Perfor. of Summer forage legumes in Pothowar
Pak. J. Agri. Sci., Vol. 48(4), 289-294; 2011
ISSN (Print) 0552-9034, ISSN (Online) 2076-0906
http://www.pakjas.com.pk
SYMPTOM DEVELOPMENT AFTER ARTIFICIAL INOCULATION OF
BOTRYODIPLODIA THEOBROMAE, A POSSIBLE CAUSAL ORGANISM TO
QUICK DECLINE IN MANGO TREES
Shafqat Saeed*, Muhammad Ijaz Khan and Asad Masood
Department of Entomology, University College of Agriculture, Bahauddin Zakariya University, Multan, Pakistan
*
Corresponding author e.mail: bumblebeepak@gmail.com
The fungus, Botryodiplodia theobromae is one of the causal agents of quick decline which was artificially inoculated to
mango varieties to see quick decline symptoms. The most famous varieties, i.e. cv. Ratol-12, black chaonsa, white chaonsa,
Fajri, Dosehri, Langra, Sindhri and Summer Bahisht were established in earthen pits under complete randomized design
(CRD) and was observed up to three months for their establishment. Three months after transplanting, B. theobromae was
artificially inoculated in the test plants which showed that Dosehri variety was comparatively tolerant to the disease as
compared to others. Regarding the appearance of percent disease symptoms, Ratol-12 showed the highest disease symptoms
followed by Langra, Fajri and then black Chounsa. So it is concluded that B. theobromae is not the major causal organism of
this disease and other sources may also be involved in the development of disease.
Keywords: Mango, varietal resistance, fungi, Botryodiplodia theobromae, quick decline
INTRODUCTION
Mango (Mangifera indica L.) is the most important fruit of
the tropical world. It is indigenous to India and Southeast
Asia. Growing countries are India, Pakistan, China, Mexico,
Bangladesh, Thailand, Philippine, Indonesia, Nigeria, Brazil,
Florida and Oman. Mango is one of the most important
foreign exchange earning fruit of Pakistan, through exports
to many countries, mainly to England, Saudi Arab, Dubai,
Germany, France, Holland, Thailand, Bangladesh,
Singapore, Italy and Malaysia. In Pakistan mango is grown
mainly in Punjab and Sindh provinces. There are many
varieties of mango grown in Pakistan but most famous of
these are Sindhri, Dosehri, Summer Bahisht, Chounsa,
Anwer Ratol, Langra and Malda (Masood et al., 2010).
According to FAO, Pakistan stood fourth during 1995
among mango growing countries of the world and come
after India, Mexico and China (Khalid et al., 2002). In 2004
Pakistan stood sixth instead of fourth among the mango
growing countries of the world. The main reason of
reduction in the ranking of Pakistan is due to the increased
number of mango orchards being cutting away because of
mango insect pests and disease problems and competition
with other crops like cotton (Anonymous, 1996). The
mango production is also vulnerable by the attack of number
of diseases (Khalid et al., 2002). Among all diseases
infecting mango, the mango quick decline is the most severe
threat to the Pakistan mango industry. Recently, incidence of
this menace was found 20 and more than 60 percent in
Punjab and Sindh Provinces of Pakistan, respectively and 60
percent in Al Batinah region of Oman (Al-Adawi et al.,
2006; Saeed et al., 2006). Typical symptoms of mango
decline include terminal and marginal necrosis of leaves,
which ultimately lead to the death of leaf blade. The dieback
gradually progresses to large branches with eventual
reduction in the number of secondary roots (Ramos et al.,
1991). The bark of affected tree is discolored and darkened
at a certain distance from the tip. Usually in young green
twigs, the disease may lead to the browning of leaves and
upward rolling of thin margins resulting in fall of leaves,
ultimately in the drying and death of twigs (Anonymous,
1995). In India the disease is caused by Botryodiplodia
theobromae (Rawal, 1998) while in Oman by
Botryodiplodia theobromae and Ceratocystis fimbriata (AlAdawi et al., 2003; Al-Adawi et al., 2005). In Brazil
Ceratocystis fimbriata causes the disease in mango (Batista,
1960). Although, B. theobromae is frequently isolated from
diseased mango tree and also give rise to mild symptoms
especially gummosis in inoculated plants (Al Adawi et al.,
2006).
The main objective of the study is to observe resistance of
different mango varieties against Botryodiplodia theobroma
and to study the expression of disease symptoms especially
gummosis.
MATERIALS AND METHODS
One year old healthy plants of eight different varieties of
mango were collected from a commercial nursery and
planted in pits at a depth of 3 feet at Agricultural Farm of
University College of Agriculture, Bahauddin Zakariya
University, Multan. Before transplanting the nursery plants,
Saeed, Khan & Masood
earthen pits were made with tractor mounted digger and also
filled with water to leach down the salts deep into the soil.
After 15 days when salts were leached down into the soil,
pits were filled with mixture of farm yard manure (FYM)
and silt in which mango plants were planted under complete
randomized design (CRD) with 5 plants for each variety.
Finally, 3 plants of each of the eight varieties were treated
with fungus and remaining 2 plants for each variety were
treated as control. The most famous mango varieties, i.e.
Ratol-12, black chaonsa, white chaonsa,Fajri, Dosehri,
Langra, Sindhri and Summer Bahisht were selected due to
presence of the symptoms of gummosis on large areas
during the field surveys.
After transplanting the nursery in the field, establishment of
plants was observed for a period of three months during
which dead plants were replaced with healthy plants. After
establishment of plants, these were inoculated with a fungus
species Botryodiplodia theobromae arranged from Mango
Laboratory, National Agriculture Research Center (NARC),
Islamabad, Pakistan. Nursery was managed by proper
agronomic practices, i.e. hoeing, timely irrigation, and NPK
(Nitrogen, Phosphorous, Potash) application. Before
inoculation all the equipments were sterilized in order to
prevent the chances of infection by any other pathogen.
Inoculation of fungus (B. theobromae) was made by
insertion a piece of fungal colony (5 mm²) growth on Potato
Dextrose agar (PDA) plate and inserted in slanting cuts (Fig.
1 and 2) in collar portion of each plant with sterilized scalpel
and covered with a polythene sheet (Fig. 3) (Mullen et al.,
1991). In plants declared as control treatment, only agar
slant without any fungal growth was inserted in slanting Tshaped cuts under the bark.
Disease evaluation: The presence or absence of disease
(quick decline) in the plants was recorded in different mango
varieties. The severity of disease symptoms in twigs,
branches, leaves and stem of individual plant was rated
using a scale from 1 to 5 (Ramos et al., 1997) corresponding
to percent disease severity from 0 to 100 % which has been
described as under:
1. Plants free of disease = 0%
2. An early stage of infection characterized by browning of
leaf petioles and mid-veins and presence of distal or
marginal leaf blade necrosis in one or two branches =
25%
3. The presence of dead leaves, which may remain
attached, in the tips of several branches, vascular
browning, and evidence of pathogen invasion of
vascular tissues = 50%
4. Dead leaves and progressive defoliation extending too
many larger branches = 75%
5. Sever decline or dieback that extended to major portions
of the plant = 100%
The disease development symptoms were observed in
inoculated and control plants of each variety on monthly
basis for a period of four months.
RESULTS
Bark splitting was the most frequent disease symptom of
quick decline disorder. It was observed that 100% plants of
Ratol-12 showed this symptom both in treated and control
conditions followed by 33% in Sindhri in both cases (treated
and control) while 33% plants in treated Summer Bahisht
and Fajri but control was devoid of bark splitting symptom.
Remaining varieties did not show the bark splitting
symptoms. Gummosis produced in treated plants of Sindhri
was 66% whereas there was no gummosis in control
treatment. Similar disease incidence was recorded in treated
and control plants of Langra. About 33% plants of Fajri
showed gummosis symptoms in control and treated plants.
Similar symptoms of gummosis were also observed on
treated plants of Black Chaonsa. Gummosis symptoms were
not observed on the plants of Summer Bahisht and white
Chaonsa. Stem rottening (cankers and black streaks) was
absolutely absent in all the plants of mango varieties.
Symptoms of healthy plants were also recorded by observing
new braches/inflorescences. It was found that 100% plants
of Sindhri produced new leaves followed by 66% of Ratol12 and Chaonsa treated plants whereas 33% of other
varieties plants except treated Fajri and white Chaonsa plant
and same were the case in control plants of Dosehri and
Langra. As the disease progressed, leaves turned yellow
because physiology of plants is affected when disease attack
occurs. Among the observed varieties of mango, only 66%
Fajri and 33% Ratol-12 treated plants leaves turned yellow
while all other plants had no yellowing of leaves. All plants
of Sindhri and Fajri while treated and control plants of
Ratol-12 and Dosehri had no any defoliation, respectively.
But 66% treated plants of black Chonsa and Langra
exhibited defoliation while 0% and 33% plants showed
curling correspondingly. Some of the varieties were found
without any symptoms of disease like Sindhri, Dosehri,
Summer Bahisht and white Chonsa. Marginal necrosis was
recorded as 0% on untreated plants of all varieties except
33% of Fajri and other treated plants of all varieties
excluding that of Ratol-12 with 100% plants (Table 1).
In case of observed symptoms of plants, 50% disease
symptoms were found on treated plants of Sindhri and black
Chaonsa compatible with untreated plants of Ratol-12.
Results showed that Dosehri variety was comparatively
tolerant to the disease as compared to other varieties of
mango whereas Ratol-12 showed the highest disease
symptoms followed by Langra, Fajri and black Chaonsa. No
significant difference was observed in the treated and
untreated plants of white Chonsa showing 38% disease
symptoms of quick decline (Table 2).
290
Variety response against quick decline pathogen
Table 1. Evaluation of disease in Botryodiplodia
theobromae inoculated varieties of mango
Disease symptoms (%)
Sr. #
Varieties
Plants as
Treated plants
control
1 Sindhri
50
25
2 Ratol 12
75
50
3 Fajri
62
38
4 Black Chonsa
50
38
5 Dosehri
38
0
6 Langra
63
25
7 Summer Bahisht
63
25
8 White Chaunsa
38
38
DISCUSSION
In present study, the disease symptoms were not only
produced by inoculated plants but also appeared in the
untreated healthy plants which may be infested due to the
spread of inoculums through different means of
dissemination, i.e. air, water or insect. So it is possible that
Botryodiplodia theobromae may not be the main causal
organism of this disease. Although, B. theobromae is
associated with quick decline mango tree and also give rise
to one or more of the symptoms of quick decline in
inoculated plants. But, it is an opportunistic pathogen and
becomes more virulent in combination with others fungi, i.e.
Ceratocystis fambriata and Fusarium aesculi (Ploetz et al.,
1996; Al Adawi et al., 2006). Recently, Shahbaz et al.
(2009) investigated that B. theobromae (Pat.) Griffon and
Maubl was relatively more frequently isolated from trees
showing symptoms of decline in Pakistan. Therefore, mango
quick decline is considered as a disease complex in which
pathogenic fungi, bark beetle and cultivation method are
involved (Malik et al., 2005; Al Adawi et al., 2006; Masood
et al., 2009). Early authors, (Westerdijk and Buisman, 1929)
supposed MSDS might be transmitted and spread by various
dissemination means, i.e. air, water, and insects. In spite of
other means of disease dissemination, bark beetle species are
expected to be involved as putative vectors (Ribeiro, 1980;
Al Adwai et al., 2006; Masood et al., 2008). The elm bark
beetle, Scolytus mutistriatus and S. scheveyrewi (Coleptera:
Scolytidae) has been reported as the primary vector of Dutch
elm disease caused by fungus, Ophiostoma novoulmi which
can be transmitted into healthy elm trees and also re-isolated
the same fungus from adult beetles as well as from infested
tree (Jacobi et al., 2007).
Although, bark beetle,
Hypocryphalus mangiferae was formerly reported as an
indigenous wood borer in mango as secondary pest
(Mohyuddin and Mahmood, 1993) but now due to its role in
disease transmission as a vector it has gained the status of
primary pest, i.e. due to transmission of Ceratocystis
fimbriata and Lasiodiplodia theobromae, the causal
organisms of mango sudden death syndrome (Al Adawi et
al., 2006; Masood et al., 2009; Masood et al., 2010).
However, research is needed to further investigate other
means of disease dissemination and other cultural practices
in the etiology of this disease in Pakistan.
The evaluation of disease symptoms due to the inoculation
of fungus Botryodiplodia theobromae, the possible casual
organism of mango quick decline was described to assess the
most tolerable available mango variety against the attack of
quick decline. According to our studies, cv. Dosheri and
white Chaonsa was found more tolerant while cv. Ratol-12
followed by Summar Bahisat was observed more susceptible
in the development of disease symptoms, i.e. gummosis,
bark splitting, leaf yellowing and defoliation. These findings
would be helpful in development of resistant mango
varieties keeping in view disease scale based on the
characteristic symptoms of mango quick decline. These
symptoms of mango quick decline observed are also
reported alone or in combination of more symptoms in
different mango orchards in Oman, Brazil or Pakistan
(Ploetz et al., 1996; Al Adwai et al., 2006; Iqbal et al.,
2007). Initial symptoms of this disease include gummosis
from the bark, bark splitting, yellowing and withering of
leaves. On scrapping of diseased tree trunk, usually vascular
discoloration, cankers and in some cases oozing of bad
smelled liquid were observed (Masood et al., 2010). Ramos
Table 2. Evaluation of disease symptoms (%) in treated and untreated mango varieties
Disease symptoms (%) in treated mango
Disease symptoms (%) in untreated healthy
varieties (n=9)
mango varieties (n=9)
Disease
Symptoms
T1
T2
T3
T4
T5
T6 T7 T8
T1
T2
T3
T4
T5
T6 T7 T8
100 33
33
0
0
0
0
33
33
33
0
0
0
0
0
0
Bark splitting
0
66
33
33
0
66
0
0
0
0
33
33
0
0
0
0
Gummosis
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Stem rottening
66 100
0
66
33
33
33
0
66 100 33
33
0
0
33
33
Leaf appearance
33
0
66
0
0
0
0
0
0
0
0
0
0
0
0
0
Leaf yellowing
0
0
0
66
0
66
33
0
0
0
0
0
0
0
0
0
Defoliation
100 100 100 100 100 100 100
0
0
33
0
0
0
0
0
Marginal necrosis 0
T1= Ratol-12; T2= Sindhari; T3= Fajiri; T4= Black Chonsa; T5= Doshari; T6=Langra; T7= Summar bahisat; T8= White Chonsa
291
Saeed, Khan & Masood
Figure 1. An inverted
T-shape cut
Figure 2. Inoculation of
fungus
Figure 3. Wrapped by
polythene
Figure 4. Gummosis
Figure 5. Black streak
in stem
Figure 6. Cankers/stem
rots
Figure 7. Bark splitting
Figure 8. Yellowing and
drying
Pictorial representation of inoculation and disease symptoms on mango plants
292
Variety response against quick decline pathogen
et al. (1997) determined the resistance of Mangifera indica
to tip dieback caused by Botryosphaeria ribis, anamorphic
state Fusicoccum sp., on 361 trees of 122 cultivars of
mango. Three trees from each cultivar were selected and the
disease was evaluated on a 1 to 5 scale ranging from those
free of tip dieback to those with extensive branch necrosis.
At the end they found that two Mangifera species (M.
odorata and M. zeylanica) showed the least mean disease
rating which shows that field resistance to tip dieback may
be present in some mango cultivars. Khalid et al. (2002)
gave the assessment keys for some important diseases of
mango and demonstrated that these are important in any
study relating disease severity to disease losses and
subsequent management tactics.
Al-Adawi et al. (2003) studied that 60% of the trees of
mango were infected in the parts of Al Batinah region of
Oman. They observed that trees showed gummosis from the
trunk, wilting and eventual browning of leaves on the single
branch. Wood of the diseased tree is stained dark brown,
spreading from point of infection. In the lesions produced on
the bark of tree, pycnidia were observed and the pathogen
Diplodia theobromae was isolated from the infected trees.
Van Van Wyk et al. (2005) studied the DNA based
characterization of Ceratocystis fimbriata which is
associated with the mango decline in Oman. They obtained
the sequence data for the internal transcribed spacer 1 and 2
regions and the 5.8 S rRNA gene regions and compared it
with the C. fimbriata from several hosts and geographic
areas. So the isolates from Oman were reported to represent
C. fimbriata sensu lato that were most closely related to an
isolate from mango in Brazil. They concluded that mango
pathogen in Oman might have originated in Brazil. Rawal
(1998) reviewed that dieback in mango is due to
Botryodiplodia
theobromae
and
Colletotrichum
gloeosporioides. He told that during the attack of dieback
pathogens epidermal and sub epidermal cells shriveled in the
early stage. The areas of cambium and phloem show brown
discoloration and yellow gum like exudates flows out of the
cells. This concept is elaborated by Aslam (2005) that
mango is indigenous to India and Southeast Asia and told
that the causal organism of mango decline in India is
Botryodiplodia theobromae Pat. and Alternaria alternate,
Colletotrichum gloeosporioides, Lasiodiplodia theobromae,
Phomopsis spp. and other fungi caused mango decline in
USA. He also described that the deficiencies of
micronutrients may predispose the trees to infection by
fungal pathogens. In this study, there were no differences
observed in the treated and untreated plants of white Chonsa
showing 38% disease symptoms of quick decline and some
healthy plants showed disease symptoms without
inoculation. So it is concluded that Botryodiplodia
theobromae is not the major causal organism of this disease
and other factors are also involved in the development of
disease. Therefore, it is necessary to investigate the other
disease facilitating factors and means of disease
dissemination so that a sustainable management strategy can
be devised for suppression of mango quick decline.
ACKNOWLEGEMENT
We acknowledge for the technical support of Mr. Munawar
Raza Kazmi, (NARC) Mr. Faisal Sohail Fateh (NARC) and
Mr. Muhammad Tariq Malik (MRS). This study was funded
by PARC project “Etiology and Management of Sudden
Death Phenomenon in Mango Orchards” and Mango ASLP
project.
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