# Management of Major Seed-Borne Fungi of Cowpea (Vigna unguiculata (L.) Walp) with Four Selected Botanical Extracts

Authors: Eric Gyasi; Daniel A. Kotey; Barnabas A. Adongo; Fuleratu K. Adams; Edmund O. Owusu; Abdul Mohammed
Journal: Advances in Agriculture
 (2022)

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## Abstract

Seed-borne fungal pathogens present significant constrain to the production and conservation of many seed crops including cowpea. Infection of mature seeds by such pathogens could result in mycotoxin contamination, loss of viability, and decay of seeds. This study aimed to identify seed-borne fungi on 200 accessions of cowpea under cold storage at CSIR-Plant Genetic Resources Research Institute (PGRRI), Ghana. Also, the antifungal effect of seeds ofPiper nigrum, Xylopia aethiopica, Aframomum melegueta, and fresh leaves of Cymbopogon citratus aqueous extracts (100% w/v) on the major seed-borne fungi identified on the cowpea seeds was determined. Seven fungal species belonging to five genera were identified from the seeds of the cowpea accessions evaluated. However, the diversity and infection levels of the pathogenic fungi recorded on the seeds were lower than that of the saprophytic fungi indicating minimal capacity of the seeds to spread pathogenic fungi on the field. Aqueous extract of Aframomum melegueta inhibited the growth of Fusarium verticillioides by 98.40%, Colletotrichum sp. by 97.83%, Aspergillus niger by 94.70%, and Aspergillus flavus by 63.38%. The only other aqueous extract that inhibited the colony growth above 60% was that of Piper nigrum which inhibited colony growth of Fusarium verticillioides by 71.7% and Colletotrichum sp. by 63.47%. Due to the benign effect of Aframomum melegueta extract on the environment and non-target organisms, its use as a seed protectant is highly recommended. Further studies to establish the spectrum of activity and dose levels of Aframomum melegueta extract are recommended.


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## Body

## 1. Introduction

Cowpea (Vigna unguiculata (L.) Walp) is a leguminous crop which due to its high protein content, is of nutritional benefit in most parts of the world [1, 2]. In Sub-Saharan Africa, the production of the crop serves as a source of income for millions of smallholder farmers and traders [3]. In Ghana, cowpea is an important staple crop and is cultivated extensively in the savanna and transitional agroecological zones of the country [4]. Whilst 26 cowpea varieties have been released and registered in Ghana, the majority of the seeds used for cultivation are recycled seeds of landraces. As a way of preventing the diversity of cowpea in Ghana from being irreversibly lost, the CSIR-PGRRI, the national genebank of Ghana, maintains a collection of cowpea germplasm at its seedbank at Bunso in the Eastern Region of Ghana. At the CSIR-PGRRI, the genetic resources of cowpea are conserved as seeds in cold storage.Amongst biotic factors that constrain production, seed-borne fungi have been identified as significant in many production areas. According to [5], cowpea seeds in the field or in storage are susceptible to infection by seed-borne pathogens which usually result in mycotoxin contamination, loss of viability, and seed rot. The planting of infected seeds may also result in disease outbreaks and loss of yield. Due to these adverse impacts, seed-borne pathogens are a serious threat to cowpea seed conservation and cultivation. Seed-borne fungi are either saprophytic or pathogenic. Saprophytic seed-borne fungi affect seeds during storage and cause seed discoloration, reduced seed weight, and seed germination. Pathogenic seed-borne fungi, on the other hand, infect seeds in the field and reduce seed vigour, weaken the plant at its initial growth stages and cause disease epidemics in the field [6, 7]. Various seed-borne fungal species have been reported to be associated with cowpea seeds in different parts of the world. Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Alternaria alternata, and Penicillium sp. were found to be associated with cowpea seeds in India [8]. The authors in [9] identified Fusarium verticillioides, Fusarium solani, Macrophomina phaseolina, and Phoma sp. on cowpea seeds in Sierra Leone. Aspergillus niger, Aspergillus flavus, Botryodiplodia theobromae, Fusarium oxysporum, Penicillium sp., and Rhizopus stolonifer have also been reported to be associated with cowpea seeds [5, 10, 11]. The control of seed-borne fungi using synthetic fungicides is widely practiced in Ghana. Although the use of synthetic fungicides is effective, the approach has the tendency to result in the unintended accumulation of toxic fungicide residues in the ecosystem which may also induce resistance in pathogens [12–14]. Due to the potential adverse effects resulting from the use of synthetic fungicides, there is a shift in focus to the identification of alternative approaches, including the use of botanical extracts, to effectively manage seed-borne pathogens without any harm to the ecosystem and applicators. The use of botanical extracts in the control of seed-borne fungi has been reported as effective, low-cost, readily available, and environmentally safe [15–18]. Several studies have been carried out to evaluate the antifungal effects of aqueous extracts of black pepper (Piper nigrum), Ethiopian pepper (Xylopia aethiopica), lemon grass (Cymbopogon citratus), and grains of paradise (Aframomum melegueta) on fungal pathogens [19–25]. [21] studied the antifungal activity of extracts of scent leaf (Ocimum gratissimum) and alligator pepper (Aframomum melegueta) at 100% concentration on the postharvest decay of carrots and reported that Aframomum melegueta aqueous extract possessed a stronger antifungal property against the mycelial growth of Fusarium species and other fungi. The authors in [23] and [26] also found that Aframomum melegueta extract showed high toxicity against some plant fungal pathogens. The authors in [19] used seeds of black pepper (Piper nigrum), rhizomes of ginger (Zingiber officinale), leaves of neem (Azadirachta indica), leaves of pawpaw (Carica papaya), and leaves of tobacco (Nicotiana tabacum) extracts to control Penicillium expansum and concluded that Piper nigrum showed high toxicity against the test fungus at 90% concentration. [27] also reported that Piper nigrum showed high antimicrobial activity against plant pathogens. [20] and [22] reported that Xylopia aethiopica showed some level of toxicity against some seed-borne fungi on cowpea seeds. The authors [28] evaluated the antifungal efficacy of 21 plant extracts against the seed-borne fungus Colletotrichum lindemuthianum and reported that lemon grass was less effective compared to the other plant extracts. This study aimed (i) to identify seed-borne fungi of 200 cowpea accessions in cold storage at the CSIR-PGRRI, Bunso and (ii) to assess the antifungal effects of black pepper (Piper nigrum), Ethiopian pepper (Xylopia aethiopica), lemon grass (Cymbopogon citratus), and grains of paradise (Aframomum melegueta) extracts on the major seed-borne fungi identified.


## 2. Materials and Methods

### 2.1. Collection of Cowpea Seed Samples and Seed Health Testing

Samples of seeds of 200 randomly selected cowpea accessions were obtained from the stock conserved under cold storage conditions (−20°C) at the CSIR-PGRRI, Bunso in February, 2019. The selected seed samples were examined for the presence of seed-borne fungi using the blotter method [29]. One hundred (100) seeds, randomly selected from each of the cowpea accession, were surface sterilised in 5% sodium hypochlorite solution for 2–3 minutes and rinsed three times with sterilised distilled water. Ten (10) seeds were then plated on sterilized, moistened three-ply blotter paper in Petri dishes and replicated ten times (Figure 1). The plates were placed on a laboratory bench under a 12 h light and a 12 h darkness photoperiod for seven (7) days. On the eighth day, each incubated seed was examined under a stereo-binocular microscope. All the seed-borne fungi on plated seeds were identified based on the colony morphology, colour of mycelium, and the shape and other characteristics of conidia produced with the aid of the laboratory manuals developed by [30] and [31]. The diversity of fungi, the incidence of each fungus, and the infection range were recorded. The incidence of each type of fungus identified was calculated using the following formula:(1)fungal incidence=number of infected samplestotal number of samples tested×100%.Figure 1
Cowpea seeds plated on moistened filter papers in Petri dishes.The infection level of each fungus for each accession was also calculated using the formula:(2)infection level=number of seeds infected by each fungus in an accessiontotal number of seeds tested per accession×100.


### 2.2. Culturing of Identified Seed-Borne Fungi on PDA

Fusarium verticillioides, Colletotrichum sp., Aspergillus flavus, and Aspergillus niger identified on the cowpea seeds were the fungal isolates used in the efficacy studies of the botanical extracts. Small portions of mycelia of the four fungal isolates were placed on potato dextrose agar (PDA) in Petri dishes and incubated at 28 ± 2°C for seven (7) days. Single conidia culturing was carried out to obtain pure cultures of the four fungal isolates for further studies.


### 2.3. Sources of Plant Products (Botanical Materials)

The seeds of the black pepper (Piper nigrum), Ethiopian pepper (Xylopia aethiopica), and grains of paradise (Aframomum melegueta) used in this study were obtained from the conservation fields of the CSIR-PGRRI, Bunso. Fresh leaves of lemon grass (Cymbopogon citratus) were also obtained from households within the Bunso community (Figure 2).Figure 2
Botanical materials used: (a) black pepper (Piper nigrum), (b) Ethiopian pepper (Xylopia aethiopica), (c) grains of paradise (Aframomum melegueta), and (d) lemon grass (Cymbopogon citratus).
(a)(b)(c)(d)


### 2.4. Preparation of the Botanical Aqueous Extracts

#### 2.4.1. Preparation of Botanical Seed Extracts

Seeds of black pepper (Piper nigrum), Ethiopian pepper (Xylopia aethiopica), and grains of paradise (Aframomum melegueta) were washed thoroughly with distilled water and soaked in a 5% sodium hypochlorite solution for 10 minutes. The seeds were then rinsed three times using sterilised distilled water in the laboratory and air dried in a laminar flow hood for 30 minutes. Fifty grams (50 g) of the seeds of each of the botanicals were weighed and ground using an electric blender (ELBEE, 2 in 1, LB-47) until a fine powder was obtained. The powder obtained from each of the plant species was separately poured into different beakers, after which 50 ml of sterilised distilled water was added to each and stirred thoroughly with a sterilised glass rod. After stirring, the contents of each beaker were left to stand for one hour. Each of the extract was then filtered through folds of sterilised cheese cloth to obtain an aqueous extract with a concentration of 100% (w/v) (Figures 3(a), 3(c), and 3(d)). The procedure described above was carried out in the laminar flow hood.Figure 3
Botanical aqueous extracts: (a)Aframomum melegueta, (b) Cymbopogon citratus, (c) Piper nigrum, and (d) Xylopia aethiopica.


#### 2.4.2. Preparation of the Botanical Leaf Extract

Fresh leaves of lemon grass (Cymbopogon citratus) were cut into pieces with a knife and washed thoroughly with distilled water. The cut leaves were soaked in a 5% sodium hypochlorite solution for 10 minutes and rinsed in sterilised distilled water three times in the laboratory. The leaves were air dried in the laminar flow hood for 30 minutes. After air drying, 50 g of the leaves were weighed and blended using an electric blender (ELBEE, 2 in 1, LB-47) in the laboratory. The paste obtained from blended leaves was put into a beaker and 50 ml of sterilised distilled water was added to it, stirred thoroughly with a sterilised glass rod and left to stand for one hour. The resulting solution was then filtered through folds of sterilised cheese cloth to obtain an aqueous extract with a concentration of 100% (w/v) (Figure 3(b)).


### 2.5. Management of Fungal Isolates with Prepared Aqueous Botanical Extracts

A modified poisoned food technique method by [32] was used to determine the effect of the aqueous botanical extracts on fungal growth. Sixty presterilised Petri dishes were divided into four equal sections by drawing two perpendicular lines at the bottom of each Petri dish before dispensing potato dextrose agar (PDA) into each. About 5 ml of the aqueous extract of each plant species was separately introduced into each Petri dish, and 20 ml of PDA was added and shaken gently until uniform mixtures were obtained in each dish placed in a laminar flow hood. The contents of each dish were allowed to solidify, after which a seven-day old disc (4 mm in diameter) of the pure culture of fungal isolates (Fusarium verticillioides, Colletotrichum species, Aspergillus flavus, and Aspergillus niger) was placed on the solidified botanical extract and PDA mixture just at the point of intersection of the two lines drawn at the bottom of each Petri dish. Control experiments were set up without the addition of any botanical aqueous extract. The experimental design used was factorial arranged in a completely randomised design with three replications. The effectiveness of the botanical aqueous extracts was recorded in terms of percentage colony inhibition of the fungal isolates and calculated according to the formula [33]:(3)growth inhibition%=DC−DTDC×100%,where DC = average diameter of control; DT = average diameter of fungal colony with treatment.


### 2.6. Data Analysis

The data obtained from the evaluation of the botanical materials was subjected to an analysis of variance (ANOVA) using the Statistix statistical package, edition 10.0. Differences in treatment means were compared for significance using the least significance difference (LSD) at a probability level of 5%.


## 2.1. Collection of Cowpea Seed Samples and Seed Health Testing

Samples of seeds of 200 randomly selected cowpea accessions were obtained from the stock conserved under cold storage conditions (−20°C) at the CSIR-PGRRI, Bunso in February, 2019. The selected seed samples were examined for the presence of seed-borne fungi using the blotter method [29]. One hundred (100) seeds, randomly selected from each of the cowpea accession, were surface sterilised in 5% sodium hypochlorite solution for 2–3 minutes and rinsed three times with sterilised distilled water. Ten (10) seeds were then plated on sterilized, moistened three-ply blotter paper in Petri dishes and replicated ten times (Figure 1). The plates were placed on a laboratory bench under a 12 h light and a 12 h darkness photoperiod for seven (7) days. On the eighth day, each incubated seed was examined under a stereo-binocular microscope. All the seed-borne fungi on plated seeds were identified based on the colony morphology, colour of mycelium, and the shape and other characteristics of conidia produced with the aid of the laboratory manuals developed by [30] and [31]. The diversity of fungi, the incidence of each fungus, and the infection range were recorded. The incidence of each type of fungus identified was calculated using the following formula:(1)fungal incidence=number of infected samplestotal number of samples tested×100%.Figure 1
Cowpea seeds plated on moistened filter papers in Petri dishes.The infection level of each fungus for each accession was also calculated using the formula:(2)infection level=number of seeds infected by each fungus in an accessiontotal number of seeds tested per accession×100.


## 2.2. Culturing of Identified Seed-Borne Fungi on PDA

Fusarium verticillioides, Colletotrichum sp., Aspergillus flavus, and Aspergillus niger identified on the cowpea seeds were the fungal isolates used in the efficacy studies of the botanical extracts. Small portions of mycelia of the four fungal isolates were placed on potato dextrose agar (PDA) in Petri dishes and incubated at 28 ± 2°C for seven (7) days. Single conidia culturing was carried out to obtain pure cultures of the four fungal isolates for further studies.


## 2.3. Sources of Plant Products (Botanical Materials)

The seeds of the black pepper (Piper nigrum), Ethiopian pepper (Xylopia aethiopica), and grains of paradise (Aframomum melegueta) used in this study were obtained from the conservation fields of the CSIR-PGRRI, Bunso. Fresh leaves of lemon grass (Cymbopogon citratus) were also obtained from households within the Bunso community (Figure 2).Figure 2
Botanical materials used: (a) black pepper (Piper nigrum), (b) Ethiopian pepper (Xylopia aethiopica), (c) grains of paradise (Aframomum melegueta), and (d) lemon grass (Cymbopogon citratus).
(a)(b)(c)(d)


## 2.4. Preparation of the Botanical Aqueous Extracts

### 2.4.1. Preparation of Botanical Seed Extracts

Seeds of black pepper (Piper nigrum), Ethiopian pepper (Xylopia aethiopica), and grains of paradise (Aframomum melegueta) were washed thoroughly with distilled water and soaked in a 5% sodium hypochlorite solution for 10 minutes. The seeds were then rinsed three times using sterilised distilled water in the laboratory and air dried in a laminar flow hood for 30 minutes. Fifty grams (50 g) of the seeds of each of the botanicals were weighed and ground using an electric blender (ELBEE, 2 in 1, LB-47) until a fine powder was obtained. The powder obtained from each of the plant species was separately poured into different beakers, after which 50 ml of sterilised distilled water was added to each and stirred thoroughly with a sterilised glass rod. After stirring, the contents of each beaker were left to stand for one hour. Each of the extract was then filtered through folds of sterilised cheese cloth to obtain an aqueous extract with a concentration of 100% (w/v) (Figures 3(a), 3(c), and 3(d)). The procedure described above was carried out in the laminar flow hood.Figure 3
Botanical aqueous extracts: (a)Aframomum melegueta, (b) Cymbopogon citratus, (c) Piper nigrum, and (d) Xylopia aethiopica.


### 2.4.2. Preparation of the Botanical Leaf Extract

Fresh leaves of lemon grass (Cymbopogon citratus) were cut into pieces with a knife and washed thoroughly with distilled water. The cut leaves were soaked in a 5% sodium hypochlorite solution for 10 minutes and rinsed in sterilised distilled water three times in the laboratory. The leaves were air dried in the laminar flow hood for 30 minutes. After air drying, 50 g of the leaves were weighed and blended using an electric blender (ELBEE, 2 in 1, LB-47) in the laboratory. The paste obtained from blended leaves was put into a beaker and 50 ml of sterilised distilled water was added to it, stirred thoroughly with a sterilised glass rod and left to stand for one hour. The resulting solution was then filtered through folds of sterilised cheese cloth to obtain an aqueous extract with a concentration of 100% (w/v) (Figure 3(b)).


## 2.4.1. Preparation of Botanical Seed Extracts

Seeds of black pepper (Piper nigrum), Ethiopian pepper (Xylopia aethiopica), and grains of paradise (Aframomum melegueta) were washed thoroughly with distilled water and soaked in a 5% sodium hypochlorite solution for 10 minutes. The seeds were then rinsed three times using sterilised distilled water in the laboratory and air dried in a laminar flow hood for 30 minutes. Fifty grams (50 g) of the seeds of each of the botanicals were weighed and ground using an electric blender (ELBEE, 2 in 1, LB-47) until a fine powder was obtained. The powder obtained from each of the plant species was separately poured into different beakers, after which 50 ml of sterilised distilled water was added to each and stirred thoroughly with a sterilised glass rod. After stirring, the contents of each beaker were left to stand for one hour. Each of the extract was then filtered through folds of sterilised cheese cloth to obtain an aqueous extract with a concentration of 100% (w/v) (Figures 3(a), 3(c), and 3(d)). The procedure described above was carried out in the laminar flow hood.Figure 3
Botanical aqueous extracts: (a)Aframomum melegueta, (b) Cymbopogon citratus, (c) Piper nigrum, and (d) Xylopia aethiopica.


## 2.4.2. Preparation of the Botanical Leaf Extract

Fresh leaves of lemon grass (Cymbopogon citratus) were cut into pieces with a knife and washed thoroughly with distilled water. The cut leaves were soaked in a 5% sodium hypochlorite solution for 10 minutes and rinsed in sterilised distilled water three times in the laboratory. The leaves were air dried in the laminar flow hood for 30 minutes. After air drying, 50 g of the leaves were weighed and blended using an electric blender (ELBEE, 2 in 1, LB-47) in the laboratory. The paste obtained from blended leaves was put into a beaker and 50 ml of sterilised distilled water was added to it, stirred thoroughly with a sterilised glass rod and left to stand for one hour. The resulting solution was then filtered through folds of sterilised cheese cloth to obtain an aqueous extract with a concentration of 100% (w/v) (Figure 3(b)).


## 2.5. Management of Fungal Isolates with Prepared Aqueous Botanical Extracts

A modified poisoned food technique method by [32] was used to determine the effect of the aqueous botanical extracts on fungal growth. Sixty presterilised Petri dishes were divided into four equal sections by drawing two perpendicular lines at the bottom of each Petri dish before dispensing potato dextrose agar (PDA) into each. About 5 ml of the aqueous extract of each plant species was separately introduced into each Petri dish, and 20 ml of PDA was added and shaken gently until uniform mixtures were obtained in each dish placed in a laminar flow hood. The contents of each dish were allowed to solidify, after which a seven-day old disc (4 mm in diameter) of the pure culture of fungal isolates (Fusarium verticillioides, Colletotrichum species, Aspergillus flavus, and Aspergillus niger) was placed on the solidified botanical extract and PDA mixture just at the point of intersection of the two lines drawn at the bottom of each Petri dish. Control experiments were set up without the addition of any botanical aqueous extract. The experimental design used was factorial arranged in a completely randomised design with three replications. The effectiveness of the botanical aqueous extracts was recorded in terms of percentage colony inhibition of the fungal isolates and calculated according to the formula [33]:(3)growth inhibition%=DC−DTDC×100%,where DC = average diameter of control; DT = average diameter of fungal colony with treatment.


## 2.6. Data Analysis

The data obtained from the evaluation of the botanical materials was subjected to an analysis of variance (ANOVA) using the Statistix statistical package, edition 10.0. Differences in treatment means were compared for significance using the least significance difference (LSD) at a probability level of 5%.


## 3. Results

### 3.1. Identified Seed-Borne Fungi on the Cowpea Seeds

Seven fungal species belonging to five genera were identified from the 200 cowpea accessions evaluated. These included five saprophytic fungi;Aspergillus flavus, Aspergillus niger, Aspergillus tamari, Penicillium sp., and Rhizopus stolonifer, and two pathogenic fungi such as Fusarium verticillioides and Colletotrichum sp. The saprophytic fungus, Aspergillus flavus, recorded the highest fungal incidence of 54.5%, with an infection level ranging from 3.0 to 57.0%. The lowest fungal incidence was recorded by Aspergillus tamari (1.0%), with an infection range of 6.0–10.0%. Lower diversity and infection levels of the pathogenic fungi were recorded on the cowpea seeds as compared to the saprophytic fungi (Table 1).Table 1
Incidence of infection (%) and infection level (%) by fungal species from cowpea seed samples.
Fungal speciesTotal no. of accessions evaluatedNumber of infested accessionsIncidence of fungi (%)Infection level (%)Aspergillus flavus20010954.53.0–57.0Aspergillus niger2005226.03.0–100.0Penicillium sp.2003718.53.0–14.0Aspergillus tamari20021.06.0–10.0Rhizopus stolonifer20042.03.0–10.0Fusarium verticillioides2009748.53.0–34.0Colletotrichum sp.20042.06.0–10.0


### 3.2. Antifungal Effect of the Aqueous Botanical Extracts on the Major Seed-Borne Fungi

Significant differences were observed between the botanical aqueous extracts and the control.Aframomum melegueta aqueous extract inhibited the colony growth of Fusarium verticillioides by 98.4%. This was followed by black pepper (Piper nigrum) aqueous extract (71.7% inhibition), Ethiopian pepper (Xylopia aethiopica) aqueous extract (25.6% inhibition), and lemon grass (Cymbopogon citratus) aqueous extract (17.23%). However, there was no significant difference between the colony inhibition of Fusarium verticillioides by Xylopia aethiopica and Cymbopogon citratus aqueous extracts.ForColletotrichum species inhibition by the aqueous botanical extracts, there were significant differences between the botanical extracts and the control. Aframomum melegueta aqueous extract recorded the highest inhibition of 97.83% of Colletotrichum species colony growth. This was followed by black pepper (Piper nigrum) aqueous extract with a 63.47% inhibition and then Xylopia aethiopica extract with a 32.17% inhibition. The lowest inhibition (10%) of colony growth of Colletotrichum species was recorded by aqueous lemon grass (Cymbopogon citratus) extract.Significant differences were also observed between the aqueous botanical extracts and the control in their inhibition ofAspergillus niger. Grains of paradise (Aframomum melegueta) aqueous extract recorded the highest inhibition of Aspergillus niger colony growth of 94.7%. This was followed by Ethiopian pepper (Xylopia aethiopica) aqueous extract (35% inhibition) then lemon grass (Cymbopogon citratus) aqueous extract (21.83%). The lowest inhibition (3.0%) was recorded by black pepper (Piper nigrum) aqueous extract.With regards to the inhibition ofAspergillus flavus by the aqueous botanical extracts, significant differences were also observed between the botanical aqueous extracts and the control. The highest inhibition of colony growth of Aspergillus flavus (63.38%) was recorded by Aframomum melegueta extract. This was followed by lemon grass (Cymbopogon citratus) aqueous extract with 14.37% inhibition then Xylopia aethiopica aqueous extract with 8% inhibition. The lowest colony growth inhibition (1.7%) of Aspergillus flavus was recorded by black pepper aqueous extract (Figure 4; Table 2).Figure 4
Growth inhibition byAframomum melegueta extract and their corresponding controls: (a) Fusarium verticillioides, (b) Colletotrichum species, (c) Aspergillus niger, and (d) Aspergillus flavus.
(a)(b)(c)(d)Table 2
Inhibition of fungal growth by the aqueous botanical extracts.
TreatmentPercentage inhibition of fungal growth (%)F. verticillioidesColletotrichum speciesA. nigerA. flavusAframomum98.40a97.83a94.70a63.38aBlack pepper71.70b63.47b3.00d1.70dXylopia25.60c32.17c35.0b8.00cLemon grass17.23c10.00d21.83c14.37bControl (PDA)0.00d0.00e0.00e0.00eLsd (5%)15.512.411.191.58CV (%)19.343.152.044.80Each value is the mean of three replicates. Values with different letters are significantly different atp<0.05.


## 3.1. Identified Seed-Borne Fungi on the Cowpea Seeds

Seven fungal species belonging to five genera were identified from the 200 cowpea accessions evaluated. These included five saprophytic fungi;Aspergillus flavus, Aspergillus niger, Aspergillus tamari, Penicillium sp., and Rhizopus stolonifer, and two pathogenic fungi such as Fusarium verticillioides and Colletotrichum sp. The saprophytic fungus, Aspergillus flavus, recorded the highest fungal incidence of 54.5%, with an infection level ranging from 3.0 to 57.0%. The lowest fungal incidence was recorded by Aspergillus tamari (1.0%), with an infection range of 6.0–10.0%. Lower diversity and infection levels of the pathogenic fungi were recorded on the cowpea seeds as compared to the saprophytic fungi (Table 1).Table 1
Incidence of infection (%) and infection level (%) by fungal species from cowpea seed samples.
Fungal speciesTotal no. of accessions evaluatedNumber of infested accessionsIncidence of fungi (%)Infection level (%)Aspergillus flavus20010954.53.0–57.0Aspergillus niger2005226.03.0–100.0Penicillium sp.2003718.53.0–14.0Aspergillus tamari20021.06.0–10.0Rhizopus stolonifer20042.03.0–10.0Fusarium verticillioides2009748.53.0–34.0Colletotrichum sp.20042.06.0–10.0


## 3.2. Antifungal Effect of the Aqueous Botanical Extracts on the Major Seed-Borne Fungi

Significant differences were observed between the botanical aqueous extracts and the control.Aframomum melegueta aqueous extract inhibited the colony growth of Fusarium verticillioides by 98.4%. This was followed by black pepper (Piper nigrum) aqueous extract (71.7% inhibition), Ethiopian pepper (Xylopia aethiopica) aqueous extract (25.6% inhibition), and lemon grass (Cymbopogon citratus) aqueous extract (17.23%). However, there was no significant difference between the colony inhibition of Fusarium verticillioides by Xylopia aethiopica and Cymbopogon citratus aqueous extracts.ForColletotrichum species inhibition by the aqueous botanical extracts, there were significant differences between the botanical extracts and the control. Aframomum melegueta aqueous extract recorded the highest inhibition of 97.83% of Colletotrichum species colony growth. This was followed by black pepper (Piper nigrum) aqueous extract with a 63.47% inhibition and then Xylopia aethiopica extract with a 32.17% inhibition. The lowest inhibition (10%) of colony growth of Colletotrichum species was recorded by aqueous lemon grass (Cymbopogon citratus) extract.Significant differences were also observed between the aqueous botanical extracts and the control in their inhibition ofAspergillus niger. Grains of paradise (Aframomum melegueta) aqueous extract recorded the highest inhibition of Aspergillus niger colony growth of 94.7%. This was followed by Ethiopian pepper (Xylopia aethiopica) aqueous extract (35% inhibition) then lemon grass (Cymbopogon citratus) aqueous extract (21.83%). The lowest inhibition (3.0%) was recorded by black pepper (Piper nigrum) aqueous extract.With regards to the inhibition ofAspergillus flavus by the aqueous botanical extracts, significant differences were also observed between the botanical aqueous extracts and the control. The highest inhibition of colony growth of Aspergillus flavus (63.38%) was recorded by Aframomum melegueta extract. This was followed by lemon grass (Cymbopogon citratus) aqueous extract with 14.37% inhibition then Xylopia aethiopica aqueous extract with 8% inhibition. The lowest colony growth inhibition (1.7%) of Aspergillus flavus was recorded by black pepper aqueous extract (Figure 4; Table 2).Figure 4
Growth inhibition byAframomum melegueta extract and their corresponding controls: (a) Fusarium verticillioides, (b) Colletotrichum species, (c) Aspergillus niger, and (d) Aspergillus flavus.
(a)(b)(c)(d)Table 2
Inhibition of fungal growth by the aqueous botanical extracts.
TreatmentPercentage inhibition of fungal growth (%)F. verticillioidesColletotrichum speciesA. nigerA. flavusAframomum98.40a97.83a94.70a63.38aBlack pepper71.70b63.47b3.00d1.70dXylopia25.60c32.17c35.0b8.00cLemon grass17.23c10.00d21.83c14.37bControl (PDA)0.00d0.00e0.00e0.00eLsd (5%)15.512.411.191.58CV (%)19.343.152.044.80Each value is the mean of three replicates. Values with different letters are significantly different atp<0.05.


## 4. Discussion

### 4.1. Identified Seed-Borne Fungi on the Cowpea Seeds

The seed-borne fungi identified from cowpea seeds have also been reported to be associated with cowpea seeds in different studies.Aspergillus niger, Aspergillus flavus, and Penicillium sp. were found to be associated with cowpea seeds in a study by [8] in India. [9] identified Fusarium verticillioides together with some other fungal species on cowpea seeds in their studies on seed-borne fungi of cowpea in Sierra Leone. The authors [20] in their studies on seed-borne mycoflora of stored cowpea in Nigeria identified Colletotrichum species, Fusarium species, and Aspergillus species. Aspergillus niger, Aspergillus flavus, Penicillium species, Colletotrichum species, and Rhizopus stolonifer have also been reported to be associated with cowpea seeds [5] in Botswana, [10] in Pakistan, and [11] in Nigeria.


### 4.2. Antifungal Effect of the Aqueous Botanical Extracts onFusarium verticillioides, Colletotrichum sp., Aspergillus flavus, and Aspergillus niger

Aframomum melegueta aqueous extract performed best at inhibiting both the pathogenic and saprophytic fungi identified on the cowpea seeds compared with the other aqueous extracts. This finding is in line with previous reports by other authors on the antifungal effects of Aframomum melegueta aqueous extract on plant fungal pathogens. The author [34] who evaluated the antifungal effects of 12 different plant species on the growth of Fusarium verticillioides causing rot disease of maize, concluded that, Aframomum melegueta and Piper guineense aqueous extracts were the best botanicals found to inhibit the growth of Fusarium verticillioides in his studies. The authors [21], also found that Aframomum melegueta aqueous extract possessed a stronger antifungal property against the colony growth of Fusarium species tested at 100% concentration. The authors [35] who evaluated the antifungal effects of different spices in controlling fungal pathogens isolated from rotten okra reported that Aframomum melegueta aqueous extract was effective in controlling Fusarium species, Aspergillus niger, Aspergillus flavus, Rhizopus stolonifer, Penicillium species, and Lasiodiplodia theobromae. Bamidele [36] in his study on the antifungal potency of Aframomum melegueta seed extracts on postharvest rot fungi of two citrus species also found that Aframomum melegueta aqueous extract was effective in inhibiting the colony growth of Aspergillus niger, Aspergillus flavus, Rhizopus stolonifer, and Rhizoctonia species. In their studies in Nigeria, [23], the authors found that Aframomum melegueta extract showed high toxicity against Aspergillus niger, Penicillium digitatum, Mucor piriformis, and Helminthosporium solani. Okwu and Njoku [26] similarly found that Aframomum melegueta extract showed high toxicity against some plant pathogens in their studies. Aside from its antifungal effect, Aframomum melegueta has been reported by several studies [37–40] to possess a broad spectrum of biological activities against arthropods. These effects, which may be initiated through contact, ingestion, or fumigation [41], include feeding and oviposition deterrence, repellence, growth disruption, reduced fitness, and sterility. [42] reported that essential oils of Aframomum melegueta have a repellent effect on storage insect pests. This, coupled with the findings of the current study, makes Aframomum melegueta an ideal botanical for the integrated management of storage insect pests and diseases of cowpea in smallholder settings.Although black pepper (Piper nigrum) was less effective in inhibiting the colony growth of the saprophytic fungi Aspergillus niger and Aspergillus flavus, it was more effective against the pathogenic fungi Fusarium verticillioides and Colletotrichum species. It inhibited the colony growth of Fusarium verticillioides by 71.70% and Colletotrichum species by 63.47%. These findings concur with those of [19] and [27], who found that Piper nigrum has high antimicrobial activity against plant pathogens.Xylopia aethiopica and Cymbopogon citratus also showed some level of effectiveness in the inhibition of the colony growth of the tested cowpea seed-borne fungi. It was, however, not as effective as Aframomum melegueta aqueous extract. The authors in [20] and [23] reported that Xylopia aethiopica showed some level of toxicity against some seed-borne fungi on cowpea seeds. In the studies by [28], it was found that lemon grass was less effective in inhibiting the colony growth of Colletotrichum lindemuthianum. The authors in [43] studied the antifungal activity of lemon grass against Aspergillus niger and Penicillium digitatum identified from maize seeds. They also reported that lemon grass was less effective in inhibiting the colony growth of Aspergillus niger.


## 4.1. Identified Seed-Borne Fungi on the Cowpea Seeds

The seed-borne fungi identified from cowpea seeds have also been reported to be associated with cowpea seeds in different studies.Aspergillus niger, Aspergillus flavus, and Penicillium sp. were found to be associated with cowpea seeds in a study by [8] in India. [9] identified Fusarium verticillioides together with some other fungal species on cowpea seeds in their studies on seed-borne fungi of cowpea in Sierra Leone. The authors [20] in their studies on seed-borne mycoflora of stored cowpea in Nigeria identified Colletotrichum species, Fusarium species, and Aspergillus species. Aspergillus niger, Aspergillus flavus, Penicillium species, Colletotrichum species, and Rhizopus stolonifer have also been reported to be associated with cowpea seeds [5] in Botswana, [10] in Pakistan, and [11] in Nigeria.


## 4.2. Antifungal Effect of the Aqueous Botanical Extracts onFusarium verticillioides, Colletotrichum sp., Aspergillus flavus, and Aspergillus niger

Aframomum melegueta aqueous extract performed best at inhibiting both the pathogenic and saprophytic fungi identified on the cowpea seeds compared with the other aqueous extracts. This finding is in line with previous reports by other authors on the antifungal effects of Aframomum melegueta aqueous extract on plant fungal pathogens. The author [34] who evaluated the antifungal effects of 12 different plant species on the growth of Fusarium verticillioides causing rot disease of maize, concluded that, Aframomum melegueta and Piper guineense aqueous extracts were the best botanicals found to inhibit the growth of Fusarium verticillioides in his studies. The authors [21], also found that Aframomum melegueta aqueous extract possessed a stronger antifungal property against the colony growth of Fusarium species tested at 100% concentration. The authors [35] who evaluated the antifungal effects of different spices in controlling fungal pathogens isolated from rotten okra reported that Aframomum melegueta aqueous extract was effective in controlling Fusarium species, Aspergillus niger, Aspergillus flavus, Rhizopus stolonifer, Penicillium species, and Lasiodiplodia theobromae. Bamidele [36] in his study on the antifungal potency of Aframomum melegueta seed extracts on postharvest rot fungi of two citrus species also found that Aframomum melegueta aqueous extract was effective in inhibiting the colony growth of Aspergillus niger, Aspergillus flavus, Rhizopus stolonifer, and Rhizoctonia species. In their studies in Nigeria, [23], the authors found that Aframomum melegueta extract showed high toxicity against Aspergillus niger, Penicillium digitatum, Mucor piriformis, and Helminthosporium solani. Okwu and Njoku [26] similarly found that Aframomum melegueta extract showed high toxicity against some plant pathogens in their studies. Aside from its antifungal effect, Aframomum melegueta has been reported by several studies [37–40] to possess a broad spectrum of biological activities against arthropods. These effects, which may be initiated through contact, ingestion, or fumigation [41], include feeding and oviposition deterrence, repellence, growth disruption, reduced fitness, and sterility. [42] reported that essential oils of Aframomum melegueta have a repellent effect on storage insect pests. This, coupled with the findings of the current study, makes Aframomum melegueta an ideal botanical for the integrated management of storage insect pests and diseases of cowpea in smallholder settings.Although black pepper (Piper nigrum) was less effective in inhibiting the colony growth of the saprophytic fungi Aspergillus niger and Aspergillus flavus, it was more effective against the pathogenic fungi Fusarium verticillioides and Colletotrichum species. It inhibited the colony growth of Fusarium verticillioides by 71.70% and Colletotrichum species by 63.47%. These findings concur with those of [19] and [27], who found that Piper nigrum has high antimicrobial activity against plant pathogens.Xylopia aethiopica and Cymbopogon citratus also showed some level of effectiveness in the inhibition of the colony growth of the tested cowpea seed-borne fungi. It was, however, not as effective as Aframomum melegueta aqueous extract. The authors in [20] and [23] reported that Xylopia aethiopica showed some level of toxicity against some seed-borne fungi on cowpea seeds. In the studies by [28], it was found that lemon grass was less effective in inhibiting the colony growth of Colletotrichum lindemuthianum. The authors in [43] studied the antifungal activity of lemon grass against Aspergillus niger and Penicillium digitatum identified from maize seeds. They also reported that lemon grass was less effective in inhibiting the colony growth of Aspergillus niger.


## 5. Conclusion

Seven fungal species belonging to five genera were identified from the 200 cowpea accessions in this study. However, the diversity and infection levels of the pathogenic fungi recorded on the cowpea seeds were low as compared to the saprophytic fungi, an indication of good phytosanitary practice on the regeneration fields. Evaluation of the antifungal effects of the four different botanical aqueous extracts against the major seed-borne fungi identified from the cowpea seeds showed that grains of paradise (Aframomum melegueta) aqueous extract (100% w/v) was effective in inhibiting the colony growth of the two pathogenic fungi, Fusarium verticillioides and Colletotrichum species, and the two saprophytic fungi, Aspergillus niger and Aspergillus flavus. Black pepper (Piper nigrum) aqueous extract was moderately effective, whilst Ethiopian pepper (Xylopia aethiopica) and fresh leaves of lemon grass (Cymbopogon citratus) aqueous extracts were less effective in controlling the major seed-borne fungi on cowpea accessions at the CSIR-PGRRI, Bunso.


### 5.1. Recommendations

(i)
Due to the benign effect ofAframomum melegueta on the environment and non-target organisms, the use of its extract as a seed protectant is highly recommended(ii)
Further studies to establish the spectrum of activity and dose levels ofAframomum melegueta extract are recommended


## 5.1. Recommendations

(i)
Due to the benign effect ofAframomum melegueta on the environment and non-target organisms, the use of its extract as a seed protectant is highly recommended(ii)
Further studies to establish the spectrum of activity and dose levels ofAframomum melegueta extract are recommended



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*Source: 3125240-2022-05-11.xml*