CHAPTER ONE

INTRODUCTION
1.1 Background Information
Artemisia (Artemisia annua L.) is commonly known as annual wormwood, sweet worm
wood or sweet annie, which is a highly aromatic annual herb of Asiatic and Eastern European
origin widely dispersed throughout the temperate region (Simon, 1990). Artemisia, one of the
largest genera of the Asteraceae family belongs to a useful group of aromatic and medicinal
plants comprising about 300 species which are distributed throughout the world (Bertea, et al.,
2005).
Artemisia annua otherwise known as Qinghao by Chinese has been used for many
centuries in the treatment of fever and malaria (Brown, et al., 2003). The plant species is a
source of both essential oil (1.4-4.0%) and other substances such as Sesquiterpene lactones,
Flavonoids, Polyalkenes and Coumarins (Botsaris, 2007).

1.2 Statement of the Problem

There has been an increase in the re-evaluation of traditional medicinal plants worldwide,
with extensive research on various therapeutic properties being carried out on Artemisia annua .
Report of antibacterial activity of A. annua L has been reported. One challenge of agriculturalist
is losses due to infection by fungal pathogens. There is need to carry out a control trial on fungal
isolates with this plant.

1.3 Justification
Artemisia annua species is an important herb which is grown in Jigawa and Kaduna
states of Nigeria. However, there has been little research on crop losses that can be directly
attributed to fungi; for example, Oecke and Dehne (2004) estimated that worldwide, weeds
caused up to 32% losses, pest 18% and pathogens about 15% for individual crops. Fungal losses
can be 100% if a susceptible cultivar is planted or the climate is favorable in any year and down
to 0% if resistant varieties are planted and fungicides used and good husbandry employed
(Oluyemi et al., 1976). So far the researchers have not come across any control trial on fungi
using this plant. Hence these attempt.

1
1.4 Aim and Objective
The aim of the study is to evaluate the anti-fungal properties of Artemisia annua with the
following Objective:

To carry out a control trail on the effect of Artemisia annua L extract on two (2) species of fungi
;Aspergillus brasiliensis(A. niger) and Penicillium chrysogenum.

2
 CHAPTER TWO
LITERATURE REVIEW

2.1 Scientific Classification of A. annua L.

Kingdom - Plantae
Division - Angiosperms
Unranked - Eudicots
Class - Asterids
Order - Asterales
Family - Asteraceae
Genus - Artemisia
Species - A. annua
Artemisia annua is native to temperate Asia, but naturalized in many countries including
scattered parts of North-America (Flora of China, Flora of Pakistan, Altervista Flora Italiana,
Assenzio annual Artemisia annua L.) Artemisia annua belongs to the plant family of Asteraceae
and is an annual short-day plant. Its stem is erect brownish or violet brown. The plant itself is
hairless and naturally grows from 30-100cm tall, although in cultivation it is possible for plants
to reach a height of 200cm. The leaves of A. annua have a length of 3-5 cm are divided by deep
cuts into two or three small leaflet. The intensive aromatic scent of the leaves is characteristic
(AnonymsArtemisia annua (sweet worm wood)) Royal Botanic Gardens (Retrieved Nov. 25,
2015). The application of salicyclic acid on the leaves shortly before harvesting the plant can
raise its artemisinin content (^Pu,Gao-Bin et al .;2009).
The proposed mechanism of action of Artemisinin involves cleavage of endo-peroxide
bridges by iron, producing free-radicals (hypervalent iron-oxo species, epoxides, aldehydes and
dicarbonyl compound). Which damage biological macromolecules causing oxidative stress in the
cells of the parasite (Cumming JN; PolyPadethP; posner G.H. 1997). Malaria is caused by
apicomplexans, primarily Plasmodium falciparum, which largely reside in red blood cells and
itself contains iron rich heme-groups (in the form of hemozoin) (^Gary, H. Posner and Paul.
Neil, M.O. 2004).
In 2015, artemisinin was shown to bind to a large number targets suggesting that it acts in a
non-selective manner (Wang et al.; 2015).

3
In 1971, scientists demonstrated the plant extracts had anti-malaria activity in primate models
and in 1972, the active ingredient, artemisinin (formerly referred to as arteannuin), was isolated
and its chemical structure described. Artemisinin may be extracted using a low boiling point
solvent, such as diethyl ether, and is found in the glandular trichromes of the leaves, stems and
inflorescences, and it is concentrated in the upper portions of plant within new growth (^Duke
S.O, Paul, R.N. 1993).
The first isolation of Artemisinin from the herb occurred from a military project known
as project 523, following the study of traditional medicine pharmacopoeias performed by Tu
Youyou and other researchers with the project (Phillips, 2015). Apart from the active compound
Artemisinin, recent studies show that A. annua is one of the four medicinal plants with the
highest Oxygen Radical Absorbance Capacity (ORAC) level (^Brisibe et al., 2009). Artemisia
annua possess the capacity to produce high phenolic compound which results in high antioxidant
activity. Five major groups (Coumarins, Flavones, Flavonoids, Phenolic acids and
Miscellaneous). Containing over 50 different phenolic compounds were identified analyzing A.
annua (Ferreira et al., 2010).
Flavonoids are generally known for their redox properties involved in the delay or
inhibition of the initiation or propagation in oxidizing chain reaction. (Ferreira et al., 2010).
Even though the beneficial effects of these phenolic compounds in association of a great number
of disease is often discussed, different studies show beneficial effects of flavonoids compounds
produced by A. annua. It has been stated that there is a negative correlation between the presence
of the mentioned components and cardiovascular disease, cancer and parasitic disease such as
malaria (Ferreira et al., 2010).

2.2 Anti-cancer properties

Several studies show that flavonoids assimilation beverages treatments such as tea might
prevent, delay or help to cure cancer. Recent investigations linked the influence of flavonoids
with different enzymes involved in drug metabolism and in chemical carcinogenesis process.
This induces to a therapeutic potential (^Kale et al., 2008). Many studies show anti-cancer result
analyzing different flavonoids, such as flavones and flavonols. In general it has been shown that
specific flavonoid compounds can inhibit specific cancer cell growth as well as cell proliferation.
Furthermore, these flavonoids induce cell apoptosis (Ferreira et al., 2010).

4
It is proven, that Artemisinin has anti-cancer activity as well, because it contains an endo-
peroxide group. Artemisinin has high anti-cancer activity due to its interaction with iron
complexes (Efferth et al., 2004).
In the blood, this shows that artemisinin derivatives induce apoptosis of cancer cells as
well (Nakase, et al., 2009). The Chinese medicinal plant Artemisia annua is the only source of
the sesquiterpene, Artemisia annua (Qinghaosu) which is used in the treatment of malaria.
Artemisinin is highly oxygenated sesquiterpene, containing a unique 1, 2, 4- trioxane ring
structure, which is responsible for the antimalarial activity of this natural product (Royal Botanic
Garden Kew 2015). The earliest record of the herbs clinical use dates back at least 2,000 years to
the Wushi Er Bring tang (Prescription for fifty two diseases) which was unearthed from the Ma
Wang Diu tomb at Changsha, Hunan in 1973. In the 4th century, the medicinal use of the herb for
fever was prescribed in Chineese Handbookof prescriptions for emergency treatment (Dhingra et
al., 2000). Artemisinin, the most studied derivatives and its semi-synthetic derivatives; arteether,
artemether, and artesunate, have been clinically evaluated and are the only anti-malarial drugs to
which clinical resistance has never been documented (Moore et al., 1995).
Artemisinin was isolated from A. annua in 1972. Its structure was elucidated in 1979
(Mueller et al., 2000). In 2004, the Roll Back Malaria partnership issued a statement in response
to antimalarial drug resistance, recommending that treatment policies for falciparum malaria in
all countries experiencing resistance to monotherapies should be combination therapies,
preferably those containing an Artemisinin derivative (Ashley et al., 2005). A. annua (Sweet
wormwood) is also valued for its essential oil which is used in cosmetics and perfumes; the oil
has been reported to have anti-microbial activity. It serves as insecticide, and expelling parasitic
worm. The oil is also used for digestive disorders and stimulates the imagination (Royal Botanic
Garden Kew 2015).

5
 CHAPTER THREE
MATERIALS AND METHOD
3.1 Study Area
This study was conducted in the laboratory of Plant Science Department of Modibbo
Adama University of Technology (MAUTECH) Yola, Located at latitude 90 14N and 120 27E
(Adebayo 1999), where the isolation, identification and control was carried out.

3.2 Sources of Samples

Artemisia annua was collected from the Institute for Agricultural Research; IAR, ABU-
Zaria. The sample was conveyed to the laboratory in a sterile polythene bag, where 309g of the
sample were dried and pounded.

3.3 Sterilization
Sterilization of the laboratory environment was carried out to avoid contamination using
100% ethanol, UV light switched on for 30 minutes before carrying out inoculations. The
inoculation needle was also sterilized by flaming and deeping into methylated spirit to cool. The
inoculation of the organisms was carried out in the inoculating chamber.

3.4 Preparation of Potato Dextrose Agar (PDA)

Twenty (20g) of Potato Dextrose Agar (PDA) was dissolved in 500mls of distilled water
and was autoclaved at 121oc pressure for 15 minutes. One capsule of chloramphenicol was added
to the sterilized media, just before pouring into petri-dishes, to prevent bacterial growth
(Sulaiman and Michael, 2013).

3.4.1 Pouring of media

The prepared media was poured in an inoculating chamber; 20ml of the PDA was poured
in 9cm sterile petri-dishes, Each plates was poisoned with 2mls of plant extracts and allowed to
cool and solidify (Smith and Onion, 1994) for the isolation.

3.5 Preparation of Plant Extract

Leaf, stem, and root of A .annua was dried under shade to maintain its composition and
powdered with mortar and pistil. The powdered sample was extracted with ethanol and distilled
water.

6
3.5.1 Aqueous extract
The dried leaf, stem and root parts of A. annua in powdered form was Dissolved in sterile
water for 14 days and was filtered with filter paper.

3.5.2 Ethanol extract

The above listed parts were dissolved in Ethanol for14days and were filtered

3.5.3 Control Trial In vitro

In vitro control was carried out using food poisoning method as suggested by Suleiman
and Michael, (2013). The Potatoes Dextrose Agar (PDA) was amended with extracts of
Artemisia annua parts before inoculation. Inoculating the pathogen, singular method was made
to serve as control all in replicate of three and incubated at room temperature for ten (10) days.

3.6 Experimental Design and Statistical Analysis

Completely Randomized Design of three (3) treatments which include leaf, stem and
root of A.annua in two (2) solvents (Ethanol and Distilled water). There were three replications
alongside control. All the data was analyzed using Analysis of Variance (ANOVA) according to
Gomez and Gomez (1984). Significant means was separated using least square differences
(LSD) at P = 0.05 with statistical software SAS according to (Ogbulu., 2009).

7
 CHAPTER FOUR
RESULTS

4.1 Influence of the other part of A. annua extracts on the growth of Aspergillus brasiliensis.
The extract of stem (ethanol) inhibits the growth of A. brasiliensis by 30%, extracts of
leaf (ethanol) inhibits the growth of A. brasiliensis by 40%, extracts of root (ethanol) inhibits the
growth of A. brasiliensis by 20%, extract of stem (aqueous) inhibits the growth of A. brasiliensis
by 85% and extract of root (aqueous) inhibits growth of A. brasiliensis by 15 %.
From the above percentage of growth inhibitory activity listed, it is evident that stem
(aqueous extracts), has the highest inhibitory activity and root (aqueous) has the lowest
inhibitory activity against Aspergillus brasiliensis.

8
Table 1: Mean table for Aspergillus brasiliensis ethanol (extract)
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf ethanol 0.00 0.00 49.33 59.67

Root ethanol 0.00 0.00 62.00 81.33

Stem ethanol 0.00 0.00 51.67 71.00

Control 14.67 31.00 68.33 90.00

LSD(P=0.05) 0.58 1.73 4.42 4.67

9
Table 2: Mean table for Aspergillus brasiliensis aqueous (extract)
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf aqueous 4.67 9.67 41.67 51.67
Root aqueous 5.00 9.67 60.33 83.00
Stem aqueous 4.33 9.67 9.67 13.67
Control 15.00 30.00 69.00 90.00
LSD (P = 0.05) 0.94 0.58 3.25 4.80

10
4.2 Influence of the other part of A. annua extracts on the growth of Penicillium
chrysogenum.
The extracts of stem (ethanol) inhibits the growth of Penicillium chrysogenum by 100%, extracts
of leaf (ethanol) inhibits the growth of Penicillium chrysogenum by 40%, extracts of root
(ethanol) inhibits the growth of Penicillium chrysogenum by70%, extracts of stem (aqueous)
inhibits the growth Penicillium chrysogenum by 85%, extracts of leaf (aqueous) inhibits the
growth of Penicillium chrysogenum by 85%, and extract of root (aqueous) inhibit.
The result showed that stem (ethanol extracts) and root (aqueous extracts) had the highest
inhibitory activity on the growth of Penicillium chrysogenum by 100%, and leaf (ethanol extract)
had the lowest inhibitory activity on the growth of Penicillium chrysogenum by 40%.

11
Table 3: Mean table for Penicillium chrysogenum ethanol (extract)
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf ethanol 0.00 0.00 41.67 61.67
Root ethanol 0.00 0.00 18.33 30.67
Stem ethanol 0.00 0.00 0.00 0.00
Control 15.00 31.00 69.00 90.00
LSD (P = 0.05) 0.00 0.10 4.81 1.67

12
Table 4: Mean for Penicillium chrysogenum aqueous (extract)
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf aqueous 3.00 9.33 9.00 14.67
Root aqueous 0.00 0.00 0.00 0.00
Stem aqueous 3.00 9.00 9.33 14.00
Control 15.00 31.00 69.00 90.00
LSD (P = 0.05) 1.30 1.73 1.29 1.15

13
Table 5: Mean Table for Aspergillus brasiliensis combined
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf ethanol 0.00 0.00 49.33 59.67
Stem ethanol 0.00 0.00 51.67 71.00
Root ethanol 0.00 0.00 62.00 81.33
Leaf aqueous 4.67 9.67 41.67 51.67
Stem aqueous 4.33 9.67 9.67 13.67
Root aqueous 5.00 9.67 60.33 83.00
Control 15.00 30.00 69.00 90.00
LSD (P = 0.05) 0.60 0.55 3.44 4.43

14
Table 6: Mean Table for Penicillium chrysogenum combined
Mean
Plant material Day 1 Day 3 Day 5 Day 10
Leaf ethanol 0.00 0.00 41.67 61.67
Stem ethanol 0.00 0.00 0.00 0.00
Root ethanol 0.00 0.00 18.33 30.67
Leaf aqueous 3.00 9.33 9.00 14.67
Stem aqueous 3.00 9.00 9.33 14.00
Root aqueous 0.00 0.00 0.00 0.00
Control 15.00 31.00 69.00 90.00
LSD (P = 0.05) 0.91 1.10 3.11 1.55

15
 CHAPTER FIVE
DISCUSSIONS, CONCLUSION AND RECOMMENDATION
5.1 Discussions
The result showed that extracts of Artemisia annua; stem (aqueous) inhibit the growth
of Aspergillus brasiliensis by 85%, root (aqueous) by 20%, leaf (aqueous) by 50%, and stem
(ethanol) inhibit the growth of Aspergillus brasiliensis by 30%, root (ethanol) by 20%, leaf
(ethanol) by 40%.
The extracts of Artemisia annua; stem (aqueous) inhibit the growth of Penicillium
chrysogenum by 85%, root (aqueous) by 100%, leaf (aqueous) by 85%, and stem (ethanol) by
100%, root (ethanol) by 70%, leaf (ethanol) by 40%.
It is evident that among all the extracts, stem (ethanol) and root (aqueous) had the highest
controlling and inhibiting properties on the growth of fungal pathogen 100%. Whereas others,
suppressed the growths of pathogens at different percent or level.
Also, the result showed that the extracts of Artemisia annua had significantly greater
inhibitory activity against Penicillium chrysogenum compared to Aspergillus brassiliensis.
Control of Penicillium chrysogenum at 100% using extracts of A. annua stem (ethanol)
and root (aqueous) showed that the two extract had a highly significant impact on the growth of
fungus, leading to complete inhibition of growth.

5.2 Conclusion
The plants parts extracts that better controlled the growth considering Penicillium
chrysogenum were stem (ethanol) and root (aqueous)
For the leaf (ethanol), root (ethanol), stem (aqueous) and leaf (aqueous) extracts of A.
annua although it has not had satisfactory or complete inhibitory result, but were able to
suppressed the growths of pathogens.
The result of the different parts of Artemisia annua extracts, i.e stem (ethanol) and root
(aqueous) demonstrated strong antifungal potential overall.

5.3 Recommendations
Considering the scarcity and effectiveness of these medicinal plants in controlling the
growth of fungal pathogens, researchers needed to grow this plant in abundance and further
studies to explore the potentials of these plants.

16
 Based on the results of this research, the stem (ethanol) and root (aqueous) can be useful
to farmers for complete control of disease of plants associated with Penicillium chrysogenum.
And other extracts of Artemisia annua can effectively suppress disease of plant associated with
Aspergillus brasiliensis.
Effective control of plant diseases associated with Aspergillus brasiliensis and Penicillium
chrysogenum can be achieved by spraying with stem, leaf and root extracts of ethanol to diseased
plants at three (3) days intervals, as the growth of fungal isolates were fully controlled by the
extracts at three (3) days of incubation.

17
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Bertea, C.M., Freije,J.R., Woude,H.V.D., Verstappem, F.W.A., Perk, L., Marquez, V., De, J.W.
Oecke and Dehne (2004).

Botsaris, A.S. (2007 of flora ). Plants used traditionally to treat malaria in Brazil: the achieves
medicinal. Journal of Ethnomedicine. 3(1):3-18.

Brown, G. D., Liang G. Y., Syl (2003). Terpenoids from the seeds of Artemisia
annua.Phytochems 64:303-323.

Cumming, J.N.; PloyPradeth P., Posner G.H. (1997)Antimalarial activity of artemisinin

(Qinghaosu) related trioxanes;mechanism and (s) of action. Adv.Pharmacol. Advances
inPharmacology.37:253-97.doi:10.1016/S1054-3589(08)60952-7.
ISBN.9780120329380.PMID8891104.

Dhingra, V., Vishweshwar, R.K., Lakshmi, N.M., Current status of artemisinin and its
derivatives as antimalarial drugs life sci 2000;66:279-300. Flora of China, Flora of
Pakistan,Altervista Flora Italiana, annual Artemisia annua L.

Gary, H. Posner and Paul M. Neil, O. (2004).Knowledge of the proposed chemical mechanism
of action and cytochrome (P450) Metabolism of Antimalarial Trioxanes like Artemisinin
Allows Rational Design of New Antimalarialperoxides.Acc.chem.Res.37(6):397-
404.doi:10.1021/ar020227u.PMID15196049.

Kale, Anup; Gawande, Sonia; Kotwal, Swati (2008-05-01). Cancer phytotherapeutiecs: role
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Nakase,Ikuhiko; Gallis, Byron; Takatani-Nakase, Tomoka; Oh, Steve; Lacoste, Eric; Singh,
Narendra, P., Goodlett, David, R., Tanaka, Seigo; Futaki, Shiroh (2009).
Transferrin receptor-dependent cytotoxicity of artemisinin-transferrin conjugates on
prostate cancer cells and induction of apoptosis. Cancer Letters.274(2):290-298.
doi:10.1016/j.canlet.2008.09.o23. PMID 19006645.

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Pu,Gua-Bin; et al., 2009. Salicyclic acid activates artemisinin biosynthesis in Artemisia
annua. (PDF) Plantcellreports. 28(7):1127-1135.doi:10.1007/s00299-009-0713-3.PMID
19521701.

Simon, J.E., Charles, D., Cebert, E., Grant, L., Janick, J., and Whipkey,A., (1990). A.annua L: A
promising aromatic and medicinal plant. P.522-526. In:Janick , J.,and Simon J.E.(eds),
Advances in new crops. Timber Press, Portland, OR.

Tom,Phillips.(October6,2015).
Tu Youyou: how Maos challenge to the malaria pioneer led to Nobel prize
Guuardian.

Wang, J.,Zhang, C.J., Chia W.N., Loh, C.C., Li, Z., Lee,, He, Y., Yuan, L.X., Lim, T.K., Liu,
M., Liew, C.X.,Lee, Y.Q., Zhang, J., Lu, N., Lim, C.T., Hua, Z.C., Liu, B., Shen, H. M.,
Tan, K.S., Lin, Q. (2015). Heme-activated promiscuous targeting of artemisnin in
Plasmodiumfalciparum.Naturecommunications.6:10111.doi:10.1038/ncomms10111.P
MC 4703832.PMID26694030.

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 APPENDICES

Appendix i. Effect of Artemisia annua on Aspergillus brasiliensis growth

Days Stem Leaf Root Control Stem Leaf Root Control
Ethanol Ethanol Ethanol (mm) Aqueous Aqueous Aqueous
(mm) (mm) (mm) (mm) (mm)
1 0 0 0 15 5 4 5 15
1 0 0 0 14 4 5 5 15
1 0 0 0 15 4 5 5 15
3 0 0 0 30 10 10 10 30
3 0 0 0 33 9 9 9 30
3 0 0 0 30 10 10 10 30
5 50 47 60 69 10 40 60 69
5 55 50 61 68 9 45 60 70
5 50 51 65 68 10 40 61 68
10 70 57 80 90 15 50 84 90
10 75 60 81 90 12 55 80 90
10 68 62 83 90 14 50 85 90

20
Appendix ii. Effect of Artemissia annua on Pennicillium chrysogenum growth
Days Stem Leaf Root Control Stem Leaf Root Control
Ethanol Ethanol Ethanol (mm) Aqueous Aqueous Aqueous (mm)
(mm) (mm) (mm) (mm) (mm) (mm)
1 0 0 0 15 3 4 0 15
1 0 0 0 15 4 3 0 15
1 0 0 0 15 2 2 0 15
3 0 0 0 30 10 9 0 15
3 0 0 0 31 9 10 0 15
3 0 0 0 32 8 9 0 15
5 0 40 20 70 10 9 0 64
5 0 45 15 69 9 10 0 68
5 0 40 20 68 9 8 0 70
10 0 60 30 90 15 15 0 90
10 0 63 32 90 14 14 0 90
10 0 62 30 90 13 15 0 90

21
Appendix iii. ANOVA Table for Aspergillus brasiliensis ethanol
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 5 96.83** 432.75** 144.87** 311.23**

Plant 2 161.3** 720.75** 237.89** 515.22**

Rep 2 0.08ns 0.75ns 5.33ns 5.25ns

Error 6 0.08 0.75 4.89 5.47

22
Appendix iv. ANOVA Table for Aspergillus brasilensis aqueous
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 5 48.18** 186.35** 1243.17** 2186.65**

Plant 2 80.31** 310.08** 2070.56** 3644.53**

Rep 2 0.000ns 0.75ns 2.08ns 0.33ns

Error 6 0.22 0.08 2.64 5.78

23
Appendix v. ANOVA Table for Penicillium chysogenum ethanol
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 5 101.25** 432.55** 1603.88** 2719.75**

Plant 2 168.75** 720.75** 2672.97** 4531.86**

Rep 2 0.000ns 0.25ns 0.25ns 1.58ns

Error 6 0.00 0.25 5.80 0.69

24
Appendix vi. ANOVA Table for Penicillium chrysogenum aqueous
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 5 79.95** 312.43** 1813.83** 2994.53**

Plant 2 132.75** 520.67** 3022.33** 4990.67**

Rep 2 0.75ns 0.08ns 1.08ns 0.33ns

Error 6 0.42 0.75 0.42 0.33

25
Appendix vii. ANOVA Table for Aspergillus brasiliensis combined
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 7 63.90** 256.25** 866.36** 1534.26**

Plant 5 85.21** 341.52** 1152.75** 2045.00**

Rep 2 0.00ns 0.43* 7.19ns 2.05ns

Error 11 0.11 0.09 3.75 6.21

26
Appendix viii . ANOVA Table for Penicillium chrysogenum combined
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 7 67.61** 293.05** 1062.77** 2585.43**

Plant 5 540.00** 390.71** 1950.16** 3447.10**

Rep 2 0.86ns 0.05ns 0.61ns 0.43ns

Error 11 0.26 0.38 3.06 0.76

Highly Significant (**)

Significant (*)
Non-Significant (ns)

27