BIBLIOGRAPHY COYASAN, JILL C. APRIL...
BIBLIOGRAPHY
COYASAN, JILL C. APRIL 2011. Characterization of Colletotrichum spp. Associated to
Berries, Leaves, and Stems of Arabica Coffee. Benguet State University, La Trinidad, Benguet.
Adviser: Andres A. Basalong, MSc.
ABSTRACT
The study was conducted primarily to describe the symptoms of Colletotrichum species
associated to stems/twig, leaves and berries of Arabica coffee and determine the cultural and
morphological characteristics of the fungi.
Result of the study showed that stems/twig infected by Colletotrichum gloesporioides
designated as isolate A showed brown lesion along the length of the stems/twig and can also
produce cankers on petioles that causes severe defoliation. Leaves infected by Colletotrichum
coffeanum designated as isolate B showed symptoms of small and irregular, brown, dark-brown
or black spots. The infected part darkens as it ages and characterized generally as large brown
leaf blight while berries infected by Colletotrichum kahawae designated as isolate C showed
symptoms of circular watery, dark sunken lesion that appear on the surface of the pulp.
The colony diameter of Colletotrichum gloesporioides in PDA after 7 days was 35.7mm
while Colletotrichum coffeanum and Colletotrichum kahawae grew up to 40.5mm and 32.3mm.
Colletotrichum gloesporioides exhibited a color of white mycelia in PDA, while Colletotrichum
coffeanum and Colletotrichum kahawae had white to black mycelia. The size of conidia of
Colletotrichum gloesporioides is 11.3µm x2.19 µm for Colletotrichum coffeanum is 11.6µm
x2.16µm and Colletotrichum kahawae is 11.3µm x1.6µm.
Three species of Colletotrichum are associated to Arabica coffee should be validated
through pathogenicity test.
TABLE OF CONTENTS
Page
Bibliography………………………………………………………………… i
Abstract …………………………………………………………………….. i
Table of Contents …………………………………………………………… ii
INTRODUCTION ………………………………………………………….. 1
REVIEW OF LITERATURE ………………………………………………. 3
MATERIALS AND METHOD ……………………………………………... 7
RESULTS AND DISCUSSION ……………………………………………. 9
Anthracnose Symptom on Leaves …………………………………… 9
Anthracnose Symptom on Stem ……………………………………... 9
Anthracnose on Coffee Berries ……………………………………… 9
Cultural Characteristics of Anthracnose isolates ……………………. 10
Morphological Characteristics of
Colletotrichum Isolates ……………………………………………... 10
Mycelia ………………………………………………………………. 10
Conidia ………………………………………………………………. 13
SUMMARY, CONCLUSION, AND RECOMMENDATION …………….. 14
Summary …………………………………………………………….. 14
Conclusion …………………………………………………………... 14
Recommendation ……………………………………………………. 14
LITERATURE CITED ……………………………………………………… 15
1
INTRODUCTION
Coffee has the great demand in the world market and it is good for business.
Coffee is the most important raw material traded throughout the world behind crude oil,
and the most important export article for the nations that grow it. Coffee is the most
precious beverage by Filipinos (Daguio, 2000).
Coffee is one of the major products which is favored by people all over the world.
It has become the major source of foreign exchange after oil for many developing
countries, and it has been sustained in their economies. It supports lives of people who
cultivate it.
The universal demand for coffee is due to the invigorating effect and to some
extent the medicinal value derived from it. Coffee assists in assimilation and digestion, it
reduces the amount of blood circulation, and whopping cough (Cresencia, 1979). The
impact of coffee to human health revolves largely around caffeine. Caffeine is the most
active stimulant of xanthine alkaloids. It also acts as diuretic, vasodilator, laxative and
appetite suppressant. Caffeine is sold commercially in medications, diet pills, as the
counter stimulants, and medicines administered to hyperactive children, where ironically,
it has a calming effect.
Coffee contains the alkaloid caffeine (1, 3, 7 trimethylxanthine), which is the
most active stimulant among the xanthine alkaloids. Caffeine is sold commercially in
pain medications, stimulants and it has a calming effect to hyperactive children.
Moreover, moderate consumption of coffee has been reported to reduce risk of colon
cancer, cirrhosis, gallstones, Parkinson’s disease and asthma (Vinson, 2001).
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
2
Farmers started growing coffee many years ago but up to the present there is still
very low supply due to attack of insect pests and diseases. Anthracnose is one of the
major diseases infecting coffee. It is caused by several species or strains of
Colletotrichum. This pathogen is capable of infecting leaves, berries, stem bark and twigs
of the coffee plant. Colletotrichum coffeanum invades the main body of the plant but does
so without any signs of disease. However, when the plant set fruit, the fungus becomes
aggressive and the diseases become apparent. Coffee coffeanum affects green or
immature coffee berries and the coffee flower at any stage in its development. Infected
berries often show dark sunken pits that spread and coalesce to cover the whole berry. As
the fungus sporulates, a pale pink crust of conidia appears on the lesion. Depending upon
the timing of the infection, the bean can also become infected. Some cases, a milder scab
like disease occurs. Losses of up to 75 % of a crop have been reported.
Previous reports of the Colletotrichum spp. infecting coffee in some growing
areas indicated that species associated to the leaves do not infect coffee berries. Likewise
species found infecting berries cannot infect the leaves.
The Study aimed to describe the symptoms of Colletotrichum spp. infection on
leaves, stems, and berries of Arabica coffee and determine the species of Colletotrichum
infecting different parts of Arabica coffee through morphological and cultural
characterization.
Diseased samples were collected at the Benguet State University Agroforestry
Laboratory from October to February 2011 while the activities were conducted at the
Department of Plant Pathology laboratory.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
3
REVIEW OF LITERATURE
Coffee plants belong to the Rubiaceae family. The commercially grown species
are Coffee Arabica, Coffee canefora (Robusta), Coffee liberica, and Coffea exelsa
(Maistri and Rena, 2001). However, several species were mentioned by other authors.
Economically, the most important species are Coffea Arabica and Coffee canephora, with
Arabica consisting 70% of the coffee production worldwide (Gilman, 1999).
An understanding of the coffee plant, it make- up and how it grows is essential to
understanding how to manage the coffee tree. Management, like the environment and the
variety planted, has a very big influence on coffee quality and yield. It is necessary to
stress the importance of growing techniques (pricking-out in nurseries, mulching, and
irrigation and planting layouts) on the distribution and functions of roots (Winston,
2005). Ringor and Versola (1981) revealed that weeds compete for nutrients and water
with newly planted coffee plants. They reduce growth to as much as 50 percent. If left
uncontrolled in the plantation area, weeds can delay production for as much as one year.
Moreover, they are host of fungal diseases and act as pest shelters, host to insects that
transmit diseases, restrict the movement of the water through the plantation area, and
hinder efficient harvesting in general. Pest and diseases greatly reduce the coffee yields
and its quality. Thus measures should be put in place to prevent and or control pest and
diseases. Since coffee is perennial crop, their communities were attack by insects. The
control of coffee pests and diseases, therefore, require careful selection of the predator
(O’ Farrill, 2000) and recommends four means of controlling pest and diseases such as
cultural that is by means of weed control, pruning, mulching etc, mechanical method
which is done by picking and destroying mechanically infested diseased part of the plant
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
4
(Anonymous, 2006) and biological method which involves using predators or parasites to
destroy the coffee pests and diseases Since cultural method is the cheapest, safest and
easily affordable it should be the first control measure to be used. Other measures should
be used only when the cultural measure fails.
Importance of Coffee
Coffee is one of the great social drinks of human culture. Coffee assumption has
always been associated with work and activities requiring thought and attention
(Macanes, 1990).
Svilaas (2004) also reported that coffee is the number one source of antioxidants.
Which have been linked to a number of potential health benefits, including protection
against heart disease and cancer.
The day may come when coffee will be gold. This must be the reason for renewed
interest to promote coffee in the region. Potentials in the coffee industry may transform
the Cordillera as the Arabica coffee capital of the country. Market for coffee in the region
is not a problem for current local production could not even supply the growing demands
of multiplying shops in Baguio City and other areas in the region (Mondonedo, 2008).
Arabica coffee can also be used as a component of watershed conservation and
rehabilitation of denuded watershed as a result of over- logging and swidden farming and
can be promoted through the adoption of coffee cultivation as Agro-forestry crop
(Alladiw, 2008).
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
5
General Characterization of Colletotrichum
The fungus Colletotrichum produces colorless or hyaline, one-celled, ovoid,
cylindrical, oblong or falcate and sometimes curved or dumbbell shaped conidia in
acervuli, Colletotrichum acervuli are disc-shaped or cushion shape and waxy having
dark, long, sterile hair like (Agrios, 1997 and Quimio, 1999).
Colletotrichum coffeanum is an ascomycete, a member of the sac fungi. One of
the features of these fungi is that they generate spores, called conidia that can be easily
dispersed by the wind and splashing rain. Spread of C. Coffeanum is dependent on water
but it can also be spread by animals, by coffee pickers being a particular problem.
Colletotrichum invades plants but does so without any sign of disease. However when the
plants set fruit, the fungus becomes aggressive and the disease becomes apparent (Silva,
2006).
Coffee berry disease Colletotrichum coffeanum attacks the green tissues at the
beginning stage of berry development and often penetrating into interior of the berry
destroying the bean. It appears at the berries then move up to the stalk. The berry will
have brown patches that seem to have small punctures. Lesions form darker areas of the
berry and in time the berry turn ash gray. The disease results in the production of poor
berries and difficulty in pulping.
According to Phuong (2010), filamentous fungi of the genus Colletotrichum and
its teleomorph Glomerella are among the most important plant pathogens world-wide.
Colletotrichum gloesporioides is so far the most predominant Colletotrichum pathogen
and can attack about 470 different host genera, Sutton (1980) and Dodd (1992).
Colletotrichum attacks fruits during development in the field as well as the post harvest.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
6
Infections at the fruiting stage on these crops caused the highest yield losses (Waller,
1992).
Coffee berry disease (CBD) is the anthracnose of green and ripe coffee berries
caused by Colletotrichum kahawae (Waller and Bridge, 2001). The fungus attacks all
parts of the plant including flowers, berries and occasionally, also the branches and
leaves. The infected berries mostly shed at an early stage of infection or remain
mummified on the stems (Waller, 1992). Colletotrichum kahawae also causes “brown
blight” symptoms in association with C. gloesporioides on ripening berries (Hakiza,
1993). The highest losses are due to premature berry shedding. This disease is the major
threat to the production of Arabica coffee in Africa.
Colletotrichum coffeanum is regarded as a saprophyte or weak pathogen of coffee
and has been described as being synonyms with C. gloesporioides Waller (2007) and
Hyde (2009a). This species is very closely related to C. gloesporioides, as indicated by
their genetic similarities, Screenivasaprasad (1993) and Cannon (2000) . However C.
kahawae can be distinguished from C. gloesporioides by its inability to utilize citric acid
and ammonium tartrate as sole carbon sources, by its high pathogenicity on coffee and by
molecular tools.
Coffee berry disease has caused severe infection in Kenya and Congo. Varieties
of Arabica coffee such as Jamaican Blue Mountain showed infection to these diseases.
Control of this disease is choosing the right environment. This disease is a cold
temperature disease and may be avoided if temperature never drops to 18.33 oC.
Chemical sprays like Perenox and other copper fungicide in four applications during the
year tends to reduce the disease.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
7
MATERIALS AND METHODS
Collection of the Diseased Specimens
Collection of infected coffee plant parts with symptoms exhibiting Colletotrichum
spp. infection was done on the coffee plantation established at the Benguet State
University Agroforestry Project at Bektey, Longlong, La Trinidad Benguet. Infected
leaves, stems and berries were randomly collected and were contained separately in
plastic bags and labeled for laboratory diagnosis.
Preparation of Culture Medium and Isolation
The Water agar (WA) as culture medium was prepared following the standard
procedure. Infected stems, leaves, and berries were cut into 2-3 mm2 sections which
included diseased and healthy tissues, disinfected with 10 % Chlorox for one minute,
rinsed with sterile distilled water for 3 times, blot dried in sterile tissue paper, planted in
WA and incubated at room temperature. Once fungal growth was observed which usually
happens after three days, agar block was cut and transferred into another PDA plate for
cultural and morphological characterization of the colonies. Samples of fungal structures
were mounted in microscope for characterization of vegetative structures and conidia.
Conidia Measurement
To be able to get the exact measurement of the conidia, a calibrated microscope
was used. The ocular micrometer was inserted on the eyepiece of the microscope. While
the stage micrometer was placed on the stage of the microscope. Focusing was done on
the stage micrometer scale using the low power objective (LPO) and then on the high
power objective (HPO) of the microscope. Zero point of the stage micrometer was set to
coincide with that of the ocular micrometer. The ocular divisions that cover the space
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
8
between the zero and coincident lines were counted. The ocular divisions that cover the
space between the zero and coincident lines were counted. Calibration factor (CF) or
calibration constant (CC) was calculated using this formula, 10 conidia were been
measured by their length and width.
CF = n divisions of stage micrometer x 10 units / divisions
n divisions of ocular micrometer
Data Gathered
1. Diseases symptoms. The appearance of symptoms on infected tissues was
characterized.
2. Cultural characteristics of the pathogens. The growths of the pathogen was
observed based on the color, and diameter of the colonies observed and the number of
days to colony diameter.
3. Morphological characterization of the pathogens. The fungal structures was
determined through microscopy based on the appearance and color of the hyphae,
mycelium and presence of septation, color of spores in terms of shapes, color and sizes.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
9
RESULTS AND DISCUSSION
Anthracnose Symptoms on Leaves
Anthracnose first appears as small and irregular yellow, brown, dark-brown, or
black spots. The spots can expand and merge to cover the whole affected area. The color
of the infected part darkens as it ages and characterized generally as large brown leaf
blight (Figure 1).
Anthracnose Symptoms on Stem
Symptom on stem or twig appeared as brown lesion along the length of the stem
or twig. The disease can also produce cankers on petioles and on stems that causes severe
defoliation (Figure 2).
Anthracnose on Coffee Berries
Infected berries exhibited circular watery, dark sunken lesion that appear on the
surface of the pulp (Figure 3). The observed symptoms appear to be similar to the scab
lesion as describe by Ferreire and Boley (1991).
Figure 1. Coffee twig with Figure 2. Coffee leaf with Figure 3. Coffee berries with
Anthracnose Anthracnose Anthracnose
(Colletotrichum (Colletotrichum (Colletotricum
gloesporioides) coffeanum) kahawae)
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
10
Cultural Characteristics of Colletotrichum Isolates
Colony size. Isolates differed in the diameter of their colony growth in culture
media within 1 week (Table 1). Colletotrichum coffeanum attained the largest colony
diameter of 40.5 mm, followed by Colletotrichum gloesporioides at 35.7 mm and the
least was isolate Colletotrichum kahawae at 32.3mm. Based on the size of colony,
Colletotrichum coffeanum obtained from infected leaves was fast growing compared to
the other isolates.
Colony color. The colony color of Colletotrichum coffeanum and Colletotrichum
kahawae were white to black (Figure 5 and 6) while Colletotrichum gloesporioides was
white (Figure 4).
Rate of growth. Colletotrichum coffeanum was observed to be fast growth as
compared to Colletotrichum gloesporioides and Colletotrichum kahawae which were
observed to be moderately fast (Table 1).These observed growth characteristics conforms
to their difference in colony size in one week culture.
Morphological Characteristics of Colletotrichum Isolates
Table 2 summarizes the morphological characteristics of the Colletotrichum spp.
isolates.
Mycelia. The mycelia of Colletotrichum gloesporioides were observed to be
hyaline, septated, and large- walled (Figure 7). Mycelia of Colletotrichum coffeanum
were hyaline branched, septated, and elongated (Figure 8) while mycelia of
Colletotrichum kahawae (Figure 9) were slender, septated and hyaline.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
11
Table 1. Cultural characterization of one to four week old isolates of Colletotrichum spp.
at temperature range of 24- 26 oC on PDA
COLLETOTRICHUM SPECIES
CRITERIA
C. gloesporioides
C. coffeanum
C. kahawae
Colony diameter
35.7 mm
40.5 mm
32.3 mm
(mm)
Colony color
white
white to black
white to black
Rate Growth
m
oderately fast
fast growing
moderately fast
in one week
Figure 4. One week old culture of Figure 5. One month old culture of
Colletotrichum gloesporioides Colletotrichum coffeanum
in PDA in PDA
Figure 6. One month old culture of Colletotrichum kahawae in PDA
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
12
Figure 7. Mycelia of one Figure 8. Mycelia of one Figure 9. Mycelia of one
week old culture of week old culture of week old culture of
C. gloesporioides C. coffeanum (400 X) C. kahawae (400X)
(400X)
Table 2. Morphological characterization of one to four week old isolates of
Colletotrichum spp. at temperature range of 24- 26 oC on PDA
COLLETOTRICHUM SPECIES
CRITERIA
C. gloesporioides
C. coffeanum
C. kahawae
Presence of septa
on
Present
Present
Present
Mycelia
Shape of conidia
Elliptic
Oblong
Cylindrical
Color of conidia
Hyaline
Hyaline
Hyaline
Size of conidia
11.3 µm x 2.19 µm 11.6 µm x 2.16 µm
11.3µm x 1.6 µm
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
13
Conidia. Shape of conidia varied between isolates (Table 2). Conidia of
Colletotrichum gloesporioides was elliptic and oblong, while Colletotrichum coffeanum,
cylindrical and for Colletotrichum kahawae, conidia color were observed similar to all
the isolates as hyaline. The elliptic spore and shape observed conforms with the findings
of Farreira and Boley (1991) that the spores of coffee berry disease vary from oval to
elliptic to some what irregularly clavate. In terms of the conidia size, isolate A has a size
of 11.3 µm and a width of 2.19 µm, isolate B is 11.6 µm x 2.16 µm (Figure 10 and 11)
and Isolate C 11. 3 µm in length and width of 1.6 µm (Figure 12).
Figure 10. Conidia of isolate Figure 11. Conidia of isolate Figure 12. Conidia of isolate
C. gloesporioides
C. Coffeanum from C. kahawae from
from one week from one week old one week old
old culture (100X) old culture (100X) culture (100X)
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
14
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted from October to February 2011 at Benguet State
University Agroforestry Project. The study found out that there were three different
Colletotrichum spp. infecting Arabica coffee. Based on their cultural and morphological
characteristics the Colletotrichum spp. Possess a characteristics similar to that of
Colletotrichum gloesporioides, Colletotrichum coffeanum, and Colletotrichum kahawae.
There were cases where two or three types of conidia appeared from a single lesions and
in the isolation process.
Conclusion
Based on the finding of the study, it could be concluded that there are three
different Colletotrichum spp. associated to Arabica coffee. Isolate A which is close to
Colletotrichum gloesporioides is mounted from the (leaf), isolate B Colletotrichum
coffeanum from the (twig) and isolate C Colletotrichum kahawae from (berries). All the
isolates differed in their cultural and morphological characteristics. However, specificity
of these isolates in infecting leaves, twigs/stems, and berries were not ascertained.
Recommendation
It is recommended that Pathogenicity test will be done to update earlier
identification of the isolates. Pathogenicity shall be done to determine their specificity to
the leaves, twigs/stems, and berries of coffee as observed in their result and claimed by
previous studies.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
15
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Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
COYASAN, JILL C. APRIL 2011. Characterization of Colletotrichum spp. Associated to
Berries, Leaves, and Stems of Arabica Coffee. Benguet State University, La Trinidad, Benguet.
Adviser: Andres A. Basalong, MSc.
ABSTRACT
The study was conducted primarily to describe the symptoms of Colletotrichum species
associated to stems/twig, leaves and berries of Arabica coffee and determine the cultural and
morphological characteristics of the fungi.
Result of the study showed that stems/twig infected by Colletotrichum gloesporioides
designated as isolate A showed brown lesion along the length of the stems/twig and can also
produce cankers on petioles that causes severe defoliation. Leaves infected by Colletotrichum
coffeanum designated as isolate B showed symptoms of small and irregular, brown, dark-brown
or black spots. The infected part darkens as it ages and characterized generally as large brown
leaf blight while berries infected by Colletotrichum kahawae designated as isolate C showed
symptoms of circular watery, dark sunken lesion that appear on the surface of the pulp.
The colony diameter of Colletotrichum gloesporioides in PDA after 7 days was 35.7mm
while Colletotrichum coffeanum and Colletotrichum kahawae grew up to 40.5mm and 32.3mm.
Colletotrichum gloesporioides exhibited a color of white mycelia in PDA, while Colletotrichum
coffeanum and Colletotrichum kahawae had white to black mycelia. The size of conidia of
Colletotrichum gloesporioides is 11.3µm x2.19 µm for Colletotrichum coffeanum is 11.6µm
x2.16µm and Colletotrichum kahawae is 11.3µm x1.6µm.
Three species of Colletotrichum are associated to Arabica coffee should be validated
through pathogenicity test.
TABLE OF CONTENTS
Page
Bibliography………………………………………………………………… i
Abstract …………………………………………………………………….. i
Table of Contents …………………………………………………………… ii
INTRODUCTION ………………………………………………………….. 1
REVIEW OF LITERATURE ………………………………………………. 3
MATERIALS AND METHOD ……………………………………………... 7
RESULTS AND DISCUSSION ……………………………………………. 9
Anthracnose Symptom on Leaves …………………………………… 9
Anthracnose Symptom on Stem ……………………………………... 9
Anthracnose on Coffee Berries ……………………………………… 9
Cultural Characteristics of Anthracnose isolates ……………………. 10
Morphological Characteristics of
Colletotrichum Isolates ……………………………………………... 10
Mycelia ………………………………………………………………. 10
Conidia ………………………………………………………………. 13
SUMMARY, CONCLUSION, AND RECOMMENDATION …………….. 14
Summary …………………………………………………………….. 14
Conclusion …………………………………………………………... 14
Recommendation ……………………………………………………. 14
LITERATURE CITED ……………………………………………………… 15
1
INTRODUCTION
Coffee has the great demand in the world market and it is good for business.
Coffee is the most important raw material traded throughout the world behind crude oil,
and the most important export article for the nations that grow it. Coffee is the most
precious beverage by Filipinos (Daguio, 2000).
Coffee is one of the major products which is favored by people all over the world.
It has become the major source of foreign exchange after oil for many developing
countries, and it has been sustained in their economies. It supports lives of people who
cultivate it.
The universal demand for coffee is due to the invigorating effect and to some
extent the medicinal value derived from it. Coffee assists in assimilation and digestion, it
reduces the amount of blood circulation, and whopping cough (Cresencia, 1979). The
impact of coffee to human health revolves largely around caffeine. Caffeine is the most
active stimulant of xanthine alkaloids. It also acts as diuretic, vasodilator, laxative and
appetite suppressant. Caffeine is sold commercially in medications, diet pills, as the
counter stimulants, and medicines administered to hyperactive children, where ironically,
it has a calming effect.
Coffee contains the alkaloid caffeine (1, 3, 7 trimethylxanthine), which is the
most active stimulant among the xanthine alkaloids. Caffeine is sold commercially in
pain medications, stimulants and it has a calming effect to hyperactive children.
Moreover, moderate consumption of coffee has been reported to reduce risk of colon
cancer, cirrhosis, gallstones, Parkinson’s disease and asthma (Vinson, 2001).
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
2
Farmers started growing coffee many years ago but up to the present there is still
very low supply due to attack of insect pests and diseases. Anthracnose is one of the
major diseases infecting coffee. It is caused by several species or strains of
Colletotrichum. This pathogen is capable of infecting leaves, berries, stem bark and twigs
of the coffee plant. Colletotrichum coffeanum invades the main body of the plant but does
so without any signs of disease. However, when the plant set fruit, the fungus becomes
aggressive and the diseases become apparent. Coffee coffeanum affects green or
immature coffee berries and the coffee flower at any stage in its development. Infected
berries often show dark sunken pits that spread and coalesce to cover the whole berry. As
the fungus sporulates, a pale pink crust of conidia appears on the lesion. Depending upon
the timing of the infection, the bean can also become infected. Some cases, a milder scab
like disease occurs. Losses of up to 75 % of a crop have been reported.
Previous reports of the Colletotrichum spp. infecting coffee in some growing
areas indicated that species associated to the leaves do not infect coffee berries. Likewise
species found infecting berries cannot infect the leaves.
The Study aimed to describe the symptoms of Colletotrichum spp. infection on
leaves, stems, and berries of Arabica coffee and determine the species of Colletotrichum
infecting different parts of Arabica coffee through morphological and cultural
characterization.
Diseased samples were collected at the Benguet State University Agroforestry
Laboratory from October to February 2011 while the activities were conducted at the
Department of Plant Pathology laboratory.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
3
REVIEW OF LITERATURE
Coffee plants belong to the Rubiaceae family. The commercially grown species
are Coffee Arabica, Coffee canefora (Robusta), Coffee liberica, and Coffea exelsa
(Maistri and Rena, 2001). However, several species were mentioned by other authors.
Economically, the most important species are Coffea Arabica and Coffee canephora, with
Arabica consisting 70% of the coffee production worldwide (Gilman, 1999).
An understanding of the coffee plant, it make- up and how it grows is essential to
understanding how to manage the coffee tree. Management, like the environment and the
variety planted, has a very big influence on coffee quality and yield. It is necessary to
stress the importance of growing techniques (pricking-out in nurseries, mulching, and
irrigation and planting layouts) on the distribution and functions of roots (Winston,
2005). Ringor and Versola (1981) revealed that weeds compete for nutrients and water
with newly planted coffee plants. They reduce growth to as much as 50 percent. If left
uncontrolled in the plantation area, weeds can delay production for as much as one year.
Moreover, they are host of fungal diseases and act as pest shelters, host to insects that
transmit diseases, restrict the movement of the water through the plantation area, and
hinder efficient harvesting in general. Pest and diseases greatly reduce the coffee yields
and its quality. Thus measures should be put in place to prevent and or control pest and
diseases. Since coffee is perennial crop, their communities were attack by insects. The
control of coffee pests and diseases, therefore, require careful selection of the predator
(O’ Farrill, 2000) and recommends four means of controlling pest and diseases such as
cultural that is by means of weed control, pruning, mulching etc, mechanical method
which is done by picking and destroying mechanically infested diseased part of the plant
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
4
(Anonymous, 2006) and biological method which involves using predators or parasites to
destroy the coffee pests and diseases Since cultural method is the cheapest, safest and
easily affordable it should be the first control measure to be used. Other measures should
be used only when the cultural measure fails.
Importance of Coffee
Coffee is one of the great social drinks of human culture. Coffee assumption has
always been associated with work and activities requiring thought and attention
(Macanes, 1990).
Svilaas (2004) also reported that coffee is the number one source of antioxidants.
Which have been linked to a number of potential health benefits, including protection
against heart disease and cancer.
The day may come when coffee will be gold. This must be the reason for renewed
interest to promote coffee in the region. Potentials in the coffee industry may transform
the Cordillera as the Arabica coffee capital of the country. Market for coffee in the region
is not a problem for current local production could not even supply the growing demands
of multiplying shops in Baguio City and other areas in the region (Mondonedo, 2008).
Arabica coffee can also be used as a component of watershed conservation and
rehabilitation of denuded watershed as a result of over- logging and swidden farming and
can be promoted through the adoption of coffee cultivation as Agro-forestry crop
(Alladiw, 2008).
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
5
General Characterization of Colletotrichum
The fungus Colletotrichum produces colorless or hyaline, one-celled, ovoid,
cylindrical, oblong or falcate and sometimes curved or dumbbell shaped conidia in
acervuli, Colletotrichum acervuli are disc-shaped or cushion shape and waxy having
dark, long, sterile hair like (Agrios, 1997 and Quimio, 1999).
Colletotrichum coffeanum is an ascomycete, a member of the sac fungi. One of
the features of these fungi is that they generate spores, called conidia that can be easily
dispersed by the wind and splashing rain. Spread of C. Coffeanum is dependent on water
but it can also be spread by animals, by coffee pickers being a particular problem.
Colletotrichum invades plants but does so without any sign of disease. However when the
plants set fruit, the fungus becomes aggressive and the disease becomes apparent (Silva,
2006).
Coffee berry disease Colletotrichum coffeanum attacks the green tissues at the
beginning stage of berry development and often penetrating into interior of the berry
destroying the bean. It appears at the berries then move up to the stalk. The berry will
have brown patches that seem to have small punctures. Lesions form darker areas of the
berry and in time the berry turn ash gray. The disease results in the production of poor
berries and difficulty in pulping.
According to Phuong (2010), filamentous fungi of the genus Colletotrichum and
its teleomorph Glomerella are among the most important plant pathogens world-wide.
Colletotrichum gloesporioides is so far the most predominant Colletotrichum pathogen
and can attack about 470 different host genera, Sutton (1980) and Dodd (1992).
Colletotrichum attacks fruits during development in the field as well as the post harvest.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
6
Infections at the fruiting stage on these crops caused the highest yield losses (Waller,
1992).
Coffee berry disease (CBD) is the anthracnose of green and ripe coffee berries
caused by Colletotrichum kahawae (Waller and Bridge, 2001). The fungus attacks all
parts of the plant including flowers, berries and occasionally, also the branches and
leaves. The infected berries mostly shed at an early stage of infection or remain
mummified on the stems (Waller, 1992). Colletotrichum kahawae also causes “brown
blight” symptoms in association with C. gloesporioides on ripening berries (Hakiza,
1993). The highest losses are due to premature berry shedding. This disease is the major
threat to the production of Arabica coffee in Africa.
Colletotrichum coffeanum is regarded as a saprophyte or weak pathogen of coffee
and has been described as being synonyms with C. gloesporioides Waller (2007) and
Hyde (2009a). This species is very closely related to C. gloesporioides, as indicated by
their genetic similarities, Screenivasaprasad (1993) and Cannon (2000) . However C.
kahawae can be distinguished from C. gloesporioides by its inability to utilize citric acid
and ammonium tartrate as sole carbon sources, by its high pathogenicity on coffee and by
molecular tools.
Coffee berry disease has caused severe infection in Kenya and Congo. Varieties
of Arabica coffee such as Jamaican Blue Mountain showed infection to these diseases.
Control of this disease is choosing the right environment. This disease is a cold
temperature disease and may be avoided if temperature never drops to 18.33 oC.
Chemical sprays like Perenox and other copper fungicide in four applications during the
year tends to reduce the disease.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
7
MATERIALS AND METHODS
Collection of the Diseased Specimens
Collection of infected coffee plant parts with symptoms exhibiting Colletotrichum
spp. infection was done on the coffee plantation established at the Benguet State
University Agroforestry Project at Bektey, Longlong, La Trinidad Benguet. Infected
leaves, stems and berries were randomly collected and were contained separately in
plastic bags and labeled for laboratory diagnosis.
Preparation of Culture Medium and Isolation
The Water agar (WA) as culture medium was prepared following the standard
procedure. Infected stems, leaves, and berries were cut into 2-3 mm2 sections which
included diseased and healthy tissues, disinfected with 10 % Chlorox for one minute,
rinsed with sterile distilled water for 3 times, blot dried in sterile tissue paper, planted in
WA and incubated at room temperature. Once fungal growth was observed which usually
happens after three days, agar block was cut and transferred into another PDA plate for
cultural and morphological characterization of the colonies. Samples of fungal structures
were mounted in microscope for characterization of vegetative structures and conidia.
Conidia Measurement
To be able to get the exact measurement of the conidia, a calibrated microscope
was used. The ocular micrometer was inserted on the eyepiece of the microscope. While
the stage micrometer was placed on the stage of the microscope. Focusing was done on
the stage micrometer scale using the low power objective (LPO) and then on the high
power objective (HPO) of the microscope. Zero point of the stage micrometer was set to
coincide with that of the ocular micrometer. The ocular divisions that cover the space
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
8
between the zero and coincident lines were counted. The ocular divisions that cover the
space between the zero and coincident lines were counted. Calibration factor (CF) or
calibration constant (CC) was calculated using this formula, 10 conidia were been
measured by their length and width.
CF = n divisions of stage micrometer x 10 units / divisions
n divisions of ocular micrometer
Data Gathered
1. Diseases symptoms. The appearance of symptoms on infected tissues was
characterized.
2. Cultural characteristics of the pathogens. The growths of the pathogen was
observed based on the color, and diameter of the colonies observed and the number of
days to colony diameter.
3. Morphological characterization of the pathogens. The fungal structures was
determined through microscopy based on the appearance and color of the hyphae,
mycelium and presence of septation, color of spores in terms of shapes, color and sizes.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
9
RESULTS AND DISCUSSION
Anthracnose Symptoms on Leaves
Anthracnose first appears as small and irregular yellow, brown, dark-brown, or
black spots. The spots can expand and merge to cover the whole affected area. The color
of the infected part darkens as it ages and characterized generally as large brown leaf
blight (Figure 1).
Anthracnose Symptoms on Stem
Symptom on stem or twig appeared as brown lesion along the length of the stem
or twig. The disease can also produce cankers on petioles and on stems that causes severe
defoliation (Figure 2).
Anthracnose on Coffee Berries
Infected berries exhibited circular watery, dark sunken lesion that appear on the
surface of the pulp (Figure 3). The observed symptoms appear to be similar to the scab
lesion as describe by Ferreire and Boley (1991).
Figure 1. Coffee twig with Figure 2. Coffee leaf with Figure 3. Coffee berries with
Anthracnose Anthracnose Anthracnose
(Colletotrichum (Colletotrichum (Colletotricum
gloesporioides) coffeanum) kahawae)
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
10
Cultural Characteristics of Colletotrichum Isolates
Colony size. Isolates differed in the diameter of their colony growth in culture
media within 1 week (Table 1). Colletotrichum coffeanum attained the largest colony
diameter of 40.5 mm, followed by Colletotrichum gloesporioides at 35.7 mm and the
least was isolate Colletotrichum kahawae at 32.3mm. Based on the size of colony,
Colletotrichum coffeanum obtained from infected leaves was fast growing compared to
the other isolates.
Colony color. The colony color of Colletotrichum coffeanum and Colletotrichum
kahawae were white to black (Figure 5 and 6) while Colletotrichum gloesporioides was
white (Figure 4).
Rate of growth. Colletotrichum coffeanum was observed to be fast growth as
compared to Colletotrichum gloesporioides and Colletotrichum kahawae which were
observed to be moderately fast (Table 1).These observed growth characteristics conforms
to their difference in colony size in one week culture.
Morphological Characteristics of Colletotrichum Isolates
Table 2 summarizes the morphological characteristics of the Colletotrichum spp.
isolates.
Mycelia. The mycelia of Colletotrichum gloesporioides were observed to be
hyaline, septated, and large- walled (Figure 7). Mycelia of Colletotrichum coffeanum
were hyaline branched, septated, and elongated (Figure 8) while mycelia of
Colletotrichum kahawae (Figure 9) were slender, septated and hyaline.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
11
Table 1. Cultural characterization of one to four week old isolates of Colletotrichum spp.
at temperature range of 24- 26 oC on PDA
COLLETOTRICHUM SPECIES
CRITERIA
C. gloesporioides
C. coffeanum
C. kahawae
Colony diameter
35.7 mm
40.5 mm
32.3 mm
(mm)
Colony color
white
white to black
white to black
Rate Growth
m
oderately fast
fast growing
moderately fast
in one week
Figure 4. One week old culture of Figure 5. One month old culture of
Colletotrichum gloesporioides Colletotrichum coffeanum
in PDA in PDA
Figure 6. One month old culture of Colletotrichum kahawae in PDA
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
12
Figure 7. Mycelia of one Figure 8. Mycelia of one Figure 9. Mycelia of one
week old culture of week old culture of week old culture of
C. gloesporioides C. coffeanum (400 X) C. kahawae (400X)
(400X)
Table 2. Morphological characterization of one to four week old isolates of
Colletotrichum spp. at temperature range of 24- 26 oC on PDA
COLLETOTRICHUM SPECIES
CRITERIA
C. gloesporioides
C. coffeanum
C. kahawae
Presence of septa
on
Present
Present
Present
Mycelia
Shape of conidia
Elliptic
Oblong
Cylindrical
Color of conidia
Hyaline
Hyaline
Hyaline
Size of conidia
11.3 µm x 2.19 µm 11.6 µm x 2.16 µm
11.3µm x 1.6 µm
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
13
Conidia. Shape of conidia varied between isolates (Table 2). Conidia of
Colletotrichum gloesporioides was elliptic and oblong, while Colletotrichum coffeanum,
cylindrical and for Colletotrichum kahawae, conidia color were observed similar to all
the isolates as hyaline. The elliptic spore and shape observed conforms with the findings
of Farreira and Boley (1991) that the spores of coffee berry disease vary from oval to
elliptic to some what irregularly clavate. In terms of the conidia size, isolate A has a size
of 11.3 µm and a width of 2.19 µm, isolate B is 11.6 µm x 2.16 µm (Figure 10 and 11)
and Isolate C 11. 3 µm in length and width of 1.6 µm (Figure 12).
Figure 10. Conidia of isolate Figure 11. Conidia of isolate Figure 12. Conidia of isolate
C. gloesporioides
C. Coffeanum from C. kahawae from
from one week from one week old one week old
old culture (100X) old culture (100X) culture (100X)
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
14
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted from October to February 2011 at Benguet State
University Agroforestry Project. The study found out that there were three different
Colletotrichum spp. infecting Arabica coffee. Based on their cultural and morphological
characteristics the Colletotrichum spp. Possess a characteristics similar to that of
Colletotrichum gloesporioides, Colletotrichum coffeanum, and Colletotrichum kahawae.
There were cases where two or three types of conidia appeared from a single lesions and
in the isolation process.
Conclusion
Based on the finding of the study, it could be concluded that there are three
different Colletotrichum spp. associated to Arabica coffee. Isolate A which is close to
Colletotrichum gloesporioides is mounted from the (leaf), isolate B Colletotrichum
coffeanum from the (twig) and isolate C Colletotrichum kahawae from (berries). All the
isolates differed in their cultural and morphological characteristics. However, specificity
of these isolates in infecting leaves, twigs/stems, and berries were not ascertained.
Recommendation
It is recommended that Pathogenicity test will be done to update earlier
identification of the isolates. Pathogenicity shall be done to determine their specificity to
the leaves, twigs/stems, and berries of coffee as observed in their result and claimed by
previous studies.
Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
15
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Characterization of Colletotrichum spp. Associated to Berries, Leaves, and Stems of
Arabica Coffee.COYASAN, JILL C. APRIL 2011
Document Outline
- Characterization of Colletotrichum spp. Associated toBerries, Leaves, and Stems of Arabica Coffee
- BIBLIOGRAPHY
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
- MATERIALS AND METHODS
- RESULTS AND DISCUSSION
- SUMMARY, CONCLUSION AND RECOMMENDATION
- LITERATURE CITED