KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY

COLLEGE OF AGRICULTURE AND NATURAL RESOURCES FACULTY OF AGRICULTURE DEPARTMENT OF HORTICULTURE

Investigation into fungal seedborne pathogens of farmer-saved seed maize (Zea mays L.) collected from three ecological zones of Ghana and efficacy of plant extracts in controlling the pathogens

Joseph Adjei February, 2011

Investigation into fungal seedborne pathogens of farmer-saved seed maize (Zea mays L.) collected from three ecological zones of Ghana and efficacy of plant extracts in controlling the pathogens

A THESIS SUBMITTED TO THE SCHOOL OF POST GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE DEGREE IN SEED SCIENCE AND TECHNOLOGY AT THE DEPARTMENT OF HORTICULTURE OF THE FACULTY OF AGRICULTURE, COLLEGE OF AGRICULTURE AND NATURAL RESOURCES, KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY, KUMASI, GHANA

JOSEPH ADJEI

FEBRUARY, 2011 CERTIFICATION

I hereby declare that this submission is my own work towards the MSc (Seed

Science and Technology) degree and that, to the best of my knowledge, it contains no material previously published by another person nor material which has been accepted for the award of any other degree of the University, except where due acknowledgment has been made in the text.

Adjei Joseph ...... Student Signature Date

Certified by:

Prof. (Mrs) N. S. Olympio ...... Supervisor Signature Date

Dr. E. Moses ...... Co-supervisor Signature Date

Dr. B. K. Maalekuu ...... Head of Department Signature Date

ii

ABSTRACT

Seedborne fungal pathogenic infections of maize (Zea mays L.) were studied using fifty-four (54) seed samples, collected from locations in the Forest, Transitional and

Guinea Savannah ecological zones in Ghana. The deep-freeze blotter method was used to detect fungal pathogens on the seed maize. Four pathogens identified were

Acremonium strictum (infection ranging from 0.5 to 22%), Bipolaris maydis (0.5 to

1.5%), Botryodiplodia theobromae (1 to 19%) and Fusarium moniliforme (5 to

84.5%). Aqueous extracts of Cymbopogon citratus (lemongrass), Chromolaena odorata (siam weed) leaf and Azadirachta indica (neem) seeds were tested for their inhibitory activity against seedborne fungal pathogens in the infected seed maize, using 50% (w/v) concentrations for 1, 3, 6 and 24 hours each. Neem seed extract exhibited the best control, reducing infection of F. moniliforme from 68 to 18% with the 24 hours of treatment and eliminating Acremonium strictum at 1 and 24 hours of treatment. Botryodiplodia theobromae infection was eliminated at all the soaking periods. Neem had no effect on germination but reduced vigour slightly.

Chromolaena odorata leaf extract also exhibited control on F. moniliforme reducing infection from 68 to 36.5% at 24 hours of treatment but had no significant effect on germination and vigour of seeds. Lemongrass extract did not have significant control effect on F. moniliforme but increased germination and vigour of seeds. There is therefore a potential of using neem seed extract as seed maize disinfectant against F. moniliforme, Acremonium strictum and Botryodiplodia theobromae.

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ACKNOWLEDGEMENT

I wish to express my sincere appreciation to my supervisor, Dr. (Mrs) N. S. Olympio, of the Department of Horticulture, Faculty of Agriculture, Kwame Nkrumah

University of Science and Technology, whose advice, suggestions, patience and objective criticism guided me throughout the period of the project work. I am very grateful for her invaluable assistance especially her motherly advice and care that propelled me through the work successfully.

In a similar manner I extend my heartfelt gratitude to my co-supervisor, Dr. E.

Moses, of Crop Research Institute (CRI), Fumesua, Kumasi for his advice, suggestions and guidance during the laboratory work and writing of the thesis.

I must thank the Head, Dr. B. K. Maalekuu and all the lecturers of the Department of

Horticulture whose constructive criticism during seminar presentations finally straightened up the research.

I owe a word of special thanks to my course-mate, Mr. David Sackey, for his immense assistance in providing blotter papers and other materials without which the work would not have been possible. I also thank Dr. Robert Asuboah of Grains and

Legumes Development Board, Kumasi, for making their seed laboratory accessible to me to carry out the germination and vigour tests.

I express my sincere appreciation to the staff of Seed Pathology laboratory, CRI, Fumesua – Mr. Samuel Nyarko, Mrs Zippora Appiah-Kubi, Ms Rose Barfi, Madam Comfort Nkrumah, Mrs Esther Dwirah and the National Service Personnels for their assistance.

Above all, I thank Almighty God who gave me strength and protection to carry out the research.

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TABLE OF CONTENTS

CERTIFICATION ...... ii

ABSTRACT ...... iii

ACKNOWLEDGEMENT ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

LIST OF APPENDICES ...... x

LIST OF ACRONYMS ...... xi

1.0 INTRODUCTION ...... 1

2.0 LITERATURE REVIEW...... 4

2.1 Origin and Botany of Maize...... 4

2.2 Importance of Maize ...... 5

2.3 Maize Production Trends ...... 6

2.4 Maize production constraints ...... 7

2.5 Diseases of Maize ...... 7

2.5.1 Major Fungal Diseases of Maize ...... 8

2.5.2 Downy mildews ...... 8

2.5.3 Black Bundle Disease and Late Wilt ...... 9

2.5.4 Anthracnose Stalk Rot ...... 9

2.5.5 Southern leaf blight ...... 10

2.5.6 Charcoal Stalk Rot ...... 10

2.5.7 Horse’s tooth ...... 11

2.5.8 Aspergillus Ear Rots ...... 11

2.5.9 Gray Ear Rot ...... 11

2.5.10 Common Smut ...... 12

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2.5.11 Botryodiplodia or Black Kernel Rot ...... 12

2.5.12 Fusarium Stalk Rot ...... 12

2.5.13 Fusarium Ear Rot ...... 13

2.6 Effect of Seedborne Fungal Diseases on Man and Animal ...... 14

2.7 Control of Seedborne Diseases ...... 15

2.7.1 Types of Seed Treatment ...... 15

2.7.2 Physical ...... 15

2.7.3 Biological Control Agents ...... 17

2.7.4 Chemical method (Use of Fungicides) ...... 18

2.7.5 Effects of Chemical Seed Treatments ...... 19

2.7.6 Plant or Botanical Extracts with Fungicidal Properties ...... 20

2.7.7 Effect of Neem on Fungi ...... 21

2.7.8 Effect of Lemongrass (Cymbopogon citratus) on Fungi ...... 22

2.7.9 Chromolaena odorata ...... 22

2.7.10 Mancozeb (Chemical fungicide) ...... 23

3.0 MATERIALS AND METHODS ...... 24

3.1 Seed Samples ...... 24

3.2 Seed Health Analysis ...... 25

3.2.1 Plating of Seeds...... 25

3.2.2 Freezing and Incubation of Seeds ...... 25

3.2.3 Examination of Incubated Seeds and Recording of Infections ...... 26

3.2.4 Isolation of Fungal Pathogen ...... 26

3.3 Treatment of Seeds ...... 26

3.3.1 Preparation of Botanical Extracts ...... 27

3.3.2 Seed Treatment and Health Analysis of Treated Seeds ...... 28

3.3.3 Effect of the Botanical Extracts on Germination and Vigour ...... 29

3.4 Statistical Analysis of Data ...... 29

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4.0 RESULTS ...... 30

4.1 Infection by Fungal Pathogens in Seed Maize from the Forest, Transition and Guinea Savannah Zones of Ghana ...... 30

4.1.1 Seed Health before Seed Treatment ...... 30

4.2 Effects of Plant Extracts on Seedborne Fungal Pathogens of Maize ...... 35

4.2.1 Effects of Neem Seed Extract on Seedborne Pathogens ...... 35

4.2.2 Effects of Lemongrass Extract on Seedborne Pathogens ...... 36

4.2.3 Effects of Chromolaena odorata Leaf Extract on Seedborne Pathogens ..37

4.3 Effects of Plant Extracts on Germination of Seed Maize ...... 38

4.3.1 Effects of Neem Seed Extract on Germination of Seed Maize ...... 38

4.3.2 Effects of Lemongrass Extract on Germination of Seed Maize ...... 39

4.3.3 Effects of Chromolaena odorata Leaf Extract on Germination of Seed Maize ...... 39

4.4 Effect of Plant Extracts on Vigour of Seed Maize ...... 40

4.4.1 Effects of Neem Seed Extract on Vigour of Seed Maize ...... 40

4.4.2 Effects of Lemongrass Extract on Vigour of Seed Maize ...... 40

4.4.3 Effects of Chromolaena odorata Leaf Extract on Vigour of Seed Maize .41

5.0 DISCUSSION...... 42

5.1 Infection by Fungal Pathogens in Seed Maize from the Forest, Transition and Guinea Savannah Zones of Ghana ...... 42

5.2 Effects of Botanical Extracts on Seedborne Fungi Pathogens, Germination and Vigour of Seed Maize...... 45

6.0 CONCLUSION AND RECOMMENDATIONS ...... 48

6.1 Conclusion ...... 48

6.2 Recommendations ...... 49

REFERENCES ...... 50

APPENDICES ...... 61

vii

LIST OF TABLES

Table 4.1: Frequency of Infection of Seed Maize by Fungal Pathogens (Forest

Zone) ...... 32

Table 4.2: Frequency of Infection of Seed Maize by Fungal Pathogens (Transition

Zone) ...... 33

Table 4.3: Frequency of Infection of Seed Maize by Fungal Pathogens (Guinea

Savannah Zone) ...... 34

Table 4.4: Mean Frequency of Infection by Fungal Pathogens ...... 35

Table 4.5: Effect of Treatments on the incidence of Acremonium strictum (%) .....37

Table 4.6: Effect of Treatments on the incidence of Botryodiplodia

theobromae (%) ...... 38

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LIST OF FIGURES

Figure 4.1: Effects of Treatments on the incidence of Fusarium moniliforme (Fm) .36

Figure 4.2: Effects of Treatments on Germination of Seed Maize ...... 40

Figure 4.3: Effects of Treatments on Vigour of Seed Maize ...... 41

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LIST OF APPENDICES

Appendix 1: ANOVA for Frequency of Infection of Seed Maize by Fungal Pathogens (Forest Zone) ...... 62

Appendix 2: ANOVA for Frequency of Infection of Seed Maize by Fungal Pathogens (Transition Zone) ...... 62

Appendix 3: ANOVA for Frequency of Infection of Seed Maize by Fungal Pathogens (Guinea Savannah Zone) ...... 63

Appendix 4: ANOVA for Effects of Treatments on the incidence of Fusarium moniliforme, Acremonium strictum and Botryodiplodia theobromae ...... 63

Appendix 5: ANOVA for Effects of Treatments on Germination and Vigour of Seed Maize ...... 64

x

LIST OF ACRONYMS

APG Angiosperm Phylogeny Group

CABI Centre for Agriculture and Biosciences International

CIMMYT International Maize and Wheat Improvement Centre (Centro

Internacional de Mejoramiento de Maízy Trigo)

HGCA Home Grown Cereals Authority

IARC International Agency for Research on Cancer

ISAAA International Service for the Acquisition of Agri-biotech Applications

ISTA International Seed Testing Association

USDA United States Department of Agriculture

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1.0 INTRODUCTION

Maize is an important cereal in many developed and developing countries of the world. It is widely used for animal feed and industrial raw material such as starches, acids and alcohols in the developed countries whereas the developing countries use it in general for food. Recently, there has been interest in using maize for the production of ethanol in the United States and China, a substitute for petroleum based fuels (Corn India, 2010). In Ghana maize is the most popular food crop in the domestic market. It fills in for other food crops like sorghum, millet, plantain, yam and cocoyam when they are in short supply. It is the main feedstuff for poultry and other livestock (Asante, 2004).

Maize is widely cultivated throughout the world, and a greater weight of it is produced each year than any other grain. Worldwide production was around 800 million tonnes in 2007, just slightly more than rice (~650 million tonnes) or wheat

(~600 million tonnes). In 2007, over 150 million hectares of maize were planted worldwide, with a yield of 4970.9 kilogram/hectare (ISAAA, 2010). In Ghanaian agriculture, maize with annual production of around 1.6 million tonnes in 2009

(Ghanaweb, 2010), occupies a prominent position in staple food supply and each part of the maize plant is put to one or the other use and very little goes as waste.

Despite its importance, maize has many production constraints which prevent farmers from getting maximum yield. Drought, fire, flood, poor soil fertility, high labour cost, transportation problems and poor agronomic practices are some of the production constraints of maize (Ekasingh et al., 2004). Pests and diseases are also some of the problems that characterise maize production in the world. According to

Neergaard (1988), Drechslera turcica, northern leaf blight, caused considerable

1 losses in 1970 in maize amounting to approximately one billion dollars in maize- growing areas of United States. In Africa Diplodia spp. and Fusarium spp. caused losses of more than 10 per cent in Lesotho and Rhodesia, and less than 10 per cent in

Tanzania (Neergaard, 1988). In developing countries with poor farming practices seedborne pathogens are among the major constraints. Two-thirds of total losses of maize due to diseases are caused by seedborne diseases, and of these Diplodia and

Fusarium graminearum account for about 25 per cent and Drechslera spp. for almost

20 per cent of the total loss due to diseases (Neergaard 1988).

To set up a healthy field that will give a good yield, healthy seeds are needed because seedborne pathogens bring about poor germination, poor vigour, poor crop establishment and crop stands, non-healthy plants, lodging and poor yield (Wiese,

1984). Moreover, farmer-saved seed maize planted by most farmers are infected with seedborne fungal pathogens (Tagne et al., 2008). Efforts have been employed to reduce crop losses due to diseases in different parts of the world to ensure higher yield. Efforts made include breeding for disease resistant varieties, the use of chemicals in reducing seedborne pathogens and the use of biological control agents to reduce plant pathogens. Chemical methods involving seed treatment with fungicides have been employed to improve germination, vigour, crop establishment, crop stands, and yield. However, indiscriminate use of chemicals for controlling plant diseases has resulted in environmental pollution and health hazards (Chapman and Harris, 1981). Moreover, haphazard use of chemicals breaks down the natural ecological balance by killing beneficial soil microbes (Neergaard, 1988). In some cases farmers in developing countries cannot afford high cost of chemical pesticides.

As a result of these problems there is an increasing attention towards the development of safer methods of disease control. This has resulted in the

2 development of botanical fungicides for the control of seedborne pathogens of food crops. Botanical fungicides that are effective have little or no adverse effect on the environment.

In this work, therefore, an attempt was made to develop botanical products that can reduce incidence of seedborne fungal pathogens of seed maize of farmers in Ghana.

Certain botanicals with reported fungicidal properties were investigated to determine their efficacy in reducing seedborne fungal pathogens of farmer-saved seed maize.

The specific objectives of the research were:

 To determine incidence of seedborne fungal pathogens in seed maize

collected from the Forest, Transition and Guinea Savannah zones of Ghana.

 To determine the efficacy of three plant extracts for reduction or control of

seedborne fungal pathogens of seed maize.

 To determine the effect of plant extracts on germination and vigour of seed

maize.

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2.0 LITERATURE REVIEW

2.1 Origin and Botany of Maize

Maize (Zea mays L.), also known in some countries as corn, is a cereal grain domesticated in Mesoamerica and subsequently spread throughout the American continents (Sauer, 1993). Maize was domesticated in Mexico about 7000 years ago and by the time Columbus arrived in the New World in 1492 AD, there were already many varieties. It was introduced to Africa in the 16th century and over time came to replace sorghum as the staple food in all but the drier areas (Biodiversity, 2010).

Maize is a direct domesticate of the teosinte, Zea mays sp. parviglumis, native to the

Balsas River Valley of southern Mexico (Eurekalert, 2009), with up to 12% of its genetic material obtained from Zea mays sp. mexicana through introgression. Zea is a genus of the family Poaceae (Graminae), commonly known as the grass family.

Maize (Zea mays L.) is a tall, monoecious annual grass with overlapping sheaths and broad conspicuously distichous blades. Maize plants have staminate spikelets in long spike-like racemes that form large spreading terminal panicles (tassels) and pistillate inflorescences in the leaf axils, in which the spikelets occur in 8 to 16 rows, approximately 30cm long, on a thickened, almost woody axis (cob). The whole structure (ear) is enclosed in numerous large foliaceous bracts and a mass of long styles (silks) protrude from the tip as a mass of silky threads (Hitchcock and Chase,

1971). The grains are about the size of a pea, and adhere in regular rows round a white pithy substance, which forms the ear. An ear of maize contains from two to four hundred grains, and is from six to ten inches in length. The grains are of various colours, violet, red, white and yellow. Maize makes excellent flour, of which it yields much more, with much less bran, than wheat does (Bambooweb, 2009).

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2.2 Importance of Maize

According to the International Institute for Tropical Agriculture (IITA) maize serves as staple food for more than 300 million of Africa’s most vulnerable inhabitants, and the most important staple food on the continent (Essiet, 2010). Maize as cornmeal

(maize flour) constitutes a staple food in many regions of the world. Maize can be harvested and consumed in the unripe state, when the kernels are fully grown but still soft. The cooked unripe kernels may also be shaved off the cob and served as a vegetable in side dishes, salads, garnishes, etc (Wikipedia, 2009). The primary uses for maize in North America are the production of corn sweeteners like corn syrup, as a feed for livestock, and the production of ethanol. Ethanol, a type of alcohol, is mostly used as an additive in gasoline to increase the octane rating. Maize cobs are used as a biomass fuel source. Maize is relatively cheap and a home heating furnace has been developed which uses maize kernels as a fuel (Bambooweb, 2009). Starch from maize can also be used in the manufacturing of plastics, fabrics, adhesives, and many other chemical products. The corn steep liquor, a plentiful watery by-product of maize wet milling process, is widely used in the biochemical industry and research as a culture medium to grow many kinds of microorganisms (Wikipedia, 2010a).

Maize is the most widely consumed staple food in Ghana. A nationwide survey carried out in 1990 revealed that 94% of all households had consumed maize during an arbitrarily selected two-week period (Alderman and Higgins, 1992). An analysis based on 1987 data showed that maize and maize-based foods accounted for 10.8% of household food expenditures by the poor, and 10.3% of food expenditures by all income groups (Boateng et al., 1990). Maize in Ghana is consumed in a variety of forms. In the north, it is commonly eaten as a thick gruel, similar to the way that sorghum and millet are consumed. In the south, it is frequently used to prepare

5 porridges and more solid dishes made from fermented or unfermented dough such as kenkey, banku, akple, tuozaafi, and aprapransa (Morris et al., 1999). It also serves as the main feed for livestock and poultry.

2.3 Maize Production Trends

Most of the maize grown in Ghana is cultivated in association with other crops, particularly in the coastal savannah and forest zones, so planting densities are generally low (Morris et al., 1999). Average grain yields of maize are correspondingly modest when expressed per unit land area, averaging less than 2 t/ha. According to official statistics, the area annually planted to maize in Ghana currently averages about 675,000 ha with an estimated total output of 1.6 million tonnes in 2009 (Ghanaweb, 2010). Both of the two key determinants of production

(area planted and yield) have increased over the longer term, although the upward trends have been characterized by high year-to-year variability typical of rainfed crops. Following a pattern that has been observed throughout West Africa, the transition zone has become increasingly important for maize production (Smith et al.

1994). The rising importance of the transition zone as a source of maize supply can be attributed to a combination of factors, including the presence of favourable agro- ecological conditions (such as annual rainfall, type of soil, soil fertility, and temperature), availability of improved production technology, a relative abundance of underutilized land, and a well-developed road transport system (Morris et al.,

1999). The relative abundance of arable land in the transition zone has attracted many migrant farmers, particularly from the north of the country, who have moved to the zone to pursue commercial food farming. Globally, the latest estimates from the

USDA put world maize production at a new record of 803.7 million tonnes (HGCA,

2010).

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2.4 Maize production constraints

Maize production has many constraints which prevent farmers from getting maximum yield. Drought, fire, flood, poor soil fertility, high cost of labour, input/output transportation problems and poor agronomical practices are some of the production constraints of maize (Ekasingh et al., 2004). Chronic difficulties in agriculture, brought on by economic problems and accentuated by unfavourable climatic conditions, have accumulated over several years, and continue to seriously undermine domestic food production in Ghana (CABI, 2007). Specifically maize production is facing extensive problems due to damage inflicted by agricultural pests including the Larger Grain Borer (Prostephanus truncatus), which causes yield losses in the range of 10 to 30%, depending on the year and location; although extremely high damage levels of up to 35% have been known to occur in maize storage in East Africa (Infonet-biovision, 2010).

2.5 Diseases of Maize

The average yield of maize in Ghana is lower in comparison with that of the world though its production area is increasing day by day. There are many causes of low maize yield of which diseases play a significant role. Moreover, seedborne diseases cause enormous losses both in storage as well as in the field. A total of 112 diseases are known to occur in maize crops (USDA, 1996) and among them, more than 70 are seedborne. Important seedborne diseases of maize are leaf spot, leaf blight, Collar rot, kernel rot, scutellum rot, seedling blight, anthracnose and head smut

(Richardson, 1990).

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2.5.1 Major Fungal Diseases of Maize

Seedborne diseases caused by fungi in maize include Fusarium and Gibberella stalk rots, head smut, false head smut, black bundle disease and late wilt (McGee, 1988).

Others include Anthracnose stalk rot, charcoal stalk rot, Penicillium ear rots,

Aspergillus ear rots, Fusarium and Gibberella ear rots, charcoal ear rot, Nigrospora ear rot, gray ear rot, common smut, Botryodiplodia or black kernel rot, Downy mildews, Horse's tooth, Acremonium zeae stalk rot, and Alternaria leaf blight

(CIMMYT, 2004). However, Anthracnose rots, Black bundle, Black kernel rot,

Brown stripe downy mildew, Crazy top downy mildew, Fusarium stalk, ear and root rot, Horse's tooth, Java downy mildew, Nigrospora ear rot, Penicillium ear rot,

Philippine downy mildew, and Sorghum downy mildew are seedborne and seed transmitted (McGee, 1988).

2.5.2 Downy mildews

Several species of the genera Peronosclerospora, Sclerospora, and Sclerophthora are responsible for downy mildews (CIMMYT, 2004). These include Crazy top downy mildew (Sclerophthora macrospora), Brown stripe downy mildew (Sclerophthora rayssiae var. zeae), Java downy mildew (Peronosclerospora maydis), Philippine downy mildew (Peronosclerospora philippinensis), and Sorghum downy mildew

(Peronosclerospora sorghi) (McGee, 1988).

Symptom expression is greatly affected by plant age, pathogen species, and environment. Usually, there are chlorotic stripings or partial symptoms in leaves and leaf sheaths, along with dwarfing (Adenle and Cardwell, 2000). Downy mildew becomes conspicuous after development of a downy growth on or under leaf surfaces. This condition is the result of conidia formation, which commonly occurs in the early morning (CIMMYT, 2004). The diseases are most prevalent in warm,

8 humid regions. Some species causing downy mildew also induce tassel malformations, blocking pollen production and ear formation. Leaves may be narrow, thick, and abnormally erect (CIMMYT, 2004).

2.5.3 Black Bundle Disease and Late Wilt

Black bundle disease is caused by Cephalosporium acremonium and is widely distributed (McGee, 1988). Serious economic losses from late wilt disease, caused by

C. maydis, have been reported in Egypt where 100% infection occurs in some fields, and in India with incidence as high as 70% and economic losses up to 51% (Johal et al., 2004). Both diseases kill the plants near flowering time. They are most common in humid, heavy soils in hot areas. The pathogens are soil and seedborne (McGee,

1988). Infected plants do not show symptoms until they reach tasseling stage and start wilting, generally beginning from the top leaves. Diseased plants produce only nubbins or ears with underdeveloped, shrunken kernels. When split, diseased stalks show brown vascular bundles starting in the underground portion of the roots

(CIMMYT, 2004).

2.5.4 Anthracnose Stalk Rot

The causal pathogen of anthracnose stalk rot disease is Colletotrichum graminicola

(Mathur and Kongsdal, 2003). The fungus causes both stalk rot and leaf blight. The stalk rot is found mostly in warm, humid areas throughout the world (CIMMYT,

2004). Anthracnose stalk rot is characterized by a distinctive blackening of the lower stalks as a result of black streaks that appear late in the season. The pith turns dark brown and has a shredded appearance. Numerous black spiny asexual fruiting bodies

(acervuli) form on the surface of the dead tissue (Reid and Zhu, 2005). In infected plants, there is premature wilting due to the complete destruction of pith tissue, with shredded vascular bundles turning dark brown. Because this and other fungi 9 overwinter in infected maize tissues, conservation agriculture practices involving mulches reportedly increase the incidence of the disease (CIMMYT, 2004).

2.5.5 Southern leaf blight

This disease is caused by Bipolaris maydis (Mathur and Kongsdal, 2003). Southern maize leaf blight is prevalent in hot, humid, maize growing areas. The fungus requires slightly higher temperatures for infection (CIMMYT, 2004). Leaves show greyish, tan, and parallel straight sided or diamond shaped, 1-4 cm long lesions with buff or brown borders or with prominent colour banding or irregular zonation

(Ullstrup, 1985). Symptoms may be confined to leaves or may develop on sheaths, stalks, husks, ears and cobs. The lesions are longitudinally elongated typically limited to a single inter vascular region, often coalescing to form more extensive dead portions (Ullstrup, 1985). Young lesions are small and diamond shaped. As they mature, they elongate. Growth is limited by adjacent veins, so final lesion shape is rectangular and 2 to 3cm long. Lesions may coalesce, producing a complete burning of large areas of the leaves (CIMMYT, 2004).

2.5.6 Charcoal Stalk Rot

The causal pathogen of this disease is Macrophomina phaseolina (McGee, 1988).

Charcoal stalk rot is most common in hot, dry environments. Incidence increases rapidly when drought and high temperatures prevail near tasseling stage. The pathogen invades seedling roots. After flowering, initial symptoms are the abnormal drying of upper leaf tissue. When plants approach maturity, the internal parts of stems show a black discoloration and vascular bundles shred, mainly in lower stalk internodes (CIMMYT, 2004). Careful examination of rind and vascular bundles reveals small, black, fungal structures known as sclerotia that can overwinter and

10 infect the next crop. The fungus may also infect kernels, blackening them completely. Many crops can serve as hosts for this pathogen (CIMMYT, 2004).

2.5.7 Horse’s tooth

This disease is caused by the fungus Claviceps gigantea (Mathur and Kongsdal,

2003), and endemic to high, cool, humid areas. Infected kernels grow into large fungal structures known as sclerotia along with normal healthy kernels (CIMMYT,

2004). In early stages of infection, sclerotia are pale coloured, soft and slimy, finally hardening toward harvest time. These sclerotia do not produce the black powder characteristic of common smut (CIMMYT, 2004). When sclerotia are dropped on the ground, they germinate and develop many head-like structures (stromata) that release new spores when the maize plants silk the following season (CIMMYT, 2004).

2.5.8 Aspergillus Ear Rots

Aspergillus ear rot is caused by Aspergillus flavus, Aspergillus niger and other

Aspergillus spp. (Mathur and Kongsdal, 2003). The disease may be a serious problem when infected ears are stored at high moisture contents (CIMMYT, 2004).

Several species of Aspergillus can infect maize in the field. Aspergillus niger is the most common; it produces black, powdery masses of spores that cover both kernels and cob (CIMMYT, 2004). In contrast, A. glaucus, A. flavus and A. ochraceus normally form yellow-green masses of spores. Aspergillus parasiticus is ivy green and less common in maize (McGee, 1988).

2.5.9 Gray Ear Rot

The causal pathogen of this disease is Physalospora zeae syn. Botryosphaeria zeae

(McGee, 1988). Hot humid weather for several weeks after flowering favours development of this ear rot (CIMMYT, 2004). Early symptoms are very similar to

11 those caused by stenocarpella ear rot, where a white-gray mycelium develops between kernels and husks, which become bleached and glued together but in latter stages ears have a distinct black colour; the mycelium is also dark and develops small sclerotia (specks) scattered throughout the cob (CIMMYT, 2004).

2.5.10 Common Smut

Common smut is caused by the fungus Ustilago maydis (CIMMYT, 2004). The fungus is Seed-borne but not seed transmitted (CABI, 2007; McGee, 1988). It occurs throughout most maize growing regions, but can be more severe in humid, temperate environments than in hot, humid, tropical lowlands (CIMMYT, 2004). The fungus attacks ears, stalks, leaves, and tassels. Conspicuous closed white galls replace individual kernels. In time the galls break down and release black masses of spores that will infect maize plants the following season (Pataky and Snetselaar, 2006). The disease is most severe in young, actively growing plants and may stunt or kill them.

This is easily distinguished from head smut by the lack of host vascular bundles that appear as fibres in smut-infected ears (Pataky and Snetselaar, 2006).

2.5.11 Botryodiplodia or Black Kernel Rot

The causal organism for transmission of this disease is Botryodiplodia theobromae

(McGee, 1988). Affected ears develop deep black, shiny kernels, and husk leaves can also turn black and be shredded (CIMMYT, 2004). Seed to seedling transmission of the disease is up to 90% (CABI, 2007). The same fungus can produce stalk rot with a conspicuous black discoloration in moist, hot environments (CIMMYT, 2004).

2.5.12 Fusarium Stalk Rot

This disease is caused by Fusarium moniliforme syn. Fusarium verticillioides

(McGee, 1988; Mathur and Kongsdal, 2003). Fusarium moniliforme is most common

12 in dry, warm areas. It is particularly severe if it begins just before tasseling. It is one of the most potentially damaging stalk-rotting agents (CIMMYT, 2004). A higher incidence of stalk rot is common when conditions that tend to encourage early senescence occur. Two such conditions are water stress and foliar diseases. Insect or hail injury may also result in more stalk rot as will high plant populations and imbalanced fertility, ie. high N to K ratio (Partridge, 2008). Symptoms produced by these pathogens resemble those caused by Stenocarpella or Cephalosporium, and cannot be differentiated until spore-producing structures are observed. Wilted plants remain standing when dry, and small, dark-brown lesions develop in the lowest internodes. When infected stalks are split, the phloem appears dark brown, and there is a general conspicuous browning of tissues. In the final stages of infection, pith is shredded and surrounding tissues become discoloured (CIMMYT, 2004).

2.5.13 Fusarium Ear Rot

The causal organism for transmission of fusarium ear rot disease is Fusarium moniliforme, syn. F. verticillioides (McGee, 1988). Fusarium moniliforme ear rot is likely the most common pathogen of maize ears throughout the world (CIMMYT,

2004). F. moniliforme occurs mainly on individual kernels or on limited areas of the ear. Infected kernels develop a cottony growth or may develop white streaks on the pericarp and germinate on the cob. Ears infested by earworms are usually infected with F. moniliforme (CIMMYT, 2004). The effect Fusarium moniliforme on seed maize germination and seedling development is not pronounced (McGee, 1988), but has serious effect on yield. The fungus produces mycotoxins known as fumonisins, which are harmful to several animal species (CIMMYT, 2004).

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2.6 Effect of Seedborne Fungal Diseases on Man and Animal

For the safety of human food, food-borne bacteria constitute the greatest hazard, followed by mycotoxins. Conversely, in terms of livestock feeds, mycotoxins pose the greatest threat. Mycotoxins have attracted worldwide attention due to the significant losses associated with their impact on human and animal health, and consequent national economic implications (Bhat and Vashanti, 1999).

Mycotoxicosis is the consequence or effect (disease or pathological abnormalities) of ingesting toxin-contaminated foods by man and animals. It may also result indirectly from consumption of animal products such as milk from livestock exposed to contaminated feed. Over 300 'mycotoxins' have been reported (Coker, 1979).

However, based on extensive analytical studies (IARC, 1993) and detailed study of the distribution of fungi in nature, the five agriculturally important toxins from fungi are aflatoxins, fumonisins, ochratoxin A, zearalenone and deoxynivanelol. Fungal toxins can cause acute or chronic intoxications, depending on the animal, sex, breed and dosage (Coker, 1979). The only toxin that has gained prominence in scientific literature in food products from the West African sub-region is aflatoxin, while there are few studies conducted on fumonisin and ochratoxin A (Bankole and Adebanjo,

2003).

Fusarium spp. can produce a number of toxic compounds including fusaric acid, fusarins, gibberellins, moniliformin, and fumonisins (Marasas et al., 1981; Nelson,

1992; Nelson et al., 1993). Recently, fumonisins have received the most attention, because fumonisin B1 is the most common toxin found in maize infected by F. moniliforme (Leslie et al., 1992; Nelson et al., 1993). Fumonisin B1 has been shown to cause a number of health problems in livestock and laboratory animals (Nelson et al., 1993, Osweiler et al., 1992; Riley et al., 1993) and has been associated with an

14 increased incidence of human oesophageal cancer in some parts of the world (Nelson et al., 1993). Fumonisins can be found in moldy and symptomless infected kernels of maize (Bullerman and Tsai, 1994; Munkvold and Stahr, 1994; Nelson, 1992; Nelson et al., 1993).

2.7 Control of Seedborne Diseases

Seedborne diseases are controlled by seed treatment practices. Seed treatment is the oldest practice in plant protection. Its origins can be traced to the 18th century with use of brine for the control of cereal smuts (Neergaard, 1988). The modern era of seed treatments began with the introduction of organo-mercury fungicides in 1912 which were widely used for several decades (McGee, 1995).

2.7.1 Types of Seed Treatment

Seed treatments can be classified as physical, biological, chemical and botanical.

Regardless of type, successful seed treatment practices must satisfy the following biological requirements (McGee, 1995).

 Consistently effective.

 Safe to operators during handling and planting.

 Safe to wildlife.

 Compatible with other materials used on seeds.

 Should not produce harmful residues on plant or soil.

 Chemical or biological methods should have desirable qualities with respect to

application and retention on the seeds.

2.7.2 Physical

Physical treatments usually apply heat to the seeds in order to kill the seedborne pathogen but cause minimal damage to the seed tissues. Hot water treatment has

15 been used since the 1920’s, and, before the advent of systemic fungicides in the

1960’s, was the only treatment available to eradicate deep-seated infections of seeds

(McGee, 1995). This methodology is still being exploited for eradication of internal infection by Xanthomonas campestris pv malvacearum in cotton (Honervogt and

Lehmann-Danziger, 1992). The treatment has limitations in that it has not always been successful in eliminating low infections of bacteria that are of epidemiological significance such as Xanthomonas campestris pv campestris in cabbage seeds

(McGee, 1995). It also can have detrimental influence on seed germination, which is a special concern in high value seeds such as hybrid cabbage (McGee, 1995).

Adverse effects of hot water on germination of rooibos tea seeds was minimized without compromising the efficacy of pathogen control by reducing the length of exposure to hot water and following treatment with scarification (Smit and Knox-

Davies, 1989). Hot vegetable oil treatment gave some control of Phomopsis longicolla infection of soybean seeds, but usually caused loss in germination (Pyndjii et al., 1987).

Treatment of seeds with dry heat also has been investigated since the 1920’s, but frequently has been only partially effective and has been little used in practice

(Neergaard, 1988). Dry heat, applied with a microwave oven, successfully eradicated several bacterial and fungal pathogens from cassava seeds (Lozano et al., 1986).

Microwave heating also reduced transmission of soybean mosaic virus, with little reduction in germination in seeds treated at 8.5% moisture content, but germination was considerably reduced in seeds treated at 16% (Jolicoeur et al., 1982). A technique using low energy electronic beams eliminates Tilletia caries and Septoria nodorum from wheat seeds in the pericarp and testa without adverse effects on germination (Burth et al., 1991).

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2.7.3 Biological Control Agents

In plant pathology, the term biological control agent applies to the use of microbial antagonists to suppress diseases (Pal and McSpadden, 2006). Biological control of plant disease, in general, has a history of inconsistency that has greatly restricted its practical application (Harman and Nelson, 1994). Public concerns over chemical pesticides in the past 20 years have stimulated scientific interest in bio-control agents

(Cook, 1993). Significant advances have been made, because attention has been paid to the mechanisms of control, particularly in the soil environment (Cook, 1993;

Harman and Nelson, 1994).

Schroth and Hancock (1981) described increase yield in several crops with

Pseudomonas seed treatment. They suggested that it results from displacement of root-infecting fungi and bacteria that, in total, reduce plant growth. There are numerous reports of potentially valuable biological control microorganisms, some of which are supplied as seed treatments, but the developmental process to bring these into commercial practice is long and arduous (Cook, 1993). As of 1994, only six microorganisms were commercially available, one of which, Bacillus subtilis, marketed as the seed treatment, Kodiak, by Gustafson Inc. Dallas, Texas

(Rhodes and Powell, 1994). As Cook (1993) points out, development of biological control agents is hindered by the expectation that it will function across a broad range of environments as most successful chemical pesticides do, when, in fact, the ecological niche in which a biological control agent can function may be relatively narrow.

Naturally occurring microorganisms also can be exploited for biological control.

Seed-transmitted endophytes, Acremonium coenophialum and A. lolii, cause animal toxicity problems in pastures, but infested grasses often have superior growth habits

17 due to insect and pathogen control (Siegel et al., 1987). Endothytes are now introduced into turf grass varieties to improve agronomic performance.

2.7.4 Chemical method (Use of Fungicides)

A survey in 1991 of seed treatment product segments throughout the world showed that fungicides dominated the market with a 68% share, followed by insecticides at

11% (Schwinn, 1994). The range of products on world markets today are quite different from those in the early part of the twentieth century when organo- mercurials and dithiocarbamates, e.g. thiram, and heterocyclics, eg. captan, that acted by direct contact with the pathogens, were the primary products used in seed treatment. Organo-mercurials are now banned in most countries because of toxicity to animals and humans, and use of captan and thiram has been restricted in use in some countries (Reigart and Roberts 1999). The latter materials, however, still are the mainstay of seed treatment chemicals for many crop species. They have broad spectrum of activity, are easily applied, and are relatively inexpensive. Some of the common chemicals used for seed treatment in the world include dithiocarbamates, mancozeb, maneb fludioxonil, shavit, prothioconazole, picoxstrobin, and fluoxastrobin (Leadbitter et al., 1994; Morton and Staub, 2008).

The change in the landscape of fungicide seed treatment chemicals can be attributed to the need to replace older chemicals because of environmental concerns and the discovery of systemic compounds that provided new opportunities to control foliar and systemic pathogens by seed treatment (McGee, 1995). The primary active ingredients that have come into common use include: carbithiins for control of rusts, smuts, and Rhizotonia; carboxin which is particularly effective against loose smut; benzimidazoles for control of Leptosphearia maculans in crucifers; metalaxyl, which is effective against Phycomycetes such as Phytophthora and downy mildews; and 18 ethirimol and triademenol forcontrol of the powdery mildews. Other systemic chemical that has potential for use seed as treatment includes iprodone and imazilil

(McGee, 1995).

2.7.5 Effects of Chemical Seed Treatments

The effect of seed treatment is one of several factors that influence the cost, risk and benefits of seed treatments. For some crops, such as corn, peanuts, rice and cereals, fungicide seed treatment is routine and there is little argument that seed treatment is a necessary and effective means of protecting seeds and seedlings from seed and seedborne pathogens. After almost 30 years of continual use of captan seed treatment of maize, Pedersen et al. (1986) re-evaluated the need for this practice and reached the conclusion that it was essential to assure stand establishment in the Corn Belt of the U.S.A.

While inexpensive organo-mercury seed treatments were in use in the U.K, cereal diseases were rare or unknown (Yarham and Jones, 1992). The replacement of organo-mercurials with more expensive products has led to several studies of benefits of seed treatment of cereals in the U.K., (Richardson, 1986; Sutherland et al., 1994). In all of these reports, no benefit to yield was evident in plots grown from treated compared to untreated seeds. At individual sites, however, significant differences were evident. Because control of seedborne pathogens is a major reason for seed treatment, it was suggested that decisions to apply seed treatments should be determined by seed health test results (Brodal, 1993).

Many of the inconsistencies in efficacy of seed treatment can be related to a lack of understanding of the epidemiological conditions under which the benefits of seed treatment can be realized. McGee (1981) criticized seed treatment research in

19 general with the following statement: “-- Application practices are usually determined by relating seed treatment rates to subsequent emergence and yield in a series of field tests in different locations and under a variety of environmental conditions. If repeated often enough, this type of experiment may provide reasonably reliable information on application rates, but very little information is obtained about disease epidemiology. As a result, failures in control practices cannot be explained and more fungicides tend to be used than are necessary.--” At least one major crop failure, resulting in multimillion dollar court settlements, has since been attributed to the inadequacies of research into efficacy of the product (McGee, 1995).

2.7.6 Plant or Botanical Extracts with Fungicidal Properties

Considerable research activity has occurred in the Asian-Pacific region on the potential for plant extracts to control seedborne fungi. The oils of cassia and clove inhibited the growth of established seedborne infections of Aspergillus flavus,

Curvularia pallescens and Chaetomium indicum in maize (Chatterjee, 1990). Seeds of the Neem tree (Azadirachta indiica) contain Azadirachtin which is believed to have antifungal properties. Aqueous extracts of Azadirachta indiica seeds, garlic bulbs, ginger rhizomes and basil leaves were used to control Alternaria padwickii in rice seeds (Shetty et al., 1989), while extracts from peppermint and garlic reduced rice seed infection by Cochliobolus miyabeanus (Alice and Rao, 1986). Garlic bulb extract inhibited the spore germination and mycelial growth of seedborne fungal pathogens of jute, including Macrophomina phaseolina, Botryodiplodia theobromae and Colletotrichum corchori (Ahmed and Shultana, 1984). Homeopathic drugs, Filix mas and Blatta orientalis, completely suppressed the population of Fusarium oxysporum in the seed mycoflora of wheat (Rake et al., 1989). Aspergillus ruber infection and weevil oviposition of Zabrotes subfaciatus were reduced by mineral oil

20 and soybean oil treatment of dry beans stored in Ecuador (Hall and Harman, 1991).

Soybean oil, applied at a rate used to suppress grain dust, reduced storage fungi growth in maize and soybeans during 12 months in field storage bins in Iowa

(McGee et al., 1989, White and Toman, 1994). Somda et al., (2007) controlled

Fusarium moniliforme and other seedborne pathogens of sorghum with lemongrass extract.

2.7.7 Effect of Neem on Fungi

Neem is a member of the mahogany family, Meliaceae. It is today known by the botanic name Azadirachta indica A. Juss. (NRC, 1992). Neem contains azadirachtin which has demonstrated antifungal activity (Natarajan et al., 2003). Two more neem components, nimbin and nimbidin, have been found to have antifungal activity

(Joshi, 2006). Should this prove widely applicable, the availability of a natural fungicide that can be grown, extracted and applied by farmers themselves could be of great consequence to worldwide agriculture and food supply. Not a lot is known about neem’s practical use against rots, smuts, wilts, mildews, die-backs, and other fungal plant disease. However, several tests have indicated considerable promise

(NRC, 1992).

In one test, neem oil protected the seeds of chickpeas against the serious fungal diseases Rhizoctonia solani, Sclerotium rolfsii and Sclerotinia sclerotiorum (NRC,

1992). It also slowed the growth of Fusarium oxysporum but did not kill it. In addition, neem cake incorporated into the soil completely blocked the development of the resting forms of Rhizoctonia solani thereby interfering with the long-term survival of this devastating fungus. In another, neem seed extracts showed beneficial effects against leaf fungi (NRC, 1992).

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2.7.8 Effect of Lemongrass (Cymbopogon citratus) on Fungi

Lemongrass belongs to the grass family, Poaceae. It is today known by the botanic name Cymbopogon citratus (DC.) Stapf. (Valencia and Myers, 1998). Fresh C. citratus contains approximately 0.4% volatile oil. The oil contains 65% to 85% citral, a mixture of two geometric isomers, geraniol and neral. Related compounds geraniol, geranic acid, and nerolic acid have also been identified (Torres, 1993; Masuda et al.,

2008). Other compounds found in the oil include myrcene (12% to 25%), diterpenes, methylheptenone, citronellol, linalol, farnesol, other alcohols, aldehydes, linalool, terpineol, and more than a dozen other minor fragrant components (Kasumov and

Babaev, 1983). Non-volatile components of C. citratus consist of luteolins, caffeic acid, fructose, sucrose, octacosanol, and others (De Matouschek, 1991). Proximate analysis of lemongrass also revealed the presence of potassium, zinc, iron and manganese (Livestrong.com, 2010). Lemongrass as well as many other plants have been successfully used to control important seedborne fungal pathogens (Amadioha,

2000). In Nigeria, lemongrass powder and essential oil have been successfully used to protect cowpea and maize against storage fungi and Macrophomina phaseolina

(Tassi) Goid (Adegoke and Odesola, 1996). Aqueous extract of lemongrass was also effective against seedborne fungal pathogens of melon (Bankole and Adebanjo,

1995).

2.7.9 Chromolaena odorata

Chromolaena odorata (L) King and Robinson (also Eupatorium odoratum L.), belongs to the family Asteraceae (APG II, 2003). Its common names in Ghana include “Acheampong”, “Busia”, or Siam weed (Timbilla and Braimah, 1990).

Although, native to South and Central America it is has spread throughout the tropics, Nigeria inclusive (Okon and Amalu, 2003). Chemical analysis of

22

Chromolaena odorata extracts reveals the presence of flavone and flavonoid, tannin, alkaloid, saponins, sesquiterpenes, and other phenolic compounds (Ardcharawan,

2005).

2.7.10 Mancozeb (Chemical fungicide)

Mancozeb is classified as contact fungicide with preventive activity (SinoHarvest,

2010). It has a chemical name manganese ethylenebis (dithocarbamate) (polymeric) complex with zinc salt and empirical formula (C4H6MnN2S4)x(Zn)y (Yellow River,

2010). It inhibits enzyme activity in fungi by forming a complex with metal- containing enzymes including those involved in production of adenosine triphosphate

(ATP) (SinoHarvest, 2010). Mancozeb is used to protect many fruits, vegetables, nuts and field crops against a wide spectrum of fungal diseases, including potato blight, leaf spot, scab on apples and pears, and rust on roses. It is also used for seed treatment of cotton, potatoes, corn, safflower, sorghum, peanuts, tomatoes, flax and cereal grains (Yellow River, 2010).

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3.0 MATERIALS AND METHODS

The research was carried out at the Plant Pathology Laboratories of Crops Research

Institute at Fumesua near Kumasi and Seed Laboratory of Grains and Legumes

Development Board in Kumasi. The research was in the following phases;

 health analysis of the seed maize,

 treatment of the seeds with botanicals,

 health analysis of treated seeds, and

 germination test for treated and untreated seeds.

3.1 Seed Samples

Fifty-four (54) seed samples collected from the Forest, Transition and Guinea

Savannah zones of Ghana were investigated. A survey was conducted in the middle of the major cropping season to identify farmers who produce maize in the Forest,

Transition and Guinea Savannah zones of Ghana. From each ecological zone, three districts were covered. From each district three locations (town/village) popularly known for production of maize crops were covered, and from each location two farmers were identified for the seed collection.

The seed samples were collected after the seed was harvested and stored by the farmers. Some seeds were collected from market where the market was known to be one of the main sources of planting seed in that location. One kilogram primary sample was taken from each selected farmer per location. The seed samples were labelled and put in plastic bags and tightly sealed and stored at room temperature

(25°C) in transit. Seed samples were then stored in a cold room of 10 to15°C at Crop

Research Institute for good preservation prior to seed health analysis and treatment.

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3.2 Seed Health Analysis

The purpose of the seed health analysis was to determine fungal pathogens on the seed samples. The seed samples were analysed using the Deep-freeze blotter method

(Mathur and Kongsdal, 2003) as described in sections 3.2.1 to 3.2.3. The design for the analysis was Completely Randomized Design (CRD)

3.2.1 Plating of Seeds

On a clean and disinfected working table the required number of sterilized 90mm diameter Petri dishes per sample were collected and accession number of each sample, the date of inspection and plate number were written on the cover. A set of three blotter papers were dipped in distilled water and placed in the lower dish. The seeds were poured into a tray and samples for plating taken at random using the spoon method as described by Mathur and Kongsdal, (2003). Ten seeds per dish were plated using a pair of forceps. Four hundred (400) seeds of a hundred (100) seeds per replicate were used for each sample (Mathur and Kongsdal, 2003; ISTA,

2007).

3.2.2 Freezing and Incubation of Seeds

All the dishes of each sample were gently collected on one tray and transferred to the incubation room. The plated seeds were incubated at a temperature of 22oC for 7 days in alternating cycles of 12 hours darkness and 12 hours light. However, the plated seeds were kept in a deep-freezer after 24 hours of incubation for 12 hours of freezing at a temperature of -20ºC after which incubation continued. The source of light used in the incubation room was near ultraviolet (NUV) (Mathur and Kongsdal,

2003; ISTA, 2007).

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3.2.3 Examination of Incubated Seeds and Recording of Infections

After incubation for 7 days, the dishes were removed and arranged serially. Moving from one Petri dish to the other, each seed was examined under a stereomicroscope.

Habit characters of each fungus were observed and used to identify the fungi that grew on seeds. Slide preparations of fruiting structures and spores of certain fungi were made and examined under compound microscope to further confirm their identities by consulting mycological literature and experienced seed health analysts.

The abbreviation for the identified fungus was written on the wet blotter beside the seed with green blotter pencil. Different fungi in each Petri dish were counted by crossing the abbreviations. Count of each fungus from each dish was entered in working recording sheets immediately after examination of the dish (Mathur and

Kongsdal, 2003; ISTA, 2007).

3.2.4 Isolation of Fungal Pathogen

The main fungal pathogens which were identified during the examinations were sub- cultured on to PDA (potato dextrose agar) into pure cultures for preservation for further studies. Sub-culturing was repeated until pure isolates of single species was obtained on PDA. The PDA used was prepared according to the manufacturer’s

(Acumedia Manufacturers Inc.) instructions. The pure cultures were preserved in refrigerator at 5ºC until required.

3.3 Treatment of Seeds

The seed maize sample with the highest infection of fungal pathogens as revealed by the Deep-freeze blotter study was selected and treated with extracts from neem

(Azadirachta indica) seed, lemongrass (Cymbopogon citratus) and Chromolaena

26 odorata leaves. Untreated seeds and treatment with mancozeb, a synthetic fungicide served as controls.

3.3.1 Preparation of Botanical Extracts

Fresh leaves of lemongrass (Cymbopogon citratus) and Chromolaena odorata and neem (Azadirachta indica) seeds were collected as treatment materials for the experiment. Aqueous extracts of each of the plant materials were then prepared.

3.3.1.1 Preparation of Neem Seed Extract

The shells and testa of dry seeds were removed and seeds grounded to form paste using an electric blender. Fifty gram (50g) weight of the paste was mixed with 100 ml of distilled water to prepare 50% concentration of the neem seed extract. The liquid was then squeezed through four layers of cheese cloth into a conical flask to obtain the extract (Moses, 2010).

3.3.1.2 Preparation of Lemongrass Extract

The leaves of fresh lemongrass were thoroughly washed in tap water, air dried and then used for the preparation of fresh extract. The extracts were prepared by cutting

50g of fresh leaves and blending leaves in 100 ml of distilled water, using electric blender, to form 50% concentration of lemon grass extract. The pulverized mass of the leaves was squeezed through four layers of cheese cloth into a conical flask to obtain the extract (Moses, 2010).

3.3.1.3 Preparation of Chromolaena odorata Leaf Extract

Fresh leaves of Chromolaena odorata were thoroughly washed in tap water, air dried and then used for the preparation of fresh extract. The extracts were prepared by blending 50g of fresh leaves in 100 ml of distilled water, using electric blender, to form 50% concentration of the Chromolaena odorata extract. The pulverized mass

27 of the leaves was squeezed through four layers of cheese cloth into a conical flask to obtain the extract (Moses, 2010), as was done for the lemongrass leaf extract.

3.3.2 Seed Treatment and Health Analysis of Treated Seeds

The seed sample with high infection by fungal pathogens as determined by the Deep- freeze blotter method, were treated with each of the prepared plant extracts (at 50% concentrations) by soaking the seeds in them for 1, 3, 6 and 24 hours. Seeds were also soaked in 2.5% mancozeb for 1, 3 and 6 hours and together with untreated seeds that served as controls. Treated seeds were collected on blotter sheets and air-dried in a LaminAir flow chamber for 1 hour before plating on a wet blotter.

The required number of sterilized Petri dishes per treatment was collected and the code number of each treatment, the date of inspection and plate number were written on the cover. A set of three blotter papers were dipped in distilled water and placed in the Petri dish. The treated seeds were poured into a tray and ten seeds per dish were plated using a pair of forceps. Four hundred (400) seeds of a hundred (100) seeds per replicate were used for each treatment (Mathur and Kongsdal, 2003; ISTA,

2007).

All the dishes of each treatment were gently collected on one tray and transferred to the incubation room. The plated treated seeds were incubated at a temperature of

22oC for 7 days in alternating cycles of 12 hours darkness and 12 hours light.

However, the plated treated seeds were kept in a deep-freezer after 24 hours of incubation for 12 hours of freezing at a temperature of -20ºC after which incubation continued. The source of light used in the incubation room was near ultraviolet

(NUV) (Mathur and Kongsdal, 2003; ISTA, 2007).

28

Observation for the incidence of fungal pathogens was made under stereomicroscopes. In some cases fruiting bodies and spores were examined using the compound microscope to achieve accurate identification.

3.3.3 Effect of the Botanical Extracts on Germination and Vigour

Four hundred (400) seeds per treatment (100 seeds per replicate) were used for germination and vigour tests. Seeds were treated as described above and placed in sterilized paper towel and put in a germinator using Completely Randomized Design

(CRD) to determine percent germination and seed vigour. Untreated seeds were also subjected to similar germination test to serve as control. Germinated seeds were counted on 4th and 7th days to evaluate vigour and germination respectively (ISTA,

2007).

3.4 Statistical Analysis of Data

The data on average incidence of fungal pathogens and their response to the botanical treatment was subjected to ANOVA using GenStat Discovery Edition 3 and means separated by Least Significant Difference (LSD).

29

4.0 RESULTS

4.1 Infection by Fungal Pathogens in Seed Maize from the Forest,

Transition and Guinea Savannah Zones of Ghana

4.1.1 Seed Health Analysis before Seed Treatment

All the samples tested were found to be infected by fungal pathogen but at different levels. The frequency of infection by different fungal pathogens in the collected seed samples from the Forest, Transition and Guinea Savannah ecological zones are presented in Tables 4.1, 4.2, 4.3 and 4.4. In all, four pathogenic fungi were found and recorded in the ecological zones namely; Fusarium moniliforme, Botryodiplodia theobromae, Acremonium strictum and Bipolaris maydis. Fusarium moniliforme was the most frequent pathogen present in all the 54 samples investigated. However, their frequency of occurrence varied from sample to sample with Transition zone showing the highest average infection of 49.4% with least average infection of 37.1% for the samples collected from the Forest zone (Table 4.4). Botryodiplodia theobromae showed an average infection of 6.1%, 4.6% and 1.8% in Guinea Savannah, Forest and Transition zones respectively with Acremonium strictum showing average infection of 3.8%, 1.9% and 0.6% in Transition, Forest and Guinea Savannah zones respectively (Table 4.4). Frequency of infection by Bipolaris maydis was 0.1% in both Forest and Guinea Savannah zones. There was no Bipolaris maydis infection in the Transition zone (Table 4.4).

Analysis of the seed health results on ecological basis revealed that although the seed sample with highest infection of F. moniliforme was from the Guinea Savannah zone

(ie. MS56 with 84.5%), it was the Transition zone that showed the highest average infection (Tables 4.1 & 4.2). In the Transition zone, 13 out of 18 samples analysed

30 showed F. moniliforme infection above 40%, while 12 out of 18 seed samples were above 50% infection (Table 4.2). Nine out of 18 seed samples in the Guinea

Savannah zone showed F. moniliforme infection above 40%, while infection in 3 out of 18 samples was above 50% (Table 4.3). The results also indicated that 8 out of 18 samples in the Forest zone showed F. moniliforme infection above 40% while 5 out of 18 were above 50% (Table 4.1). Every seed sample analysed carried an infection of F. moniliforme.

Botryodiplodia theobromae infection was prevalent in the Guinea Savannah zone infecting all the samples analyzed from the area. Only three of the samples have infection above 10% but below 20% with the remaining below 10% (Table 4.3).

Three of the seed samples from the Forest zone showed B. theobromae between 10 and 20% with five samples showing no infection (Table 4.1). Eleven samples from the Transition zone showed B. theobromae infection with only one sample showing

10% infection. The remaining seven samples did not show B. theobromae infection

(Table 4.2).

Acremonium strictum was prevalent in the Transition zone with 14 of the samples showing infection but only one sample was above 10% (Table 4.2). The Transition zone however, did not show Bipolaris maydis infection. Seven samples were infected with A. strictum in the Forest zone with only one sample with infection of 22% and the remaining six below 10%. The Guinea Savannah zone showed A. strictum infection in 10 samples (rate of infection was 2% and lower) Table 4.3. Both the

Forest and Guinea Savannah zones have Bipolaris maydis infection in 1 and 3 samples respectively with infection rates below 2% in each sample (Table 4.1 & 4.3).

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Table 4.1: Frequency of Infection of Seed Maize by Fungal Pathogens (Forest Zone)

Location Seed Fusarium Botryodiplodia Acremonium Bipolaris Sample moniliforme theobromae strictum maydis Sekyeredumase MS36 78.5 1.5 1.5 0.0

Ve-Koloenu MS12 71.0 3.5 6.5 0.0

Drobong MS34 69.5 0.0 0.0 0.0

Hohoe MS18 63.0 0.0 0.0 0.0

Ve-Koloenu MS6 51.5 0.0 1.0 1.0

Nkranpo MS32 49.0 14.0 0.0 0.0

Nkranpo MS35 44.0 19.0 0.0 0.0

Gbi-Godenu MS11 43.5 2.0 0.5 0.0

Ahomahomasu MS2 39.5 5.5 0.0 0.0

Abourso MS5 34.5 3.5 2.0 0.0

Abourso MS3 32.5 6.5 0.0 0.0

Sekyeredumase MS33 31.5 7.5 0.0 0.0

Otuater MS8 17.5 0.5 0.0 0.0

Otuater MS1 11.0 3.5 22.0 0.0

Gbi-Godenu MS9 10.5 0.0 0.0 0.0

Hohoe MS14 9.0 16.0 0.0 0.0

Hohoe MS13 7.0 0.5 1.0 0.0

Gbi-Godenu MS7 5.0 0.0 0.0 0.0

Mean 37.1 4.6 1.9 0.1

LSD (0.05%) 23.46 5.16 3.62 0.70

CV (%) 30.1 52.9 89.9 600.0

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Table 4.2: Frequency of Infection of Seed Maize by Fungal Pathogens (Transition Zone)

Location Seed Fusarium Botryodiplodia Acremonium Bipolaris Sample moniliforme theobromae strictum maydis Wenchi MS80 76.0 3.0 2.0 0.0

Kuntunso MS41 74.5 0.5 0.0 0.0

Kintampo MS85 72.5 0.0 2.5 0.0

Nkoranza MS38 66.5 2.5 1.0 0.0

Tanoso MS42 65.5 5.0 0.0 0.0

Wenchi MS78 63.0 3.5 2.0 0.0

Nkoranza MS37 60.0 0.0 7.0 0.0

Techiman MS26 55.5 0.5 5.5 0.0

Terchire MS29 54.0 10.0 1.5 0.0

Grumakrom MS31 53.0 0.0 7.5 0.0

Koasi MS83 48.5 1.0 2.0 0.0

Hansua MS39 48.0 0.0 0.5 0.0

Koasi MS84 47.0 0.5 5.5 0.0

Tanoso MS40 30.5 0.0 6.0 0.0

Techiman MS43 26.0 1.5 16.0 0.0

Terchire MS27 25.0 5.0 8.5 0.0

Donkro Nkwanta MS28 12.5 0.0 0.0 0.0

Ayerede MS30 11.5 0.0 0.0 0.0

Mean 49.4 1.8 3.8 0.0

LSD (0.05%) 15.97 3.97 7.53 0.0

CV (%) 15.4 102.0 96.6 0.0

33

Table 4.3: Frequency of Infection of Seed Maize by Fungal Pathogens (Guinea Savannah Zone)

Location Seed Fusarium Botryodiplodia Acremonium Bipolaris Sample moniliforme theobromae strictum maydis Yiworgu MS56 84.5 1.5 0.5 0.0

Pissi MS45 62.5 4.0 2.0 0.5

Chorwu MS60 52.5 2.5 0.5 0.0

Kafabiara B. MS48 48.5 9.5 0.0 0.0

Kanshigu MS57 48.5 9.5 1.0 1.5

Nyeshei MS49 48.0 8.5 0.0 0.0

Lahagu MS53 47.0 10.5 0.5 0.0

Noori MS58 43.5 6.0 0.0 0.0

Bamahu MS46 42.5 2.0 0.0 0.0

Kafabiara B. MS47 36.0 3.5 0.0 0.0

Nyeshei MS49 35.5 10.0 1.0 0.0

Yiworgu MS54 35.0 18.0 2.0 0.0

Pissi MS44 33.5 3.0 0.5 0.0

Savalugu MS52 32.0 2.5 0.0 0.0

Noori MS59 25.5 6.0 0.0 0.0

Lahagu MS55 24.5 2.0 0.5 0.0

Dufaa MS50 22.0 1.5 2.5 0.0

Dufaa MS51 11.5 8.5 0.0 0.5

Mean 40.7 6.1 0.6 0.1

LSD (0.05%) 16.78 6.81 2.27 1.16

CV (%) 19.6 53.5 176.7 398.0

34

Table 4.4: Mean Frequency of Infection by Fungal Pathogens

Ecological Fusarium Botryodiplodia Acremonium Bipolaris zone moniliforme theobromae strictum maydis . (%) (%) (%) (%)

Forest 37.1 4.6 1.9 0.1

Transition 49.4 1.8 3.8 0.0

Guinea Savannah 40.7 6.1 0.6 0.1

LSD @5% 9.90 2.27 1.97 0.17

CV (%) 49.90 116.00 201.20 567.30

4.2 Effects of Plant Extracts on Seedborne Fungal Pathogens of Maize

Three major fungal pathogens were found on the seed maize sample selected for treatment. These are Fusarium moniliforme (68.0%), Acremonium strictum (3.5%) and Botryodiplodia theobromae (2.0%).

4.2.1 Effects of Neem Seed Extract on Seedborne Pathogens

Seed maize treated with neem seed extract for 24 hours showed very significant (P<

0.001) reduction in incidence of Fusarium moniliforme (18.0%) as compared with untreated seeds (68.0%) and seeds treated with mancozeb for 1 hour (21.5%) (Fig.

4.1). Seeds treated for 1, 3 and 6 hours also showed significant decrease in incidence of Fusarium moniliforme (28.0, 27.5 25.5% respectively) as compared with untreated seeds. There was also not much difference between them and mancozeb treatment for

1 hour (21.5%) (Fig. 4.1). The treatment reduced the incidence of Acremonium strictum from 3.5% in untreated seeds to 0.5 and 2.0% in soaking for 3 and 6 hours respectively (Table 4.6). Soaking of seeds for 1 and 24 hours however, eliminated

Acremonium strictum completely (Table 4.5). Neem seed extract treatment

35 completely eliminated Botryodiplodia theobromae from the seed maize at all soaking periods (Table 4.6).

80

70 68.567.5 68.0 65.5 64.0

60

(%) 55.5 50.5 50

39.0 40 36.5

F. moniliforme F. 30 28.027.5 25.5 21.5

20 18.0 Incidence of of Incidence

10 4.0 3.5

0 Neem Seed Lemongrass Chromolaena Mancozeb Untreated Seeds Extract Extract odorata Extract Treatments 1 Hour 3 Hours 6 Hours 24 Hours

Figure 4.1: Effects of Treatments on the incidence of Fusarium moniliforme (Fm)

4.2.2 Effects of Lemongrass Extract on Seedborne Pathogens

Treatment of seed maize with lemongrass extract for 24 hours showed significant

(P< 0.001) reduction in incidence of Fusarium moniliforme (39.0%) as compared with untreated seeds but the reduction is not closer to that of mancozeb treatment for

1 hour (21.5%) (Figure 4.1). However, seeds treated for 3 and 6 hours did not show any significant (P< 0.001) reduction in incidence of Fusarium moniliforme as compared with untreated seeds. One hour treatment with lemongrass, on the other hand, saw slight increase in incidence of Fusarium moniliforme (68.5%) as compared

36 with that of untreated seeds, 68.0% (Figure 4.1). The incidence of Acremonium strictum increased from 3.5% in untreated seeds to 4.0, 8.5 and 5.0% for 1, 3 and 6 hours soaking respectively. Twenty-four hours of soaking in lemongrass extract however, showed that Acremonium strictum infection decreased to 1.5%. Treatment with lemongrass extract completely eliminated Botryodiplodia theobromae from the seed maize at all soaking periods except for 6 hours where the infection was reduced to 0.5% (Table 4.6).

Table 4.5: Effect of Treatments on the incidence of Acremonium strictum (%)

Treatments 1 Hour 3 Hours 6 Hours 24 Hours

Neem Seed Extract 0.0 0.5 2.0 0.0

Lemongrass Extract 4.0 8.5 5.0 1.5

Chromolaena odorata Extract 6.5 1.5 5.5 0.5

Mancozeb 0.0 0.0 0.0

Untreated Seeds 3.5

4.2.3 Effects of Chromolaena odorata Leaf Extract on Seedborne Pathogens

Seed maize treated with Chromolaena odorata leaf extract showed significant reduction in the incidence of Fusarium moniliforme (P< 0.001) as compared with the untreated seeds except for those treated for 1 hour in which the reduction was not significant from the untreated (Figure 4.1). Seeds treated with Chromolaena odorata leaf extract for 1 and 6 hours showed an increase in Acremonium strictum infection to 6.5 and 5.5% respectively. Three (3) and 24 hour treatment showed a reduction in the infection to 1.5 and 0.5% respectively as compared with 3.5% in the untreated

37 seeds (Table 4.6). There was slight decrease in the incidence of Botryodiplodia theobromae to 1.5, 1.0 and 0.5% in seeds treated with Chromolaena odorata leaf extract for 1, 6 and 24 hours respectively (Table 4.6). However, seeds soaked for 3 hours showed no Botryodiplodia theobromae infection (Table 4.6).

Table 4.6: Effect of Treatments on the incidence of Botryodiplodia theobromae (%)

Treatments 1 Hour 3 Hours 6 Hours 24 Hours

Neem Seed Extract 0.0 0.0 0.0 0.0

Lemongrass Extract 0.0 0.0 0.5 0.0

Chromolaena odorata Extract 1.5 0.0 1.0 0.5

Mancozeb 0.0 0.0 0.0

Untreated Seeds 2.0

4.3 Effects of Plant Extracts on Germination of Seed Maize

4.3.1 Effects of Neem Seed Extract on Germination of Seed Maize

Seeds treated with neem seed extract for 1 and 3 hours showed an increase in germination (75.5 and 76.0% respectively) as compared with the untreated (67.5%) but there was slight increase of 72.5% by those treated for 6 hours as compared with the untreated (67.5%) (Figure 4.2). Seeds treated for 24 hours however, showed a very small decrease in germination (66.5%). Although there was increase in germination in seeds treated with mancozeb for 3 and 6 hours (80.5 and 79.5%), it was not significantly higher at P < 0.005 than the corresponding treatment with neem seed extract (76.0% and 72.5%) (Figure 4.2).

38

4.3.2 Effects of Lemongrass Extract on Germination of Seed Maize

Treatment of seeds with lemongrass for one hour showed significant (P< 0.005) increase in germination (79.0%) as compared with the 67.3% of the untreated seeds

(Figure 4.2). Seeds treated for 3 and 6 hours showed (73.0 and 72.0) increase in germination but not significantly (P< 0.005) different from the untreated seeds

(Figure 4.2). Lemongrass treatment for 24 hours however, showed decrease in germination (66.5%) but not significantly (P< 0.005) different from 67.3% of the untreated seeds (Figure 4.2).

4.3.3 Effects of Chromolaena odorata Leaf Extract on Germination of Seed

Maize

Seeds treated with Chromolaena odorata leaf extract for one hour showed significant

(P< 0.005) increase in germination (76.0%) as compared with 67.3% of the untreated seeds (Figure 4.2). Seeds treated for 3 and 6 hours also showed an increase in germination (73.0 and 73.5% respectively). Chromolaena odorata treatment for 24 hours however, showed decrease in germination (65.0%) but also not significantly

(P< 0.005) different from 67.3% of the untreated seeds (Figure 4.2). Changes in germination that occurred as a result of Chromolaena odorata treatment are not significantly different from that of mancozeb treatment 1 hour (70.0%), 3 hours

(80.5%) and 6 hours (79.5%).

39

90

80.5 79.0 79.5 80 75.576.0 76.0 73.5 72.5 73.072.0 73.0 70.0 70 66.5 66.5 67.5 65.0 60

50

40 Germination (%) Germination 30

20

10

0 Neem Seed Lemongrass Chromolaena Mancozeb Untreated Seeds Extract Extract odorata Extract Treatments 1 Hour 3 Hours 6 Hours 24 Hours

Figure 4.2: Effects of Treatments on Germination of Seed Maize

4.4 Effect of Plant Extracts on Vigour of Seed Maize

4.4.1 Effects of Neem Seed Extract on Vigour of Seed Maize

Vigour decreased at all duration of neem seed extract treatment (19.0, 8.0, 30.0 and

23.5% for 1, 3, 6 and 24 hours respectively), but the treatments for 6 and 24 hours were not significantly (P< 0.001) different from the untreated seeds, 31.0% (Figure

4.3). Seeds treated for 1 and 3 hours however, showed significant (P< 0.001) decrease in vigour as compared with the untreated seeds (Figure 4.3). The vigour for all the neem seed extract treatments was significantly lower than the corresponding treatments with mancozeb (Figure 4.3).

4.4.2 Effects of Lemongrass Extract on Vigour of Seed Maize

Apart from one hour treatment of seed maize with lemongrass that showed reduction in vigour (29.5%), treatment for 3, 6 and 24 hours showed significant P< 0.001)

40 increase in vigour as compared with the untreated seeds (Figure 4.3). Three-hour lemongrass treatment showed higher vigour (55.5%) than three-hour treatment with mancozeb (54.5%).

4.4.3 Effects of Chromolaena odorata Leaf Extract on Vigour of Seed Maize

There was a significant (P< 0.001) increase in vigour (48.0 and 51.5%) in seeds treated with Chromolaena odorata leaf extract for 3 and 6 hours respectively, but the increase in vigour for the 24 hour treatment (31.5%) was not significant as compared to 31.0% of the untreated seeds (Figure 4.3). Chromolaena odorata leaf extract treatment for one hour however, showed decrease in vigour (15.5%) as compared with 31.0% of the untreated seeds (Figure 4.3). There was also an appreciable increase in vigour in 3 and 6 hours treatments as compared with that of mancozeb treatment (Figure 4.3).

70 63.5

60 55.5 54.5 51.5 50 48.0

40 37.0 35.0

(%) Vigour 31.5 31.0 30.0 29.5 30 25.5 23.5

20 19.0 15.5

10 8.0

0 Neem Seed Lemongrass Chromolaena Mancozeb Untreated Seeds Extract Extract odorata Extract Treatments 1 Hour 3 Hours 6 Hours 24 Hours

Figure 4.3: Effects of Treatments on Vigour of Seed Maize

41

5.0 DISCUSSION

The aim of this study was to investigate the incidence of seedborne fungal pathogens in seed maize collected from the Forest, Transition and Guinea Savannah ecological zones of Ghana and to determine the efficacy of plant extracts in controlling seedborne fungal pathogens in maize. The occurrence of the various fungal pathogens varied in the three ecological zones. The Transition zone recorded the highest average frequency of Fusarium moniliforme and Acremonium strictum but lowest frequency of Botryodiplodia theobromae. The Guinea Savannah zone however, recorded the highest frequency of Botryodiplodia theobromae. The Forest zone showed moderate frequency of all the pathogens identified indicating differences in occurrence of the pathogens in the ecological zones.

Treatment with neem seed extract reduced the incidence of the fungal pathogens significantly at all soaking periods. It also had increased effect on germination but reduced vigour slightly. Moreover, all the three plant extracts had increasing effect on germination except for 24 hours of treatment.

5.1 Infection by Fungal Pathogens in Seed Maize from the Forest,

Transition and Guinea Savannah Zones of Ghana

All the fifty-four samples of seed maize studied were infected by pathogenic and saprophytic fungi regardless of their ecological origins. This could be attributed to the fact that fungi thrive well in warm humid areas and Ghana’s location in the humid tropics facilitates the growth of these pathogens. Percent infection was variable according to location and seed samples. The variation of infection at location may be due to different farming practices and some of the practices such as

42 continuous cultivation of the same kind of crop which leads to build up of pathogens

(Pickett and Pruitt, 2010).

Fusarium moniliforme was present in all samples and its infection percentages were very high. This high incidence of Fusarium moniliforme may be due to build-up as a result of continuous cropping as reported by Pickett and Pruitt (2010). F. moniliforme is a widely distributed pathogen of maize (Zea mays L.), causing seedling diseases, root rot, stalk rot, and ear or kernel rot. It is the most commonly reported fungal species infecting maize (Kommedahl and Windels, 1981; Nelson,

1992; Nelson et al., 1993). In Ghana, Amoah et al., (1995) detected F. moniliforme in seeds of maize, rice and sorghum collected from the various regions of the country. Furthermore, toxins like fumonisins which are produced by this fungus in maize kernel (Leslie et al., 1992; Nelson et al., 1993) cause oesophageal cancer in humans. Reducing the infection of F. moniliforme in maize, which is the staple food in Ghana, will help to reduce health hazards caused by the pathogen. It is therefore important to define appropriate measures for maintaining maize seed quality.

Attention should be paid to avoid the build-up of seedborne inoculum of this pathogen in both certified and farmer-saved seed maize.

Acremonium strictum was present in samples tested from Forest and Transition zones. Due to the bimodal distribution of rainfall in these zones (Morris et al., 1999), maize is cultivated in two seasons within a year, enabling the pathogen to survive. It is however, not able to survive as much in the Guinea Savannah zone owing to unimodal rainfall distribution in the area. It is most common in humid, heavy soils in hot areas (CIMMYT, 2004), however, soil in Guinea Savannah zone is not as heavy as those in Forest and Transition zones. It is reported to cause stalk rot called black bundle disease and kernel rot (CIMMYT, 2004). Seed of maize can rot when heavily 43 infected by A. strictum (Richardson, 1990). Although detected in all the seed samples examined, this fungus has not yet been reported to cause any disease on field in

Ghana. However, as A. strictum infection has been detected, it is important to perform field observations in maize-growing areas to evaluate the incidence of black bundle disease caused by this fungus.

Botryodiplodia theobromae was also found abundantly in the Forest and Guinea

Savannah zones. Their presence in abundance in these regions may be due to alternate host plants such as cacao, citrus, mango, avocado, Carica papaya, pigeonpea, banana and plantain (Wikipedia, 2010b) in the regions. The pathogen therefore has enough propagating places when maize is not in cultivation. It is known to cause black kernel rot disease in maize (CIMMYT, 2004). It is also responsible for causing black kernel rot disease of maize (Nelson et al., 1993). As B. theobromae infection percentages were high in these ecological zones, it is important to perform field observations in maize-growing areas in Ghana to evaluate the incidence of diseases caused by this fungus.

Apart from one seed sample from Forest zone and three samples from Guinea

Savannah zone which were slightly infected, seed samples from Transition zone were free of Bipolaris maydis, the causal agent of southern leaf blight (Mathur and

Kongsdal, 2003). It has been found to be the most damaging fungal disease of maize in Burkina Faso, which shares border with Ghana, according to Sanou (1996). There is therefore, the need to strengthen control of this pathogen particularly in the

Northern agro-ecological zones of Ghana. B. maydis has been known to be seedborne exclusively in maize but it has been shown to be borne on seeds of non-susceptible crops such as lettuce, watermelon, cowpea and sorghum (Fatima et al., 1974).

44

However, these crops are rarely grown in combination with maize in Ghana, accounting for the low infection of the pathogen.

5.2 Effects of Botanical Extracts on Seedborne Fungal Pathogens,

Germination and Vigour of Seed Maize.

Aqueous extracts of neem (Azadirachta indica) seed, lemongrass (Cymbopogon citratus) leaves and Chromolaena odorata leaves were tested for their efficacy in controlling seedborne fungal pathogens. Aqueous extract of neem seed treatment for

24 hours was active in reducing infection by Fusarium moniliforme in seed maize by reducing it from 68% to 18%. This drastic reduction is likely to be the result of the antifungal effect of azadirachtin in the neem seed extract. Similar results were obtained by Masum et al. (2009) in which neem seed extract reduced incidence of F. moniliforme and other seedborne fungal infection in sorghum. The result also showed that neem seed extract’s effectiveness in controlling F. moniliforme is closer to that of the chemical fungicide (mancozeb). Neem seed extract treatment for 1 and

24 hours eliminated Acremonium strictum. It also eliminated Botryodiplodia theobromae at all soaking periods indicating effectiveness of azadirachtin against the two fungal species. Germination in seeds treated with neem seed extract was higher than the untreated seeds except for the 24 hours treatment which was slightly lower.

The increase in germination may be due to a reduction in fungal pathogen infection of the seeds by azadirachtin in the neem seed extract. However, germination decreased as soaking time in neem seed extracts increased. Over long periods of treatment, the active ingredients in the extract may become toxic to the delicate developing embryo. Similar report was given by Latif et al. (2006) in which germination of mustard seeds treated with neem extract decreased gradually with time of treatment (ie. soaking period). Vigour was found to be much lower in neem

45 seed extract treated seed maize (8.0 and 30.0% for 3 and 6 hour treatments respectively) compared with mancozeb treated seed maize (54.5 and 63.5% for 3 and

6 hour treatments respectively) but not highly different from that of untreated seeds.

Low vigour in seeds treated with neem seed extract has been reported to be due to presence of terpenoids in neem seeds. These terpenoids are known to inhibit coleoptile development (Saab et al., 1992).

There was a slight increase in incidence of F. moniliforme when seed maize was treated with lemongrass extract for one hour (68.5%) compared with the untreated seeds (68%). There was also a remarkable increase in Acremonium strictum incidence in treatment for 1 hour (4.0%), 3 hours (8.5%) and 6 hours (5.0%) compared with the untreated seeds (3.5%). This observed increase in incidence is a possible indication of certain growth factors being present in lemongrass that support fungal growth. De Matouschek (1991) reported that lemongrass contains fructose, sucrose, and octacosanol and these are nutritive to fungi. Yasmin et al. (2008) also reported that some plant extracts stimulate growth of F. moniliforme at a given concentration and soaking period. However, seeds treated with lemongrass extract for 3, 6 and 24 hours showed decrease in F. moniliforme incidence with 24 hours treatment showing significant decrease to 39% compared with the untreated seeds.

Somda et al. (2007) also found out that lemongrass extract reduced F. moniliforme infection in sorghum. Lemongrass oil was also reported to possess antifungal activity capable of controlling postharvest pathogens (Tzortzakis and Economakis, 2007).

Treatment with lemongrass increased germination percentage to 79% compared to the untreated. Mancozeb gave a comparable germination percentage of 80%.

Lemongrass contains compounds such as citral, citronellal, geranic acid and other monoterpenes that may promote coleoptile development or results in enhanced

46 germination and vigour (Kasumov and Babaev, 1983; Torres, 1993; and Masuda et al., 2008). Seed treatment with lemongrass extract again resulted in increased in vigour with 3 hours treatment providing the most vigorous seedlings of 55.5% which is comparable to mancozeb treatment. The increase in seedling vigour may be as a result of the presence of potassium in lemongrass (Livestrong.com, 2010) that induced vigour. These results are similar to that of Somda et al., (2007) where treatment with lemongrass extract promoted seedling emergence in sorghum.

Seed maize treated with Chromolaena odorata leaf extract showed reduction in the incidence of F. moniliforme compared with the untreated seeds. It also showed reduction in incidence of A. strictum and B. theobromae for 1 hour compared with the untreated seeds (Tables 4.5 & 4.6). C. odorata has antifungal properties due to presence of sesquiterpenes in the leaves. These compounds may be responsible for the antifungal activities exhibited against the fungal pathogens in this study. The presence of these sesquiterpenes compounds as antifungal in C. odorata has been reported by Ardcharawan (2005). This antifungal activity exhibited by C. odorata agrees with a report by Begum et al. (2007) in which six phytopathogenic fungi including Fusarium equiseti studied were reduced more than 50%. Germination of seed maize treated with C. odorata leaf extract was very high (76.0, 73.0, and 73.5 % for 1, 3 and 6 hour treatments respectively ) which is closer to seeds treated with chemical fungicide, mancozeb, and decreases as soaking period increases. Seedling vigour was also higher than that of the untreated seeds with vigour percentages of 48,

51.5 and 31.5 percent for 3, 6 and 24 hours of treatment respectively.

47

6.0 CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusions

Fusarium moniliforme, Botryodiplodia theobromae, Acremonium strictum and

Bipolaris maydis are the seedborne fungal pathogens of seed maize collected from the ecological zones. The incidence of these fungal pathogens varied in the three ecological zones with Fusarium moniliforme as the commonest seedborne fungal pathogen in the ecological zones studied.

Generally, incidence of seedborne pathogenic fungi infection reduces as soaking period increased at a specific weight to volume concentration, w/v of a given plant extract. It was observed that the incidence of F. moniliforme, B. theobromae and A. strictum generally decreased with increasing soaking period which means that the pathogens were controlled better at 50% w/v botanical extract if the treatment period is increased. Neem seed extract provided the best treatment for the control of pathogens found in the seed maize among the three botanicals. Chromolaena odorata leaf extract provided moderate control of F. moniliforme and B. theobromae.

On the other hand, inhibition of germination was increased as soaking period increased from 3 to 24 hours for almost all the treatments. Chromolaena odorata leaf extract provided high germination of seed maize and moderate seed vigour in maize seedling. Neem seed extract showed high germination rate as compared with the controls, untreated seeds and mancozeb treated seeds, but its effect on vigour was low. Although seed treatment with lemongrass gave the best germination and vigour that can be compared with chemical seed treatment, its control of F. moniliforme and

A. strictum infection was poor.

48

6.2 Recommendations

It is recommended that neem seed extract could be used for treating seed maize infected by F. moniliforme, B. theobromae and A. strictum. It must be noted however, that it does not disinfect infected seeds completely.

Chromolaena odorata leaf extract could be used to treat seeds against moderate infections of F. moniliforme particularly in localities where neem seed is hard to come by.

It is recommended that field trials should be performed to ascertain the effects of environmental factors on the efficacy of botanical extracts before they are recommended to farmers.

49

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APPENDICES

Appendix 1: ANOVA for Frequency of Infection of Seed Maize by Fungal Pathogens (Forest Zone)

Variation in Forest zone

Parameter F-values LSD @5% CV (%) SD

Fusarium moniliforme 9.04 23.46 30.10 7.90 P < 0.001

Botryodiplodia theobromae 11.67 5.16 52.90 1.74 P < 0.001

Acremonium strictum 18.55 3.62 89.90 1.22 P < 0.001

Bipolaris maydis 1.00 0.70 600.00 0.24 P < 0.001

Appendix 2: ANOVA for Frequency of Infection of Seed Maize by Fungal Pathogens (Transition Zone)

Variation in Transition zone

Parameter F-values LSD @5% CV (%) SD

Fusarium moniliforme 14.45 15.97 15.40 5.38 P < 0.001

Botryodiplodia theobromae 4.07 3.93 102.00 1.32 P < 0.002

Acremonium strictum 2.73 7.53 96.60 2.54 P < 0.020

Bipolaris maydis 0 0 0 0

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Appendix 3: ANOVA Frequency of Infection of Seed Maize by Fungal Pathogens (Guinea Savannah Zone)

Variation in Guinea Savannah zone

Parameter F-values LSD @5% CV (%) SD

Fusarium moniliforme 8.60 16.78 19.60 5.65 P < 0.001

Botryodiplodia theobromae 3.74 6.81 53.50 2.29 P < 0.004

Acremonium strictum 1.09 2.27 176.70 0.76 P < 0.430

Bipolaris maydis 0.93 1.16 398.00 0.39 P < 0.562

Appendix 4: ANOVA for Effects of Treatments on the incidence of Fusarium moniliforme, Acremonium strictum and Botryodiplodia theobromae

Variation in treatments

Parameter F-values LSD @5% CV (%) SD

Fusarium moniliforme 28.45 12.91 15.10 4.30 P < 0.001

Acremonium strictum 2.30 5.41 104.60 1.80 P < 0.055

Botryodiplodia theobromae 3.57 0.99 136.10 0.33 P < 0.008

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Appendix 5: ANOVA for Effects of Treatments on Germination and Vigour of Seed Maize

Variation in treatments

Parameter F-values LSD @5% CV (%) SD

Germination 2.25 9.63 6.20 3.21 P < 0.050

Vigour 17.20 11.40 15.40 3.80 P < 0.001

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