Greener
Journal of Biological Sciences Vol. 9(1), pp. 16-21 2019 ISSN: 2276-7762 Copyright ©2019, the copyright of this article is retained by the
author(s) DOI Link: http://doi.org/10.15580/GJBS.2019.1.071018095 http://gjournals.org/GJBS |
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Pathological Studies on Lasiodiplodia theobromae the Causal
Agent of Gummosis Infected Khaya
grandifiolia
Department of Forestry and
Environment, Faculty of Agriculture, Rivers State University, Port Harcourt,
Nigeria
ARTICLE INFO |
ABSTRACT |
Article No.: 071018095 Type:
Research DOI: 10.15580/GJBS.2019.1.071018095 |
The objective of
this research was carried out the pathological studies on Lasiodioplodia theobramae the causal
agent of gummosis infected African mahogany. The experiment was laid out in a
completely randonized design (CRD) with five replicates. The effects of
temperature and light and darkness on mycelial growth of L. theobromae were evaluated. The fungus grew from 20-40oC
with optimum growth observed at 25-35oC on potato dextrose agars.
On the 10th day, mycelial growth at 25-35oC was (15.6mm
± 0.0 - 30.6mm ± 0.05; 18.4mm ± 0.28 - 32.5mm ± 0.10). There was significant effects of light and darkness
on the mycelial difference (P> 0.05) on the growth of L. theobromae. It is recommended from this research work that
environmental factor such as temperatures; light/darkness significantly
supported the growth of Lasiodioplodia
theobramae. |
Submitted: 10/07/2018 Accepted: 17/07/2018 Published: |
|
*Corresponding
Author Chukunda, F. A. E-mail: onyifrank2002@
yahoo.com Phone:
08037501179 |
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Keywords:
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INTRODUCTION
African
Mahogany (Khaya grandifiolia): Local Names English (Nigerian
mahogany, ivory Coast mahogany, Gold Coast mahogany, African mahogany); French
(acajoud’ Affique, acajou); German (rotes-Khaya, mahagoni); Indonesian (kaya);
Trade name African mahogany.
(Opuni-Frimpong, 2006, Lemmens, 2008).
Khaya grandifiolia is distributed from Côte d’lvoire east to Cameroon and
south to Cabinda (Angola); it possibly also occurs in Guinea, Liberia, the
Central African Republic and Congo. It is fairly widely grown in plantations
within its natural area of distribution but also in tropical Asia and tropical
America.(Abdelgaleil et al., 2005)
Evergreen or deciduous or monoecious large to very large tree up to 60 m tall,
bole branchless for up to 30 m, usually straight and cylindrical, up to 160-210
cm in diameter, with large buttresses up to 2-4m high. Sometimes extending into
prominent surface roots; bark surface brown and slightly rough, exfoliating in
small circular scales leaving a pock-marked, mottled greyish brown and orange
brown surface, inner bark pink to reddish; crown massive and rounded twigs
glabrous. (Andre, 2011; Lemmens, 2008).
Leaves
arranged spirally but clustered near ends of branches, paripinnately compound
with 3-7 pairs of leaflets; stipules absent; petiole 1-4cm long, rachis 6-20cm
long: petiolules 0.5-1cm long; leaflets opposite, oblong to oblong-elliptical,
5-14cm x 2-6cm, cuneate to obtuse and slightly asymmetrical at base, distinctly
acuminate at apex, margins entire, leathery, glabrous, pinnately veined with
5-10 pairs of lateral veins inflorescence an axillary panicle up to 20cm long.
However,
since 1999 a high incidence of leaf spot caused by the fungus; Thanatephorus cucumers (teleomorph of Rhizoctonia
solani) has been observed, causing
numerous lesions on leaves of larger trees and 100% leaf fall in seedlings.
Seeds are commonly attacked by seed-boring beetles and eaten by small rodents.
Attacks of living trees by wood borers (Apate
spp.) have been observed. The bark of saplings is sometimes eaten by porcupines
and squirrels, which can kill the plants. In nurseries in Cote d’lvoire
seedlings are frequently attacked by psyllids (Phacosema spp.), bugs and scale insects, after which they are
infested by secondary fungal pathogens, resulting in a smut blackening the
leaves. (Abdelgaleil et al., 2005 and
Nwoboshi, 1982).
Fungal gummosis was initially observed in the
United States near Fort Valley, Georgia, during the 1960s. It subsequently spread
to other production areas of the Southeast. The disease was independently
discovered at about the same time in Japan, where it is described as peach
blister canker or ibokawa byo, and was later reported in China and
Australia. This disease, characterized by symptoms associated with bark
lenticels, contributes to a general decline of trees. (Weaver, 1974; 1979;
Beckman et al., 2003). Symptoms of fungal
gummosis are caused by a physiological race of Botryosphaeria dothidea (Moug.
Fr.). Other Botryosphaeria species have been reported to cause peach
gummosis in the United States (Pusey and Bertrand, 1993). However, these species are primarily wound
invaders that are not known to cause infections at lenticels. B. rhodina (Cooke)
Arx is relatively rare on peach. B. obtusa (Schwein.). Shoemaker
is very common in the Southeastern United States, but its importance as a peach
pathogen is unclear. B. obtusa is localized in dead tissue and outer
bark (rhytidome) and is absent or in the newly infected cortex and phloem
(Pusey et al., 1995; Weaver, 1974).
The earliest fungal gummosis symptoms appear
on young bark of vigorous trees as blisters 1 to 6 mm in diameter, generally
each with a lenticel at its center. These raised areas are due to an abnormal
multiplication of plant cells (Hyperplasia) in response to the causal organism
at the lenticel. Removal of outer bark
with a knife reveals diseased tissue at the lenticel margin surrounded by the
hyperplastic tissue. Blisters may be observed late in the season when infection
occurs or the following spring. By the end of the second season, the area of
necrosis surrounding lenticels has enlarged, and the hyperplasia is often less
visible or absent. Some second season necrotic lesions exude resins. Lesions
that appear in the second season after infection may or may not be preceded by
the formation of blisters (Pusey 1993; Pusey and Bertrand, 1993).
Beginning when trees are 2 or 3 years old,
sunken necrotic lesions encircling lenticels can be seen on the trunk and major
branches. Typically, copious resin exudate is associated with lesions at
multiple sites. Lesions of 2 cm or more in diameter on the oldest bark may
coalesce to form large cankers (Figure 2.1). Phloem and cortex are primarily
affected however, necrosis may extend to the xylem. Peach branch with blisters
caused by the fungus Botryosphaeria dothidea (Pusey et al., 1986)
The fungus overwinters in diseased bark and
in dead and dying wood, where it produces an abundance of spores. It spreads
within the orchard mainly by dispersal of conidia in rainwater. In the
Southeastern United States, asexual spores of the fungus are present from March
through October. Infections at lenticels develop from March through August, but
May through July is the key infection period. The fungus also invades through
wounds, causing cankers. Cankers may remain active for more than one year and
lead to secondary infections at lenticels. Blossoms, leaves, and fruits are not
infected (Weaver, 1974).
Regrettable
there are limited information about the diversity of Lasiodiplodia theobromae. Lack of information on host range of L. theobromae on the trees found in
Aboretum of Forestry and Environment, Rivers State University, Nkpolu-Oroworukwo,
Port Harcourt. Therefore, the present study was undertaken to observe the
influence of environmental factors on the mycelial growth of Lasiodiplodia theobromae.
This
research is aimed at investigating the pathological studies on Lasiodiplodia theobromae the causal
agent of gummosis infected African mahogany.
Specific
objectives of this research were to:
(i)
isolate and identify fungal pathogens
associated with infected leaves and stem bark of African mahogany.
(ii)
determine effect of temperature on the
mycelial growth of Lasiodipodia
theobromae.
(iii)
assess the effect of light and darkness on
mycelial growth of Lasiodiplodia theobromae.
MATERIALS AND METHODS
Study Area
The study was carried
out at the laboratory of Forestry and Environment (Pathology Unit) and Food
Science and Technology, Rivers State University, Nkpolu Oroworukwo, Port
Harcourt.
Sample Collection
The infected diseased
plant parts showing typical symptoms of gummosis disease were collected from
stem bark and leaves portions of diseased African mahogany tree from Aboretum
of Forestry and Environment, Rivers State University, Port Harcourt.
Figure
1: Infected Stem and leaf of African mahogany
Isolation
and Identification of Pathogen
The associated
pathogens of African mahogany were isolated from the infected stem barks and
leaves as seen in Plate 1 (Pathak, 1987). The infected bark tissues (lesions)
and leaves were cut into small pieces 3-4mm in size with help of sterilized knife.
The pieces were surface sterilized in 2% Sodium hypochlorite solution for 3
minutes followed by five washing with sterile distilled water. The surface
sterilized pieces of stem barks and leaves were then placed on three layers of
moistened fitter paper in Petri dishes (Saleem and Nasir, 1991). All Petri
dishes were incubated at 28±20C, for 3-5 days (Punithalingam, 1976).
The fungi were examined under microscope and maintained on pure culture plates
at room temperature (Ukomia and Chukunda 2016; Chukunda and Offor, 2015; Pathak, 1987). The frequency of
occurrence of fungi was calculated using the formula:
% Frequency
occurrence = (Chukunda,
2014, Ukoima et al., 2013).
Effect
of Temperature on the Growth of Lasiodiplodia
theobromae
Five millimeter
culture disc of L. theobromae were
cut with sterilized Cork borer from advancing margin colonies of the fungus and
inoculated on PDA plates. The effect of temperature on mycelial growth of L. theobromae was evaluated on potato dextrose agar (PDA).
The inoculated plates were placed in an inoculating chamber and incubated at
15, 20, 25, 30, 35oC in the dark. Each treatment was replicated
three times. At each temperature the plates were arranged in a completely
randomized design (CRD). Colony diameters were measured along two axes perpendicular
to one another. The measurement of the mycelial growth was calculated after 5,
10 and 15 days of inoculation (Ukoima and Chukunda, 2016).
Effect of
light and darkness on mycelial growth of L. theobromae
The effect
of light and darkness on mycelial growth of isolated fungus 5mm culture discs
were cut with the sterilized cork borer from advancing margin of the colonies
of L. theobromae and inoculated on
PDA plates at 5 days interval for 15 days. Carbon paper was used to wrap the
Petri dishes for darkness, while unwrapped Petri dishes were used for light
exposure. All the Petri dishes were incubated at 28 ±
Experimental Design and Statistical Analysis
The
experiment was laid out in a Completely Randomized Design (CRD). The treatments
were replicated three time. Data collected were analyzed by analysis of
variance (ANOVA) using SPSS Genstat software as described by Steel and Torrie
(1980).
RESULTS
Fungi
Isolated from Infected Parts of African mahogany
The results on the
frequency of occurrence of fungal isolates of infected parts of leaves and stem
bark tissues were of obtained from the Aboretum of Department of Forestry and
Environment, Rivers state University, Nkpolu-Oroworokwo, Port Harcourt are
presented in Table 1 and Plates 1. The result showed that Lasiodiplodia theobromae and Fusarium
solani were found in the stem bark
and leaves of Khaya grandifiola, L.
theobromae (86.25% ± 2.20;
45.26% ± 0.28) was the most frequently occurring fungus isolated from both
plant parts, while Fusarium solani
had (50.26% ± 0.28; 45.20%± 0.22).
Table
1: Fungi isolated from infected parts of African mahogany (Mean ± SD)
Fungal isolate (%) |
Infected
plant parts (%) |
|
|
Stem bark tissues |
Leaves |
Lasiodiplodia
theobromae |
86.25 ± 2.20 |
45.26 ± 0.25 |
Fusarium
solani |
45.26 ± 0.28 |
45.20 ± 2.22 |
Mean ± SD (n=5)
Plate 1: Fungi isolated from infected stem bark
tissues and leaves of African mahogany
Effect of different temperature in the
mycelial growth of Lasiodiplodia
theobromae
The results on the
effect of different temperatures on Lasiodiplodia
theobrammae mycelial growth are presented in Table 2. The result showed
that different temperature and culture media influenced the mycelial growth of L. theobrammae. The relative increase in
fungus mycelial growth increased with the increase in temperature. It was observed
that the temperature range of 25-35oC was optimum for mycelial
growth in the PDA medium (15.6 ± 0.02mm
– 30.6 ±
0.05mm; 18.4 ± 0.28mm
– 32.5 ± 0.10mm).
Table 2: Effect of Different Temperature on
the mycelial Growth of Lasiodiplodia
theobromae (Mean ± SD)
Temperature (toC) |
Incubation period/mycelial growth (mm)/days |
|
|
||
|
PDA 5 10 |
|
|
|
|
20 |
12.0 ± 0.01 |
14.6 ± 0.81 |
25 |
15.6 ± 0.02 |
18.4 ± 0.28 |
30 |
20.5 ± 0.03 |
24.0 ± 0.22 |
35 |
30.6 ± 0.05 |
32.5 ± 0.10 |
40 |
21.5 ± 0.06 |
23.5 ± 0.22 |
Mean
± SD (n=5) * PDA = Potato dextrose agar
Effect
of light and darkness on mycelial growth of Lasiodiplodia
theobromae on potato dextrose agar (PDA) incubated at room temperature (28 ± 2oC)
The result on the
effect of light and darkness in Lasiodiplodia
theobromae growth on stem bark tissues and leaves portions of Khaya ivorensis are shown in Table 3.
The result indicated that light and darkness significantly (p≤ 0.05)
affected the growth of L. theobromae at
different days. On the 5th day of incubation, L. theobromae under continuous darkness mycelial growth on PDA was
(13.5 ± 0.20mm – 18.2 ± 0.30mm). In continuous light, L. theobramae mycelial growth was (12.3 ± 0.02mm – 16.0 ± 07mm). Generally, the highest growth was observed after 10 days for
light and darkness on both PDA medium.
Table 3: Effect of light and darkness on
mycelial Growth of Lasiodiplodia
theobromae on PDA incubated at room temperature 28 ± 2oC (Mean ± SD)
Light and darkness |
Incubation
period/mycelial growth (mm)/days |
|
PDA 5 10 |
||
Continuous light |
12.3 ± 0.02 |
16.8 ± 0.22 |
Continuous darkness |
13.5 ± 0.20 |
18 ± 0.30 |
* PDA = Potato dextrose agar
DISCUSSION
The
results on the frequency of occurrence of fungal pathogens of infected stem
bark tissues and leaves of African mahogany (Table 1) indicated that Lasiodiplodia theobromae and Fusarium
solani were found to be responsible for the stem and leaves rot of African
mahogany collected from the forest arboretum. The stem and leaves pathogens of African mahogany have earlier been
implicated by some researchers. Ukoima and Chukunda (2016) reported on
the influence physiological factors on mycelia growth of B. thobromae, and Rhizopus
stolonifer caused serious rotting in Annoan muricata. Similarly, Ukoima et al., (2013) isolated Aspergillus
niger from seeds of Jatropha. This is in conformity with present research
work.
Chukunda (2014) found Aspergillus
niger, Aspergillus flavus, Rhizopus stolonifer, Fusarium pallidoroseum,
Botryodiplodia theobromae, Colletotrichum gloeosporoides, Penicillum expansum and
Botrytis cinera to be responsible for
the serious decay of avocado pears obtained in the Niger Delta ecosystem.
The
mycelia growth of lasiodiplodia
theobromae (Table 2) showed a variable trend in response to temperature
change using potato dextrose agar (PDA). Mycelia growth increased as
temperature increased from 20-35OC and then decreased with further
increase temperature. However, optimum mycelia growth of test fungus occurred
at 25-35OC. This results agreed with those reported by Quroshi and
Meah (1991) and Alam et al., (2001)
who reported that 25-30OC temperatures was optimum for the colony
growth and sporulation of lasiodiplodia
theobromae.
The
results of the effects of light and darkness on fungal growth (Table 3) revealed
that there was an increase in growth of L.
theobromae in both light and darkness. Rewal and Grewal (1989) studied the
effect of light on conidial germination of three strains of Botrytis cinerea infecting chickpea, and
found that conidia of B. theobromae germinated best under continuous
light and strain B2, B. theobromae of germinated well under
light and darkness treatment. From the study, it implied that light and
darkness are necessary for growth and sporulation of test fungi. This is in agreement with Ahmed (1985) who
observed that light promoted the growth and sporulation of Collectotrichum gloeosporoides.
Similarly, Marshi et al., (1959) reported that fungi exhibited varying response to
light depending on the light intensity, quality and duration of exposure. Prota (1992), Oladiran and Iwu (1993) and
Pihet et al., (2009) reported that
ultra violet (UV) radiation or sunlight affected the survival of fungal spores,
sclerotia and pycndia. However, some fungi need light to sporulate whereas
other fungi sporulate better in darkness.
In their investigation, Aspergillus
ornatus produced abundant conidia when grown in continuous light and
virtually none when grown in dark while Cleistothecia and ascospores are
produced in the dark whereas neither is produced in continuous light
(Schwemmin, 1960).
Hill
(1976) further explained that light inhibits glucose uptake and phosphorylation
which caused starvation and retards fungi growth and conidia formation.
Conversely the growth of Mycospharella
pinodes, Aspergillus niger increased when exposed to darkness. (Ukioma and
Chukunda, 2016).
On
the contrary, Alam et al., (2001)
reported that light is not necessary for growth and sporulation of B. theobromae,
but it enhances the growth and the number of conidia formation which is in
partial agreement with the observation of Rewal and Grewal (1989). The increasing
glucose in medium, the fungus could utilize it in a certain level and grow
properly, and after that level, the fungal physiology does not permit the
utilization of glucose for growth of the pathogen. There the fungus might utilize the glucose by
different ways instead of growth and formed more pigmentation using more
glucose.
According
to Cochrane (1958), temperature range permitting reproduction is usually
narrower than that permitting growth.
Earlier, Leach (1979) had reported variations in optimum temperature
requirements within the same species for light induced sporulation at
continuous light and continuous darkness Alam et al., (2001) obtained more growth of L. theobromae under continuous light and less in continuous darkness.
These findings agreed with the present research work where L. theobromae test fungi had a good growth performance for both
light and darkness.
However,
Teyegaga and Clerk (1972) earlier demonstrated the relationship between Cercospora canescens conidia longevity and storage humidity, and observed that
in the dark there was longest survival of conidia at low humidity than those
under light. Generally the spores
stored in the darkness appeared to be more viable than those in light. This may be due to metabolic disruption by
light or that light inhibited the spores of test fungi thus reducing their
percentage conidial germination.
CONCLUSION AND
RECOMMENDATIONS
Conclusion
Results from the
study revealed that Lasiodiplodia
theobromae is a major serious disease affecting the production of Khaya granifiolia from seedling level.
Temperature and light/darkness also played an important role in L. theobromae growth particularly
influenced by the culture medium growth kinetic. Results showed that L. theobromae was highest at a
temperature of 30-35OC.
Recommendations
Based on the present
findings the following recommendations are made;
1.
To reduce yield losses a good establishment
of host range of trees that are not susceptible to this pathogen should be
planted.
2.
Silvicultural practices will help reduce
re-occurrence of Lasiodiplodia theobromae
infection on the plantation.
3.
Preventing wounds on the test plant is the
best way to minimize the spread of L.
theobromae.
4.
Cankered branches of Khaya ivorensis should be pruned to reduce the inoculum from
initiating new infection.
5.
It is revealed from the study that
temperature, light and darkness significantly (P≤0.05) affected the
mycelial growth of L. theobromae.
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