The experiment was done to assess incidence and severity; variations in Colletotrichum kahawae isolates and resistance levels in Hararghe coffee. Assessment was conducted in Bedeno, Boke, Habro and Darolebu districts August to September 2010. Incidence and severity were recorded on 50 and 10 randomly selected coffee trees/ farm respectively. Pathogenic variability was studied by cultural and morphological characteristics, and placed into 4 based on colony color. Reactions of 11 landraces and checks were evaluated through hypocotyl inoculation. Mean disease incidence was 51 % at Darolebu and 75 % at Bedeno, and mean disease severity 26 % at Boke and 50 % at Bedeno. Mean radial colony growth of isolates was between 4.05 and 5.35 millimeter/24 hour on malt extract agar and potato dextrose agar, respectively. Conidia width and length ranged from 2.12-4.24 and 10.51-15.78 μm, respectively. Significant (P < 0.05) variation was observed among isolates in sporulation capacity. Highly significant variations (P < 0.0001) was observed among selections H-05/02, H-568/02, H-87/02 and the resistant check in percent mean incidence and percent mean severity, and were classified as moderately susceptible indicating presence of resistance sources. Hence, important to conserve and conduct intensive selection from more diverse populations.

1. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
IJPBCS
Coffee berry disease (Colletotrichum kahawae): status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia Berhanu Tamiru Emana
Addis Ababa University, College of Veterinary Medicine and Agriculture, P.O.Box 245, Fiche, Ethiopia
Email: btamirue@gmail.com, Tel.: 251-910073375, Fax number: 251- 111352910 The experiment was done to assess incidence and severity; variations in Colletotrichum kahawae isolates and resistance levels in Hararghe coffee. Assessment was conducted in Bedeno, Boke, Habro and Darolebu districts August to September 2010. Incidence and severity were recorded on 50 and 10 randomly selected coffee trees/ farm respectively. Pathogenic variability was studied by cultural and morphological characteristics, and placed into 4 based on colony color. Reactions of 11 landraces and checks were evaluated through hypocotyl inoculation. Mean disease incidence was 51 % at Darolebu and 75 % at Bedeno, and mean disease severity 26 % at Boke and 50 % at Bedeno. Mean radial colony growth of isolates was between 4.05 and 5.35 millimeter/24 hour on malt extract agar and potato dextrose agar, respectively. Conidia width and length ranged from 2.12-4.24 and 10.51-15.78 μm, respectively. Significant (P < 0.05) variation was observed among isolates in sporulation capacity. Highly significant variations (P < 0.0001) was observed among selections H-05/02, H-568/02, H-87/02 and the resistant check in percent mean incidence and percent mean severity, and were classified as moderately susceptible indicating presence of resistance sources. Hence, important to conserve and conduct intensive selection from more diverse populations. Key words: Hararghe coffee, Colletotrichum kahawae, Landrace, sporulation capacity, coffee trees
INTRODUCTION
Coffee is one of the most important commodities in the international agricultural trade, representing a significant source of income to several coffee producing countries including Ethiopia. It ranks second in global trade, rated after petroleum products (ICO, 2007). It remains a backbone of the Ethiopian economy and currently contributes more than 60 % of the annual foreign currency in Ethiopia; followed by oil seeds, pulses, gold, chat (Catha edulis), flower, fruits, cotton, textiles, livestock and minerals (EAFCA, 2010).
The coffee types of Ethiopia that are distinguished for their very fine quality, unique aroma and flavor include Harar, Sidamo, Yirgacheffe, Ghimbi and Limu types (Workafes and Kassu, 2000). This invaluable genetic resource is under severe threat of extinction due to population pressure, settlements and land degradation and over exploitation of forest products and biotic factors (Feyera et al., 2008). So, there is no question that coffee improvement should continue to meet the increasing demands for coffee in terms of both quantity and quality.
Coffee berry disease (CBD) is a disease caused by fungal pathogen, an anthracnose of green and ripe coffee berries, which is induced by Colletotrichum kahawae, taxonomically, belongs to order Melanconiales of the fungi imperfection (Agrios, 2005). From the diseases coffee prone to, CBD alone is known to reduce coffee International Journal of Plant Breeding and Crop Science Vol. 1(2), pp. 018-027, October, 2014. © www.premierpublishers.org. ISSN: 2167-0449
Research Article

2. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Emana BT 018
yields between 25-30 % (Eshetu et al, 2000). Survey
results of 1996/97 and 1997/98; showed that severity of
CBD was 31 % and 32 %, respectively. In both years the
highest severity (57 %) was recorded in East Hararghe,
followed by west Hararghe (55.30 %) (Arega et al, 2008).
Harar coffee cultivation has been hampered by wide
spread of CBD, which decreased the annual coffee
production, by an average of 47 % on some of the
individual small holder’s farms (Berhanu, 1998).
Hararghe coffee is susceptible to CBD; as a result it is
under threat of genetic erosion mainly because of the
losses caused by the disease and farmers prefer growing
alternative cash crops such as chat to planting coffee
(Arega et al, 2008).
The aim of the Ethiopian coffee research program was to
search for improved coffee cultivars with wider adaptation
by giving more emphasis to the southwestern part of the
country.
A number of improved and disease (CBD) resistant
varieties have been developed and distributed to coffee
farmers in the southwestern part of the country. However,
this research direction failed in providing varieties that are
suitable for eastern parts of Ethiopia.
Some efforts were made to introduce into the eastern
part of the country those cultivars have been released for
planting since 1977 that are especially suitable for the
western parts of the country. However, these cultivars
have not performed well in the coffee growing areas of
the Hararghe districts. Recently, Jima Agricultural
Research Center collected 200 coffee landraces from
major coffee producing districts of Hararghe zones (as
part of 2004 national coffeegermplasm collection
program). These accessions are currently under field
condition at Mechara Agricultural Research Center, West
Hararghe.
Breeding and improvement activities so far undertaken
were not exhaustive. Moreover, factors contributing to
disease intensity and susceptibility of Hararghe coffee to
CBD are not yet understood. This may be because of the
differences in pathogen populations, the host, and
weather condition or production system in the region that
needs to be critically addressed. More importantly, the
accessions which are currently under field conditions at
Mechara Agricultural Research Center were not yet
evaluated against CBD.
Therefore, this study was proposed with the
following objectives:
To assess the incidence and severity of coffee
berry disease in major coffee producing districts of
Hararghe,
To study variations in Colletotrichum kahawae
isolate populations,
To evaluate resistance levels in Hararghe coffee
landraces to coffee berry disease in the region.
MATERIALS AND METHODS
Description of Study Sites
The study was conducted both in the field and laboratory.
The field studies were conducted in the major coffee
producing districts of Hararghe: Bedeno (East Hararghe),
Boke, Habro and Darolebu (West Hararghe). In each
district disease assessment and sampling were
conducted and laboratory works were conducted at
Haramaya University.
Disease Survey
The survey of coffee berry disease was undertaken in
major coffee producing districts of Hararghe, namely
Darolebu, Habro, Boke and Bedeno. These districts were
purposively selected for their coffee production and
disease prevalence. Based on the secondary information
from the district BoARD, the major coffee producing
districts were divided into lowland, midland and highland.
From each agro ecological zone, three to five farms and
a total nine to fifteen were surveyed from each district.
Fields were sampled at intervals of about 5-10 km along
the roads and the distance between sample fields was
based on the topography and the relative importance of
coffee production within each district.
Data Collected
Two types of assessment methods were conducted on
the same tree following procedures used by Tesfaye and
Ibrahim (2000).
(a) Disease incidence: Fifty trees per farm were randomly
observed and diagnosed for presence and absence of
the disease on each tree and disease incidence was
calculated as:
Table 1.
(b) Berry count and Disease severity: Ten trees / farm
were randomly selected and each tree was divided into 3
strata of branches (top, middle and bottom). From each
stratum one middle branch was selected to determine
berry count. CBD damaged and healthy berries were
counted and then percentage of diseased berries over
total counted berries calculated. Moreover, disease
severity including fallen and remaining berries attached
on branches and still showing symptoms was also
recorded separately.
Disease Sample Collection
To study the population variation in the CBD pathogen in
Hararghe, fresh green coffee berries (with active CBD
lesions) were collected from each of the surveyed district.

3. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Int. J. Plant Breeding Crop Sci. 019 Table 1. Weather Variables in coffee berry disease surveyed districts of Hararghe
District
Altitude (m)
Latitude
Longitude
Total annual Rainfall (mm)
Max &min Tem (0C)
Bedeno
1600-2260
9008`N
41038`N
1294
16-21
Boke
1762-1855
8042`N
40044`N
700
24-27
Habro
1621-1772
8042`N
40028`N
1200
21-27
Darolebu
1658-1833
8036`N
40019`N
1000
18.1-33.3
Samples of 15-20 green coffee berries with active lesions were collected during August-September 2010. Samples were collected in a sterilized plastic bag, kept in icebox and transported to the Haramaya University Pathology Laboratory and maintained at 4 0C until they were used for isolation of the pathogen.
Isolation and Identification
Isolation of fungi from berries was carried out following the procedures used by Tefestewold (1995). The berries from each farm were washed in 5 % sodium hypochlorite solution for 1 minute and rinsed 3 times (1 minute each) with sterilized distilled water, placed on sterilized moist tissue paper in disinfected plastic boxes with lid to induce conidial production. The boxes were maintained at room temperature (23-24 0C) until conidial mass and / or fungal growths were observed. Conidia producing berries were aseptically transferred to 100 ml beaker containing sterilized distilled water and conidia were harvested by gentle shaking manually and suspension was plated on PDA amended with 100-ppm (Parts per million) streptomycin sulfate.After identification, the isolates of C. kahawae were subcultured on PDA for 10–14 days in an incubator adjusted to 22- 25 oC using completely randomized design. Pure cultures from isolates were preserved at room temperature (18-25 oC) for later.
Cultural and morphological characterization of C. kahawae isolates
Cultural and morphological characteristics of representative C. kahawae isolates were studied following the methods and procedures used by Tefestewold (1995).
Cultural Appearances
Ranges of cultural variations (Colony radial growth, Colony color) in C. kahawae population were examined using representative isolates of Hararghe, including isolates from CBD hot-spot area of southwestern (Gera) (for comparison) by culturing on potato dextrose agar (PDA) and malt extract agar (MEA). Colony (mycelial) radial growth Cultures of C. kahawae isolates from Hararghe coffee landraces were inoculated on PDA and MEA with three replications. Mycelial (colony) radial growth (mm) of each isolate was measured with ruler, colony diameter from two perpendicular planes on the reverse side of the Petri- dishes. Colony color Colony (mycelia) color on obverse side and types of pigments from the reverse side of each C. kahawae isolate were determined on PDA and MEA using RGB color chart (Anonymous, 2005).
Morphological characteristics
Representative isolates of C. kahawae were cultured on PDA adjusted at pH 5.5 ± 0.1 and incubated at 25 oC in darkness and replicated three times per isolate. Parameters, viz. Conidia sizes, Sporulation capacity and Shapes of conidia frequency were taken from cultures. Conidial Size C. kahawae isolates were incubated on PDA medium for 14 days replicated 3 times per isolate. All types of shapes and most frequent sizes were included at random to minimize further measurement biasness. Conidial size (length and width) was measured with ocular micrometer (μm). Table 2. Shape of conidia Frequency of conidial shapes was tallied from 14 days cultures of C. kahawae isolates that were incubated on PDA. Conidial shape of representative C. kahawae isolates were described using binocular compound microscope and the most frequent 5 conidial shapes which were standardized (Tefestewold, 1995). Sporulation Capacity
Ten days old cultures of each C. kahawae isolate, incubated

4. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Emana BT 020 Table 2. Percent of necrotic lesion of C. kahawae isolates on detached faba bean leaf
0= no infection
None aggressive
1= 1-25 % leaf area damaged
Less aggressive
2= 26-50 % leaf area damaged
Medium aggressive
3= 51-75 % leaf area damaged
Aggressive
4= >75 % leaf area damaged
Highly aggressive
5= dead leaves (100 %) leaf area damaged
Percent of necrotic lesion (%NL) was scored at 10 days post inoculation by using 0 to 5 scoring scale as done by Fernandez and Chen (2005).
on PDA was washed by flooding with 10 ml sterilized distilled water, rubbed with sterilized scalpel and transferred to 50 ml sterilized beaker and thoroughly stirred for 10-15 minutes with magnetic stirrer to extract the spores from the interwoven mycelia and then filtered into another sterilized beaker through double layer cheese clothes. The number of conidia per milliliter was counted using haemocytometer as it was done by (Tefestewold, 1995). Aggressiveness Test The aggressiveness of C. kahawae isolates was studied in the laboratory using detached faba bean leaf as suggested by Hanounik (1986). Young, fully expanded leaflets of similar age were detached and surface sterilized. Two leaflets per plate were laid on moist filter paper in petriplates (one leaf in upper surface and the other lower surface) were inoculated with C. kahawae isolate. Leaflets inoculated with distilled water were used as control. The petriplates were arranged in completely randomized design with one faba bean genotype x 24 isolates. The plates were placed in incubator, dropped with distilled water to maintain high relative humidity for infection. Disease development was scored at 10 days post infection.
Evaluation of some Coffee Landraces for Resistance to CBD
Hararghe coffee collections established at Mechara Agricultural Research Center together with resistant (Code No741) and susceptible (Code No 370) checks were inoculated with most fast growing isolate obtained from Bedeno. Mycelial colonies of the isolate were carefully slashed with a scalpel from the PDA medium while washing with distilled sterilized water to harvest conidia from 14 days old cultures. The suspension of the isolate was stirred with magnetic stirrer for 5 minutes and filtered through double layers of cheese clothes. After repeating the procedure again the spore concentration was adjusted to 2 x 106 conidia/ml. Completely randomized design (CRD) (most fast growing CBD pathogen isolate (from Bedeno) x 11 coffee landraces with resistant and susceptible checks x 3 replications) was employed to see the host-pathogen interactions. The interaction of selected cultivars and CBD isolate was evaluated following the procedures used by Van der Graaff (1981) and Bayetta et al. (2000). Coffee seedlings were raised from freshly harvested seeds of each cultivar. To obtain seedlings, ripe cherries of each cultivar were picked from field and seeds of each coffee cultivar were prepared by removing the parchment and soaked in sterilized distilled water dried under shade after removing the pulp by hand. Thereafter, seeds were sown (25 seeds / box) in sterilized and moist sand soil in plastic boxes. Two days before inoculating the hypocotyls at unfolding stage, temperature was adjusted to 20 oC and seedlings were sprayed with sterilized water and covered with plastic sheet for 48 hours to obtain 80- 90 % relative humidity. Batches of 3 plastic boxes from each coffee selection containing 15 seedlings / box were inoculated by wrapping strips of absorbent cotton wool dipped in inocula round the seedlings, below the cotyledons. After inoculation the trays of inoculated seedlings were covered with closely fitting plastic sheets. They were kept for 48 hours and relative humidity was maintained at high level (80-95 %). After 48 hours the seedlings were uncovered and the stripes of cotton wool carefully removed before placing the trays in a cooled illuminated room made of sacks at 19-21 0C for the next three weeks as done by (Hakiza, 1985). The time taken for seedlings to develop symptoms at the point of inoculation was recorded. At the end of 28 days, number of infected seedlings was recorded. The reaction of each seedling hypocotyl against the pathogen was assessed 15, 21 and 28 days after inoculation using the symptom classification and scoring scale of Van der Graaff (1981).
RESULTS AND DISCUSSION
Occurrence of CBD in Hararghe
The incidence and severity of CBD varied among and within Hararghe coffee. It was observed in Bedeno and Habro consistently. In Bedeno it was also found at low altitude with higher intensity than other districts but with

5. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Int. J. Plant Breeding Crop Sci. 021 Figure 1. Severity of CBD % in Hararghe major coffee producing districts.
lower intensity than the higher altitude. In Boke and Darolebu the disease was found to be severe in pocket (limited areas) coffee areas. The disease incidence ranged up to 100 %, 80 %, 95 % and 90 % was observed in Bedeno, Boke, Habro and Darolebu districts respectively. The mean incidence ranged between 51.5 % at Darolebu and 75 % at Bedeno where as the severity of the disease varied between 26 and 50 % as indicated in figure 1 at Boke and Bedeno, respectively. Similarly, Bayetta (2001) explained high CBD occurrence related with high humidity with high altitude around Gera. High incidence of CBD may be explained by the particularly high rainfall found in relatively high altitudes of Bedeno and to some extent in Habro. As Cook (1975) explained that high rainfall, high humidity or wetness and relatively low temperatures that persist for long periods favor CBD development and the disease is invariably severe at higher altitudes where these conditions generally prevail. The incidence and severity of the disease varied from one district to other. Similarly, the variation in severity and incidence of CBD in other coffee production systems also reported by many authors. In 1994 crop season, prevalence of CBD was conducted in Oromiya Region and Southern Nations Nationalities and Peoples Region (SNNPR) and the result indicated 38.8 and 17.2 % of mean percent prevalence of the disease, respectively (IAR,1997). All the fields in the surveyed districts for CBD showed up to 95, 90, 80 and 100 % incidence in Habro, Darolebu, Boke and Bedeno districts respectively which are located in 1500-2000 m a s l except five fields located in Bedeno in altitudes above 2000 m a s l showed up to 100 % disease incidence. The annual mean rainfall, minimum and maximum temperature data recorded from representative BoARD of the surveyed areas were obtained. Generally, high rainfall, high humidity or wetness, and relatively low temperatures that persist for long periods favor CBD development and the disease is invariably severe at higher altitudes where these conditions generally prevail (Cook, 1975 and Bayetta, 2001). Scrutiny of the minimum and maximum temperatures of the districts revealed that in Boke, the minimum and maximum temperatures were 24-27 0 C implying that less favorable environmental conditions were prevalent which could be one cause for the low severity observed in the district. In the other districts, temperatures mainly in the range of 16-33.3 0C prevailed suggesting that the temperature requirements of the pathogen were available in those districts. Data on rainfall also showed that there was availability of moisture for conidial germination to occur and for CBD to develop extensively.
Detailed classifications of coffee types or cultivars grown in Hararghe are not known. However, farmers in the area have their own local names to distinguish the coffee types grown on their farms. Eight local names representing variations in coffee types were given in 4 districts surveyed (Table 3). The results of the survey conducted in Hararghe coffee producing districts depicted that at least eight types of coffee landraces are produced in the districts. Hararghe coffee farmers were able to identify coffee landraces by their morphological feature and their reaction to disease such as coffee berry disease (CBD). Farmers give names to landraces based on several reasons (Table 3). For instance based on names of the place from where it was first obtained,

6. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Emana BT 022
Table 3. Coffee types and their characteristics in Hararghe (According to farmers` classification)
Local name
Districts
Reaction to CBD
Features
Bedeno
Boke
Habro
Darolebu
Abadir
-
-
+
+
Susceptible
High yielder than other types
Buna Gurracha
-
+
-
+
Tolerant
Irregularly, biennial bearing. Tolerant to drought
Buna qallaa
-
+
+
-
Tolerant
Very high yielding but irregular
Maxee
-
-
+
-
No response
No response
Shumbure
-
+
+
+
Tolerant
High yielder at early age, produces small beans, tolerant to drought, has short life span for over bearing
Shunkuyi
+
-
-
-
No reponse
No response No response
Torbi
-
-
-
+
No response
No response
Wogere
-
+
-
-
No response
No response
+=present -=Absent Table 4. Colony color of C. kahawae isolates on PDA and MEA observed from 10 days culture
Isolate
Colony Color on Media
PDA
MEA
TOP
REV
TOP
REV
Ge1
Dark gray
Gray
Light gray
Dark gray
Ge2
Gray
Dark gray
Light gray
Green
Ge3
Dark gray
Gray
Light gray
Green
Ge4
Gray
Dark gray
Light gray
Dark gray
Ge5
Dark gray
Gray
Light gray
Dark gray
Be1
Dark gray
Rosy brown
Light gray
Rosy brown
Be2
Gray
Dark gray
Light gray
Rosy brown
Be3
Light gray
Rosy brown
Gray
Green
Be4
Gray
Dark olive green
Gray
Dim gray
H1
White
Light gray
Gray
Green
H2
White
Light gray
Light gray
Dark olive green
H3
Gray
Dark gray
Light gray
Gray
Bo1
White
Light gray
White
Rosy brown
Bo2
White
Light gray
White
Rosy brown
Bo3
Gray
Gray
Gray
green
Bo4
Light gray
Dark olive green
Gray
Gray
DL1
Gray
Dim gray
Light gray
Gray
DL2
Gray
Dark olive green
Gray
Gray
DL3
Gray
Gray
White
Dim gray
Five (Ge1, Ge2, Ge3, Ge4, Ge5), Three (H1, H2, H3), Four (Be1, Be2, Be3, Be4), Four (Bo1, Bo2, Bo3, Bo4) and Three (DL1, DL2, DL3) Colletotrichum kahawae isolates were collected from Gera, Habro, Bedeno, Boke and Daro Lebu respectively. TOP: Colony color from above: REV: Colony color from the reverse side of the petri-plate.
names of individuals and other reasons. Accordingly, the coffee landrace ‘Abadir’ was named after the name of an individual that was a famous religious leader in the region.

7. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Int. J. Plant Breeding Crop Sci. 023
Table 5. Mean radial mycelial (colony) growth (mm) of C. kahawae isolates on PDA and MEA incubated at 25 0C
Media PDA MEA
Isolate
7
10
15
7
10
15
Be2
59.59 a
78.75 ab
92.19 a
30.00 cd
48.00 e
66.00 de
Be5
59.19 a
79.24 a
91.50 ab
H3
56.19 a
75.49 b
88.50 b
26.19cde
36.99 fg
51.18 g
Be3
51.15 b
69.49 c
80.29 cd
27.99cde
39.99 f
55.20 f
H5
50.49 b
69.99 c
79.99 cd
18.30 f
28.74 h
43.24 h
Be1
49.99 b
69.49 c
81.49 c
24.99 de
34.50 g
49.09 g
Be4
48.99 b
68.49 c
79.50 cd
D1
42.69 c
61.99 d
77.37 de
41.79 a
67.99 a
87.00 a
Bo3
39.19 cd
58.50 e
68.49 f
24.60 e
34.99 g
51.69 fg
H6
37.20 de
56.49 efg
66.30 gf
12.50 g
35.49 g
49.50 g
Bo1
36.90 de
57.00 ef
67.99 f
36.79 b
59.50 c
76.29 c
H2
36.49 de
55.50 efg
74.89 e
23.37 e
34.50 g
49.50 g
H1
36.30 de
55.50 efg
74.89 e
38.70 ab
65.49 ab
85.29 ab
Bo2
36.00 de
55.50 efg
66.30 fg
30.00 cd
38.49 fg
55.20 f
Bo4
35.10 def
54.49 fg
63.69 ghi
25.20 de
35.49 g
51.61 fg
D4
33.69 defg
52.99 gh
64.99 fgh
35.17 b
51.99 d
67.39 d
Ge2
32.28 efgh
49.52 h
60.82 ij
27.49cde
37.50 fg
51.97 fg
G1
30.65 fgh
49.98 h
61.50 hi
18.81 f
30.00 h
42.93 h
G4
28.99 gh
49.99 h
57.79 j
25.74cde
36.00 fg
51.00 g
D3
27.99 h
42.48 i
51.48 k
25.08 de
34.50 g
49.50 g
G3
27.94 h
42.42 i
48.69 kl
24.99 de
34.99 g
49.50 g
D2
19.50 i
33.49 j
45.48 l
30.49 c
48.00 e
63.14 e
H4
17.35 i
54.00 fg
63.79 ghi
36.30 b
61.99 bc
82.84 b
CV (%)
8.70
4.16
3.54
11.20
6.00
4.21
Be, H, Bo, D and Ge Colletotrichum kahawae isolates collected from Bedeno, Habro, Boke, Daro-Lebu and Gera (used as check) respectively.
Means with the same letter are not significantly different according to DMRT at 0.05 alpha level. Hindorf and Muthappa (1974) found 13.1 x 3.8 μm and (11 – 23) x 3.4 – 4.7) μm average and range of conidia sizes from Kenyan C. kahawae isolates.
Of the coffee landraces grown in Hararghe, landraces Abadir, Shumbure, Buna qallaa and Buna gurrachaa are the dominant ones. Abadir is the most preferred coffee landrace in the districts, according to farmers, due to its yielding potential and bigger bean size. Basic features of the different coffee landraces that are being produced in the districts are summarized in Table 3.
Cultural and Morphological Characteristics of C. kahawae Isolates from Hararghe
Cultural (colony) appearances
Pure culture of C. kahawae isolates representing the four Hararghe districts and representative isolates from Gera were examined for colony (mycelia) aerial growth, pigmentation, sectoring and radial growth of colony.
Isolates were placed into four based on their colony color on PDA and MEA culture plates viz., light gray , dark gray , gray and white mycelia (Table 4). Isolates from Gera (Ge1, Ge2, Ge3, Ge4, and Ge5), Bedeno (Be1, Be2), Habro (H2, H3) Darolebu (DL1) on MEA and Bedeno (Be3) and Boke (Bo4) showed distinct light gray mycelium. In this group, C. kahawae isolates had white mycelial color up to the first 4-6 incubation days and then after changed into light gray mycelia on both PDA and MEA media. Similar result was reported by Hindorf (1970); it was observed that C. kahawae isolates initially white mycelium changes after 4-6 days to gray and eventually to dark olive brown. Similarly, Tefestewold (1995) also reported light bluish gray colored C. kahawae isolates from Kaffa and Illubabor on PDA. In the second group, isolates Ge1, Ge3 and Ge5 (from Gera), and Be1 (from Bedeno) showed dark gray (color intensity between light gray and gray) colony color in the first 15 days incubation period. Tefestewold and Mengistu (1989) found C. kahawae isolates from Hararge isolates that were grayish mycelium form (light grayish to white).
On the reverse side of culture plates, colonies of the first group revealed whitish yellow or light yellow colors in the first 4-6 incubation days and then changed into various combinations of colors. The second group manifested light gray color in the first 4-6 incubation days and then

8. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Emana BT 024
Table 6. Comparison of conidia production of C. kahawae isolates on PDA after 10 days of incubation at 25 0C.
Isolate
Place of collection
Mean of Conidia production (×10000/ml)
B2
Bedeno
260.85 a
B3
Bedeno
155.59 b
B1
Bedeno
149.58 b
H3
Habro
122.43 c
H1
Habro
114.43 cd
G3
Gera
93.24 de
G2
Gera
87.61 e
Bo1
Boke
82.59 ef
D3
Darolebu
82.20 ef
G1
Gera
80.77 ef
D2
Darolebu
69.09 efg
D1
Darolebu
59.61 fgh
Bo2
Boke
59.61 fgh
H2
Habro
54.66 gh
Bo3
Boke
39.53 h
Cv (%)
15.5
changed into various colors combination. The third group showed light orange and/or rosy brown color and then changed into various colors combinations. There was significant (P < 0.05) difference among isolates in their radial colony growth both on PDA and MEA. The result revealed that the radial colony (mycelial) growth of isolates was faster on PDA than MEA. The mean colony diameter of C. kahawae isolates were 38.86, 58.30 and 69.91 mm on PDA, and 27.83, 42.63 and 58.55mm on MEA after 7, 10, and 15 days of incubation, respectively (Table 5). Mean radial colony (mycelial) growth rate of C. kahawae isolates was varied on MEA and PDA, i.e. 4.05 and 5.35 mm /24 hr, respectively. The result was similar with that of Tefestewold and Mengistu (1989) report that they recorded 6.5 and 6.7 mm/24hr growth rates on PDA (25 ± 1 oC) for dark mycelia and grayish mycelia isolates of CBD pathogen, respectively (Tefestewold, 1995). Morphological Characteristics Size of conidia The morphological characteristics of the C. kahawae isolates were observed on PDA. The sizes and shapes of conidia were variable. The average size of conidia was 13.50 x 4.10 μm. Conidia width and length ranged as 2.12 – 4.24 μm and 10.51 – 15.78 μm, respectively. In agreement with those of Hindorf (1973) and Hindorf and Muthappa (1974) the range of conidial sizes was observed from 7 days cultures. Hindorf (1973) recorded 13.1 ± 0.6 x 3.8 ± 0.2 μm and (10.8 – 23.0) x (3.4 – 4.7) μm average and range of conidia sizes, respectively. Hindorf and Muthappa (1974) found 13.1 x 3.8 μm and (11 – 23) x 3.4 – 4.7) μm average and range of conidia sizes from Kenyan C. kahawae isolates. Sporulation Capacity / Conidia production Conidial production that was taken from 10 days old culture showed significant difference (P<0.05) among isolates .It varied in average between 39.53 x104 conidia / milliliter produced by isolate Bo3 from Boke and 260.85 x 104 conidia / milliliter produced by isolate B2 from Bedeno. Isolate B2 produced significantly (p=0.05) high amount of conidia, followed by isolates from same district (B3, B1), Habro (H3) and Habro (H1) which had produced 155.59 x 104, 149.58 x 104, 122.43 x 104 and 114x104 conidia/ml, respectively (Table 6). Tefestewold (1995) observed (12-52) x 104 conidia/ml and (684-1720) x 104 conidia/ml production from 6 CBD pathogen isolates on PDA. Means followed with the same letter are not significantly different according to DMRT at 0.05 alpha level. Isolates from Gera (Ge) used as check. Shape of conidia
The result indicated that more than 50 % of conidial shape frequency of each isolate failed under conidial shape of type 1 except isolates Be2, Ge3 and H3 (Table 7). Isolate Ge3 produced almost type 1 and 2 conidia shapes in equal proportion. Isolate Be2 produced all types of conidia shapes except type 5 and H3 produced all types of conidia shapes but both produced dominantly type 1. Conidia shape variability of C. kahawae also reported by Hindorf (1970). From this experiment, as described by Hindorf (1973) and Tefestewold (1995), the 5 types of conidia shapes were frequently observed in different proportion when detected from each isolate. But conidia shape of type 1 (cylindrical and round at both

9. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Int. J. Plant Breeding Crop Sci. 025
Table 7. Comparisons of frequencies of different kinds of conidia shapes (%) produced by C. kahawae isolates on PDA. Isolates 1 2 3 4 5
Ge1
55
32
7
6
0
Ge2
60
10
25
5
0
Ge3
49
49
1
0
1
Be1
53
16
31
0
0
Be2
40
20
33
7
0
Be3
76
8
15
1
0
Bo1
66
20
6
2
6
Bo2
66
22
10
1
1
Bo3
81
6
12
1
0
H1
50
16
31
3
0
H2
50
37
12
1
0
H3
49
28
15
4
4
DL1
51
17
16
6
0
DL2
50
25
20
2
0
DL3
58
24
14
1
1
**Conidial shapes: 1= cylindrical and round at both ends, 2= cylindrical acute at one and round at the other end, 3= clavate-round at both ends starts attenuating from one fourth of its length, 4= reniform or kidney shaped, 5= Oblong-elliptical. * Isolates from Gera used as check
ends) was dominantly observed from all isolates and accounted a proportion which ranged between 40 and 81 %.
Aggressiveness Test
Isolates were classified into 5 aggressiveness groups (0=None aggressive; ≤1=Less aggressive; ≤2= Medium aggressive; ≤3= Aggressive; ≤4 to 5= Highly aggressive). Differences were observed among the isolates in aggressiveness (Table 8) and the most aggressive isolates are Be2, Be3, Be5, Be6, H1, H2, H3, D3, D4, D5, Bo2, Bo3, and G1 isolates produced greater % NL in detached leaf test (Table 8). This indicates that more aggressive isolates able to invade and colonize host tissue rapidly more leaf damage. Mean while isolate Be1 and H5, G2 and Control, Be4, D2, Bo4 classified as medium, less and none aggressive groups respectively. The remaining ones were grouped as aggressive. Milus et al. (2009) showed that isolates having greater % NL are considered to be more aggressive than isolates having lesser % NL. Besides this study, the results of other workers show that necrotic lesion can be used as a better measure of aggressiveness.
Evaluation of some Hararghe Coffee Landraces for Resistance to CBD
Seedlings of 11 top yielding Hararghe coffee landraces (H-759/02, H-762/02, H-01/02, H-67/02, H-570/02, H- 569/02, H-662/02, H-87/02, H-05/02, H-566/02, and H- 568/02) and resistant (Code No 741) and susceptible (Code No 370) checks were inoculated. There existed highly significant (p<0.0001) differences among the selections in seedling percent infection in reaction to CBD (Table 9). Three of the tested selections and the resistant check (Code No 741), H- 05/02, H- 568/02 and H-87/02) revealed low level of mean seedling infection compared to the standard susceptible check and classified as moderately susceptible. Susceptible seedling selection of H-01/02, H-566/02, H-662/02, H- 569/02, H-759/02, H- 570/02, 762/02 and H-67/02 cotyledons initially had small, shining brownish orange spots which produced circular to irregular shaped brown pigmented lesion progressing to blight. Hypocotyls of the susceptible coffee selections formed small brown spots coalesced to form dark brown lesions causing a wilt of the upper part of the seedlings and collapse or rots with varying speed between the first 15 to 28 days. Results of this experiment confirmed that the variations were mainly due to the existence of difference in genetic make up of the selected lines in reaction to CBD. A search for resistance among 200 arabica cultivars, which had been introduced from abroad earlier in 1969, indicated that none of them were found resistant except Rume Sudan, which showed some level of resistance, although not as resistant as the local types (Bayetta, 2001; Hindorf and Arega, 2006 ).
CONCLUSIONS AND RECOMMENDATIONS
Coffee berry disease was present in all assessed districts but the incidence and severity varied from one district to other depending on environmental condition. Landraces Abadir, Shumbure, Buna gurracha and Buna qallaa are the dominant coffee landraces grown in the region.

10. Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia
Emana BT 026 Table 8. Scoring Scale of lesion size C. kahawae isolates and aggressiveness group on detached faba bean leaf under laboratory condition
Isolates
Scoring scale
Aggressiveness group
Control
0
Non- aggressive
Be1
2
Medium aggressive
Be2
5
Highly aggressive
Be3
4
Highly aggressive
Be4
0
Non- aggressive
Be5
4
Highly aggressive
Be6
4
Highly aggressive
H1
5
Highly aggressive
H2
5
Highly aggressive
H3
4
Highly aggressive
H4
3
Aggressive
H5
1
Less aggressive
D1
3
Aggressive
D2
0
None aggressive
D3
4
Highly aggressive
D4
5
Highly aggressive
D5
4
Highly aggressive
Bo1
3
Aggressive
Bo2
4
Highly aggressive
Bo3
4
Highly aggressive
Bo4
0
Non- aggressive
G1
5
Highly aggressive
G2
1
Less aggressive
G3
3
Aggressive
0=No infection; 1=1-25 % leaf area damaged; 2=26-50 % leaf area damaged;3=51-75 % leaf area damaged; 4= >75 % leaf area damaged; 5=dead leaves 100 % leaf area damaged.
Considerable variation was observed among C. kahawae isolates in their conidia production / sporulation capacity indicating differences in aggressiveness. Highly significant Variations (p<0.0001) were detected among Hararghe coffee selections in resistance / tolerance to CBD. This indicated the presence of resistance sources in Hararghe coffee germplasms that may be exploited for coffee improvement purposes. So, it is important to conserve both insitu and exsitu and use sustainably the Hararghe coffee germplasms by conducting intensive collection and selection from more diverse coffee population and evaluation for resistance to coffee berry disease where as, selections namely H- 67/02, H-762, H-570/02, H-759/02, H-569/02, H-662/02, H-566/02, H-01/02 showed narrow range of variation as compared to H-05/02, H-568/02 and H-87/02.
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Citation: Emana BT (2014). Coffee berry disease
(Colletotrichum kahawae): Status, pathogenic variability
and reactions of coffee landraces in Hararghe, Eastern
Ethiopia. International Journal of Plant Breeding and
Crop Science, 1(2): 018-027.
Copyright: © 2014. Emana BT. This is an open-access
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