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S. THAHIR BASHA, M. NAGALAKSHMI DEVAMMA* AND N.P. ESWARA REDDY
Department of Botany, Sri Venkateswara University, Tirupati – 517502, India
Environmental problems have raised great interest in ecofriendly sustainable agricultural practices. Mango (Mangifera
indica L.) is one of the most important fruit crops in India and the state of Andhra Pradesh is leading in production and productivity
is severely affected with the devastating anthracnose disease caused by Colletotrichum gloeosporioides reducing the fruit quality
and responsible for 30 to 60 per cent of postharvest losses. Of all the 276 (bacteria – 247 + fungi – 29) putative leaf endophytes
screened against the aggressive pathogenic isolate Cg25, the complete inhibition (100%) of the C. gloeosporioides pathogen was
recorded by the bacterial endophytes EB07, EB35, EB39, EB99, EB57 and EB59. The potential bacterial leaf endophyte EB35
along with its compatible fungicide thiophanate methyl (25 ppm) proved to be the best combination in combating the anthracnose
disease both in vitro and in field trials and delayed the ripening of mango fruits upto 14 days was identified as Bacillus subtilis
(EB35) based on 16S rRNA analysis. The talc based formulations of these endophytes evaluated upto 90 days were viable at 4°C
and remained potent against C. gloeosporioides in dual culture study. The suppressive effect of these beneficial endophytes may
also be affected by environmental conditions. In this context research findings on development of cost effective and ecofriendly
region specific formulations against C. gloeosporioides are herewith proposed.
KEYWORDS: Colletotrichum gloeosporioides, anthracnose, endophytes, formulations.
Mango (Mangifera indica L.) is considered as one
of the most popular and choicest fruit trees grown
throughout the tropics and subtropics worldwide known
as “King of fruits” (Shad et al., 2002) is grown more
than 90 countries in the world and the global production
of the mango has doubled in the past thirty years. The
total global area under mango is 43.69 lakh ha and the
global production is to the tune of 312.51 lakh tones. India
ranks first among top world’s mango producing countries
contributing nearly 49.62 per cent of world’s area
accounting for 47.80 per cent of the global production
(National Horticulture Board, New Delhi). Most of the
Indian mango varieties have specific eco-geographical
requirements for optimum growth and yield. The major
mango growing states are Andhra Pradesh, Uttar Pradesh,
Karnataka, Bihar, Gujarat and Tamil Nadu. Andhra
Pradesh ranks second in mango production with a share
of 15.23 per cent and highest productivity. The country
has exported 42,998.31 MT of fresh mangoes to the world
for the worth of ` 302.54 crores during the year 2014-15.
The major export destinations during 2014-15 are United
Arab Emirates (54.98%), Nepal (22.78%), United
Kingdom (4.12%), Saudi Arabia (3.85%), Qatar (2.80%),
Kuwait and Bahrain (2.06%), Oman (1.17%) and
Singapore (1.60%). Although a lion’s share of Indian
mango goes to the Gulf countries, efforts are being made
to exploit European, American and Asian markets too
(Agricultural and Processed Food Products Export
Development Authority (APEDA) and National
Horticulture Board, New Delhi). Among all the diseases,
mango anthracnose caused by Colletotrichum
gloeosporioides (Penz.) Penz. and Sacc., (teleomorph:
Glomerella cingulata ) is the most serious disease (Ploetz,
1999) and is the most important disease of mango in
humid production areas (Arauz, 2000; Dodd et al., 1997;
Lim and Khoo, 1985; Ploetz and Freeman, 2009).
Anthracnose is also the major postharvest disease of
mango in all mango producing areas of the world (Dodd
et al., 1997) and (Swart et al., 2002). The fungus prefers
warm humid environment for spreading the anthracnose
disease uniformly and effectively. It is the major pre and
postharvest disease of mango (Arauz, 2000), and can
result in serious decay of fruit during marketing and after
sale. Over recent decades there has been increase in public
outcry to minimize the use of synthetic fungicides in
agriculture products and their presence in the
environment. Synthetic fungicides are the primary means
to control postharvest diseases (Eckert, 1990) either used
alone, combined in mixtures, or applied separately in
sequence (Ismail and Zhang, 2004). These fungicides are
at risk for resistance development because a change in
the pathogen at this point can render the fungicide less
effective or ineffective. Several fungicides are reported
to reduce disease development, but are uneconomical and
also cause environmental pollution (Misra and Pandey,
1999). Endophytic bacteria are referred to as those which
can be detected at a particular moment within the tissues
of apparently healthy plant hosts (Hallmann et al., 1997).
Many endophytic bacteria possess a number of plant
beneficial traits in vitro; few of those exhibit them in
planta and only a small number of endophytes proved to
be very effective plant growth promoting biocontrol
agents (Scherwinski et al., 2008). All surfaces of living
plants leak nutrients that may support microbial growth
and thus endophytes which colonize in internal plant
tissues, thereby gain direct access to nutrients within a
protected environment. The intent of this study is to
provide insights into the enormous benefits of leaf
endophytes, the products they make, and how some of
these organisms assure to show potential use in agriculture
that draws much attention as a sustainable alternative to
synthetic fungicides and resistance related issues. Hence,
efforts are made in order to reduce identify the potential
leaf endophytes against C.gloeosporioides causing mango
anthracnose that add value to ecofriendly agriculture and
can easily adapt the specific agro climatic regions.
MATERIAL AND METHODS
Isolation and identification of pathogen
The pathogen was isolated from the mango leaves
showing typical symptoms of anthracnose disease by
tissue segment method (Rangaswami and Mahadevan,
1999) on potato dextrose agar medium (PDA). The fungal
colonies developed were purified by single spore isolation
method (Rangaswami and Mahadevan, 1999) and the
pathogen was identified based on its mycelial and conidial
characteristics as per standard mycological keys (Barnett
and Hunter, 1972).
Pathogenicity test
One year old Baneshan mango grafts were infected
by pin prick method (Bhuvaneswari and Rao, 2001)
followed by spraying spore containing a load of 2.0 × 104
conidia ml-1. Alcohol washed hand atomizer was used
separately for spraying inoculum suspension of each
isolate. The inoculated seedlings were covered with
polythene bags for two days to ensure high humidity and
sterile distilled water served as control. Three replications
were maintained for each isolate and the disease severity
was calculated based on the 0-4 disease rating scale after
8-10 days of inoculation (Agostini et al., 1992).
Isolation and screening for potential endophytes
against mango anthracnose
Five grams of healthy leaves were surface sterilized
for five minutes with 70 per cent ethanol and homogenized
in 20 ml of sterilized phosphate buffer using a mortar
and pestle. Appropriate dilutions (10-4 for fungi and 10-6
for bacteria) of these suspensions are plated on PDA and
NA for the isolation of fungi and bacteria, respectively.
The plates were incubated at 28 ± 2°C for the development
of colonies (Kishore et al., 2005a). Dual culture technique
was employed to identify the potential antagonistic leaf
endophytes (Bhuvaneswari and Rao, 2001). Mycelial
discs measuring 6 mm diameter from four days old
cultures of both fungal antagonist and the test pathogen
were placed at equidistant on sterile Petri plate containing
PDA medium. One day old cultures of bacteria were
streaked on opposite side of the pathogen on PDA medium
and the Petri plates.
Compatibility of antagonistic endophytes with
different fungicides
Spectrophotometric method (Kishore et al., 2005b)
was used to determine the compatibility of antagonistic
bacterial isolates with the fungicides. Five hundred
microlitres of antagonistic bacterial cultures grown in
nutrient broth (NB) for 16 hours at 28 ± 2°C at 180 rpm
were added to 50 ml of NB in 250 ml flasks containing
different fungicides under study. Inoculated flasks were
incubated at 28 ± 2°C and the bacterial growth was
recorded at 600 nm after 24 hours of incubation.
Mass multiplication of potential bacterial leaf
endophytes
The talc based formulations of fungicide compatible
potential leaf endophytic bacteria were prepared as
described by Vidhyasekharan and Muthamilan (1995).
Overnight grown 10 ml of antagonistic bacterium was
inoculated into one litre of NB and grown in rotary at
150 rpm for 48 h and 72 h at 28 ± 2°C respectively. One
kg of talc powder (montmorillonite) was taken in a metal
tray under aseptic conditions and pH was adjusted to 7.0
by adding calcium carbonate (CaCO3
) @ 15 g/kg. 10 g of
carboxymethyl cellulose (CMC) was added to 1 kg of
talc, mixed well and the mixture was autoclaved for 30
minutes at 121°C for 2 successive days. 400 ml of the
bacterial suspension containing 1 × 108
cfu/ml was mixed
with carrier cellulose mixture under aseptic conditions.
After drying (35% moisture content) overnight under
aseptic conditions, the mixture was packed in a
polypropylene bag, sealed and stored both at room
temperature (28 ± 2°C) and refrigerator (4°C).
Shelf life and efficacy of bacterial formulations
The talc based formulations developed were
evaluated periodically in order to determine the shelf life
and efficacy of the potential endophytes for every 15 days
upto 90 days.
Identification of potential bacterial leaf endophytes
by16S rRNA analysis
The 16S rRNA analysis has been selected for
identification of potential bacterial leaf endophytes
(Marchesi et al., 1998. As a part of this, PCR technique
has been standardized using 63F as forward primer (51
–
CAG GCC TAA CAC ATG CAA GTC – 31
) and 1387R
(51
– GGG CGG (AT) GT GTA CAA GGC – 31
) as reverse
primer (Marchesi et al., 1998). PCR amplifications were
carried out in 0.2 ml eppendorf tubes with 25 μl reaction
mixture which consists of 2.5 μl of 10x Taq buffer, 2.0 μl
of 25 mM MgCl2
, 2.0 μl of respective primer (10
picomoles / μl), 1.0 μl of 10 mM dNTP mix, 1.25 μl of
Taq polymerase enzyme (conc. 3 U μl-1) and 11.25 μl of
sterile PCR water (Genei, Bangalore) and 2 μl (40-50
ng) of DNA sample. Amplification was carried out by 5
minutes of initial denaturation at 94°C followed by 30
cycles of denaturation of 94°C for 1 minute; annealing at
56.8°C for 1 minutes; extension at 72°C for 1.5 minutes
with final elongation at 72°C for 5 minutes. Amplified
PCR products were subjected to 1.0 per cent agarose gel
electrophoresis with 1.0 × TBE as running buffer. The
banding patterns were visualized under UV transilluminator
with ethidium bromide (10 mg ml-1) staining.
The DNA banding profiles were documented in the gel
documentation system (Alpha Innotech) and compared
with 1 kb DNA ladder (Genei, Bangalore).
RESULTS AND DISCUSSION
Twenty eight isolates of C. gloeosporioides were
isolated from the mango leaves showing typical
anthracnose symptoms appeared oval, brown to black
spots with greyish centre on the leaves. The results are in
agreement with the earlier findings reported by Banos et
al., 2003 and Linh, 2007. Conidia produced on branch
terminals, mummified inflorescences, flower bracts and
leaves (most important) are significant sources of
inoculum (Dodd et al., 1991; Fitzell, 1979). They are
produced most abundantly when free moisture is
available, but also at relative humidities as low as 95 per
cent. Conidia are dispersed by rain splash and infection
requires free moisture (Jeffries et al., 1990).
The pathogenicity of different isolates of
C.gloeosporioides was tested by spray inoculation method
on one year old baneshan mango grafts revealed that the
maximum incidence of disease was recorded in the isolate
Cg25 (56.14%) and the least percent disease was observed
in case of Cg13 (25.48%). This highly virulent isolate
Cg23 was chosen for further experiments. Based on per
cent disease incidence the isolates were classified into
highly virulent, moderately virulent and less virulent (data
not shown). Sampath kumar et al., (2007) also used the
same method for studying the pathogenic variability
among the isolates of C. gloeosporioides. Shivakumar et
al., (2015) reported the maximum PDI was recorded in
isolate Cg2 (46.89%) followed by Cg5 (46.71%) and
classified as highly virulent. This experiment was able to
reveal the degree of disease severity by the pathogen
which depends on the fungal pathotype. Therefore, the
information on pathogenic variability which was
important to find the differences in disease severity and
disease management was successfully carried out.
Among the 247 bacterial isolates tested under in
vitro, complete inhibition (100%) of the C. gloeosporioides
was recorded by the bacterial endophytes EB07, EB35,
EB39, EB99, EB57 and EB59 and the least per cent of
inhibition was observed in EB1 (7.28%) compared to all
other antagonists. Statistically there was significant
difference among the isolates. Much attention and efforts
on mango anthracnose control are concentrated on the
use of chemical fungicides as the disease is difficult to
control in wet seasons when blossom blight is serious
(Pope, 1924). Apart from guidelines of use of strategy of
at-risk fungicides that may be helpful for preventing an
managing resistance development, there is a need to
devise means for long term sustainable management of
resistance so that adequate disease control is assured of
which, Biocontrol, using antagonistic organisms offers a
reliable approach either alone or in integration with other
disease management practices (Patibanda and Prasad,
2004) and is in compliance with sustainable environment
issues during recent years (Patel and Patel, 1998).
Several microorganisms are reported to be
antagonists against plant pathogens. In such approach,
fungicides need to be used with biocontrol agents without
any toxic effect on antagonists (Papavizas and Lumsden,
1980). Spectrophotometric method was employed to test
the compatibility of the six potential antagonistic bacterial
endophytes viz., EB07, EB35, EB39, EB99, EB57 and
EB59 with the commonly used fungicides viz.,
carbendazim, thiophanate-methyl, propioconazole,
hexaconazole, mancozeb and copper oxychloride as all
the six endophytes have enormously inhibited the growth
of highly virulent pathogenic isolate Cg25 under in vitro
when compared to other antagonists. The higher OD value
at 600 nm, indicates high compatibility of the antagonist
with the fungicide. From the results it is evident that all
the isolates were significantly differing with each other.
Among the fungicides, thiophanate-methyl was
found to be highly compatible with all the six bacterial
antagonists followed by propioconazole, mancozeb,
hexaconazole and copper oxychloride. The potential
bacterial leaf endophyte EB35 was more compatible with
thiophanate-methyl (98.25%) followed by hexaconazole
(81.22%), carbendazim (73.42%) and COC (69.72%) and
less compatible with propioconazole (55.74%) compared
to other fungicides. The other fungicides under the study
were able to inhibit the growth of the biocontrol agent to
some extent reveals that all the antagonists which may
be efficient in controlling the pathogen individually may
not integrate with the fungicide in integrated management
strategies. Hence, a compatibility of biocontrol agent with
the recommended fungicide plays a major role in
inhibiting the growth of the pathogen. The mean
compatibility of bacterial endophytes with different
fungicides was highest in EB35 followed by EB39, EB57,
EB99, EB59 and EB 07. In general, application of mixture
of strains may results in higher biocontrol and lower
variability of biocontrol, as it has been reported in other
studies on different pathosystems. Application of more
than one antagonist with different ecological requirements
would increase the reliability and decrease the variability
of biocontrol. The combination of biocontrol agents plus
fungicide shall increase the effect of their biocontrol
mechanism may be superior.
Talc based formulations of fungicide compatible
potential bacterial endophytes viz., EB07, EB35, EB39,
EB99, EB57 and EB59 were developed against C.
gloeosporioides under aseptic conditions and stored both
at room temperature (28 ± 2°C) and refrigerator (4°C)
respectively. The talc based formulations developed have
been evaluated periodically in order to determine the
efficacy and shelf life period of the potential endophytes
for every 15 days upto 90 day. The bacterial population
was checked on day one prior to packing in polythene
bags and found to be sufficient (16.51 × 107
). In the due
course of storage at different temperatures, gradual
decline in bacterial populations were noticed in the
formulations stored at room temperature. However, the
bacterial populations were almost intact in the
formulations stored in refrigerator upto 120 days. Dual
culture technique was employed to evaluate the efficacy
of endophytes both stored at two different temperatures
after the shelf life of 120 days. From the results it is
evident that the potential endophytes (EB35, EB39 and
EB99) stored in refrigerator were effective in inhibiting
the pathogen compared to other antagonists stored at room
temperature. Thus, the findings reveal that the bacterial
formulations stored at 4°C has superior shelf life and
retain their antagonistic potential intact in inhibiting the
growth of the pathogen compared to formulations stored
at room temperature.The antagonistic bacterial isolates viz., EB35, EB39,
EB99 and EB35 leaf endophytes having different degrees
of antagonistic activity were amplified with the 63F and
1387R primers to to produce 1300 bp fragment product
of 16S rRNA region, cloned into the pTZ57/RT vector
using TA cloning kit. The plasmid was isolated from the
transformed white colonies were analyzed for the
presence of 1300 bp insert by restriction analysis using
EcoR I and Hind III in order to release insert from the
transformants. Alternatively, transformed white colonies
were subjected to Colony PCR and finally sequenced.
The fungicide compatible leaf endophyte EB35 was
identified as Bacillus subtilis. Most broadly, biological
control by novel bioagents is the suppression of damaging
activities of one organism by one or more other organisms,
often referred to as natural enemies. With regards to plant
diseases, suppression can be accomplished in many ways.
Integrated management of postharvest mango anthracnose
under tropical conditions requires knowledge of the
biology of the pathosystem and the technologies available
for control, their economical feasibility, and ecological
acceptability. Because the plant host responds to
numerous biological factors, pathogenic and nonpathogenic,
induced host resistance might be considered
a form of biological control. More narrowly, biological
control refers to the purposeful utilization of introduced
or resident living organisms, other than disease resistant
host plants, to suppress the activities and populations of
one or more plant pathogens