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American Journal of  
Life Science and Innovation (AJLSI)

Microorganisms Associated with Tomato (Lycopersicum Esculentum) Rot and Effect of  Neem 
(Azadirachta Indica) Extract in Rot Control in Makurdi Metropolis, Benue State, Nigeria

Igbe Abi Obed1, Innocent Okonkwo Ogbonna2*

Volume 3 Issue 2, Year 2024
ISSN: 2833-1397 (Online)

DOI: https://doi.org/10.54536/ajlsi.v3i2.3438
https://journals.e-palli.com/home/index.php/ajlsi

Article Information ABSTRACT

Received: July 17, 2024

Accepted: August 20, 2024

Published: August 24, 2024

Tomato (Lycopersicum esculentum) is a vital agricultural crop globally, yet it is significantly 
affected by various microbial pathogens leading to post-harvest rot, which threatens food 
security and economic stability. This study aimed to identify the microorganisms associated 
with tomato rot in Makurdi Metropolis and evaluate the efficacy of  neem (Azadirachta 
indica) extract as a biocontrol agent. A total of  60 tomato fruits were sampled from the 
five major markets within the Makurdi metropolis. They were cultured on Nutrient agar, 
MacConkey agar, Potato Dextrose agar, Mannitol salt agar, and Eosin Methylene blue agar. 
The organisms identified were Klebsiella sp, Salmonella sp, Proteus vulgaris, Enterobacter sp, Shigella 
sp, Staphylococcus sp, Bacillus sp, Escherichia coli, Rhizopus sp, Mucor sp, Aspergillus sp, and Fusarium 
sp. Neem extract was used for susceptibility tests on the bacterial isolates at 10%, 50%, and 
100% concentration, and it was found most effective on some bacterial isolates at higher 
concentrations of  100% with wider zones of  inhibitions. Vegetative growth of  the fungi on 
neem extract decreased with increase in concentrations. This finding proved the potentiality 
of  plant extracts for controlling the fungal rot of  tomato fruit.  A pathogenicity test was 
conducted with readings taken at intervals of  days and it was noticed that the microbial 
isolates were actually the cause of  tomato rot. This study underscores the importance of  
identifying microbial threats to tomato crops and highlights neem extract as a promising 
natural alternative for managing tomato rot, promoting sustainable agricultural practices in 
Makurdi Metropolis and Nigeria at large.

Keywords

Microorganisms, Tomato, 
Neem, Susceptibility, Zone of  
Inhibition, Bacteria Isolates, 
Food Security

INTRODUCTION 
Tomato (Lycopersicum esculentum) is a widely cultivated and 
consumed vegetable, known for its nutritional value 
and economic importance. However, its high-water 
content makes it particularly susceptible to spoilage by 
various microorganisms, including bacteria and fungi 
(Obeng, et al., 2018). The tomato fruit comprises the 
skin, pericarp and locular cavities. The locular cavities 
are filled with jelly-like parenchyma cells that surround 
the seeds. The cell walls are composed of  alpha-cellulose, 
pectin, hemicelluloses and some protein (Mautante & 
Mala, 2024). It is rich in vitamins including vitamin 
A and vitamin C, carbohydrates, proteins, fats, fibres, 
potassium, and phytochemicals (Talvas et al., 2010). It is 
rich in lycopene which has many beneficial health effects. 
Tomato is a fruit that contains the seeds and ovary of  a 
flowering plant (Ugwu et al., 2014). It is known to be a 
very profitable crop that provides high returns for small 
scale farmers in most developing countries (Lemma, et 
al., 2014). Due to its nutritive value, taste, affordability, 
and accessibility, there has been an increase in demand 
by consumers (Behravesh, et al., 2012). However, 
isolation and identification of  microorganisms that are 
associated with spoilage of  tomatoes have gained some 
research focus (Akinyele, et al., 2020). In most developing 
countries, microbial infestation of  tomatoes can occur 
during the harvesting period, post harvesting, handling, 

storage, transportation, and processing by customers 
(Barth, et al., 2009 and Yeboah et al., 2011). Baiyewu 
et al.,2018 have also reported that another means of  
bacterial contamination is by exposing them on benches 
and baskets in the open markets for customers. The 
relative humidity (dew) during temporary storage of  
tomato fruits and nature of  the storage room could 
play a great role in their contamination (Pardaev, 2022). 
The proliferation of  bacteria, especially in damaged 
tomatoes, could be considered more harmful when such 
contaminated tomatoes are consumed in improperly 
cooked food (Valadez et al., 2019).
Several studies have been performed on bacteria 
associated with tomatoes and tomato products in several 
countries. A survey by Ajayi and colleagues in the United 
States showed that Clostridium sp., Staphylococcus sp., and 
Bacillus sp. were the predominant bacteria isolated from 
canned and raw tomatoes. In India, a study conducted 
on tomato puree showed the presence of  Klebsiella sp., 
Proteus mirabilis, Vibrio sp., and Pseudomonas sp. (Garg 
et al., 2017). In Nigeria, Wogu and Ofuase isolated 
Bacillus subtilis, Klebsiella aerogenes, Pseudomonas aeruginosa, 
Salmonella typhi, Proteus mirabilis, and Staphylococcus 
aureus from spoiled tomatoes in Benin City. A similar 
study also showed that the content of  Staphylococcus sp. 
(22.5%), Bacillus sp. (20%) and Escherichia coli (15%) in 
Lagos State, Nigeria 

1 Department of  Biotechnology, I. M. Sechenov First Moscow State Medical University, Moscow, Russia
2 Department of  Microbiology, Joseph Sarwuan Tarka University, Makurdi Benue State, Nigeria
* Corresponding author’s e-mail: ogbonna09@yahoo.com



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Statement of  the Problem
Azadirachta indica (commonly known as neem) is a tree 
in the mahogany family Meliaceae. Neem products are 
believed to be antifungal, antibacterial, antidiabetics, 
antiviral, contraceptive, and sedative. Hence, it is 
particularly prescribed for treating skin diseases such 
as eczema, psoriasis, etc. (Sharma and Nupur, 2014). 
Reports has shown that compounds from plant sources 
are moderately toxic and are suitable as fungicides. 
Neem (Azadirachta indica) extract has been studied for 
its antimicrobial properties, offering a potential natural 
solution for controlling tomato rot. The extract’s 
bioactive compounds can inhibit the growth of  spoilage-
causing microorganisms, thus extending the shelf  life of  
tomatoes and reducing economic losses. By investigating 
the microorganisms associated with tomato rot and 
assessing the potential of  neem extract as a biocontrol 
agent, this study aims to advance our understanding of  
disease dynamics in agricultural systems and contribute 
to the development of  environmentally friendly strategies 
for managing tomato diseases.

MATERIALS AND METHODS 
Tomato fruit samples were collected from the five biggest 
markets in the Makurdi metropolis: Modern Market, 
Wadata Market, Wurukum Market, Northbank Market, 
and High-level Market. Each market sample was placed 
separately in a sterile plastic bag to prevent contamination. 
Samples were immediately transported to the laboratory, 
of  the Department of  Microbiology, Joseph Sarwuan 
Tarka University, Makurdi, Benue state. Culturing, 
Isolation, and Identification of  the major pathogenic 
organisms in the disease portion was carried out. All 
materials used were sterilised by appropriate methods to 
remove extraneous microbial contamination. Glass wares 
were sterilised in a hot air oven at 1200C for one hour. 
In contrast, culture media were sterilized by autoclaving 
at 1210C for 15 minutes. Inoculation loops were heated 
till red hot in a Bunsen burner flame and allowed to cool 
before being used. Work surface was identified routinely 
by cleaning with cotton wool impregnated with ethanol 
before the commencement and on completion of  work. 
The aseptic technique was maintained throughout the 
cause of  this work. 

Preparation of  Stock Sample and Serial Dilution
The diseased portion was cut, crushed and fluids from the 
five different markets were squeezed into a conical flask 
labelled against each market. Five test tubes containing 
9ml of  distilled water (DW) were set. 1ml of  tomato juice 
from the conical flask was transferred into the first test 
tube labelled against the same market, giving a dilution of  
10-1. This was mixed and 1ml was transferred to the next 
test tube (DW) marking its 10ml and giving it a dilution 
of  10-2. This process was continued till the 5th test tube 
giving a dilution of  10-5. This same process was carried out 
on the samples collected from all the markets. Ethanolic 
extracts were prepared in the laboratory by the traditional 

Method. One kilogram of  fresh neem leaves (Azadirachta 
indica) was collected from neem trees at Joseph Sarwuan 
Tarka University, Makurdi Benue State. Leaves were dried 
at room temperature and crushed in a mortar. Combining 
the crushed leaf  with ethanol and subsequently soaking 
the mixture overnight at room temperature. The next 
morning, the extract was strained through a Miracloth, 
the liquid was kept in a water bath in the laboratory and 
allowed to evaporate to have a pure concentrated extract.

Microbiological Analysis 
Media were prepared according to the standard 
preparation protocol and the media used were Nutrient 
agar, MacConkey agar, Potato Dextrose Agar (PDA), 
Mannitol Salt Agar (MSA), and Eosin Methylene Blue 
Agar (EMBA). Using a sterile pipette, aliquot of  0.1ml 
was taken from 10-5 dilution and was transferred into the 
duplicates plates of  the media used i.e. Nutrient agar, 
MacConkey, PDA, MSA and EMBA. Pour plate method 
of  inoculation was used to inoculate on the media 
according to the markets they were collected from and on 
what symptoms or disease inferred from these markets. 
Inoculated plates of  the media were incubated at 370C 
for 24 hours for isolation of  bacteria, and also at room 
temperature (25±20C) for 48–72 hours for isolation 
of  fungi. Representative colonies from all the media 
were sub-cultured on Nutrient agar for pure isolate, 
which were later subjected to biochemical analysis for 
identification.

Observation of  Fungal Characteristics and 
Identification 
The fungal isolation on PDA was identified by visual 
observation of  their growth and microscopic examination 
using a drop of  lactophenol cotton blue stain. The 
observed fungi were identified by comparison with 
diagram and keys documented by Harrigan and McCance 
(1976) as reported by Barton et al., 2006. Distinguishing 
characteristics looked for included the general 
morphology and hyphae (Septate and non-septate). The 
shape, colour and distribution patterns were also used in 
identification. The staining was done according to Pelazar 
et al. (1999) as follows:
A pin head of  fungi growth was picked using needle. This 
was placed on a slide with a drop of  lactophenol blue stain. 
A drop of  distilled water was added. The slide was covered 
with a cover slip and observed under the microscope at 
high objective of  X10 power magnifications.  

RESULTS AND DISCUSSIONS
Table 1 below shows the Mean of  the total microbial 
load of  tomatoes sold in different markets in the Makurdi 
metropolis. This investigates the levels of  microbial 
contamination present in tomatoes from various 
markets within the region. It measures and compares 
the total microbial load, including bacteria, fungi, and 
other microorganisms, to assess food safety and hygiene 
practices. The findings revealed significant differences 



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in microbial loads across different markets, highlighting 
potential health risks for consumers and the need for 

improved handling and storage practices to ensure the 
safety of  tomatoes sold in these areas.

Table 1: Mean of  total microbial load of  tomato sold in different markets in Makurdi metropolis
s/n Samples TVC (cfu/g) TCC Fungi 
1 High level 163x105 120x105 148x105

2 Wurukum 216x105 129x105 150x105

3 North Bank 160x105 109x105 132x105

4 Modern market 202x105 135x105 95x105

Key: TVC = Total Viable count: TCC = Total Colony Count: Cfu/g = Colony forming unit/g 

From the table, the bacterial count was maximum in 
Nutrient agar with a total viable count ranging from 160 
min. to 216 max., total colony count ranging from 109 
min. to 135 max. and fungi count ranging from 95 min. 
to 150 max. 
The study on the morphological and microscopic 
features of  fungi isolated from the tomato samples is 
represented in Table 2. This focuses on identifying and 
characterizing the fungal species found in tomatoes. It 
examines both the physical characteristics (morphological 

features) such as the fungi’s colour, texture, and growth 
patterns, as well as their microscopic structures, including 
spore shape, size, and arrangement. This information 
helps in understanding the types of  fungi present, and 
their potential impact on tomato quality and safety, 
and may provide insights into contamination sources 
and prevention methods. Overall, it contributes to the 
knowledge of  fungal diversity associated with tomatoes 
and its implications for food safety.

Table 2: Morphological and microscopic features of  fungi isolated from the tomato sample
Morphological characteristics Microscopic examination Suspected 

organism
Long hyphal growth which sporulated 
within two days to turn to black spores

Non-septate, branched mycelium with round-shaped 
sporangia

Rhizopus sp.

White and woolly aerial growth that darkens 
as it sporulates

Non-septate hyphae with straight sporangiophore 
with many spherical spores

Mucor sp.

Pink fluffy and spreading colonies which are 
creamy around the edges.

Septate hyphae with sickle chlamydospores at the 
hyphae.

Fusarium sp.

Velvety filament white growth that 
sporulates into black powdery spores.

Long septate hyphae with conidiophore bearing 
brown spores

Aspergillus sp.

Table 3: Microscopic and Biochemical Characteristics of  the Bacterial Isolates
Gram reaction Cat Cit Ure Ind MR VP Suspected Organism
-ve rod + + + - - - Klebsiella sp.
-ve rod + + - - - - Salmonella sp.
-ve rod + + + + + - Proteus vulgaris
-ve rod + + - + - - Enterobacter sp.
-ve rod + - - - - - Shigella sp.
+ve cocci + - + - - - Staphylococcus sp.
+ve rod + + + - - + Bacillus sp.
-ve rod + + - + + - Escherichia coli

Key: '+' = positive result; '-' = negative result

From the study, the microscopic and biochemical 
characteristics of  the bacterial Isolates which focus on 
identifying and characterizing bacterial strains isolated 
from the tomato samples are represented in table 3. The 
microscopic characteristics include the bacteria’s shape, 
size, arrangement, and staining properties., which helps 
in their classification. The biochemical characteristics 

involve various tests to determine metabolic activities, 
such as fermentation, enzyme production, and nutrient 
utilization. Together, these analyses provide insights into 
the diversity, functionality, and potential pathogenicity 
of  the bacterial isolates, contributing to a better 
understanding of  their role in the ecosystem or their 
impact on health and disease.



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Table 4 and 5 summarizes the susceptibility test of  Neem 
extract in different concentrations on bacterial isolates using 
Amoxicillin as control and the Pathogenicity test of  bacterial 
isolates on healthy tomatoes respectively. Antibacterial 
effects of  neem extract at various concentrations against 
specific bacterial isolates were identified and recorded. 
The effectiveness of  neem extract is compared to that 
of  amoxicillin, a commonly used antibiotic, serving as a 
control. The results indicate how different concentrations 

of  neem extract inhibit bacterial growth, providing 
insights into its potential as a natural antimicrobial agent. 
The pathogenicity of  isolated bacteria was evaluated by 
inoculating healthy tomato plants with these strains. The 
effects on tomatoes health, including disease symptoms 
and overall plant vigour, were observed. Findings from this 
test help determine which bacterial isolates are harmful to 
tomatoes, contributing to understanding plant-bacterial 
interactions and potential agricultural impacts.

Table 4: Susceptibility Test of  Neem extract in different concentrations on Bacterial Isolates using Amoxicillin as control
Bacterial isolates Zone of  inhibition per concentration (mm)

10% 50% 100% Control (Amoxycillin)
Klebsiella sp - - 12 15
Salmonella sp 8 12 14 17
Proteus vulgaris - 6 11 15
Enterobacter sp 11 13 17 19
Shigella sp 11 16 18 21
Staphylococcus sp 13 14 19 22
Bacillus sp - 8 13 18
Escherichia coli 10 14 16 19

Key: '-' Shows no zone of  inhibition  

Table 5: Pathogenicity Test of  Bacterial Isolates on Healthy Tomato 
Bacterial isolates Zone of  pathogen spread per day (mm)

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Klebsiella sp  - - 3 5 9 13 15
Salmonella sp  - - 4 8 12 15 16
Proteus vulgaris  - 2 6 11 13 16 18
Enterobacter sp  - 3 7 13 15 17 20
Shigella sp  - 4 8 10 14 15 19
Staphylococcus sp  - - 3 7 12 14 18
Bacillus sp  - 2 6 9 12 15 17
Escherichia coli  - - 2 7 10 13 15

Key: - Shows no zone of  spoilage 

From the table, they were no or little infection on the first 
day and there were subsequent spread of  infection and 
spoilage from the second day to maximum infection on 
the tenth day.

CONCLUSION
This study was carried out to examine the tomato 
rot causing microorganisms and the effect of  neem 
extract on rot control in the Makurdi metropolis. The 
organisms identified were Klebsiella sp, Salmonella sp, 
Proteus vulgaris, Enterobacter sp, Shigella sp, Staphylococcus sp, 
Bacillus sp, Escherichia coli, Aspergillus sp, Rhizopus sp, Mucor 
sp, and Fusarium sp. Neem extract was used to conduct 
susceptibility tests on the bacterial isolates and it was 
found most effective on some bacterial isolates at higher 
concentrations. A pathogenicity test was conducted with 
readings taken at interval of  days and it was noticed that 
the microbial isolates were actually the cause of  tomato rot.

From the result of  this study, it is found that tomato 
rot is caused by some microorganisms such as Klebsiella 
sp, Salmonella sp, Proteus vulgaris, Enterobacter sp, Shigella sp, 
Staphylococcus sp, Bacillus sp, Escherichia coli, Aspergillus sp, 
Rhizopus sp, Mucor sp, and Fusarium sp. And neem extract at 
50% and 100% concentration can be used to control the 
rot caused by some of  these organisms.
Therefore, the extracts are potentially simple 
environmentally safe alternative for use as botanical 
fungicides, and could be exploited to manage post-harvest 
diseases of  tropical fruits effectively. Neem extract, 
derived from the seeds, leaves, and bark of  the neem 
can be used as natural pesticide due to its insecticidal 
properties. It can also be used in various traditional 
and modern medicines for their anti-inflammatory, 
antibacterial, antifungal, and antiviral properties, and as 
natural food preservatives.



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