CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

The African locust bean plant (Parkia biglobosa) is a perennial tree legume belonging to the

family Fabaceae. African locust bean seeds are rich in protein and usually fermented to a tasty

food condiment called “dawadawa” which is used as a flavour intensifier for soups and stews

and also adds protein to a diet that is deficient. (Ikenebomeh and Kok 1984; Odunfa, 1986; Dike

and Odunfa, 2003). This plant is used as food especially the seeds which are used as spices in

Africa. P. biglobosa is a wild legume and many of these wild legumes are known for their

inexpensive proteins, high calorific value, essential amino acids, essential fatty acids, fibre and

vitamins; but the presence of anti-nutrients in the seeds has limited their use (Bhat & Karim,

2009).

Odunfa, (1981) reported that fermented locust bean seed is commonly consumed in Ghana,

Nigeria, Sierra Leone and Togo. It is called”Iru or “Igba in Yoruba,”dawadawa in Hausa and

“ogiri-igala in Igbo parts of Nigeria (Lamien et al., 2011).Because of their high nutritional value

and organoleptic properties they are widely used in preparation of many dishes (N’dir et al.,

2011). The sweet pulp can be licked and also used as beverage because of its sweet taste. It can

also be used in folk medicine, glaze for ceramic pots, fodder, firewood and charcoal production.

The pulp of the fruit pods is rich in sucrose and the seeds are rich in carbohydrates, proteins and

lipids thus, constituting an important source of energy. (Kourouma Koura et al., 2011). The bark

and roots have useful medicinal properties. (Booth and Wickens, 1988). The bark of the stem is

boiled in water and taken as decoction for the treatment of malaria and diarrhoea. (Asase et al.,

1
2005). The seed and the pulp is a host to a number of field-to-store pests, whose eggs are laid on

the fruit pods in the field and develop in storage causing significant economic loss. However,

despite the importance of the fruit and the effects of the pests in the field and storage, very little

is known about its pest profile and the biology and control of the pests.

In recent times, several pharmacological and nutritional benefits of P. biglobosa have been

reported (Badu et al., 2012; Olabinri et al., 2013). Consequently, considerable interest has been

directed toward exploring P. biglobosa in the prevention, treatment, and management of a

number of metabolic diseases among which is diabetes mellitus. This pharmacological and

nutritional benefit of P. biglobosa has been associated with its physicochemical properties,

which is perceived to act independently or in synergy to play a pivotal role in the maintenance of

normoglycemia by activating diverse protective mechanisms. (Ogunyinka et al., 2015). The pulp

is consumed directly or used for bakery, whereas the seed is used for production of soumbala, a

nutritious ingredient rich in proteins used as a standard condiment in rural diets. The tree has a

wide spreading crown and reaches the heights of 15–20 m. As an orthodox species, the seed can

be stored for prolonged periods, and without pre-treatment the seed may not germinate until

several weeks after sowing (Sacande and Clethero 2007). The lesser grain borer, Rhyzopertha

dominica (F., 1792) (Coleoptera: Bostrichidae), is a destructive pest of stored grain, finished

grain and consumable materials all over the world (Edde et al., 2012). Generally regarded as a

strong flier, hence it can easily disperse from one storage facility to another and create new

infestations (Stejskal et al., 2003). Females lay eggs on the surface of grain kernels, and upon

hatching, the larva enters the kernel (Neethirajan et al., 2007; Ozkaya et al., 2009) and remains

inside until maturity (Chanbang et al., 2007).

2
There has been many researches on P.biglobosa pulp including the physicochemical properties,

phytochemical content and nutritional composition but there has been few reports on the insects’

pests profile attacking the pulp however this project researches into the aspect of

entomopathogenic control of the fruit.

1.2 Statement of problem

Despite the fact that P. biglobosa is widely useful in culinary and medicinal values and there has

been various researches on P.biglobosa, it faces the irrefutable threat of various insect pests

which depletes the wholesome nutritional, medicinal and other values of this specie and

rendering it unhealthy for consumption. As a result of severe insect infestation, it could give way

for mould attack thereby causing 100 percent loss in the yield of P. biglobosa fruit if not

controlled.

1.3 Objectives of the study

Objectives of this study are to:

The main objective of the study is to evaluate the virulence of entomopathogenic fungi against

lesser grain borer, Rhyzopertha dominica.

The specific objectives are to:

1. isolate entomopathogenic fungi from soil,

2. study growth relations of the entomopathogens,

3. produce the infective spores in solid substrate,

4. develop the spores into experimental biopesticide and

5. test the biopesticide against insect pest and evaluate insect mortality.

3
 1.4 Justification of the study

In contemporary times there has been cases of pest attack on the fruit of this tree which has

reduces the overall quality of the fruit therefore in order to achieve a pest and disease free

pulp for the consumption of the populace. There is need to evaluate the biocontrol potentials,

study growth relations of the entomopathogenic fungi, and develop effective biopesticides as

control measure against insect pest attacking African locust bean, P.biglobosa in storage.

4
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Rhyzopertha dominica

R. dominica also known as the lesser grain borer (Coleoptera: Bostrichidae) is known to be an

inimical pest of grains that are in storage as well as edible foodstuffs (Edde et al., 2012). The

larvae together with the adults have the tendency to affect uninfested grain (Mayhew and

Phillips, 1994). R. dominica is a key pest of rice globally (Chanbang et al., 2008).The young

insect as well as the matured ones yield frass and originates into weight losses through nurturing

on grains, R. dominica Invasion can diminish rice to dust (Emery and Nayak, 2007). R.

dominica’s nurturing activity on seed germs decreases growth rates and vigour of the grains and

possibly will be trailed by minor pests and fungi (Bashir, 2002). Statistics gathered from wheat

warehoused in commercial grain elevators in south-central Kansas, USA, revealed that C.

ferrugineus, R. dominica and T. castaneum were the major insect species originated in composed

wheat samples, at the height of 3.7m inside the warehouse R.dominica comprises of 44% of the

insects discovered in the samples, and at the height of 3.8-12.2m it was available at 84% (Flinn

et al., 2010). In 2008, R. dominica caused severe destruction to grain in an Indonesian

government storage unit (Astuti et al., 2013). One of the most essential control method of

R.dominica is packing of grains in sealed buildings, the building must be tested with pressure to

check airtightness, Transportable enclosed storage bags are also obtainable (Garcia et al., 2013).

Solid red winter wheat comprising diverse life cycles (egg, larvae, pupae and 2-week-old adults)

of R.dominica was prevented from infestation through the medium of a flameless catalytic

infrared emitter (Khamis et al., 2010). It was concluded that the technology of applying

5
flameless catalytic infrared emitter is a promising medium for disinfestation of stockpiled wheat,

because nearly 94% mortality rate of all R. dominica life cycles occurred after 113.5g of wheat

was exposed for 60s at a distance of 8.0cm from the emitter, culminating in wheat temperatures

that ranged between 107.6± 1.4 and 113.5± 0.5ºc (Khamis et al., 2010). The ecology of the pest

other than in grain storage facilities is barely documented and the rate of development is

proportional to temperature (Edde et al., 2012). Past reports on R. dominica shows that it grows

adequately and displays oviposition in seeds of some trees and shrubs and in spoilt acorns (Jia et

al., 2008). Bostrichidae family in which R. dominica belongs has the ability to convert sapwood

into a powdery frass. Hence, they are of considerable economic importance to forestry and to

wood-using industries (Liu et al., 2008). Limited information is available pertaining to the

seasonal flight activity of R.dominica (Toews et al., 2006). R. dominica adults are strong fliers

and are capable of moving across landscapes and they can only be trapped by flight traps using

pheromone as the bait (Hagstrum, 2001).

2.2 Morphology

Adults of R. dominica are elongated and cylinder-shaped and reddish brown to dark brown in

colour. The body length of the adult beetle is 2–3 mm and it is 0.8–1 mm wide, they possess

exarate and sedentary pupae and also sexual dimorphism is noticeable at the tip of the abdomen

(Edde et al., 2012). The lowest temperature at which R. dominica can complete its life cycle is

about 18–20°C, while it is unable to survive over 39°C and under 3°C (Baldassari et al., 2005).

Therefore, dependent on temperature and humidity, the amount of generations could differ

significantly. However, in warmer regions R. dominica could complete up to six generations per

year (Pollini, 2013).

6
2.3 Life cycles

2.3.1 Egg

R. dominica egg, which has an oval shape is about 0.5-0.6 mm in length and 0.2-0.25 mm in

diameter. The chorion comprising two layers, is about 2.7 mm in thickness (Kucerova and

Stejskal, 2008). The eggs are laid in groups on grains or singly among frass formed by the

insect.it is impervious, has a white colour and with waxy appearance when recently laid, and

turns pinkish with time (Kucerova and Stejskal, 2008).

2.3.2 Larvae

The larva of R. dominica is normally C-shaped with tiny hairs existing on its dorsal figure which

has a creamy, white colour. It has three pairs of thoracic legs and uses its whole lifecycle inside

the host grains until development as matured insect. Movement of the young insects of

R.dominica obviously varies along larva intsars. First and second larval stages are mostly

moveable but later instars become immovable. Mature larva turns into pre-pupa which does not

have ocelli (simple eyes) and is less mobile and with somewhat flattened body (Guedes et al.,

1996). Freshly developed first instar larva willingly discovers the host grain for shelter and food.

It prefers to enter and feed on the germplasm rather than feeding on the endosperm portion of the

grains (Mahroof and Phillips, 2006). This is the most important stage of its life cycle, more

susceptible to ecological pressure as related to other larval instars. Female borers lay eggs

usually in or on partly infested or succulent part of the grains such as on embryo or on cracked

grains. R. dominica has four larval instars. The emerging larvae feed inside of the grain kernel,

eventually causing damage to germplasm and endosperm and also weight loss of the grain, while

young larvae do not enter into the grains normally (Mason, 2003).

7
2.3.3 Pupa

Last instar larva of R. dominica upon development pupates within nourishing shaft or hollow of

the grains and progressively undertakes the shape of an adult. Usual life span of pupal stage is

about 8 days at 25ºC and 5–6 days at 28ºC. A healthy pupa is about 3.9 mm long, having white

to brownish white colour. There are several distinct appendages on the curved end of the pupal

body, dependent on its sexual variation. In male pupa, papillae are convergent and has two

segments, while in females, they are divergent with three segments (Nguyen, 2006).

2.3.4 Adult

R.dominica adult is about 2-3 mm long and 0.8-1 mm wide. Fresh body weight ranges from 0.99

to 1.38 mg, whereas the mean of the fresh body weight is about 1.20 mg. Mean longevity of

adult male and female R. dominica fed on wheat kernels at 28 ºC and 65% Relative humidity is

26 and 17 weeks respectively (Edde and Phillips, 2006) Mean longevity values of malnourished

adult male and female were 5.7 and 4.7 days, respectively (Edde and Phillips, 2006). Adverse

effects on fertilization and movement of adult R. dominica may transpire after four days of

undernourishment. The effects of undernourishment is more evident on female R. dominica,

apparently due to more energy demand on them for fertilization (Nguyen et al., 2008).

2.4 Feeding Habits

It feeds mostly on grains from families Poeaceae (e.g. rice, wheat, sorghum, oats, pearl, millet,

malt, barley) and Fabaceae (e.g. chickpeas, peanuts, beans) (Edde et al., 2012). The larvae and

adults of R. dominica spend most of their life inside the kernel, feeding on both the germ and

endosperm, directly causing damages and changes in the grain’s physicochemical properties

(Rees, 2004). Insect-damaged kernels are characterized by the presence of irregularly shaped

8
holes (about 1 mm in diameter) of increasing depth that extends from the point of larval entry,

around the edge of the kernel to the pupation chamber (Rees, 2004). Adult feeding activities

produce large amount of dust, which often leads to the reduction of grain kernel to the pericarp.

(Nawrot et al., 2010) investigated the amount of grain consumed by R. dominica under

experimental conditions, but the losses of nutrient value varied among the authors.

2.5 Management of R. dominica

Several practices are being implemented to control stored grain insect pests including chemical,

physical, cultural, mechanical, genetic, and biological control (Phillips& Throne, 2009). Many

scientists have revised different components of integrated pest management (IPM) against stored

food insect pests.

2.5.1 Physical control

Physical control in case of R. dominica is mostly achieved through dehydrating or cooling the

grains or stored food products below 15ºC to suppress insect activities (Vincent et al., 2003).

The Range of temperature in wheat and other grain varieties during mid- summer is mostly 27–

34ºC (Jian et al., 2009). This temperature is very advantageous for the growth, development,

existence and optimum fertilization of R. dominica (Lord, 2005). Drying is a mechanical

ventilation of different stored grains aimed at sustaining constant and cool conditions within

storage infrastructure. Although it is found that R. dominica cannot be controlled totally through

drying (Liu, 2005), it is an active and viable tool for decreasing insect growth rates and is also

used to reduce the danger of fungal contamination (Arthur et al., 2011).

2.5.2 Biological control

9
Among several control methods, one of the most viable control options is the biological control

of stored grains insect pests. It contributes about 3–20% in Integrated Pest Management of stored

food products (Schöller et al., 2006).In this method, different insect predators and parasitoids

which are naturally occurring in bulk grain storage localities are used to control insect pests

(Eliopoulos et al., 2002). These predators and parasitoids are known to attack a number of insect

pests of stored grain (Hagstrum and Flinn, 1992).

2.5.3 Chemical control

Different pesticides has been applied as chemical control method against pests including R.

dominica (Collins, 2006). Due to an extended and persistent exposure to pesticides, R. dominica

has developed resistance to these stored grain and food protectants (Daglish & Nayak, 2010). For

instance, deltamethrin is found ineffective against R. dominica in several parts of the Brazil even

when it is used at a high dose (Lorini and Galley, 2000). Similarly, R. dominica has shown

resistance against other different chemicals such as malathion, pirimiphos methyl, fenitrothion,

phosphine and chlopyrifos-methyl (Song et al., 2013). Therefore, ensuring stored food quality

and quantity, an efficient and eco-friendly integrated pest management should be developed.

2.5.4 Entomopathogenic microorganisms

Bacteria, fungi, protozoans and nematodes have also been observed as a source of mortality in

R. dominica. These microorganisms attacks both the adult and larval stages of the insect. Hot and

dry conditions in stored grains often limit the population of these microorganisms. Usually these

entomopathogenic microorganisms prefer high relative humidity to be active (Moino et al.,

1998). Nevertheless, some dissimilar findings were reported that some pathogenic fungi such as

Beauveria bassiana are active against stored grain beetles even at relative humidity as low as

10
43% (Lord, 2005). In another study, (Adane et al., 1996) showed that B. bassiana infects O.

surinamensis (saw toothed beetle) at 100% relative humidity.

2.6 Symptom description and pest status

Grain invasions may happen from remaining pest populations in stowing buildings and mingling

of diseased and uninfested grain. (Hagstrum, 2001). The winged adults usually enter a grain

container through the headspace, perch on the grain surface, and slowly moves through the grain

mass in a slow downward progression (Hagstrum, 2001). R. dominica could move down into the

grain mass to a depth of 12 m, which is deeper than observations for other grain beetles (Flinn et

al., 2010).

2.7 Origin, Distribution and Taxonomy of P.biglobosa

The African locust bean plant (P. biglobosa) is a perennial tree legume belonging to the

Family Fabaceae. It is a genus of flowering plants which belongs to the sub-family Mimosoideae

and Leguminosae with the genus Parkia and botanical name P.biglobosa (Abdoulaye, 2012) P.

biglobosa (Jacq.) G. Don is a highly valued multipurpose tree species native to African

savannahs with a large distribution range extending from Senegal to Uganda (Hopkins, 1983;

Hall et al., 1997). Commonly called “Néré” or “African locust bean” it is one of the most

important agroforestry tree species in West Africa (Nikiema, 2005).

2.7.1 Natural range of P. biglobosa

The geographic distribution of P. biglobosa has been described in a range map by Hall et al.,

(1997) showing geographic boundaries overlapped with climatic zones. The natural range

11
extends over 20 African countries north of the equator. The species is mainly found in African

savannahs, particularly in the Sudanian vegetation zone, partially in the drier, more northern

Sahelian zone and the wetter, more southern Guinean zone. The range covers different habitats

usually on deep loamy and sandy soils (Arbonnier, 2004) with annual rainfall varying between

700 mm in the North to 2,600 mm in the South and exceptional levels up to 4,500 mm in Sierra-

Leone and Guinea (Hall et al., 1997). In Burkina Faso, located in the centre of species’ range,

density varies between less than one tree per hectare in the sub-Sahelian zone to up to 25 trees

per hectare in the southern part of the Sudanian zone (Ouedraogo, 1995). Due to the expansion of

agriculture, pasture land, urbanization, mining, and also the vulnerability of the species to global

environmental changes (Bouda et al., 2013), significant changes in the distribution range of P.

biglobosa may have taken place during the last 20 years, since occurrence data were assembled

for the first time (Hall et al., 1997).

Figure 1: Distribution of Parkia biglobosa in relation to mean and annual rainfall

Source: Hall et al, 1997

12
2.8 Economic importance of P. biglobosa Tree

2.8.1 Seeds

P.biglobosa (African locust bean) seeds are used for preparing popular soup condiment in

Nigeria and other West African countries it is called ‘iru’ among the Yoruba ethnic group. The

processing of African locust bean include cooking, dehauling, washing then cooking for the

second time and fermentation. Fermentation brings out the desired nutritional value and other

organoleptic properties such as taste, flavour and texture. (Aderibigbe et al., 2018).

2.8.2 Fruit (pulp)

Locust bean pulp from rain forest vegetation had the highest value for crude protein (15.34%),

crude fibre (19.45%) total ash (4.50%) and dry matter (91.1%). (Olujobi, 2012). The pulp is also

used as an ingredient in the preparation of various stews, and soups (Odebunmi et al., 2010)

Plate 1: Fresh pulp of P. biglobosa

Source: Anthony Simeon 2002

13
2.8.3 Bark

Some clinical investigations described the antibacterial properties of P. biglobosa bark and root

extracts against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae,

Escherichia coli, Shigella dysenteriae responsible for urinary tract and digestive system

infections (Alinde et al., 2014) Additionally, P. biglobosa stem bark has been discovered to

have anti-microbial effects against Bacillus subtilitis and was recommended as a natural

preservative against pharmacological contaminations. (Oguntibe et al., 2014).

2.8.4 Leaves

P. biglobosa leaf extract was described to exhibit anti-plasmodia activity against malaria

recently, antioxidant, anti-carcinogenic and anti-trypanosomic activities of the plant have been.

acknowledged (Oguntibe et al., 2014).

Plate 2: Parkia biglobosa tree.

Source: Sobande, 2013 and Ojewumi et al., 2016.

14
 CHAPTER THREE
3.0 MATERIALS AND METHOD

Materials: Petri-dishes, Cork borer, Falcon bottles, Inoculating loop, McCartney bottles, Potato

Dextrose Agar, Syringe, Coverslips, Glass slides, Microscope, Hand gloves, Spatula,

Chloramphenicol, Glass jars, Ethanol.

3.1 Study area

The study was carried out in the Laboratory of Forest Resources and Wildlife Management.

3.2 Isolation of entomopathogens from soil using insect bait method

Soil samples were collected from different locations in the school premises. The collected soil

samples were air dried on a plain white paper after which was sieved in order to remove plant

debris and stones from the soil. Samples were distributed in small quantities in petri dishes and

labelled to represent each locations.

Plate 3: Collection of soil samples in petridishes

15
3.3 Preparation of pure cultures of pathogen.

3.3.1 Preparation of media for isolation of entomopathogenic fungi.

250ml of distilled water was prepared and 9.75g of PDA was measured and poured into a

conical flask and covered with an aluminium foil and taped round to prevent contamination and

also prevent the content from pouring. It was autoclaved for 45 minutes, the agar was amended

with 1ml of 99.99% ethanol and 0.05g of chloramphenicol and then shaken thoroughly to

prevent the growth of bacteria. The agar was poured in petridishes and then allowed to solidify.

Plate 4: Preparation of PDA in conical flask Plate 5: Solidified PDA in Petri dishes.

3.2 Evaluation of growth and sporulation rates

Sterile McCartney bottle were filled with 10ml of distilled water and tween80 was added for

proper view on microscope. 1cm cork borer was used to pick 1 agar plug randomly from each

petri dish containing the cultured fungus on PDA and dropped into the labelled McCartney

bottles. It was then vortexed for 10- 15 minutes.

The conidia of the fungus were estimated using haemocytorila. 200ml of the spore suspension

was drawn using a micro pipette and loaded into the haemocytometer. The spores in the upper

and lower chambers were counted respectively with the aid of microscope.
16
 Plate 6: Spore counting of fungi

3.3 Mass production of conidia on solid substrate fermentation (SSF) system

85g of millet was weighed into two conical flasks and rinsed with water. 10ml of water was

measured into the two conical flasks containing the millet and then covered with foil and

autoclaved for two hours. Solid substrate fermentation using millet was done by preparing

conidia suspension of all entomopathogenic fungi by using a syringe to draw the water which

was then poured into a conical flask containing autoclaved millet and then covered with

sterilized cotton wool.

Plate 7: Solid substrate fermentation (Millet)

17
3.4 Preparation and standardization of conidia suspension

Conidia suspension was done by using cork borer to remove a portion of the entomopathogenic

fungi in the petri dish and placed into McCartney bottles containing sterile water and tween80

and then shaken vigorously for proper mix and few drops of the suspension were placed in each

petri dish containing PDA, the plates were taped round with parafilm. Plates were observed for

growth for 7 days.

Plate 8: Conidia suspension in McCartney bottles

3.5 Evaluation of pathogenicity

Ten adults of Rhyzopertha dominica were introduced into each petri dish system. R. dominica

adults were sprayed with different concentrations of conidia (8.20 x105, 2.05x105, 4.10x105,

4.92×104, 6.15×104.Daily cumulative mortality of the insect was recorded.

18
Plate 9: Bioassay of test insects

19
 CHAPTER FOUR

4.0 RESULT

4.1.1 Result from comparing growth rates of two isolates of entomopathogenic fungi

Table 1 shows the comparism of growth rates of two isolates of entomopathogenic fungi when

analysed, it shows that the two isolates were significantly different It was observed that the sum

of squares for the intercept which was 39.934 was higher than that of the isolates which was

6.43. The Mean square for the intercept was 39.934 having a higher value than that of the

intercept which was 6.43 according to the table, the Fcal value for the intercept which was

96.822 has a higher value than the isolates which was 15.589.the significance value for the

intercept was 0.001 which was lesser compared to that of the isolates which was 0.017. The SS

error which was 1.65 was greater than that of MS error which was 0.412.

20
Table 1: Analysis of variance table to compare growth rates of two isolates of entomopathogenic

fungi

Tests of Between-Subjects Effects

Dependent Variable: Growth rate (mm/day)

Source Type III Sum of Squares df Mean Square F Sig.
Intercept 39.934 1 39.934 96.822 0.001
Isolates 6.43 1 6.43 15.589 0.017

Error 1.65 4 0.412

Total 48.014 6

Corrected Total 8.08 5

a R Squared = .796 (Adjusted R Squared = .745)

21
4.1.2 Result from growth rates of two insect pathogenic fungi on Potato Dextrose Agar

Medium at Ambient temperature.

Table 2 shows the growth rate of two entomopathogenic fungi isolates identified as Fusarium

oxysporium (Isolate-E1) and Beauveria bassiana (Isolate-E11) when compared on Potato

Dextrose Agar (PDA) at ambient temperature in the dark .The result shows that the growth rate

of Fusarium oxysporium was faster than B. bassiana with a growth rate of 3.6151mm/day while

that of B. bassiana had a growth rate of 1.5447mm/day.

22
Table 2. Growth rates of two insect pathogenic fungi on Potato Dextrose Agar medium at
ambient temperature in the dark

Entomopathogenic fungal
isolates Growth rate (mm/day) R2 values
F. oxysporum (Isolate-E1) 3.6151a R² = 0.9976
B. bassiana (Isolate -E11) 1.5447b R² = 0.9954

23
4.1.3 Results on Conidia rates of inscect pathogenic isolates of F. oxysporium and

B. bassiana on Potato Dextrose Agar after incubation.

Figure 2 shows the comparism between conidia rates of two pathogenic isolates of

F.oxysporium and B.bassiana on Potato Dextrose Agar after incubation for 7days at ambient

temperature.It shows that the value of the conidia density measured as (conidia/ml) of

F.oxysporium which was 2.17×104 conidia/ml was higher than that of B.bassiana which was

2.14×104 conidia/ml. It also revealed that their spore production was not significantly different,

both were not comparable in terms of spore production.Therefore B.bassiana was prefered over

F.oxysporium, it can be commercialized for mass production as biopesticide due to its high

sporulation rate.

24
 Conidia density (conidia/cm2 colony area) Conidiation rates on agar
2.18E+04

2.16E+04

2.14E+04

2.12E+04
F. oxysporum B. bassiana

Entomopathogenic fungal isolates

Figure 2. Conidia rates of insect pathogenic isolates of Fusarium oxysporum and Beauveria
bassiana on PDA media after incubation for 7 days at ambient temperature

25
4.1.4 Results on Conidia rates of inscect pathogenic isolates of F. oxysporium and B. bassiana on

Solid Substrate (millet) after incubation

Figure 3 shows the compared conidia rate of two pathogenic isolates of F. oxysporium and B.

bassiana on Solid substrate (Millet) after incubation for 14days at ambient temperature. The

result shows that the value of the conidia density (conidia/ml) of F.oxysporium was 4.9×105

conidia/g was found higher than that of B.bassiana which was 1.0×105 conidia/g.

26
 Conidiation rates on solid substrate (millet)

6.00E+05
(per gram solid substrate)
Conidia concentration

5.00E+05

4.00E+05

3.00E+05

2.00E+05

1.00E+05

0.00E+00
F. oxysporum Beauveria bassiana
Entomopathogenic fungal isolates

Figure 3. Conidia yield of Fusarium oxysporum and Beauveria bassiana on solid substrate (millet) after
incubation for 14 days at ambient temperature.

27
4.1.5 Result on mortality curves of R. dominica infected by insect pathogenic strain of B. bassiana at

ambient temperature

Figure 4. Shows the result of cumulative mortality of R. dominica after the period of exposure

for 7days to pathogenic strains of B. bassiana. The result further shows that at the end of the 7

days exposure to biopropagules, treatment T5 with conidia concentration of 6.15×10 4 has the

lowest cumulative mortality of 18% when compared to Treatment T1 (8.20×10 5) which had the

highest cumulative mortality of 50%.

28
 Treatment Conidia
s Concentratio
100 ns
T1 T2 T3 T4 T5 T6 T1 8.20 x105
T2 2.05x105
80
Cummulative mortality (%)

T3 4.10x105
T4 4.92x104
60 T5 6.15 x104
T6 Water
40

20

0
1 2 3 4 5 6 7
Period of exposure to biopropagules (Days)

Figure 4. Mortality curves of Rhyzopertha dominica infected by insect pathogenic strain of Beauveria
bassiana at ambient temperature

29
4.1.6 Result showing Percentage mortality of R. dominica infesting P.biglobosa pulp after

seven days treatment with B. bassiana based biopesticide formulation at ambient

Temperature.

Result from (Figure 5) shows the percentage mortality of R. dominica infesting P. biglobosa pulp after

seven days treatment with Beauveria bassiana based biopesticide formulation at ambient temperature

.Treatment T1with the highest conidia concentration had the highest percentage mortality rate of

insects after 7days post treatment with B. bassiana having 50% mortality. Treatment T6 which

was the control had the lowest mortality rate of 5%.This result further shows that conidia

concentration of treatment T1 was more effective than all other treatments having killed 50% of

the insect population.

30
 Treatme Conidia
nts Concentratio
ns
100
T1 8.20 x105
T2 2.05x105
80 T3 4.10x105
at 7 days post-treatment T4 4.92x104
Total dead insects (%)

T5 6.15 x104
60 T6 Water
50
47

40 36
30

20 18

5
0
T1 T2 T3 T4 T5 T6
Concentrations of infective conidia
(conidia/ml)

Figure 5: Percentage mortality of Rhyzopertha dominica infesting Parkia biglobosa pulp after seven days
treatment with Beauveria bassiana-based biopesticide formulation at ambient temperature

31
4.2 DISCUSSION

4.2.1 Growth rates comparison of two isolates of entomopathogenic fungi.

The result from analysis of variance (Table 1) shows that there was significant difference in the

growth rates of the two isolates of entomopathogenic fungi which were identified as F.

oxysporium and B. bassiana

4.2.2 Comparison of growth rates of two insect pathogenic fungi on Potato Dextrose Agar

medium at ambient temperature in the dark

The result from growth rates of the two isolates of entomopathogenic fungi (Table 2) shows that

F. oxysporium had faster growth rates on Potato Dextrose Agar (PDA) than B. bassiana. This

could be attributed to the fact that PDA media has organic substrates that supports more of the

growth of F. oxysporium than B. bassiana. The result on growth rates of the two

entomopathogenic fungi which were isolated and cultured on PDA shows that the growth rate of

F. oxysporium was faster than B. bassiana. This assertion is in consonance with that of Pradeep

et.al, 2013 who revealed that mycelia growth and spore production in fungi was more in

Fusarium spp. Khan etal., (2011) revealed that Potato dextrose agar (PDA) was the best medium

for the growth and sporulation of different Fusarium isolates which may be the reason for faster

growth rate in F. oxysporium.

4.2.3 Conidia rates of insect pathogenic isolates of F.oxysporium and B.bassiana on Potato

Dextrose Agar after incubation.

The result from conidia rates of two insect pathogenic isolates (Figure2) shows that

F.oxysporium produced more spores than B.bassiana on Potato Dexrose Agar which was

32
2.17×104 conidia/ml and 2.14×104 conidia/ml respectively. This result may be due to organic

substrates such as carbohydrates and peptones present in PDA media which accelerates the

conidiation rates of F.oxysporium ahead of B.bassiana, this findings in this experiment are

similar to those reported by Silva and Teixeira, (2012) who revealed that carbohydrate-rich

media such as PDA and PSA when incubated at 24 hours induce an increase in the production of

conidia of F. solani, more than any other insect pathogenic fungi. Sharma and Pandey, (2010)

also observed more intense F. oxysporum sporulation in lignocellulose agar medium.

4.2.4 Conidia rates of inscect pathogenic isolates of F.oxysporium and B.bassiana on Solid

Substrate(millet) after incubation

The results from conidia rates of two insect pathogenic isolates (Figure 3) shows that F.

oxysporium produced more spores than B. bassiana on solid substrate (millet) with F.oxysporium

having a higher conidia rate of 4.9×105 conidia/g than B. bassiana which was 1.0×105 conidia/g.

This findings correlates with Reddy et al., (2016) who revealed that B.bassiana has 2.0 × 109

conidia/g on pearl millet solid substrate. Similar assertion has been reported by Lee et al., (2019)

that conidia productivity of six isolates of B. bassiana when evaluated on millet grain medium

produced 1.6 - 2.3×109 conidia/g at 7 days after inoculation, and produced 3.8×109 conidia/g at

14 days after inoculation.

4.2.5 Mortality curves of R. dominica infected by insect pathogenic strain of B. bassiana at

ambient temperature

The result from mortality curves of R. dominica infected by pathogenic strain of B. bassiana

shows that the highest concentration of conidia suspension (8.20×10 5) gave 50% mortality rate of

R. dominica was observed after 7 days post treatment (Figure 4). This result correlates with the

33
findings from Athanassiou and Steenberg (2007), who controlled Sitophilus granarius with

conidia suspensions of B. bassiana in stored wheat and found out that the fungus B. bassiana

caused 52% mortality in S. granarius in 7 days after treatment. Dejaegher (2018), in his findings

revealed 80% mortality rate of Cabbage looper: Trichoplusia ni when controlled with B.

bassiana with conidia concentration of 9.8×107conidia/ml after 9 days. The lowest concentration

of conidia suspension (6.15×104) gave 18% mortality rate of R. dominica when observed after 7

days. Several authors like Akmal et al., (2017) revealed 90% mortality rate of R. dominica after 7

days when controlled with B. bassiana which had conidia concentration of 5.0×108condia/ml.

4.2.6 Percentage mortality of R. dominica infesting P. biglobosa pulp after seven days

treatment with B. bassiana based biopesticide formulation at ambient Temperature.

Result from (Figure 5) shows the percentage mortality of R. dominica infesting P. biglobosa

pulp after seven days treatment with Beauveria bassiana-based biopesticide formulation

Treatment T1with the highest conidia concentration (8.20×105) had the highest percentage

(%)mortality rate of insects after 7days post treatment with B. bassiana having 50% mortality.

Control experiment Treatment T6 (water) had the lowest mortality rate of 5%. This result is in

agreement with Hassan et al., (2019), who confirmed the value of control experiment (water) of

B. bassiana against Squash beetle; Epilachna chrysomelina to be 6.67% mortality rate after 6

days post treatment.

CHAPTER FIVE
34
CONCLUSION AND RECOMMENDATION

CONCLUSION

The result obtained from this study revealed that entomopathogenic fungi has bio-control

efficacy against insect pests in storage if developed into bio-pesticide and it is safe and non-toxic

to humans and the environment compared to chemical method of control which is harmful and

toxic to humans and the environment at large . From this study Beauveria bassiana has proven to

be a potential bio-control agent against Lesser Grain Borer, Rhyzopertha dominica (Coleoptera;

Bostrichidae). The results obtained from this study revealed that the highest concentration of

conidia suspension of B. bassiana gave the highest mortality rate (50%) and the lowest

concentration gave the lowest mortality rate (18%).

Growth rates and conidia rates of F. oxysporium on Potato Dextrose Agar and Solid substrate

(Millet) was higher than that of B. bassiana. F. oxysporium will cause mortality of the insects if

used but also has the potentials of developing into a plant pathogen which will later infect the

plant thereby releasing toxic substances to the environment which may render the plants

unhealthy for consumption. However, B. bassiana was chosen for this experiment because it

serves as an endophyte in plant and does not develop into a plant pathogen.

RECOMMENDATION

The role of entomopathogenic fungi as bio-pesticides in insect management plays a minor role

due to limited scientific information and continuous use of toxic insecticides. The regulatory

bodies should create awareness on the use of bio-pesticides which are environmentally friendly,

safe to humans and animal health. This study suggests an increased need for the search of bio-

pesticides that has complete biocontrol efficacy on lesser grain borer, Rhyzopertha dominica.

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