Journal
Journal of Applied Horticulture, 14(2): 102-109, 2012
Appl
Postharvest microbial diversity on major cultivars of Indian
mangoes
S.N. Jha*, Pranita Jaiswal, K. Narsaiah, Rishi Bhardwaj, Poonam Preet Kaur, Ashish Kumar
Singh, Rajiv Sharma and R. Kumar
Agricultural Structures and Environmental Control Division, Central Institute of Postharvest Engineering & Technology,
Ludhiana 141004, India. *E-mail: snjha_ciphet@yahoo.co.in
Abstract
Microbial diversity on fruit surface of nine mango cultivars (Alphonso, Banganapalli, Chausa, Dashehri, Kesar, Langra, Mallika,
Maldah and Neelam) harvested from orchards of nine Indian states (Andhra Pradesh, Bihar, Gujarat, Karnataka, Maharashtra, Orissa,
Punjab, Tamil Nadu and Uttar Pradesh) were studied using standard methods. A total of 47 fungal and 123 bacterial isolates were
purified from 761 mango samples, which included 63 Gram positive and 60 Gram negative bacterial isolates. The relative abundance
of Gram positive, Gram negative bacteria and different filamentous fungi varied among cultivars. Gram positive bacteria dominated on
Langra of Uttar Pradesh, while Dashehri from Punjab showed dominance of Gram negative bacteria. Among total fungal isolates, the
common genera were Aspergillus and Fusarium, while among bacterial isolates, the most common genera were Bacillus, Aeromonas,
Pseudomonas, Lactobacillus, Citrobacter, Mycobacterium and Serratia. Alphonso and Kesar variety from Maharashtra showed
maximum and minimum fungal diversity, respectively. Genera and species identified include members known for spoilage of fruits;
having all types of pectinase and cellulase activities and those used in biocontrol of plant pathogens.
Key words: Bacteria, biochemical, diversity, filamentous fungi, mango, relative abundance
Mango (Mangifera indica) is an important tropical fruit and
India is the largest mango producer contributing 37% of 30.5
million tons of total global mango production. Annually, India
supplies 50,000 tons of mangoes to different parts of the world
including Japan, Middle East, Europe and United States and the
demand is increasing year by year (Pandit et al., 2009). However,
tropical and subtropical fruits such as mango present greater
problems in transportation and storage due to its perishable
nature and presence of numerous microflora on its surface (Mitra
and Baldwin, 1997) which may cause spoilage of fruits. The
postharvest loss of such perishable commodities is estimated to
be as high as 50% (Mitra and Baldwin, 1997). This can partly be
reduced by knowing the spectrum of microbial community and
devising the measures to reduce their effect.
Microorganisms associated with postharvest spoilage of fruits
have drawn attention of scientists for years (Verma et al., 1991;
Okigbo, 2001). Mango fruit is also susceptible to many postharvest
diseases such as anthracnose (Colletotrichum gloeosporoides) and
stem end rot (Lasiodiplodia theobromae) during storage under
ambient conditions or even at low temperature (Arauz, 2000).
Spoilage microbes are capable of colonizing and creating lesions
on healthy, undamaged plant tissue (Tournas, 2005). Many
spoilage microorganisms use their extra cellular lytic enzymes
to degrade plant polymers into simpler fractions which can be
used as nutrients for their growth. Fungi and many bacterial
strains produce an abundance of extracellular pectinases and
hemicellulases, which play a major role in spoilage (Miedes and
Lorences, 2004). Besides, many microbes isolated from the fruit
surface have been identified to be useful agents in postharvest
treatment (Wisniewski and Wilson, 1992; Wilson et al., 1993).
Extensive studies have been conducted on the diversity of
epiphytic microbes on annuals bearing deciduous leaves (De
Jager et al., 2001; Joshi, 2008). Recently microbial population
on long living leaves of evergreen trees like mangoes have also
been studied (Pruvost and Luisetti, 1991; De Jager et al., 2001;
Ngarmsak et al., 2006). Information on microbial diversity on
Indian varieties of mangoes is missing, thererefore the objective
of this investigation was to study the microbial diversity on
the surface of mango fruit for deciding strategies to reduce
postharvest spoilage of commercially important varieties.
Materials and methods
Complementary Copy
Introduction
Sampling procedure: Major mango cultivars (Alphonso,
Banganapalli, Chausa, Dashehri, Kesar, Langra, Maldah, Mallika
and Neelam) were collected from orchards of nine Indian states
(Andhra Pradesh, Bihar, Gujarat, Karnataka, Maharashtra,
Orissa, Punjab, Tamil Nadu and Uttar Pradesh) using complete
randomized block design (Jha et al., 2010). Fruits with 5-10 cm
stalk were manually plucked directly in pre-sterilized zip locked
plastic bags in the forenoon, from each side and also from centre
of tree canopy in random manner. Sampling schedule along
with abbreviations used for each cultivar is presented in Table
1. Zip locked plastic bags were transported to the laboratory
in the well ventilated corrugated fiber board boxes along with
partially refrigerated gel packs placed in between the two layers
of mangoes to minimize the quality loss (Jha et al., 2010).
Isolation and growth of bacterial and fungal isolates:
Microbial communities from the mango fruit were isolated by
washing and dilution plating method (Jha et al., 2010). In order
to isolate total microbial diversity from mango surface, three
Postharvest microbial diversity on major cultivars of Indian mangoes
mangoes from each cultivars were used in experimentation.
The mangoes were washed and suitable dilutions of wash water
were prepared. Inoculums from different dilutions containing
surface micro flora from mango were plated on Nutrient Agar
(NA) and Potatao Dextrose Agar (PDA, Himedia, India) plates
in triplicate (Atlas and Snyder, 2006). NA plates were incubated
at 37oC and PDA plates at 28oC. After incubation, the individual
colonies were picked up and purified with streak plate method.
The isolates were grown on their respective media in petriplates
or slants to study their morphological features. Bacterial isolates
were characterized based on shape, color, surface and edge of
the bacterial colonies while filamentous fungi were characterized
based on their hyphal and spore characteristics according to
Hawksworth et al. (1995). Identification of filamentous fungi was
further confirmed at NTCC (National Type Culture Collection
Center), Indian Agricultural Research Institute, New Delhi based
on morphological features.
Biochemical characteristics: Each bacterial isolate was
propagated in nutrient broth before use and an overnight culture
was employed in the tests. Standard staining procedures (such
as Gram’s staining, Negative staining, Acid fast staining,
Spore staining) and biochemical tests namely citrate, urease,
hydrogen sulfide production, carbohydrate utilization test,
lactose fermentation test, glucose oxidation and fermentation test,
indole production test, methyl red test, oxidase test, catalase test,
flouroscein production test, cellulose degradation test and pectin
hydrolysis test were used for characterization of microbial isolates
using commercially available media (Himedia, India). The
isolated organisms were purified and assayed as recommended
in Bergey’s manual (Holt et al., 1994).
Statistical analysis: Evenness/Relative abundance (RA):
Evenness is defined as a measure of the relative abundance of
different species which was calculated as below:
RA (%) = (N x 100) /T
Where, N = Total number of isolates belonging to one group
T = Total number of isolates belonging to all groups
Richness was calculated as number of species per sample.
Simpson’s Index (D) was calculated as below:
D = ∑ (n/N)2
Where, n = Total no. of organisms of a particular species
N = Total no. of organisms of all species
Simpson’s Index of Diversity
Simpson’s index of diversity = 1 – D
Where, D = Simpson’s index
Results and discussion
Bacterial diversity: Microbial diversity describes complexity
and variability at different levels in an ecosystem where, microbes
play a crucial role in biological organization. Special interest
in the fruit surface microorganisms exist in view of identifying
microbes responsible for spoilage of mango fruit and further
development of biosensor by generating antibodies against them
for quick identification. Further, microbes present on plant surface
have been reported to fix atmospheric nitrogen, compete with
plant parasites, produce plant growth regulator such as gibberellic
acid and produce lipases which degrade surface waxes (Chun
and Mc Donald, 1987). If such organism also resides on fruit,
they may predispose fruit to moisture loss and decay during long
term storage. In current scenario, the adverse effects of synthetic
chemical residues on human health and the environment have lead
scientists from all over the world to develop alternative control
strategies such as studies on natural microbial diversity on fruit
surface and their role in plant protection.
A total of 123 bacterial strains were isolated from the mango
fruit samples of nine major cultivars collected from nine Indian
states. All the isolates were characterized initially based on the
biochemical nature of cell wall using standard Gram staining
procedure. Results indicated that among total 123 isolates,
Gram positive and Gram negative bacterial isolates were found
to be 51.22 and 48.78%, respectively. The RA of Gram positive
bacteria varied from 11.11% in CU to 75.00 % in LU (Table 2),
while that of Gram negative bacteria varied from 25.00 % in
LU to a maximum of 100.00 % in DP. In variety BA and NT,
the RA of Gram positive and Gram negative was found to be
almost same. Results showed great variation in RA of Gram
positive and Gram negative bacteria among different mango
cultivars (except BA, MO and NT) which might be attributed
to variation in environmental condition (humidity) at the time
of sampling at different sampling site. Silva et al. (2000) also
reported that the percent composition of Gram positive and Gram
negative bacteria is mainly governed by moisture content of the
atmosphere, with Gram positive bacteria dominated in wet and
Gram negative bacteria in dry atmosphere. The percentage of
rods was found to be twice that of cocci in both Gram positive
and Gram negative bacteria in all the mango cultivars indicating
the dominance of spoilage causing microflora, as most of the
bacterial strains responsible for spoilage of fruits and vegetables
are reported to be Gram positive or Gram negative rods (Frazier
and Westhoff, 2008).
Complementary Copy
Table 1. Abbreviations used for mango cultivars harvested from different
locations
Name of cultivar
Place of procurement
Abbreviations used
Alphonso
Karnataka
AK
Alphonso
Maharashtra
AM
Banganapalli
Andhra Pradesh
BA
Banganapalli
Orissa
BO
Chausa
Punjab
CP
Chausa
Uttar Pradesh
CU
Dashehri
Punjab
DP
Dashehri
Uttar Pradesh
DU
Kesar
Gujarat
KG
Kesar
Maharashtra
KM
Langra
Uttar Pradesh
LU
Maldha
Bihar
MB
Mallika
Orissa
MO
Neelam
Tamil Nadu
NT
103
Among total bacterial isolates, the colonies of 55 isolates were
found to be pigmented (Table 3). Such chromogenic isolates
potentially have selective advantage over other inhabitants
because their pigmentation protects them from ultra violet
radiation and are frequently isolated and had been reported to
colonize plant surface in large numbers (Crosse, 1971; Hirano
and Upper, 1991; Mansvelt and Hattingh, 1987).
Biochemical characterization of all the 123 bacterial isolates
showed presence of 63 Gram positive and 60 Gram negative
isolates (Table 3). Among Gram positive isolates, 40 were found
to be rod shaped and the rest 23 were cocci form. Further, among
104
Postharvest microbial diversity on major cultivars of Indian mangoes
Table 2. Number and Relative abundance of Gram positive and Gram negative bacteria from different mango cultivars
AK
AM
BA
BO
CP
CU
DP
DU
KG
KM
LU
MB
MO
NT
Gram positive Gram positive Gram negative Gram negative Gram positive Gram positive Gram negative Gram negative
(number)
(RA)
(number)
(RA)
rods (RA)
cocci (RA)
rods (RA)
cocci (RA)
9
13
5
4
4
1
0
1
6
5
3
1
4
7
52.94
72.22
50.00
57.14
66.67
11.11
0.00
25.00
60.00
62.50
75.00
16.67
44.44
50.00
8
5
5
3
2
8
1
3
4
3
1
5
5
7
47.06
27.78
50.00
42.86
33.33
88.89
100.00
75.00
40.00
37.50
25.00
83.33
55.56
50.00
Gram positive rods, 14 isolates were found to form spores and
26 were non-spore formers. These spore forming strains were
purified under purely aerobic conditions, therefore, the isolates
identified belonged to genera Bacillus. Among the 26 non-spore
formers, 10 showed positive acid fast reaction, which may be from
genus Mycobacterium and remaining 16 showed the negative acid
fast reaction. Further, results of catalase test showed that all the
16 bacterial isolates with negative acid fast reaction belonged
to Lactobacillus genus, out of which 13 bacterial isolates
showed acid formation on glucose fermentation test, which gave
indication that these isolates may be L. casei or L. delbrueckii and
remaining 3 bacterial isolates gave no reaction, indicating that
they belong to other species of Lactobacillus genera.
Bacillus spp. as a group is one of the important components of
soil microbial community (Prescott et al., 2006). Many Bacillus
spp. had been reported to secrete wide range of degradative
enzymes such as cellulases, amylases, pectinases and proteases
(Silva et al., 2000). Both pectinase and pectate lyase had been
reported from Bacillus spp. (Soriano et al., 2000). In current
investigation many identified isolates as Bacillus (according to
Bergey’s manual) have shown presence of hydrolytic enzymes
cellulases and pectinases, which might endow them with the
advantage of colonizing the fruit surface. De Jager et al. (2001)
also reported the predominance of Bacillus pumilis on mango leaf
surface besides other bacterial genera (Cornyform, Pseudomonas,
Xanthomonas and Erwinia) found to be present on mango
phylloplane.
All 23 isolated Gram positive cocci showed negative catalase
activity indicating that they may either belong to Streptococcus or
Enterococcus genus (Table 3). Among 60 Gram negative bacterial
isolates, 37 were rod shape and rest were cocci. Among Gram
negative rods 26 isolates showed positive oxidase test, and other
11 showed negative oxidase test. The isolates showing negative
oxidase test belonged to family Enterobacteriaceae. Out of 26
isolates with positive oxidase test, 24 exhibited acid production
on glucose fermentation, further, they didn’t require presence of
sodium in the medium for growth. Therefore, isolates represent
characteristics of genus Aeromonas as given in Bergey’s manual
and rest of bacterial strains (2), although were able to ferment
glucose, didn’t produce acid in the medium. Thereby, indicating
that these isolates can belong to genus Pseudomonas. Among
the Gram negative bacterial isolates, Aeromonas was found to
35.29
38.89
40.00
42.86
50.00
0.00
0.00
25.00
30.00
62.50
50.00
0.00
11.11
35.71
17.65
33.33
10.00
14.29
16.67
11.11
0.00
0.00
30.00
0.00
25.00
16.67
33.33
14.29
29.41
22.22
30.00
28.57
16.67
44.44
100.00
50.00
20.00
37.50
25.00
50.00
33.33
28.57
17.65
5.56
20.00
14.29
16.67
44.44
0.00
25.00
20.00
0.00
0.00
33.33
22.22
21.43
be the most dominant genera as it has been isolated from 12
mango cultivars (AK10, AK11, AK17, AM04, AM06, AM07,
AM12, BA05, BA09, BA10, BO03, CP06, CU01, CU02, CU07,
DU03, KG09, KM03, KM08, MB02, MB06, LU03, NT07 and
NT10), while Pseudomonas was found on BO02, MO07 and
NT05 cultivars. Aeromonas is widely found in nature including
decomposing vegetable matter, while Pseudomonas is well known
for their ability to metabolize a variety of diverse nutrients (Frazier
and Westhoff, 2008). Many strains have been reported to play an
important role in environmental biotechnological applications
(Walsh et al., 2001; Mark et al., 2006; Ali Khan and Ahmad,
2006). However, on the contrary, many other have been reported
to be phytopathogen and are widely dispersed on plants mainly
on leaves and rhizosphere (Silva et al., 2000). Bacterial isolates
belonging to Enterobacteriaceae family were further sub grouped
based on the lactose fermentation test and results indicated that
7 (MB03, DU02, MO08, CU08, KG01, MO09 and NT01) were
able to ferment lactose (Table 3). Further MB03 showed indole
production by oxidizing an essential amino acid tryptophan,
which utilized citrate as sole carbon source and showed negative
VP test which is characteristic of genus Citrobacter. DU02 and
MO08 were able to ferment lactose, showed indole production
but did not show citrate reaction, which is characteristic of genus
Escherichia. CU08, KG01, MO09 and NT01 did not oxidize
tryptophan but KG01 showed positive reaction for both MRVP tests indicating that KG01 may be Enterobacter intermedius.
CU08 & MO09 showed positive test for methyl red only and NT01
showed positive VP test only indicating that CU08 and MO09 may
be Serratia fonticola / Klebsiella pneumoniae (subsp. Ozaenae)
/ Citrobacter freundii and NT01 may be Klebsiella pneumoniae
(subsp. pneumoniae)/ Enterobacter spp. / Erwinia caotovora /
Serratia rubidaea. The other four isolates of Enterobacteriaceae
family showing negative reaction for lactose fermentation were
able to oxidize tryptophan to indole and showed negative result for
hydrogen sulfide production test, may belong to genus Morgenella
/Providencia. Enterobacter is a common inhabitant of soil and
sewage. Erwinia spp. is another common Gram negative spoilage
microbe associated with fresh-cut vegetables. Erwinia, a genus
within the family Enterobacteriaceae are small rods and facultative
anaerobes. Erwinia is reported to cause rapid necrosis, progressive
tissue maceration called “soft-rot”, occlusion of vessel elements
called “vascular wilt,” and hypertrophy leading to gall or tumor
formation in plant tissues (Margaret et al., 2009). Brocklehurst et al.
(1987) and Manvell and Ackland (1986) identified E. carotovora as
Complementary Copy
Cultivar
Table 3. Morphological and biochemical characteristics of bacterial isolates from nine different mango cultivars
Morphological characteristic
Biochemical characteristics
Isolate
Group
G N
S
A
P
T
S
1
2
3
4
5
6
7
8
9 10 11
AK1
A
+
R
+
+
+
+
+
+
AK2
A
+
R
+
+
+
+
+
+
+
+
AK3
A
+
R
+
+
+
+
+
AK4
A
+
R
+
+
+
+
+
+
+
+
+
+
AK5
A
+
R
+
+
+
+
+
+
AK6
B
+
C
+
+
+
+
+
+
+
+
+
+
AK7
B
+
C
+
+
+
+
+
+
+
+
AK8
B
+
C
+
+
+
+
+
+
+
+
AK9
A
+
R
+
+
+
+
+
+
+
+
+
AK10
C
R
+
+
+
+
+
+
+
AK11
C
R
+
+
+
+
+
+
+
+
+
+
AK12
C
R
+
+
+
+
+
+
+
+
+
AK13
D
C
+
+
+
+
+
+
AK14
D
C
+
+
+
+
+
+
+
+
AK15
D
C
+
+
+
+
+
+
+
AK16
C
R
+
+
+
+
+
+
+
+
+
AK17
C
R
+
+
+
+
+
+
+
+
+
AM1
B
+
C
+
+
+
+
+
+
AM2
A
+
R
+
+
+
+
+
AM3
B
+
C
+
+
+
+
+
+
+
+
AM4
C
R
+
+
+
+
+
+
+
AM5
B
+
C
+
+
+
+
+
+
AM6
C
R
+
+
+
+
+
+
+
+
AM7
C
R
+
+
+
+
+
+
+
+
AM8
B
+
C
+
+
+
+
+
+
AM9
A
+
R
+
+
+
+
+
+
AM10
A
+
R
+
+
+
+
AM11
A
+
R
+
+
+
+
+
+
AM12
C
R
+
+
+
+
+
+
AM13
A
+
R
+
+
+
+
+
+
AM14
A
+
C
+
+
+
+
+
+
AM15
A
+
C
+
+
+
+
+
AM16
D
C
+
+
+
+
+
+
AM17
A
+
R
+
+
+
+
+
+
AM18
A
+
R
+
+
+
+
+
+
+
BA1
A
+
R
+
+
+
+
+
+
+
+
+
BA2
B
+
C
+
+
+
+
+
+
BA3
D
C
+
+
+
+
+
+
BA4
A
+
R
+
+
+
+
+
BA5
C
R
+
+
+
+
+
+
+
BA6
D
C
+
+
+
+
+
+
+
BA7
A
+
R
+
+
+
+
+
+
BA8
A
+
R
+
+
+
+
+
+
+
+
BA9
C
R
+
+
+
+
+
+
+
+
+
+
BA10
C
R
+
+
+
+
+
+
+
BO1
D
C
+
+
+
+
+
+
BO2
C
R
+
+
+
+
+
+
BO3
D
C
+
+
+
+
+
+
+
BO4
A
+
R
+
+
+
+
+
+
+
+
BO5
B
+
C
+
+
+
+
+
+
+
BO6
A
+
R
+
+
+
+
+
+
BO7
A
+
R
+
+
+
+
+
+
+
+
+
+
CP1
B
+
C
+
+
+
+
+
+
CP2
A
+
R
+
+
+
+
+
CP3
A
+
R
+
+
+
+
+
+
+
CP4
D
C
+
+
+
+
+
+
+
+
+
+
+
CP5
A
+
R
+
+
+
+
+
+
+
CP6
C
R
+
+
+
+
+
+
+
+
+
CU1
C
R
+
+
+
+
+
+
+
+
CU2
C
R
+
+
+
+
+
+
CU3
D
C
+
+
+
+
+
CU4
B
+
C
+
+
+
+
+
-
105
12
-
13
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
14
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
15
+
+
+
+
+
+
+
+
+
-
16
+
+
+
+
+
+
+
+
+
-
17
+
+
+
+
+
+
+
+
-
Complementary Copy
Postharvest microbial diversity on major cultivars of Indian mangoes
CU5
CU6
CU7
CU8
CU9
DP1
DU1
DU2
DU3
DU4
KG1
KG2
KG3
KG4
KG5
KG6
KG7
KG8
KG9
KG10
KM1
KM2
KM3
KM4
KM5
KM6
KM7
KM8
MB1
MB2
MB3
MB4
MB5
MB6
LU1
LU2
LU3
LU4
MO1
MO2
MO3
MO4
MO5
MO6
MO7
MO8
MO9
NT1
NT2
NT3
NT4
NT5
NT6
NT7
NT8
NT9
NT10
NT11
NT12
NT13
NT14
Postharvest microbial diversity on major cultivars of Indian mangoes
D
D
C
C
D
C
D
C
C
A
C
B
D
A
D
B
A
B
C
A
A
A
C
A
C
A
A
C
B
C
C
D
D
C
A
A
C
B
B
A
D
B
D
B
C
C
C
C
A
D
D
C
A
C
A
A
C
A
B
D
B
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
C
C
R
R
C
R
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Complementary Copy
106
G=Gram reaction, N=Negative staining, S=Spore staining, A=Acid fast staining, P=Pigmented colony, T=Transparent colony, S=Smooth edge on colony.
Biochemical characteristics: 1=Lactose fermentation 2=Casein Hydrolysis 3=Starch hydrolysis 4=Flouroscein production 5=Peroxidase reaction
6=Gelatin hydrolyis 7=Methyl red test 8=Voges-Proseuker test 9=Urease test 10= Indole production test 11=Citrate utilization test 12= Hydrogen
sulphide production test 13=Glucose fermentation test 14= Oxidase test 15 = Cellulose degradation test 16=Pectate lyase production test 17= Pectinase
production test
Postharvest microbial diversity on major cultivars of Indian mangoes
107
Table 4. Mold diversity on different mango cultivars from different parts of India
Alphonso,
Maharashtra
Isolate
AM01
AM02, 07, 10
AM08
AM03, 04, 06
AM05, 13
AM09
Alphonso,
AK02,0 4
Karnataka
AK03
Baganpalli,
BA02
Andhra Pradesh
BA08
Baganpalli, Orissa BO03
Chausa, Punjab
CP01, 03
CP02
Chausa,
CU01
Uttar Pradesh
CU04
CU03
CU06
Dashehri, Punjab DP01
DP02
DP03
DP04
Kesar, Gujarat
KG01
KG02
KG03
Kesar, Maharashtra KM01, 02
Langra,
LU01
Uttar Pradesh
LU02
LU03
Maldah, Bihar
MB01
MB03
Mallika, Orissa
MO05
MO01
MO06
MO02
MO03
Neelam,
NT01
Tamil Nadu
NT02
NT03, 4
Fungus
Evenness / Relative
Abundance, RA (%)
Species
9.09
A. fumigatus
27.27
A. flavus
9.09
A. niger
27.27
Fusarium
F. solani
18.18
Alternaria
A. alternata
9.09
Cladosporium
C .cladosporioides
66.66
Aspergillus
A. niger
33.33
A. flavus
50
Aspergillus
A.terreus
50
Pencillium
P. citrinum
100
Aspergillus
A. niger
66.67
Aspergillus
A. fumigatus
33.33
Pencillium
P. chrysogenum
25
Fusarium
F. moniliforme
25
F. oxysporum
25
Aspergillus
A. flavus
25
A. fumigatus
25
Fusarium
F. pallidoroseum
25
Aspergillus
A. niger
25
A. nidulans
25
A. fumigatus
33.33
Fusarium
F. moniliforme
Aspergillus
A. fumigatus
33.33
33.33
A. flavus
100
Aspergillus
A. niger
33.33
Alternaria
A. alternata
33.33
Aspergillus
A. niger
33.33
Heminthosporium H. spiciferum
50
Aspergillus
A. fumigatus
50
Trichoderma
T. longibrachiatum
20
Fusarium
F. pallidoroseum
20
Pencillium
P. chrysogenum
20
P. oxalicum
20
Aspergillus
A. terreus
20
A. niger
25
Aspergillus
A. flavus
25
A. terreus
50
A. niger
Genera
Aspergillus
a principal spoilage microbe of both fresh-cut and fresh vegetables.
Buick and Damoglou (1987) found that E. carotovora was the
dominant spoilage microorganism on sliced carrots packed in air,
consisting of more than 80% of total detectable microflora. Robbs
et al. (1996) identified Erwinia in 5 of 16 soft-rot samples of
fresh-cut celery. Erwinia had been reported to produce pectinolytic
enzymes, which cause degradation of cell wall leading to soft rot
disease (Lund, 1983).
All the bacterial isolates were tested for presence of hydrolytic
enzymes. Out of 123 isolates, 50 showed cellulase activity and 32
showed pectin hydrolysis out of which 17 showed production of
pectatae lyase and 15 showed production of polygalacturonase.
Such microbes having capability of producing hydrolytic
enzymes may use it to overcome plant defence mechanisms and
gain access to plant nutrients. The pectolytic enzymes, including
pectin methyl esterase (PME), polygalacturonase (PG), pectin
lyase (PNL), and pectate lyase (PL), can degrade pectins in the
middle lamella of the cell, thereby resulting in liquefaction of the
plant tissue leading to conditions such as soft rot.
Besides, many strains of Bacillus and Erwinia had been reported
to show antagonistic effect against pathogens causing black spot
Species
Richness
Simpson’s
index (D)
Simpson’s index of
Diversity (1-D)
6
0.17
0.83
2
0.56
0.44
2
0.50
0.50
1
1.00
0.00
2
0.56
0.44
4
0.25
0.75
4
0.25
0.75
3
0.33
0.66
1
1.00
0.00
3
0.33
0.66
2
0.50
0.50
5
0.20
0.80
3
0.37
0.63
Complementary Copy
Mango cultivar
of mango (Pruvost and Luisetti, 1991). Okigbo and Osuinde
(2003) reported bio-control of fungal leaf spot disease of mango
with Bacillus subtilis. In South Africa, concerted efforts had been
made to develop biocontrol agent against anthracnose disease
of mango (Burger and Korsten, 1988). Korsten et al. (1991)
reported effective reduction in the incidence of the disease when
Bacillus licheniformis was used as either pre or postharvest
application. They further reported that the efficacy of the biocontrol agent could be further improved when it was applied with
recommended fungicide used at lower concentration (Korsten et
al., 1992, Silimela and Korsten, 2001). Bacillus licheniformis is
now available in commercial formulation (Mango green), and is
reported to effectively reduce the fungal population from mango
surface (Govender et al., 2005).
Fungal diversity: Altogether 47 filamentous fungal isolates were
purified from mango fruit surface of nine mango cultivars and
among them the most abundant genera was Aspergillus followed
by Fusarium and Pencillium with a relative abundance (RA) of
60.00, 17.00 and 8.00 %, respectively. Other fungi identified
in minority belonged to genus Alternaria, Cladosporium,
Helminthosporium and Trichoderma.
108
Postharvest microbial diversity on major cultivars of Indian mangoes
Among the genus Aspergillus, the dominant species were A.niger,
A. flavus and A. fumigates., while the genus Fusarium was
dominated by F. monoliforme. Aspergillus species are highly
aerobic and widespread in nature, being found on fruits, vegetables
and other substrates which may provide nutriment, where they
commonly grow as molds on the surface of a substrate, as a
result of the high oxygen tension. Some species of this genus are
involved in food spoilage (Pelczar et al., 2008).
Acknowledgement
Aspergillus niger and Aspergillus flavus were found to be most
common on mango cultivars representing 23.00 and 15.00 % of
the total fungal isolates, respectively. Aspergillus niger is reported
to cause black mould disease on certain fruits and vegetables such
as grapes, onions and peanuts and is a common contaminant of
food. It is ubiquitous in soil and is commonly reported from indoor
environments (Frazier and Westhoff, 2008). Fungal composition
did not vary much among the cultivars however, maximum
species richness was found on AM followed by MO, CU and DP
while lowest on BO and KM. Alphonso mango harvested from
Maharastra (AM) showed presence of 11 fungi belonging to genus
Aspergillus, Fusarium, Alternaria and Cladosporium with RA
45.45, 27.23, 18.18 and 9.09 %, respectively, thereby exhibiting
highest Simpson’s index of diversity (measure of the probability
that two individuals randomly selected from a sample will belong
to the same species.) amongst various mango cultivars under
study (Table 4). While cv. BO and KM showed the presence of
Aspergillus niger only and hence, depicting lowest species richness
and Simpson’s diversity index (Table 4). Tournas and Katsoudas
(2005) also reported the dominance of Alternaria, Cladosporium,
Penicilium, Fusarium, Trichoderma, Geotrichum, Rhizopus and
A. niger in their study with citrus fruits.
Ali Khan, M.W. and M. Ahmad, 2006. Detoxification and bioremediation
potential of a Pseudomonas fluorescens isolate against the major
Indian water pollutants. J. Environ. Sci. Health Part A Toxic/
Hazardous Subsances Environ. Eng., 41(4): 659-674.
Atlas, R.M. and J.W. Snyder, 2006. Handbook of Media for Clinical
microbiology. CRC Press, London.
Arauz, L.F. 2000. Mango anthracnose: economic impact and current
options for exported mango. Plant Disease, 84: 600-611.
Barnby, F.M., F.F. Morpeth and D.L. Pyle, 1990. Endopolygalacturonase
production from Kluyveromyces marxianus. I. Resolution,
purification and partial characterization of the enzyme. Enzym.
Microb. Tech., 12: 891-897.
Brocklehurst, T.F., C.M. Zaman-Wong and B.M. Lund, 1987. A note
on the microbiology of retail packs of prepared salad vegetables. J.
Appl. Bact., 63: 409-415.
Buick, R.K. and P.A. Damoglou, 1987. The effect of vacuum-packaging
on the microbial spoilage and shelf-life of “ready-to-use” sliced
carrots. J. Sci. Food Agr., 38: 167-175.
Burger, R. and L. Korsten, 1988. Isolation of antagonistic bacteria against
Xanthomonas campestris pv. Mangiferaeindicae. South African
Mango Growers Association Yearbook, 8: 9-10.
Chun, D. and R.E. Mcdonald, 1987. Seasonal trends in the population
dynamics of fungi, yeasts and bacteria on fruit surface of grape fruit
in Florida. Proc. Florida State Hort. Soc., 100: 23-25.
Crosse, J.E. 1971. Interactions between saprophytic and pathogenic
bacteria in plant disease. In: Ecology of Leaf Surface Microorganisms,
Preece, T.F., C.H. Dickinson, (eds.). Academic Press, London. p.
283-90.
De Jager, E.S., F.C. Wehner and L. Korsten, 2001. Microbial ecology on
the mango phylloplane. Microbial Ecol., 42: 201-207.
Frazier, C.W. and C.D. Westhoff, 2008. Food Microbiology. Fourth
Edition. McGraw Hill, New Delhi.
Govender, V., L. Korsten and D. Sivakumar, 2005. Semi-commercial
evaluation of Bacillus licheniformis to control mango postharvest
disease in South Africa. Postharvest Biol. Technol., 38: 57-65.
Hawksworth, D.L., P.M. Kirk, B.C. Sutton and D.N. Pegler, 1995.
Ainsworth and Bisby’s Dictionary of the Fungi. CAB International,
Wallinford, UK.
Hirano, S.S. and C.D. Upper, 1991. Bacterial community dynamics.
In: Microbiol. Ecology of leaves. J.H. Andrew and S.S. Hirano
(eds.).Springer-Verlag, New York. p. 271-294.
Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Stanley and S.T. Williams,
1994. In: Bergey’s Manual of Determinative Bacteriology. M.D.
Baltimore, (eds.). Ninth edition. Williams & Wilkins. p. 787.
Hoondal, G.S, R.P. Tiwari, R. Tewari, N. Dahiya and G.K. Beg, 2002.
Microbial alkaline pectinases and their industrial applications - A
review. Appl. Microbiol. Biotechnol., 59: 409-418.
Jha, S.N., P. Jaiswal, K. Narsaiah, R. Bhardwaj, R. Sharma, R. Kumar
and A. Basedia, 2010. Postharvest micro-flora on major cultivars of
Indian mangoes. Scientia Hort., 125(4): 617-621.
Joshi, S.R. 2008. Influence of roadside pollution on the phylloplane
microbial community of Alnus nepalensis (Betulaceae). Rev. de boil.
Trop., 56(3): 1521-1529.
Korsten, L., J.H. Lonsdale, E. De Villiers and E.S.De Jager, 1992.
Preharvest Biological Control of Mango Diseases. South African
Mango Growers Association Yearbook, 12: 72-74.
Current study showed that mango surface harbored a huge
microbial diversity, which comprised varietal as well as regional
variation. In general, Gram positive rods showed predominance
in bacterial community and Aspergillus spp. in fungal community.
The highest fungal diversity was observed in variety AM, followed
by MO, CU, DP, KG, LU, NT, BA, MB, AK, CP, BO, KM. This
study is an important step in identification of spectrum of microbial
community on mango surface which will be helpful in devising
measures to reduce the effect of spoilage and pathogenic microbes.
Further, the available bio-resources on mango surface can be used
to screen the potential micro-flora with a role in biocontrol of plant
diseases, for production of metabolite/enzyme of commercial
importance or for the development of instrumental methods such
as biosensors, spectroscopic instruments, etc. for rapid detection
of microbial as well as physico-chemical characteristics.
References
Complementary Copy
The fungal isolates were tested for presence of hydrolytic
enzymes (cellulases and pectinases) and results indicated that
6 out of 47 isolates showed presence of one or more hydrolytic
enzymes (data not shown). Majority of these isolates belonged
to genus Aspergillus except one, which belonged to genus
Alternaria. Aspergillus is well known fungus for its hydrolytic
activity. Almost all the commercial preparartions of pectinases
are produced from fungal sources (Singh et al., 1999). Aspergillus
niger is the most commonly used fungal species for industrial
production of pectinolytic enzymes (Kotzekidov, 1991; Barnby
et al., 1990; Naidu and Panda, 1998).
This research was supported by the National Agricultural Innovation
Project (NAIP), Indian Council of Agricultural Research (ICAR)
through its subproject entitled “Development of non-destructive
systems for evaluation of microbial and physico-chemical quality
parameters of mango” Code number “C1030”.
Postharvest microbial diversity on major cultivars of Indian mangoes
Pelczar, M.J., E.C.S. Chan and N.R. Krieg, 2008. Microbilogy. 5th ed.
McGraw Hill, New Delhi, India.
Prescott, M.L., P.J. Harley and A.D. Klein, 2006. Microbilogy. 6th ed.
McGraw Hill, Singapore.
Pruvost, O. and J. Luisetti, 1991. Effect of time of inoculation with
Xanthomonas campestris pv. mangiferaeindicae on mango fruits
susceptibility. Epiphytic survival of X. c. pv. mangiferaeindicae on
mango fruits in relation to disease development. J. Phytopathol.,
133(2): 139-151.
Robbs, P.G., J.A. Bartz, G. McFie and N.C. Hodge, 1996. Causes of
decay of fresh-cut celery. J. Food Sci., 61: 444-448.
Silimela, M. and L. Korsten, 2001. Alternative methods for preventing
Pre and Post harvest diseases and sunburn on mango fruits. South
African Mango Growers Association Yearbook, 21: 39-43.
Silva, C.F., R.F. Schwan, E.S. Dias and A.E. Wheals, 2000. Microbial
diversity during maturation and natural processing of coffee cherries
of Coffea arabica in Brazil. Int. J. Food Microbiol., 60: 251-260.
Singh, S.A., M. Ramakrishna and A.G.A. Rao, 1999. Optimization of
down-stream processing parameters for the recovery of pectinase
from the fermented broth of Aspergillus carbonarious. Proc.
Biochem., 35: 411-417.
Snedecor, G.W. and W.G. Cochran, 1967. Statistical Methods. Oxford
and IBH Publishing Co. Pvt.Ltd.
Soriano, M., A. Blanco, P. Diaz, F. Jawier and I. Pastor, 2000. An unusual
pectate lyase from a bacillus species with high activity on pectin:
cloning and characterization. Microbiology (UK), 146: 89-95.
Tournas, V.H. 2005. Spoilage of vegetable crops by bacteria and fungi
and related health hazards. Crit. Rev. Microbiol., 31: 33-44.
Tournas, V.H. and E. Katsoudas, 2005. Mould and Yeast flora in fresh
berries, grapes and citrus fruits. Int. J. Food Microbiol., 105: 11-17.
Ubalua, A.O. 2007. Cassava waste: treatment options and value addition
alternatives. African J. Biotechnol., 6(18): 2065-2073.
Verma, K.S., S.S. Cheema, M.S. Kang and A.K. Sharma, 1991. Hitherto
unrecorded disease problems of mango from Punjab. Plant Dis.
Res., 6: 141-142.
Walsh, U.F., J. P. Morrissey and F.O’Gara, 2001. Pseudomonas for
biocontrol of phytopathogens: from functional genomics to commercial
exploitation. Current Opinion in Biotechnol., 12(3): 289-295.
Wilson, C.L., M.E. Wisniewski, S. Droby and E. Chalutz, 1993. A
selection strategy for microbial antagonists to control post-harvest
diseases of fruits and vegetables. Scientia Hort., 53: 183-189.
Wisniewski, M.E. and C.L. Wilson, 1992. Biological control of postharvest diseases of fruits and vegetables. Scientia Hort., 27: 9498.
Weaver, J.E., H.W. Hogmire, J.L. Brooks and J.C. Sencindiver, 1990.
Assesment of pesticide residues in surface and soil water from a
commercial apple orchard. Appl. Agr. Res., 5: 37-43.
Complementary Copy
Korsten, L., M.W.S. Van harmelen, A. Heitmann, E. DeVilliers and E.S. De
Jager, 1991. Biological Control of Postharvest Mango Fruit Diseases.
South African Mango Growers Association Yearbook, 11: 65-67.
Kotzekidov, P. 1991. Production of polygalacturonases by Byssachlamys
fulva. J. Ind. Microbiol., 7: 53-56.
Lichtenberg, E. and D. Zilberman, 1987. Regulating environmental and
human health risk from agricultural residuals. Appl. Agr. Res., 2:
56-64.
Lund, B.M. 1983. Bacterial spoilage. In: Postharvest Pathology of
Fruits and Vegetables. Dennis, C. (ed.). Academic Press, London.
p. 218-257.
Mansvelt, E.L. and M.J. Hattingh, 1987. Scanning electron microscopy of
colonization of pear leaves by Pseudomonas syringae pv. Syringae.
Canad. J. Bot., 65: 2517-2522.
Manvell, P.M. and M.R. Ackland, 1986. Rapid detection of microbial
growth in vegetable salads at chill and abuse temperatures. Food
Microbiol., 3: 59-65.
Margaret, B., R.H. Thomas, Z. Hong and B. Frederick, 2009.
Microbiological Spoilage of Fruits and Vegetables. In: Compendium
of the Microbiological Spoilage of Foods and Beverages. Food
Microbiology and Food Safety, Sperber, W.H. and M.P. Doyle, (eds.).
doi, 10.1007/978-1-4419-0826-1_6, ©Springer Science+Business
Media, LLC, 2009.
Mark, G., J.P. Morrissey, P. Higgins and F. O’gara, 2006. Molecularbased strategies to exploit Pseudomonas biocontrol strains for
environmental biotechnology applications. FEMS Microbiol. Ecol.,
56(2): 167-177.
Miedes, E. and E.P. Lorences, 2004. Apples (Malus domenstica)
and tomato (Lycopersicum) fruits cell-wall hemicelluloses and
xyloglucan degradation during pencillium expansum infection. J.
Agr. Food Chem., 52: 7957-7963.
Mitra, S.K. and E.Z. Baldwin,1997. Mango. In: Postharvest Physiology
and Storage of Tropical and Subtropical Fruits, CAB International,
West Bengal, India. p. 85-122.
Naidu, G.S.N. and T. Panda, 1998. Production of pectolytic enzymes-A
review. Bioproc. Eng., 19: 355-361.
Ngarmsak, M., P. Delaquis, P. Toivonen, T. Ngarmsak, B. Ooraikul
and G. Mazza, 2006. Microbiology of fresh-cut mangoes prepared
from fruit sanitised in hot chlorinated water. Food Sci. Tech. Inter.,
12: 95-103.
Okigbo, R.N. 2001. Occurrence, pathogenicity and survival of
Macrophoma mangiferae in leaves, branches and stems of mango
(Mangifera indica L.). Plant Prot. Sci., 37: 138-144.
Okigbo, R.N. and M.I. Osuinde, 2003. Fungal leaf spot diseases of mango
(Mangifera indica) in South Eastern Nigeria and biological control
with Bacillus subtilis. Plant Prot. Sci., 39(2): 70-77.
Pandit, S.S., H.G. Chidley, R.S. Kulkarni, K.H. Pujari, A.P. Giri and V.S.
Gupta, 2009. Cultivar relationships in mango based on fruit volatile
profiles. Food Chem., 114: 363-372.
Patil, S.R. and A. Dayanand, 2006. Exploration of regional agrowastes
for the production of pectinase by Aspergillus niger. Food Technol.
Biotechnol., 44(2): 289-292.
109
Received: February, 2012; Revised: July, 2012; Accepted: October, 2012