Coffee berry disease (Colletotrichum kahawae): status, pathogenic variability and reactions of...

Coffee berry disease (Colletotrichum kahawae): Status, pathogenic variability and reactions of coffee landraces in Hararghe, Eastern Ethiopia

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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: [email protected], 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


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.


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 41

038`N 1294 16-21

Boke 1762-1855 8042`N 40

044`N 700 24-27

Habro 1621-1772 8042`N 40

028`N 1200 21-27

Darolebu 1658-1833 8036`N 40

019`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


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 10

6 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


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,


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.


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


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 x10

4 conidia /

milliliter produced by isolate Bo3 from Boke and 260.85 x 10

4 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 10

4, 149.58 x 10

4, 122.43 x 10

4 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


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.


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


Be4 0 Non- aggressive



H1 5 Highly aggressive



H4 3 Aggressive

H5 1 Less aggressive

D1 3 Aggressive

D2 0 None aggressive

D3 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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Accepted 05 October, 2014. 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 article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited.

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