Isolation and Characterization of Phylloplane Bacteria from Papaya Plant for the Biocontrol of post-harvest Diseases in Papaya

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International Journal of Environment, Agriculture and Biotechnology
Vol-6, Issue-1; Jan-Feb, 2021
Journal Home Page Available: https://ijeab.com/
Journal DOI: 10.22161/ijeab
ISSN: 2456-1878
https://dx.doi.org/10.22161/ijeab.61.37 307
Isolation and Characterization of Phylloplane Bacteria
from Papaya Plant for the Biocontrol of post-harvest
Diseases in Papaya
Ashwini Narasimhan*, Kasturi Banerjee
Jain University, Bengaluru, India
*Corresponding author
Received: 03 Dec 2020; Received in revised form: 21 Jan 2021; Accepted: 19 Feb 2021; Available online: 28 Feb 2021
©2021 The Author(s). Published by Infogain Publication. This is an open access article under the CC BY license
(https://creativecommons.org/licenses/by/4.0/).
Abstract— Papaya is one of the most significant crops cultivated in tropical and subtropical countries all
over the world. Post-harvest diseases are the major threat to papaya fruit yield leading to huge losses. This
study focuses on controlling the fungal pathogens of papaya fruit by isolating the disease control bacteria
from the phylloplane of the papaya plant and screening them for antagonism towards the pathogens. The
fungal pathogens chosen for this study were Colletotrichum, Fusarium and Rhizopus. The three bacterial
isolates showing the maximum diameter for the zone of inhibition against these pathogens were selected
for morphological and biochemical characterization. In studies, the isolates were found to be Bacillus and
Pseudomonas. Consortium study was conducted between Bacillus and Pseudomonas which showed more
efficiency in controlling the growth of fungal pathogens when combined. Fruit assay was then performed to
establish these bacterial isolates as biocontrol agents. Papaya fruits were inoculated with fungal
pathogens and fungal pathogens along with bacterial isolates. It was observed that the papayas inoculated
with bacterial isolates showed a better shelf life than those without. The present study reports the
biocontrol ability of the bacteria which can be used as disease control agents.
Keyword— Biocontrol, papaya, post-harvest diseases, phylloplane, fungal pathogens.
I. INTRODUCTION
Papaya, (Carica papaya), also called papaw or pawpaw, is
a tropical and sub-tropical fruit that is classified under
various plant families, including Passifloraceae,
Cucurbitaceae, Bixaceae, and Papayaceae. Currently, it is
placed under Caricaceae, a plant family incorporating
species in four genera, Carica, Cylicomorpha, Jacaratia
and Jarilla [1].
An additional aspect of the mechanisms of the pesticides
through which they reduce plant growth is exhibited [2].
Thus, there is a need for new solutions to plant disease
problems that provide effective control while minimizing
negative consequences for human health and the
environment [3]. Biological control, using microorganisms
to suppress plant disease has offered a powerful alternative
to the use of synthetic chemicals [4].
The production of phytopathogen inhibitor compounds by
the biocontrol agents and their biocontrol potential was
evaluated by measuring the production of these
compounds, hydrolytic enzymes (amylases, lipases,
proteases, and chitinases) and phosphate solubilisation [5].
It has been proven that induced resistance as an alternative
for the control of postharvest diseases in fruit is effective
in both the laboratory and a few cases in the field [6].
Biocontrol of diseases in plants is a difficult subject for
understanding because these diseases mostly occur in the
non-static environment such as the interface of the plant
root and the aerial parts of plants [7].
II. REVIEW OF LITERATURE
Papaya is majorly cultivated in the region of tropics and
sub-tropics. According to a report in 2004, this fruit was

Ashwini Narasimhan Nadia et al. International Journal of Environment, Agriculture and Biotechnology, 6(1)-2021
ISSN: 2456-1878
https://dx.doi.org/10.22161/ijeab.61.38 308
produced over 6.8 million tonnes (Mt) worldwide, which is
about 389,990 Ha [8]. Fungal plant pathogens are known
to cause considerable post-harvest loss of fruit and
vegetables [9]. Papaya is susceptible to more than a dozen
fungal pathogens like Phytophthora rot (Phytophthora
palmivora) root and fruit rot, anthracnose (Collectricum
gloerosporioides), powdery mildew (Oidium caricae),
Rhizopus rot (Rhizopus stolonifer) and black spot
(Asperisporium caricae) are, however, the more important
fungal pathogens [10]. Anthracnose, caused by
Colletotrichum gloeosporioides (Penz.), primarily affects
papaya fruit and is an important postharvest disease in
most tropical and subtropical regions [11].
The widespread use of pesticides in agricultural settings,
public health, commerce, and individual households
throughout the world is an indication of the importance of
these compounds [12]. The sources of these chemicals are
houses, factories, water bodies etc which finds their
applications in public spaces; home, garden, and lawn use
and occupational association [13]. Due to the interest in
public safety concerns, the exploitation of the integrated
pest management aspect in being worked on [14].
Biocontrol microbes/micro-organisms are cellular or non-
cellular entities, capable of replication or of transferring
genetic material. The list of biocontrol agents included in
CIB for registration is many [15]. The dual activity of
Pseudomonas BCAs (i.e. direct antagonism of
phytopathogens and induction of disease resistance in the
host plant) further highlights their potential as plant
protection products (PPPs) [16]. In current times, the focus
is aimed at understanding, how Pseudomonas strains to act
as efficient biological control agents. This approach of
understanding the mechanism is helping the development
of novel strains with enhanced modified traits for its
increased biocontrol efficacy [17].
Elicitors, as a part of integrated pest management (IPM)
approach, are usually used to induce resistance against
postharvest diseases [18]. It has been proven that induced
resistance as an alternative for the control of postharvest
diseases in fruit is effective in both the laboratory and a
few cases in the field [6]. The results of various studies
confirmed the potential use of some essential oils for
protection of fruits and vegetables against postharvest
pathogens and for increasing the shelf life of plant
products [19].
III. MATERIALS & METHODS
3.1. Isolation and screening of bacteria
Potent bacterial biocontrol agents were obtained from
phylloplane of papaya.
5 different phylloplane samples were procured from the
papaya growing fields in Bangalore.
Isolation of phylloplane bacteria was carried out by the
leaf imprint method [20]. These plates were then incubated
at 37  C. The isolates obtained were maintained on nutrient
agar plates.
Fig. I. Leaf Imprint method
3.2. Procurement of potent papaya fungal pathogens
Fungal pathogens were isolated from diseased papaya
fruits which were collected from vegetable and fruit
markets. The pathogens were isolated by direct plating on
Potato Dextrose Agar. The plates were incubated at room
temperature.
The following are the pathogens that were isolated from
the diseased papaya.
1) Colletotrichum sp.
2) Fusarium (type 1)
3) Fusarium (type 2)
4) Rhizopus
5) Penicillium
3.3. Dual assay of phylloplane bacteria against papaya
fungal pathogens
To test the antagonistic potential of each isolate, the
pathogen and bacteria were inoculated 3 cm apart on
potato dextrose agar plates. Fungal growth on each plate
was observed and the zone of inhibition, if present, was
determined. The treatments were replicated in triplicates
for statistical validation. Results were expressed in terms
of percentage inhibition which was calculated as per the
given formula.
% inhibition =𝐃𝐢𝐚𝐦𝐞𝐭𝐞𝐫𝐨𝐟𝐭𝐡𝐞𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧𝐳𝐨𝐧𝐞
𝐓𝐨𝐭𝐚𝐥𝐝𝐢𝐚𝐦𝐞𝐭𝐞𝐫 x 100
3.4. Morphological characterization of the isolates
Morphological characteristics like cell and spore
morphology, motility; growth characteristics (growth in
the presence of NaCl 7%) were investigated [21]. Gram
staining was performed by standard procedures.
3.5. Biochemical characterization of the isolates

Ashwini Narasimhan Nadia et al. International Journal of Environment, Agriculture and Biotechnology, 6(1)-2021
ISSN: 2456-1878
https://dx.doi.org/10.22161/ijeab.61.38 309
Various biochemical tests were conducted based on
Bergey’s manual of systematic bacteriology and Manual of
Microbiology Methods [22].
3.6. Evaluation of Individual and Consortium biocontrol
potential
The individual biocontrol agents, as well as the consortium
of biocontrol agents, were co-inoculated into tubes
containing potato dextrose broth to evaluate and compare
the potential of biocontrol capacity. This was conducted
between the obtained isolates. The tubes were then kept for
incubation at room temperature for a week. At the end of
the incubation period, dry weight analysis was performed
by filtering out the fungi on a filter paper and subjecting it
to hot air oven mediated drying at 60֯C for 1 hour [23]. The
dry weight of the fungi was taken and inhibition % was
calculated.
3.7. Preliminary bioassay to evaluate disease control
ability of the isolates
INVITRO BIOASSAY
Disease control potential of the isolates was checked on
papaya fruits in vitro. Fruits were spot inoculated [24] and
subjected to various treatments. The treatments were
replicated in triplicates for statistical validation.
Table I. Various treatments with its abbreviations
ABBREVIATION
TREATMENT
T1
CONTROL
T2
RHIZOPUS
T3
IS-6 + RHIZOPUS
T4
IS-7 + RHIZOPUS
T5
COLLETOTRICHUM
T6
IS-6 + COLLETOTRICHUM
T7
IS-7 + COLLETOTRICHUM
T8
FUSARIUM
T9
IS-6 + FUSARIUM
T10
IS-7 + FUSARIUM
IV. RESULT & DISCUSSION
4.1. Dual Plate Assay
The bacterial isolates obtained from the phylloplane
samples were subjected to screening using a dual plate
assay method. 3 different isolates numbered IS1, IS6 and
IS7 exhibited a good percentage of inhibition against the
fungal pathogens and hence were chosen for further
studies.
Fig. II. Dual assay of phylloplane bacteria against papaya fungal pathogens
4.2. Morphological and Biochemical characterization
Isolate 1 was found to be gram-positive rods; Isolate 6 was
identified as gram-positive, spore-forming rods. The
hanging drop method confirmed it to be motile rods. It was
able to degrade starch and casein. It showed positive for
catalase test, negative for citrate and gelatin liquefaction. It
was able to grow well on a nutrient agar plate containing
7% NaCl. Isolate 7 was identified based on its colony
colour on nutrient agar, its gram character, its inability to
ferment sugars tested, MR, VP, catalase, oxidase, citrate
and gelatin liquefaction tests.
Thus, IS6 was identified as Bacillus and IS7 was identified
as Pseudomonas.
4.3. Evaluation of Individual and Consortium biocontrol
potential
0
5
10
15
20
25
% Inhibition
Fungal Pathogens
DUAL ASSAY
ISOLATE 1
ISOLATE 6
ISOLATE 7

Ashwini Narasimhan Nadia et al. International Journal of Environment, Agriculture and Biotechnology, 6(1)-2021
ISSN: 2456-1878
https://dx.doi.org/10.22161/ijeab.61.38 310
The consortia of IS6 and IS7 showed higher disease
control potential in papaya fruit against the pathogens
Fusarium (type 2) and Colletotrichum.
Fig. III. Evaluation of Individual and Consortium biocontrol potential
4.4. Invitro bioassay to determine the disease control
potential of the isolates
Fruit assay conducted to assess the shelf life of the papaya
fruits and disease control potential of the isolates under in
vitro conditions showed that fruits treated with the isolates
and the pathogen exhibited better shelf life and appeared
fresh in comparison with the control and only pathogen
inoculated fruit. The reason for the same could be
bacterial-fungal antagonism where the disease control
bacterial isolates might produce antifungal metabolites or
modify the environment such that the fungal pathogens are
unable to grow. Fruits co-inoculated with IS – 7 and the
pathogens showed the maximum freshness. This shows the
significant biocontrol ability of post-harvest diseases of the
phylloplane bacterial isolates.
Table II. Invitro bioassay to determine the disease control
potential of the isolates
TREATMENTS
INFECTION
PERCENTAGE (%)
T1
22.2 ± 1.0
T2
100 ± 1.2
T3
100 ± 1.2
T4
11.1 ± 0.7
T5
100 ± 1.2
T6
0
T7
0
T8
100 ± 1.2
T9
11.1 ± 0.7
T10
0
0
20
40
60
80
100
120
Fungal dry weight (%)
Fungal Pathogens
Consortium Study
CONTROL
ISOLATE 6
ISOLATE 7
ISOLATE 6 + ISOLATE 7

Ashwini Narasimhan Nadia et al. International Journal of Environment, Agriculture and Biotechnology, 6(1)-2021
ISSN: 2456-1878
https://dx.doi.org/10.22161/ijeab.61.38 311
Fig. IV. Invitro bioassay to determine the disease control potential of the isolates
Fig. V. Control (papaya fruits without any treatment)
Fig. VI. Papaya fruits inoculated with Rhizopus
0
20
40
60
80
100
120
12345678910
INFECTION %
TREATMENTS
INVITRO BIOASSAY

Ashwini Narasimhan Nadia et al. International Journal of Environment, Agriculture and Biotechnology, 6(1)-2021
ISSN: 2456-1878
https://dx.doi.org/10.22161/ijeab.61.38 312
Fig. VII. Papaya fruits inoculated with Rhizopus and Isolate 6
Fig. VIII. Papaya fruits inoculated with Rhizopus and Isolate 7
Fig. IX. Control (papaya fruits with any treatment)
Fig. X. Papaya fruits inoculated with Colletotrichum

Ashwini Narasimhan Nadia et al. International Journal of Environment, Agriculture and Biotechnology, 6(1)-2021
ISSN: 2456-1878
https://dx.doi.org/10.22161/ijeab.61.38 313
Fig. XI. Papaya fruits inoculated with Colletotrichum and Isolate 6
Fig. XII. Papayas fruits inoculated with Colletotrichum and Isolate 7
Fig. XIII. Control (papaya fruits with any treatment)
Fig. XIV. Papaya fruits inoculated with Fusarium

Ashwini Narasimhan Nadia et al. International Journal of Environment, Agriculture and Biotechnology, 6(1)-2021
ISSN: 2456-1878
https://dx.doi.org/10.22161/ijeab.61.38 314
Fig. XV. Papaya fruits inoculated with Fusarium and Isolate 6
Fig. XVII. Papaya fruits inoculated with Fusarium and Isolate 7
V. CONCLUSION
From the studies conducted it is observed that bacterial
isolates from the phylloplane have the ability to control
fungal pathogen growth in papaya fruits. Out of 20 isolates
studied, it can be concluded that IS 6 and IS 7 had
maximum inhibitory activity and increased shelf life of the
papaya fruits. These two isolates were found to be Gram-
positive rods with endospores and Gram-negative rods,
respectively. The maximum inhibition was seen against
Fusarium. By the Dual assay test, it was revealed that
maximum antagonistic ability was revealed by IS – 6 and
IS – 7. The papaya fruits treated with IS – 6 and are – 7
showed better shelf life and appeared fresh. The isolates
were morphologically and biochemically characterized and
identified as Bacillus and Pseudomonas respectively. Thus
it can be concluded that Bacillus and Pseudomonas
obtained from the phylloplane of the papaya plant act as
potential biocontrol agents against various post-harvest
diseases of papaya.
VI. FUTURE SCOPE
• Pathogenicity testing of the potential biocontrol
agents
• Mode of action of the biocontrol agents
• Formulation studies
• Field studies
• Extension of post-harvest biocontrol potential to
other fruits and vegetables
ACKNOWLEDGEMENT
This project consumed a huge amount of work, research
and dedication, which would not have been possible
without the support of my parents, teachers and friends.
Also, many thanks to the management of Jain University
for providing this opportunity and infrastructural support.
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https://dx.doi.org/10.22161/ijeab.61.38 315
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