Plant Archives ISOLATION, CHARACTERIZATION AND SPORULATION OF FUNGI FROM DECAYING VEGETABLES AND FRUITS OF LOCAL VEGETABLE MARKET IN HAZARIBAG INDIA

Abstract

A preliminary study was done to isolate fungal contaminants, particularly those responsible for spoilage of tomatoes, onions, cabbage and other vegetables sold at the local vegetable market of Hazaribag. The spoilt samples of tomato, onion, cabbage,, some other vegetables and some fruits were cut into pieces each with a sterile razor blade. The samples were then cultured on PDA and incubated at room temperature for 5 days after which the fungal growths were observed. The isolates were purified on Sabouroud's Dextrose Agar plates. A total of thirty five fungal isolates were obtained from the three samples. The fungi isolated and most commonly observed were A. niger, R. stolonifer, A. flavus, Mucor Spp, Penicillium Spp., A. flavus and some other common fungi were found to be associated with contamination of the above-mentioned vegetables and fruits. Based on these findings, it was observed that perishable food such as onion, tomato, and other vegetables and some fruits are susceptible to spoilage by fungi probably because the spores of these organisms are easily transmitted via the air which could lead to spoilage of these vegetables. This study, therefore, suggests that there is a need to wash as well as properly store these vegetables at the appropriate temperatures so as to minimize the level of contamination.

Full text

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Adarsh Kumar Chandra et al.
Plant Archives Vol. 22, No. 2, 2022 pp.426-430 e-ISSN:2581-6063 (online), ISSN:0972-5210
Plant Archives
Journal homepage: http://www.plantarchives.org
DOI Url : https://doi.org/10.51470/PLANTARCHIVES.2022.v22.no2.077
ISOLATION, CHARACTERIZATION AND SPORULATION OF FUNGI FROM DECAYING
VEGETABLES AND FRUITS OF LOCAL VEGETABLE MARKET IN HAZARIBAG INDIA
Adarsh Kumar Chandra, Harsh Raj, Himanshu Ranjan
, Kumar Anand*
and Puja Kumari
University Department of Biotechnology,Vinoba Bhave University
Hazaribag, Jharkhand -825301, India
*Corresponding author: kanand2901@gmail.com
(Date of Receiving : 09-08-2022; Date of Acceptance : 14-10-2022)
ABSTRACT
A preliminary study was done to isolate fungal contaminants, particularly those responsible for spoilage of
tomatoes, onions, cabbage and other vegetables sold at the local vegetable market of Hazaribag. The spoilt samples
of tomato, onion, cabbage,, some other vegetables and some fruits were cut into pieces each with a sterile razor
blade. The samples were then cultured on PDA and incubated at room temperature for 5 days after which the
fungal growths were observed. The isolates were purified on Sabouroud's Dextrose Agar plates. A total of thirty
five fungal isolates were obtained from the three samples. The fungi isolated and most commonly observed were A.
niger, R. stolonifer, A. flavus, Mucor Spp, Penicillium Spp., A. flavus and some other common fungi were found to
be associated with contamination of the above-mentioned vegetables and fruits. Based on these findings, it was
observed that perishable food such as onion, tomato, and other vegetables and some fruits are susceptible to
spoilage by fungi probably because the spores of these organisms are easily transmitted via the air which could lead
to spoilage of these vegetables. This study, therefore, suggests that there is a need to wash as well as properly store
these vegetables at the appropriate temperatures so as to minimize the level of contamination.
Keywords : Contaminants, isolates, vegetables, spoilage, perishable.
Introduction
Normally raw fruits and some vegetables are consumed
as they are the comestible part of mature ovary of flowering
plants (Ikhiwili, 2012). Fruit also includes many structures
that are not commonly called fruits such as bean pods, corn
kernels, tomatoes, and wheat grains (Ikhiwili, 2012). Fruits,
as well as vegetables, are of great importance as they provide
nutrition as well as essential growth factors such as vitamins
and minerals which are necessary for proper body
metabolism (Al-Hindi, 2011). Not only humans but also
animals have become dependent on fruits as well as
vegetables as a source of food (Lewis, 2002).
In comparison to vegetables, fruits are easily spoilt and
usually have active metabolism during the storage stage
(Singh, 2007). The high concentration of various sugars,
minerals, vitamins, amino acids, and low pH also enhances
the successful growth and survival of various parasitic and
saprophytic forms of fungi (Droby, 2006). Reports state that
almost 20% of fruits and vegetables produced are lost due to
spoilage (Barth, 2009), especially during post-harvest stages
(Singh, 2007). This has been associated with spoilage fungi
which can be toxigenic or pathogenic (Zhu, 2006). Toxin-
producing fungi have been identified and isolated from spoilt
fruits especially (Al-Hindi, 2011). Allergic reactions as well
as infections also occur due to pathogenic fungi (Monso,
2009). Aspergillus spp. especially Aspergillus flavus,
Aspergillus parasiticus produces mycotoxins and other toxic
metabolites which can be harmful to humans and animals
globally (Afsah-Hejri, 2013; Petzinger, 2002).
Among the diverse group of micro-organisms, fungi are
especially known to destroy fruits as well as vegetables and
henceforth, reducing the quantity for consumption and the
profits obtained from sales of fruits. In view of the above, we
need to identify these micro-organisms especially those that
are pathogenic to humans so as to reduce the risk of
contamination and infection arising from handling and
consumption of fruits as well as vegetables. Keeping in mind
the above fact an initiative was und ertaken to isolate and
identify fungi associated with spoilt fruits as well as
vegetables commonly sold in Hazaribag main vegetable
market.
Study area
The study was conducted in Hazaribag district of
Jharkhand. The summers are much rainier than the winters in
Hazaribag. The average ambient temperature remains
24.4
℃
,varies from 6.9
℃
to 39.2
℃
. The average relative
humidity remains around 67.6%, varies from 10.8% to
98.3%. The annual rainfall is 1255 mm (Awowole, 2007).
Materials and Methods
Rotten fruits as well vegetables were collected in sterile
polythene bags from the local vegetable market of Hazaribag.
They were then brought to University Department of
Biotechnology, Vinoba Bhave University, Hazaribag.

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Adarsh Kumar Chandra et al.
Further, studies were done to isolate and identify the fungus
from the above samples.
Isolation of fungi
About 80 different rotten fruit samples as well as
vegetable samples were undertaken for study. Some healthy
fruits and vegetables were also examined .The fruits as well
as vegetables were cut into small segments (3 mm in
diameter) with a sterilized blade, surface sterilized in 1%
hypochlorite for 2 min, plated on Potato Dextrose
Agar(PDA) media aseptically and then incubated at 28°C for
5 days.
A pure culture was obtained and maintained by sub-
culturing each of the different colonies that emerged onto the
PDA plates and incubating at 28°C for 5 days.
As a control, each of the healthy fruits and vegetables
was sterilized with 70% ethanol. The fruits were cut into
small segments (3 mm in diameter) with a sterile blade,
placed on PDA and then incubated at 28°C for 5 days.
Identification of isolated fungi
The isolated fungi were later on identified. The isolates
were then identified using cultural and morphological
features such as colony growth pattern, conidial morphology,
and pigmentation (Tafinta, 2013). Lactophenol using cotton
blue stain was used to identify the fungi using the technique
of Oyeleke and Manga (Oyeleke, 2008). The fungus
identification was done by placing a drop of the stain on
clean slide with the aid of a mounting needle, where a small
portion of the aerial mycelia from the representative fungi
cultures was removed and placed in a drop of lactophenol.
The mycelium was then spread on the slide with the needle.
A cover slip was gently placed on it. Care was taken to avoid
pressure to eliminate air bubbles. The slide was then mounted
and observed under the light microscope with ×10 and ×40
objective lenses. The morphological characteristics and
appearance of the fungal organisms seen were identified in
accordance with Adebayo-Tayo et al., 2012; Onuorah et al.,
2015; Klich, 2002; Samson and Varga, 2007.
Results
The work was conducted to isolate and identify
different fungus from isolated decaying vegetables and fruits
of local vegetable market of Hazaribag.
The table below shows the frequency of occurrence of
fungi in the various fruits and vegtables. Aspergillus niger
had the highest occurrence in vegetables as well as fruits with
a frequency of 40%. Fusarium avenaceum followed with the
frequency of occurrence of 20% in fruits as well as
vegetables while Penicillium digitatum and R. stolonifer had
the least frequency of 5% each in vegetables as well
vegetables. Other fungal species were identified as yeast
(Saccharomyces species) (10%), F. solani (10%) and
Aspergillus flavus (10%).
Table 1 : Frequency of occurrence of fungal species:
Fungi isolate Source Frequency occurrence
(%)
Aspergillus niger Fruits & vegetables 40
Fusarium avenaceum Fruits & vegetables 20
Yeast (Saccharomyces spp.) Fruits & vegetables 10
Fusarium solani Fruits & vegetables 10
Aspergillus flavus Fruits & vegetables 10
Penicillium digitatum Fruits & vegetables 5
Rhizopus stolonifer Fruits & vegetables 5
Fig. 1 : Photograph of fungal colonies isolated on PDA media from decaying vegetables
and fruits of local vegetable market of Hazaribag
Fig. 2 : Microphotographs of fungi stained with Lactophenol cotton blue grown on PDA media.

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Isolation, characterization and sporulation of fungi from decaying vegetables and fruits of
local vegetable market in Hazaribag India
Discussion
The isolation and distribution of fungi in spoilt fruits in
Hazaribag is a novel discovery which exposed array of fungi
which are pathogenic to man and animals. The identified
fungal organisms associated with spoilt fruits in the study
area include A. niger, A. flavus, F. solani, F. avenaceum, P.
digitatum, R. stolonifer, and yeast (Saccharomyces species)
suggesting that these fungal organisms could be responsible
for the fruit spoilage. This finding is in conformity with
previous works of Baiyewu et al., 2007 and Chukwuka et al.,
2010 which reported isolation of A. niger, F. avenaceum, R.
stolonifer and yeast from pawpaw in Nigeria.
Previous literature indicates that processes such as
harvesting, storing, packing and transporting, fruits may
encounter physical injury that increases post-harvest loss and
the possibility of fungal contamination. In addition, the
problem can be enhanced from poor management of fruits as
well as vegetables in Hazaribag market (Gultie, 2013).
Market conditions that favor contamination can be worsened
by poor hygiene of the vendors, using microbial unsafe
container poor handling practice and poor environmental
conditions such as sanitarily unsafe marketing environment.
The consequence of the problems could be increased loss of
fruit due to microbial spoilage and the existence of some
human pathogens (Okojie, 2014; Gultie, 2013).
Out of fungi isolated in this study, A. niger which
causes a disease called black mold on certain fruits and
vegetables and produces potent mycotoxins called
ochratoxins that can be harmful to human beings and animals
had the highest occurrence (40%) followed by F. avenaceum
(20%) while P. digitatum (5%) and R. stolonifer (5%) had the
lowest frequency of occurrence. However, Tafinta et al.,
2014 reported a frequency of occurrence of 36%, 25%, 22%,
and 17% for R. stolonifer, A. flavus, A. fumigatus, and A.
niger, respectively, from sweet oranges. These differences
could be attributed to number and type fruits examined in
both studies.
Most of the fungal organisms isolated in this study play
a pivotal role in the deterioration of food and feed systems
and some of them are also able to produce toxic compounds
for humans and animals. The mycotoxins produced by these
fungi can cause serious health hazards including
cancerogenic, immunotoxic, teratogenic, neurotoxic,
nephrotoxic and hepatotoxic effects, and Kashin-Beck
disease (Tafinta et al., 2014). F. avenaceum is well-known
for causing ear blight and root rot of cereals, blights of plant
species within genera as diverse as Pinus and Eustoma, as
well as post-harvest storage rot of numerous crops, including
potato, broccoli, apple and rutabaga. F. avenaceum has also
been described as an endophyte, and an opportunistic
pathogen of animals (Sorensen, 2009; Lysoe, 2014; Varvas,
2013 and Yacoub, 2012). P. digitatum causes a destructive
fruit rot of citrus. It is generally considered the most
important agent of post-harvest spoilage in the most citrus
species. Early symptoms include a soft water-soaked area on
the peel, followed by the development of a circular colony of
white mold, up to 4 cm diameter after 24-36 h at 24°C.
Penicillium species are common fungi in the environment
and are often considered non-pathogenic to humans (Yacoub,
2013). R. stolonifer is a significant agent of fruit disease. It is
a threadlike mold and a heterotrophic species; it depends on
sugar or starch for its source of carbon substances for food. It
uses food matter, mostly soft fruits, like grapes or
strawberries, as a food source for growth, nutrition and
reproduction (Foody, 2008).
In this study, fungal organisms were isolated from
pineapple, watermelon, pawpaw, orange and tomato. A. niger
and F. avenaceum were more widespread among all the
spoilt fruits examined followed by F. solani and yeast
(Saccharomyces species). P. digitatum and A. flavus were
isolated from only tomato. Similar findings on the isolation
fungal pathogens from fruits stored and sold in the market
have been reported by earlier researchers (Bali, 2008). Bali et
al. stated that A. niger was the cause of post-harvest spoilage
in sweet orange and acid lime at field. Okereke et al.; 2010
reported that A. niger, Alternaria species, Botryodiplodia
theobromae and Colletotrichum gloeosporioides were
isolated from the spoilt mangoes. Chukwuka et al., 2010
implicated Rhizopus nigricans, A. flavus, A. niger, Fusarium
spp., and Mucor spp. in pawpaw fruit spoilage from a farm in
Oyo state, Nigeria.
Fungal pathogens are causing losses of marketable
quality and hygiene of fruits, resulting in major economic
problem in Nigeria and the world at large. Fruit spoilage can
be prevented using physical (Boyer, 2009) and chemical
methods (Msagati, 2012), but no efficient strategy has been
proposed so far to reduce the microbial growth ensuring
public health safety. Lactic acid Bacteria (LAB) can play a
vital role as natural preservatives. The protection of fruits or
fruit products using LAB is mainly because of the production
of antifungal compounds such as carboxylic acids, fatty
acids, ethanol, carbon dioxide, hydrogen peroxide, and
bacteriocins (Pawlowska, 2012).
The control experiment showed no fungal growth on
PDA after healthy fruits were sterilized with 70% ethanol
indicating that the isolated fungi were introduced postharvest
from farms through fruit vendors and finally to consumers
(Singh, 2007). Fresh fruits recently have been identified as a
significant source of plant and human pathogens and
chemical contaminants that pose a potential threat to human
health worldwide. Because it is likely to be eaten raw by
scavenging animals especially ruminants, humans also stands
risk of getting infected with pathogenic fungi from fruits and
vegetables as a results of poor processing methods. More so,
fresh fruits pose potential food safety hazard and poor type of
microbiologically lethal processing regime could lead to
potential food safety problems. Poor handling can damage
fresh fruits, rendering their products susceptible to the
growth or survival of spoilage and pathogenic
microorganisms (Gultie, 2013).
Conclusions
This study has shown that A. niger, A. flavus, F. solani,
F. avenaceum, P. digitatum, R. stolonifer and yeast
(Saccharomyces species) were isolated from spoilt pineapple,
watermelon, pawpaw, orange, and tomato. However, some
fruits such as pineapple, watermelon, oranges, and pawpaw
are free from contamination with fungi such as A. flavus, P.
digitatum, and R. stolonifer. These pathogenic fungi species
associated with fruits spoilage are of economical and public
health significance. Care should be taken during handling of
these fruits, technology based modern preservative methods
such as pasteurization, vacuum packing, radiation, pulsed

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Adarsh Kumar Chandra et al.
electric field electroporation, high-pressure food
preservation, and bio preservation are suggested to enhance
the keeping quality of fruits.
Acknowledgments
The authors would like to acknowledge the University
Department of Biotechnology, Vinoba Bhave University,
Hazaribag for providing the lab as well as infrastructural
facilities used for this research. All coauthors have equal
contribution in completing the work, especially the students.
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