (mm) Growth reduction

Days after incubation

2 5 7 (%)

Control 12.5 a1

42.8 ab

62.3 ab

-

Carica papaya 12.0 a 46.3

a 65.8

a 6

Mangifera indica 9.5 abc

38.3 b 57.3

bc 8

Cymbopogon citratus 10.0 ab

37.3 b 55.0

c 12

Nystatin 10.2 ab

38.3 b 54.0

c 13

Citrus limon 6.5 cd

28.0 c 41.3

d 34

Xylopia aethiopica 6.8 cd

25.8 c 39.0

d 37

Citrus aurantifolia 7.3 bcd

25.5 c 36.3

d 42

Cassia alata 7.5 bc

24.3 cd

36.0 d 42

Zingiber officinale 4.4 d 19.3

d 29.5

e 53

Aframomum melegueta 0.0 e 11.3

e 16.8

f 73

Garcinia kola 0.0 e 8.3

e 12.3

f 80

Piper guineense 0.0 e 7.0

e 11.5

f 82

Std Error 0.85 1.59 1.77 - a1

= Means with different letters are significantly different at P= 0.05 (Tukey HSD all-pairwise comparisons test). Std error = Standard error for comparison of means.

Plate 1. In vitro effect of partially purified extracts of medicinal plants against Fusarium verticillioides at 7 days of incubation; A) Fusarium verticillioides (control); B) Aframomum melegueta; C) Garcinia kola; D) Piper guineense.

A B

C D

Adejumo 27

Table 3. Growth of Fusarium verticillioides on the purified methanolic extracts of medicinal plants at 100 mg/ml.

Extract

Diameter of fungal growth (mm) Growth reduction

Days after Incubation

3 5 7 (%)

Control 22.0 cde1

40.0 b 76.0

a -

Carica papaya 29.5 a 51.0

a 76.0

a 0

Mangifera indica 24.8 bc

46.0 a 76.0

a 0

Cymbopogon citratus 27.0 ab

50.0 a 72.0

b 5

Cassia alata 18.8 def

34.5 bcd

49.5 d 35

Nystatin 17.5 efg

33.0 d 49.0

d 36

Citrus limon 20.5 cde

32.0 d 48.5

d 36

Zingiber officinale 15.0 fg

30.0 d 45.0

e 41

Xylopia aethiopica 19.0 def

34.0 cd

44.0 e 42

Aframomum melegueta 14.0 g 29.0

d 43.5

e 43

Garcinia kola 13.0 g 22.5

e 35.0

f 54

Piper guineense 6.8 h 11.5

f 18.0

g 76

Std Error 1.14 1.45 0.65 - cde1

= Means with different letters are significantly different at P= 0.05 (Tukey HSD all-pairwise comparisons test).Std error = Standard error for comparison of means.

Table 4. Minimum inhibitory concentration (MIC) of plant extracts against Fusarium verticillioides.

Extract Concentration (mgL-1

) Zone of inhibition* (mm)

Aframomum melegueta 10 7.5

Citrus limon 10 9.0

Garcinia kola 10 9.0

Zingiber officinale 10 9.8

Cassia alata 10 10.0

Piper guineense 50 8.5

Xylopia aethiopica 250 12.0

*Diameter of discs= 6 mm.

inhibitory activities. These three extracts that showed large inhibitions produced reduction of mycelial growth of 76, 54 and 43 respectively (Table 3, Plate 1). The results of this assay technique may indicate the synergistic effect of the active principles present in these plant extracts.

The results of minimum inhibitory concentration (MIC) of the plant extracts showed that at 10 mgL

-1,

the extracts of A. melegueta, C. limon, G. kola, Z. officinale and C. alata have minimum antifungal activities against F. verticillioides with 7.5, 9.0, 9.0, 9.8 and 10.0 mm respectively (Table 4). P. guineense and X. aethiopica however showed the minimum activities with zones of inhibition of 8.5 and 12.0 mm at higher concentrations of 50 and 250 mgL

-1 extracts. The

results in Table 5 showed the antifungal activity of the retention factor (rf) values of the fractions of extracts of Piper guineense and Aframomum melegueta from the scraped TLC plate (Figure 1). P. guineense yielded six fractions, out of which four (rf values 0.47-0.41, 0.30-

0.27, 0.19-0.14 and 0.07-0.05) showed inhibition to F. verticillioides. Two rf values of the fractions were obtained for A. melegueta, out of which 0.66 to 0.61 showed antifungal activity against F. verticillioides (Table 5). This means that P. guineense and A. melegueta had six and two different compounds respectively, out of which 4 and 1 are respectively the active principles against the tested fungus. DISCUSSION The extracts of P. guineense, G. kola, A. melegueta, Z. officinale, C. alata, X. aethiopica and C. limon possess antifungal activities against F. verticillioides in a decreas-ing order. Various authors (Okogun and Ekong, 1974; Okogun et al., 1977; Ayitey-Smith and Addae-Mensah, 1977) reported the phytochemical investigation of P. guineense that the extracts yielded more than twenty

28 J. Microbiol. Antimicrob.

Table 5. Antifungal activity of different fractions of the retention factor (rf) of the extracts of Piper guineense and Aframomum melegueta (from scraped Thin-layer chromatographic plate).

Extract Rf range Inhibition* Fusarium verticillioides

Piper guineense 0.47-0.41 +

0.37-0.30 -

0.30-0.27 +

0.19-0.14 +

0.09-0.07 -

0.07-0.05 +

Aframomum melegueta 0.66-0.61 +

0.31-0.25 -

*Diameter of discs= 6 mm, + Presence of inhibition, -No inhibition.

Figure 1. TLC profiles of seven promising plant extracts (acetic acid + hexane 1:1 at 280nm wavelength).

different compounds; mainly amide alkaloids which have been found to possess antimicrobial activity against Klebsiella pneumoniae, Mycobacterium smegmatis and Candida albicans (Addae-Mensah, 1992). Raju and Maridass (2011) also highlighted that the high level anti-bacterial and antifungal potential against Gram positive,

Gram negative bacteria and moulds on seven Piper species used in indigenous system of medicine, might be due to the principal active constituents. In the essential oils of P. nigram, sabinene (3.9 to 18.8%), β-pinene (3.9 to 10.9%), limonene (8.3 to 19.8%) and β- caryophyllene (28.4 to 32.9%) have been found to be active consti-

A B D E F G H

A=Piper guineense

B=Zingiber officinale

D=Aframomum

melegueta

E=Citrus limon

F=Xylopia aethiopica

G=Cassia alata

H=Garcinia kola

1Acetic Acid+ hexane 1:1 at 280 nm wavelength

tuents (Uni-Graz, 2005; Pruthi and Srivas,1962). The high inhibition of the growth of F. verticillioides in this study is due to these active constituents.

Ajayi et al. (2011) reported that seeds of G. kola showed absence of alkaloid, while presence of tannin and saponin were observed. The antimicrobial activity of this plant agrees with the work of Madubunyi (1995) which showed that the petroleum ether, ethanol, the milky layer and ethyl acetate fractions of the seed extracts of this plant possessed antimicrobial properties. This might be due to the presence of a polyisoprenyl benzophenone (kolanone) in the petroleum ether extract, as well as the hydroxybiflavanonols present in the ethyl acetate fraction. The author was of the opinion that GB1 (hydroxybiflavanonol), was the main component exhibiting significant antimicrobial activity against Gram-positive and Gram-negative bacteria, C. albicans and Aspergillus flavus.

Sonibare et al., 2011 reported the presence of alkaloids and saponins, and absence of Tannin in the seed extracts. Galal (1996) also isolated 6-Paradol and 6-shogaol from the seeds extracts following bioactivity-guided fractionation. These compounds were active against Mycobacterium chelonei [M. chelonae], M. intracellulare, M. smegmatis and M. xenopi. The author further stressed that the desmethyl derivative of 6-paradol retained antimycobacterial activity, and was more active against Candida albicans than either 6-paradol or 6-shogaol. On the other hand the seed extracts of A. melegneta inhibited the growth of F. verticillioides at low MIC.

In this study, other extracts that showed antimicrobial activity include the lemongrass. Its main components have been found to be responsible for the antimicrobial activity and preservative potential. The extracts have been found to contain alkaloids, tannins and cardiac glycosides (Tzortzakis and Economakis, 2007; Adegoke and Odesola, 1996), aliphatic alcohol, geranial, neral and aliphatic aldehydes (Velluti et al., 2004). The aliphatic alcohols and phenols exhibited significant action against Aspergillus aegyptiaceus, Penicillium cyclopium and Trichoderma viride (Megalla et al., 1980). Velluti et al. (2004) reported the mycelial inhibition of F. verticillioides at 1000 mg/ml. The present study agrees with the reports that lemongrass was effective against F. verticillioides at 100 to 500 mg/ml. The antimicrobial activity of the oil is believed to be associated with the phytochemical compo-nents of lemongrass such as monoterpenes (Matasyoh et al., 2007) which diffuse into and damage cell membrane structures.

Velluti et al. (2004) highlighted that generally, one of the critical things to consider for commercial applications is that the levels of essential oils and their compounds necessary to inhibit the microbial growth were higher in foods than in culture media. This is due to interactions between the phenolic compounds and the food matrix (Nuchas and Tassou, 2000). Treatment with basil oil was found to control the crown rot and anthracnose, thus

Adejumo 29 prolonging storage of bananas (Anthony et al., 2003), while cinnamon and eucalyptus oil-enrichment reduced fruit decay and improved fruit quality of tomato and strawberries (Tzortzakis, 2007). However, the composition of the oil from the botanicals is significantly affected by the ripeness of fruits, vegetative stage of plant, storage condition and extraction methods (Njoroge et al., 2006; Chutia et al., 2009).

Further studies on these effective botanicals should include bioassay-directed fractionation of the extracts which will lead to the isolation of the compounds that is showing considerable antifungal activity. The continuation of study on the plant is essential to isolate, identify, characterize and elucidate the structure of the bioactive compounds responsible for the observed antifungal activities. From this result, it is essential to investigate the specific constituents which are responsible for this observed activity and such characterization studies are underway. The in vivo study is also required to confirm the usefulness of the obtained results. Conclusion The in vitro results of this study confirm the potentiality of P. guineense, G. kola and A. melegueta as the best sources of antifungal therapy. Hence, further work is necessary to evaluate its potentiality in in vivo studies on other pathogens. Simultaneous investigations are also needed to characterize, formulate and market the active principles of these extracts which may provide leads for the discovery of novel antimycotic compounds. These biofungicidal botanicals are environmentally safe, therefore, they could successfully replace the toxic and hazardous synthetic compounds and exploited as an ideal treatment for future plant disease management programs. ACKNOWLEDGEMENTS The financial support for this research was made possible by the Alexander von Humboldt Foundation (AvH), Germany through the Georg Forster Renewed Research Fellowship granted to Dr. T.O. Adejumo. The support is gratefully appreciated. Many thanks to Prof. Dr. Lindhauer, Dr. Hollmann, Frau Dr. Schwake-Anduschus, Frau Marie-Therese Hanneforth, Frau Meyer-Wieneke, Frau Ute Versen, Frau Lippert, Frau Brackemann and Frau Förster of Max Rubner-Institut, Detmold, Germany for your efforts and technical support. REFERENCES Abila B, Richens A, Davies JA (1993). Anticonvulsant effects of extracts

of the west African black pepper, Piper guineense. J.

Ethnopharmacol., 39(2): 113-117. Addae-Mensah I (1992). Towards a rational scientific basis for herbal

30 J. Microbiol. Antimicrob.

medicine –A phytochemist’s two decades contribution. An inaugural lecture delivered at the University of Ghana, Legon, Ghana. Universities Press, Accra, p. 63.

Adegoke GO, Odesola BA (1996). Storage of maize and cowpea and inhibition of microbial agents of biodeterioration using the powder and essential oil of lemon grass (Cymbopogon citratus), Int. Biodeterior.

Biodegrad., 6: 81−84. Adejumo TO, Hettwer U, Karlovsky P (2007a). Occurrence of Fusarium

species and trichothecenes in Nigerian maize. Int. J. Food Microbiol., 116: 350-357.

Adejumo TO, Hettwer U, Karlovsky P (2007b). Survey of maize from

south western Nigeria for Zearalenone, α- and β- Zearalenols, Fumonisin B1, and Enniatins produced by Fusarium species. Food Addit. Contam., 24(9): 993-1000.

Ajayi IA, Ajibade O, Oderinde RA (2011). Preliminary Phytochemical Analysis of some Plant Seeds. Res. J. Chem. Sci., 1(3): 58-62.

Ali BH, Blunden G, Tanira MO, Nemmar A (2008). Some phytochemical, pharmacological and toxicological properties of ginger (Zingiber officinale Roscoe): a review of recent research. Food Chem. Toxicol., 46: 409-420.

Anthony S, Abeywickrama K, Wijeratnam SW (2003). The effect of spraying essential oils Cymbopogon nardus, Cymbopogon flexuosus and Ocimum basilicum on postharvest diseases and storage life of

Embul banana. J. Hortic. Sci. Biotechnol., 78: 780−785. Ayitey-Smith E, Addae-Mensah I (1977). A preliminary pharmacological

study Winnine:,A piperine type of alkaloid from the roots of Piper guineense. W. Afr. J. Pharmacol. Drug. Res., 4: 7-8.

Boyom FF, Ngouanaa V, Zollob PHA, Menutc C, Bessiered JM, Gute J, Rosenthal PJ (2003). Composition and anti-plasmodial activities of essential oils from some Cameroonian medicinal plants. Phytochemistry, 64: 1269–1275.

Chutia M, Bhuyan PD, Pathak MG, Sarma TC, Boruah P (2009). Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India, LWT – Food Sci. Technol., 42: 777–780.

Dongmo PMJ, Tatsadjieu LN, Tchinda Sonwal E, Kuate J, Amvam Zollo PH, Menut C (2009). Essential oils of Citrus aurantifolia from Cameroon and their antifungal activity against Phaeoramularia angolensis. Afr. J. Agric. Res., 4(4): 354-358.

Engelhardt G, Barthel J, Sparrer D (2006). Fusarium mycotoxins and ochratoxin A in cereals and cereal products. Results from the Bavarian Health and Food Safety Authority in 2004. Mol. Nutr. Food Res., 50: 401–405.

Galal AM (1996). Antimicrobial activity of 6-paradol and related compounds. Int. J. Pharmacog., 34(1): 64-69.

Grover RK, Moore JD (1962). Toxicometric studies of fungicides against brown rot organisms Sclerotinia fructicola and S. laxa.

Phytopathology, 52: 876–880. Harrison LR, Colvin BM, Green JT, Newman LE, Cole JR (1990).

Pulmonary oedema and hydrothorax in swine produced by fumonisin B1, a toxic metabolite of Fusarium moniliforme. J. Vet. Diag. Investig., 2: 217–221.

Iwu M (1993). Handbook of African medicinal plants. CRC Press, Roca Raton, FL.

Kellerman TS, Marasas WFO, Thiel PG, Gelderblom WCA, Cawood M, Coetzer JAW (1990). Leukoencephalomalacia in two horses induced by oral dosing of fumonisin B1. Onderstepoort J. Vet. Res., 57: 269–275.

Li M, Lu S, Ji C, Wang Y, Wang M, Cheng S, Tian G (1980). Genetic and environmental factors in experimental and human cancer. Tokyo: Japanese Science Society Press.

Madubunyi II (1995). Antimicrobial Activities of the Constituents of Garcinia kola Seeds. Pharma. Biol., 33(3): 232–237.

Mahesh B, Satish S (2008). Antimicrobial Activity of some important medicinal plants against plant and human Pathogens. World J. Agric. Sc., 4: 839-843.

Marasas WFO, Jaskiewicz K, Venter FS, Van Schalkwyk DJ (1988). Fusarium moniliforme contamination of maize in oesophageal cancer

areas in Transkei. South Afr. Med. J., 74: 110–114. Matasyoh JC, Wagara IN, Nakavuma JL, Kiburai AM (2007). Chemical

composition of Cymbopogon citratus essential oil and its effect on mycotoxigenic Aspergillus species. Afr. J. Food. Sci., 5(3): 138-142.

Maziya Dixon B, Akinyele IO, Oguntona EB, Nokoe S, Sanusi RA,

Harris E (2004). Nigeria food consumption and survey 2001–2003 summary. International Institute of Tropical Agriculure (IITA). Ibadan, Nigeria.

Megalla SE, El-Keltawi NEM, Ross SA (1980). A study of antimicrobial action of some essential oil constituents. Herba Pol., 3: 181–186.

Nene Y, Thapilyal L (2000). Poisoned food technique of fungicides in plant disease control. 3rd Edition, Oxford and IBH Publishing Company, New Delhi.

Njoroge SM, Munga HN, Koaze H, Phi NTL, Sawamura M (2006). Volatile constituents of mandarin Citrus reticulata Blanco peel oil from Burundi. J. Essential. Oil Res., 18: 659–662.

Nuchas GE, Tassou CC (2000). Traditional preservatives-oils and spices. In Robinson RK, Batt CA, Patel PD (Eds.), Encyclopedia of food microbiology. London, UK: Academic Press, pp. 1717−1722.

Ogu EO, Agu RC (1995). Comparison of some chemical characteristics of Garcinia kola and hops for assessment of Garcinia Brewing value. Biores. Technol., 54: 1-4.

Oji O, Madubuike FN, Nwagbo LC, Oji PO (1992). Protection of stored Zea mays L., against Sitophilus zeamais L., with non-toxic natural products. Potential of Xylopia aethiopica and Piper guineense. Acta

Agron. Hungarica, 41: 131-135. Okogun JI, Ekong DEU (1974). Extracts from the fruits of Piper

guineense Schum. and Thonn. J. Chem. Soc. (Perkin I), p. 2195.

Okogun JI, Sondengram BL, Kimbu SF (1977). New Amides from the extracts of Piper guineense. Phytochemistry, 16: 1295.

Purseglove JW, Born EG, Green CL, Robbins SRJ (1981). Spices Volume 2. Longman, London and NewYork, p. 813.

Prescott LM, Harley JP (1996). Microbiology. 3rd Edition. Chicago W.C.

Brown. Pruthi JS, Srivas SR (1962). An integrated process for the recovery of

volatile oil, oleoresin and starch from ginger., Proceedings of Symposium on Spices –Role National Economy. Washington, DC.,

p.11. Raju G, Maridass M (2011). Evaluation of antimicrobial Potential of

Piper (L.) species, www.gbtrp.com. Nat. Pharmaceut. Technol., 1(1):

19-22. Reddy MVB, Angers P, Gosselin A, Arul J (1998). Characterization and

use of essential oil from Thymus vulgaris against Botrytis cinerea and Rhizopus stolonifer in strawberry fruits. Phytochemistry, 47: 1515−1520.

Sadasivam S, Manickam A (1996). Biochemical methods, 2nd

Edition. New age Publishers, New Delhi.

Sengupta S, Ghosh SN, Ghosh SB, Das AK (2008). Antimycotic Potentiality of the Plant Extract Bacopa monnieri (L.) Penn., Res. J. Bot.,

3(2): 83-89. Soliman KM, Badeaa RI (2002). Effect of oil extracted from some

medicinal plants on different mycotoxigenic fungi. Food Chem. Toxicol., 40: 1669−1675.

Sonibare MA, Lawal TO, Ayodeji OO (2011). Antimicrobial evaluation of plants commonly used in the management of psychosis opportunistic infections. Int. J. Pharmacol., 7(4): 492-497.

Strange RR, Midland SL, Sims JJ, Greg Mccollum TG (2002). Differential effects of citrus peel extracts on growth of Penicillium digitatum, P. italicum, and P. expansum. Physiol. Mol. Plant Pathol.,

61: 303-311. Taylor RSL, Manandhar NP, Hudson JB, Towers GHN (1995).

Screening of selected medicinal plants of Nepal for antimicrobial activities. J. Ethnopharmacol., 546: 153-159.

Tzortzakis NG, Economakis CD (2007). Antifungal activity of lemongrass (Cympopogon citratus L.) essential oil against key

postharvest pathogens. Innov. Food. Sci. Emerg. Technol., 8: 253–258.

Tzortzakis NG (2007). Maintaining postharvest quality of fresh produce with volatile compounds. Innov. Food Sci. Emerg. Technol., 8(1): 111-116.

Ukeh DA, Birkett MA, Pickett JA, Bowman AS, Luntz AJM (2009). Repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale, against the maize weevil, Sitophilus zeamais. Phytochemistry, 70: 751–75

Uni-Graz (2005). http://www.uni-graz.at (accessed on 2005/04/16). Valero M, Salmeron MC (2003). Antibacterial activity of 11 essential oils

against Bacillus cereus in tyndallized carrot broth. Int. J. Food Microbiol., 85: 73−81.

Velluti A, Marlın S, Gonzalez P, Ramos AJ, Sanchis V (2004). Initial screening for inhibitory activity of essential oils on growth of Fusarium verticillioides, F. proliferatum and F. graminearum on maize-based agar media. Food. Microbiol., 21: 649–656.

Viallaroya MLE, Bernal-Santos R (1976). Anthraquinones from the leaves of Cassia alata. Asian J. Pharm., 3: 10- 24.

Wagner H, Bladt S (1996). Plant drug Analysis: A thin layer Chromatography atlas. 2

nd Ed. Berlin, Springer.

Adejumo 31 Yang CS (1980). Research on esophageal cancer in China: A review.

Cancer Res., 40: 2633–2644.