CHINHOYI UNIVERSITY OF TECHNOLOGY
Isolation and Identification of seed borne fungi from farm
served seeds, affecting groundnuts in Zimbabwe.
By
Punungwe Innocent
C18134551U
A mini project submitted in partial fulfilment of the requirements
of the degree in Biotechnology
SCHOOL OF AGRICULTURAL SCIENCES AND TECHNOLOGY
DEPARTMENT OF BIOTECHNOLOGY
Supervisor : Dr Chikwambi
Submission date : 5 July 2021
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Declaration
Student declaration:
I Punungwe Innocent (C18134551U) vow that this mini project has not been submitted to any
University or Academic institution and that it is my own work conducted under the
supervision of Doctor Z Chikwambi. All the assistance towards the completion of this work
and all the academic sources used have been duly accredited.
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Acknowledgements
I would like to thank my supervisors and my lecturers for their supervision throughout the
course of this research. I would also like to acknowledge the members of the National
Biotechnology Authority of Zimbabwe for their help and support. I would also like to express
my sincere gratitude to my family and friends for their motivation. Sincere gratitude to the
Lord almighty for his grace throughout the course of this project
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ABSTRACT
To test the health of seeds for seed borne pathogens is an important step in management of
diseases in crops. This study was conducted to assess the incidence of seed borne fungi from
farm served seeds. ISTA technique, agar plating of groundnuts showed that the seed coat was
infected with Aspegillus, Rhizopus and Fusarium oxy followed by cotyledon and embryo.
These three fungi found to be responsible for seed borne . The percentage frequencies were
54.77% for Aspergillus, 33.2 for Fusarium and 29.4 for Rhizopus of the total isolates. The
results also showed that there was significant difference (p≤ 0.05)) in mean isolation
frequency of fungi.
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Table of content
CHAPTER 1: INTRODUCTION
Background......................................................................................................
1.1 Problem statement..........................................................................................
1.2 Justification......................................................................................................
1.3 Aims and Objectives.......................................................................................
1.4 Hypothesis.....................................................................................................
CHAPTER 2:LITERATURE REVIEW.........................................................................
2.1 Origin and botany of groundnuts
2.2 Yield of groundnut
2.3 Seed borne pathogens of groundnuts
2.4 Seed health testing methods
CHAPTER 3: MATERIALS AND METHODS
3.1 Collection of material.................................................................................
3.2 Study area..................................................................................................
3.3 Materials and method.................................................................................
3.3.1 Isolation of fungi on Agar plat.......................................................
3.3.2 Fungal Identification......................................................................
3.3.3 Data collection ..................................................................................
CHAPTER 4: RESULTS
4.1 Agar plate results .......................................................................................................
4.2 Microscopy
CHAPTER 5: DISCUSSION
5.1 Discussion ................................................................................................
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CHAPTER 6: CONCLUSION AND RECOMMENTATION
6.1 Conclusion.........
6.2 Recommendation
References....................................................................................................
List of figures
Figure Pages
Figure 1: Groundnut variety.............................................
Figure 2: Agar plates of growing fungi...........................
Figure 3: Aspergillus.......................................................
Figure 4: Fusarium Oxysporium.....................................
Figure 5: Rhizopus..........................................................
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List of tables
Table Page
Table 1: Percentage Frequency..........................................................
7
List of Abbreviations
PDA ................................. Protein Dextrose Agar
ISTA...................................
PCR.................................... Polymerase Chain Reaction
PF......................................... Percentage Frequency
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CHAPTER 1: INTRODUCTION
1.0 Background
Groundnut is a crop which originated from Bolivia in South America ( Kumar et al., 2018). It
is divided into two subspecies which are Archis hypogae and fastigiata (Kumar et al., 2018).
Groundnut is the second most produced crop from maize in Zimbabwe (Sabine et al,.2015).
The nuts are an important source of proteins, oils, essential minerals and vitamins in human
diet (Pretorius 2006).
Fig1: Star variety in Zimbabwe known as Illanda
The groundnut crop however is affected by various pathogens, chief among them are fungi
found on peanut pods, shells and seeds (Al-Amod, 2015 ). Seed borne fungi are responsible
for both pre and post-emergence death of grain. A number of fungal species have been
isolated from groundnut seeds fungi such as Aspergillus niger, Aspergillus flavus, Alternaria
dianthicola, Curvularia lunata, Curvularia pellescens, Fusarium oxysporum, Fusarium
equiseti, Macrophomina phaseolina, Rhizopus stolonifer, Penicillium digitatum and
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Penicillium chrysogenum. Fungal infection on groundnut seed presents an array of symptoms
such as discoloration, rotting, shrinking, necrosis of the seed. Loss in germination capacity
and presence of toxic chemicals (e.g. aflatoxins) in seed or seed products are additional signs
of seed borne fungal infection (Elwakil et al., 2001). Fusarium solani, [Link] cause
damping off of groundnut seedlings (Rasheed S et al .,20004). Aspergillus flavus attacks
germinating groundnut seed. A. niger cause crown rot disease in peanut.
There are a number of methods that are used to isolate and identify fungi infecting seeds, but
the Blotter method is the most common and affordable method (Fakhrunnisa and Ghaffa .,
2006). In the Blotter method, seeds are incubated for a certain period on agar or blotter under
specific environmental conditions to allow pathogens on the seed to grow. Morphological
features such as form, length and arrangement of conidiophores, form, size, septation and
chain formation of aconidia are used to then differentiate fungal species (Dawar, 1994).
According to Dawar (1994), the blotter technique has the ability to isolate and identify a
significantly higher number of fungi than the agar plate and deep freezing methods with
sunflower seeds.
Potato dextrose agar (PDA) is another method used for the identification, cultivation, and
enumeration of yeast and molds in foods and seeds (Rijal N, 2015). Potato dextrose agar
(PDA) contains dextrose as a carbohydrate source which serves as a growth stimulant and
potato infusion that provides a nutrient base for growth of most fungi (Rijal, 2015). Agar is
added as the solidifying agent. Yeasts will grow as creamy to white colonies and molds will
grow as filamentous colonies of various colors. (Rijal N, 2015)
There are also molecular methods which are used for fungal identification. Fungal pathogens
can be identified by using PCR at different taxonomic levels ( genus, species and strain)
depending on the specificity of the primers used ( Jeeva et al 2010). In PCR, DNA is
amplified first and this is achieved by repeated cycles of denaturation, polymerization, and
elongation at different temperatures using specific primers ( Kumar et al., 2015). The
amplified DNA fragment can be visualised by electrophoresis or flourometric assays (Kumar
et al, 2015).
1.2 PROBLEM STATEMENT
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Most smallholder farmers in Zimbabwe that practice crop production use farm saved seeds,
which are usually of poor quality and seed borne pathogens accumulate in saved seeds ( ).
From the study done by SNV in 2016, it showed that smallholder farmers who cultivate
returned seeds get an average yield of 964 kg/ha or below compared to those who use
certified seeds and get an average yield of 4 tonnes per ha (SNV.,2016). Smallholder and
marginal farmers cannot afford the agronomic cost that can reduce incidence of fungi
contamination (Dube and Maphosa, 2014)
The food security of Zimbabwe has been affected by low yield. Importation of groundnuts
from Malawi and Zambia has increased due to low yield (USAID, 2010).
1.3 Justification
It is necessary to identify seed borne fungi because they affect eventual output of the crop and
they are responsible for seed quality changes that occur during storage. In this study the fungi
are going to be isolated so that we know the fungal pathogens of groundnuts and this will
help to find measures to protect the seed and also creating an awareness to farmers. Due to
fungal diseases, smallholder farmers are failing to meet confectionary standards because the
groundnuts are small, which presents processing difficulties.
Due to financial constraints, most smallholder farmers in Zimbabwe use returned seed used
for many generations carrying with them the fungal pathogens. Pathogenic microorganisms
are the most important problem for plant growth, which eventually affects the food
production system whenever the chronic threat of pathogens had occurred (Yuttavanichakul1
et al, 2012). Seed-borne pathogen cause economic losses.
1.4.1 AIMS AND OBJECTIVES
The aim of this study is to isolate and identify seed-borne fungi associated with groundnut
seeds .
1.4.2 The Objectives are:
To isolate seed-borne fungi in farm served groundnut seed sample
To identify seed-borne fungi on groundnut varieties
1.4.3 Hypothesis
H0: Fungal seed-borne pathogens are present in both certified and farm saved
groundnuts seed.
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CHAPTER 2 LITERATURE REVIEW
2.1 Origin and botany of groundnuts
Groundnut (Arachis hypogaea L.) is an annual crop and belongs to the plant family
leguminosae (Al-Amod, 2015). It is a self-pollinated annual legume crop and it originated
from South America (Kakad et al,. 2019). Groundnut, or peanut, is commonly called the
poor man's nut (FAO, 2002). It is also called as “King of oil seeds”, widely grown for its
high quality edible oil and food use in the tropical and warm temperate regions of the world
(Kakad et al,. 2019). In developing countries, groundnut play an important role both as oil
and food crop (FAO, 2002). Groundnut is the 13th most important food crop source of edible
oil and the 3th most important source of vegetable protein (Al-Amod, 2015). The crop is
grown in more than 100 countries (Kakad et al,. 2019).West Africa is the largest groundnut
(Arachis hypogaea L.) producing region of Africa, contributing about 54% of the total
groundnut production of the continent (Subrahmanyam, 1991). The major groundnut
producers are China, India, Nigeria, USA, Senegal, Myanmar, Indonesia, Burma and Sudan.
Groundnut is adapted to varying agro-climatic conditions and soils (Kakad et al,. 2019).
2.1 Yield of groundnuts
The average groundnut yield in Zimbabwe is, however, very low (964 kg/ha) compared to
other developing countries (3500 kg/ha). Production of groundnut is low in Zimbabwe
because of many challenges such as lack of inputs, use of unimproved varieties (farm served
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seeds), pests and diseases This affects productivity resulting in the loss of Zimbabwe's export
potential to various markets.
2.3 Seed borne pathogens of groundnuts
Groundnut (Arachis hypogaea L.) crop is attacked by a variety of seed and soil borne fungal pathogen
as well as other pathogens, under field condition as well as during storage causing economical losses
(Kakad et al,. 2019). Several fungi and other microorganisms are associated with seeds but only the
ones which cause diseases in plants or seeds are considered to be as seed-borne in nature (Agarwal
and Sinclair, 1987). Seeds represent an efficient vehicle to disperse seed borne pathogens (Mancini
et al,.2016). In most cases infected seeds are symptomless ((Pellegrino et al.,2010). Seed
infection can result in severe crop losses (Mancini et al,.2016). Mancini et al (2016), stated
that the close association of fungi with seeds facilitates long-term survival and
widespread dissemination of such pathogens. The fungi infect groundnut seed through the
minute cracks on seed coat and pod walls due to mechanical injuries and abiotic stresses caused by
heat or drought ( Falad.,2018)
Groundnut is attacked by a number of pathogenic fungi of economic importance (Al-Amod,
2015). Several authors isolated the following fungi from peanut pods, shells and seeds like
Aspergillus niger, Aspergillus flavus, Alternaria dianthicola, Curvularia lunata,
Curvulariapellescens, Fusarium oxysporum, Fusarium equiseti, Macrophomina praenomina,
Rhizopus stolonifer, Penicillium digitatum and Penicillium chrysogenum causes
discoloration, rotting, shrinking, seed necrosis, loss in germination capacity and toxification
to oilseeds (Elwakil et al., 2001; Chavan and Kakde, 2008). Janardhan et al. (2011) found that
Aspergillus is a common mould in tropical and sub-tropical countries and causes aflatoxin
contamination due to bad storage of crops , such as groundnut, cereal and cotton seeds.
According to Chavan and Kakde (2008), groundnut seeds are highly susceptible to diseases,
because they serve as a source of stored nutrients for fungi such as Aspergillus niger,
Aspergillus flavus, Alternaria dianthicola,Curvularia lunata, Curvularia pellescens,
Fusarium oxysporum, Fusarium equiseti, Macrophomina phaseolina, Rhizopus stolonifer,
Penicillium digitatum and Penicillium chrysogenum causes discoloration, rotting, shrinking,
seed necrosis, loss in germination capacity and toxification to oil seeds.
Fungi growing on stored seeds such as groundnut, can reduce the germination rate along with
loss in the quantum of carbohydrate, protein and total oil content, induces increased moisture
content, free fatty acid content and enhancing other biochemical changes (Junaithal Begum,
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2013). High temperatures and high relative humidity has great impact on the reservation of
cereal grains, oil seeds and can lead to total lose of seed quality (Begum., 2013).
2.4 Seed health testing methods
Several investigators have studied seed health testing methods for detection of seed borne
pathogens. Deep freezing method was used by Mathur et al (1975) and they find it more
suitable for detection of Fusarium spp in sorghum. Blotter method and agar method were
found to be suitable for detection of Fusarium spp in rice seeds (Khan et al (1988).
Blotter test is one of the most important methods in testing for seed borne fungi. In blotter
method the seeds are placed on moistened blotter paper and incubated to promote fungal
growth (Amza., 2019). The seeds are allowed to germinate while seed borne fungi manifest
under the environmental conditions during incubation (Amza.,2018). The infection of the
seed is indicated by the presence of mycelium and fruiting bodies (Amza., 2018).
The agar plate method is another method for detection of seed born fungi even at preliminary
phase of development. Seeds are placed onto sterile agar media ( potato dextrose or malt
agars) to encourage growth of seed borne fungi (Amza., 2018). Amza., 2018 said that agar
plate may be employed to quantitatively determine the fungal load such as CFU/gm of seed
(dilution plate methods) or to qualitatively determine the species composition (direct plate
method).
There is also nucleic acid hybridization assays (southern and northern blotting), in which
DNA or RNA is transferred from an electrophoresis gel onto membrane and then the nucleic
acids are detected with a labelled probe (Amza.,2018). The nucleic acid spot hybridization
(NASH) technique, in which a labelled DNA pathogen hybridizes directly to the pathogen
DNA immobilized on a nylon membrane, can also be used without going through the PCR
stage (Rao et al., 2006).
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CHAPTER 3: MATERIALS AND METHODS
3.1 Collection of material
Groundnuts seed samples were collected from Mbare and tested for seed born pathogens.
The samples were farm served seeds.
3.2 Study area
All laboratory analyses was done at NBA Department of research and Development in
Newlands, Princess Drive Harare.
3.3 Materials and method
3.3.1 Isolation of fungi on Agar plate
300 seeds per variety of groundnut were used for the agar plate. Various seeds were sterilized
with 70% alcohol for 30 seconds, followed by 1% NaOCl for 3 minutes and then washed
with sterile distilled water. The 300 treated seeds of groundnut variety were divided
into three aliquot lots of 100 seeds. One lot of seeds was incubated on potato
dextrose agar (PDA) by ISTA method (2016).The seeds were placed into a petri dish
containing Potato Dextrose Agar (200 g of potato extract, 18 g of agar, 20 g of dextrose, and
1000 mL of distilled water), 10 seeds per petri dish. Ten replicate plates were used and
incubated for 5 days at 25(±1°C).A drop of streptomycin was added in each plate
for the suppression of bacterial growth. The second lot of seeds was dissected
individually and aseptically into component parts (seed coat, cotyledons and
embryo) and the components parts were placed on PDA medium for the growth of
fungal colonies. The third lot of seeds was kept in plastic vials, 10 seeds in a vial
plugged with cotton wool. The vials was kept at-20C for 24 hrs. The seed samples
were then put on PDA medium and incubated at 25°C (±1°C) for 5-7 days under an
alternating cycle of 12 h darkness for the growth of seed borne fungi into
discernible fungal colonies. A pure culture of each fungus was maintained on PDA for
identification purposes (Wain-Tassi et al. 2012).
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3.3.2 Fungal identification
For microscopic identification of fungi temporary slides were prepared from the fungal
colony and observed under compound microscope at 100x and 400x and identified with the
help of colony characteristics such as colour and texture of mycelia and type of pigmentation.
Microscopic characteristics of spores such as mycelium, shape, size and colour were also
used to identify the pathogens associated with the seed (Soresanto et al., 2020).
3.3.3 Data Collected
Fungal species found growing on the surface of seeds, Type and frequency of occurrence of
identified fungal species was recorded. Percentage frequency (PF) of occurrence of fungal
was calculated by using the following formula of ISTA (1996):
PF (%)= (Number of seed with fungus per plate)/(Number of total seeds per plate) × 100
CHAPTER 4: RESULTS
The agar plate method technique has proven more suitable for the detection of Aspergillus
ssp, Fusarium and Rhizopus (Table 1), the highest incidence of these pathogens were
recorded on seeds from Buhera and Mbare. Aspergillus ssp, Fusarium and Rhizopus were
predominant on most of the studied varieties. A total of three fungi species comprising three
genera namely Aspergillus ssp, Fusarium ssp and Rhizopus ssp were isolated from peanut
seed samples of Illanda (star variety) collected from DR&SS. Three species were isolated by
the agar method. Aspergillus occurred on 54.77% of the samples tested on agar plate method;
while Fusarium occurred in 34.69% of the samples tested on agar plate method, another
important storage pathogen Rhizopus occurred in 29.4% of the samples tested on blotter
method.
Aspergillus Fusarium Rhizopus Mean
Buhera white 66.3 44.4 33.0 47.9
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Buhera red 52.86 36.05 32.93 40.61
Star variety 41.3 22.10 21.49 31.96
Mbare white 51.6 33.2 29.4 38.06
LSD 4.73 2.98
Mean 54.77 34.69 38.06
LSD 2.15
Table 1: Percentage frequency of seed-borne fungi of groundnut.
4.2 Agar Plate culture
17
Fig 2: This figure shows the results of growing fungi from groundnut seeds cultured on a
PDA agar plate after seven days of incubation.
4.3 Microscopy identification
The fig 3 ,4, 5 shows different patterns of different isolates. The mycelium of different fungi
were distinguished from the other by mycelium mophology. The hyphal of Aspergillus were
dense than that of Fusarium Oxysporium and Rhizopus.
Fig 3: Aspergillus
Fig 4: Fusarium Oxysporium
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Fig 5: Rhizopus Stolonifer
CHAPTER 5
5.1 Discussion
Farmers in Zimbabwe use their own served seeds for planting and none of these farmers treat
their groundnut seeds before planting. Continuous use of farm served seed has contributed to
high incident of seed borne fungi
Results of the study have shown three important seed borne fungal which are Aspegillus,
Fusarium and Rhizopus. There are other studies which have been done by Chavan 2011 and
Oladipupo 2011 which discovers Aspergillus ssp, Penicillium, Fusarium, Rhizoctonia and
Alternaria as seed-borne fungi in stored groundnut. These fungi species reduce seed
germination, and can cause seed damage during storage. Aspegillus produce mycotoxins and
produces aflatoxin B1, B2, G1 and G2 which are hepatocarcinogenic. Mycotoxins have
health effects and an affect kidneys, liver and nervous system ( ). Janardhan et al. (2011)
found that Aspergillus is a common mould in tropical and sub-tropical countries and causes
aflatoxin contamination due to bad storage of crops , such as groundnut, cereal and cotton
seeds.
5.2 Conclusion
19
Farm served seed obtained from Buhera and Mbare had a varying degree of fungal infections
by Aspergillas ssp, Fusarium ssp and Rhizopus ssp. Awareness to farmers should be
increased and the need to seed dress before sawing should be highlighted.
5.3 Recommendation
The use of certified groundnut seeds for sowing is recommended to prevent the introduction
of disease-causing agents unto the field. Seed should be treated to control fungi present either
on the seed surface or carried internally in the seed; and to also control fungi present in the
soil, or on crop residue in the soil.
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