Variation among colletotrichum isolates associated with coffee berry disease in Ethiopia

Figures & data

Table 1. Description of sample collection areas

Region	Zone	District	Altitude(m.a.s.l)	Latitude	Longitude
Oromiya	Borena	Abaya	1520–1574	6° 09ʹ N	38° 00ʹ E
	Illubabor	Ale	1811–1947	8° 00ʹ N	35° 39ʹ E
		Bedele	1898–1983	8° 27ʹ N	36° 21ʹ E
		Chora	1533–1935	8° 19ʹ N	36° 14ʹ E
		Mettu	1583–1773	8° 18ʹ N	35° 34ʹ E
		Yayu	1386–1758	8° 09ʹ N	36° 00ʹ E
	Jimma	Gera	1939–2048	7° 39ʹ N	36° 14ʹ E
		Gomma	1786–2102	7° 49ʹ N	36° 39ʹ E
		Mana	1654–1958	7° 44ʹ N	36° 44ʹ E
	East Hararghe	Bedeno	2036–2119	9° 00ʹ N	41° 29ʹ E
		Hararzuriya	1668–1992	9° 18ʹ N	42° 07ʹ E
		Kombolcha	2127–2176	9° 24ʹ N	42° 00ʹ E
	West Wollega	Gimbi	1852–1882	9° 00ʹ N	36° 00ʹ E
		Haru	1732–1812	8° 49ʹ N	36° 00ʹ E
SNNPR^a	Gedeo	Wonago	1639–1967	6° 19ʹ N	38° 19ʹ E
		Yirgachefe	1860–2190	6° 39ʹ N	38° 29ʹ E
	Sidama	Aletawondo	1723–1821	6° 34ʹ N	38° 19ʹ E
		Aletachuko	1705–1897	6° 58ʹ N	38° 33ʹ E
		Dale	1722–1846	6° 44ʹ N	38° 19ʹ E
		Shebedino	1726–1964	6° 49ʹN	38° 29ʹ E
		Wondo Genet	1715–1822	7° 08ʹ N	38° 61ʹ E
		Wensho	1835–1956	6° 74ʹ N	38° 49ʹ E
	Kaffa	Ginbo	1500–2500	7° 19ʹ N	36° 09ʹE
	Bench magi	Sheko	950–1800	6° 49ʹ N	35° 00ʹ E

^aSNNPR, Southern Nations Nationalities and Peoples Region.

Table 2. Proportion of variation in colony color among isolates of Colletotrichum species from diseased coffee berries from Ethiopia

Colony Color	Culture media
	PDA		MEA		SDA
	Top	Reverse	Top	Reverse	Top	Reverse
Creamy white	0.00^a	0.00	14.29	0.00	5.71	0.00
Dark grey	22.86	11.43	8.57	5.71	45.71	0.00
Dark Olivia green	0.00	2.86	0.00	2.86	0.00	51.43
Dim grey	2.86	11.43	2.86	2.86	8.57	0.00
Greenish grey	0.00	17.14	0.00	45.71	0.00	17.14
Grey	31.43	5.71	48.57	2.86	25.71	8.57
Light greenish grey	0.00	20.00	0.00	11.43	0.00	0.00
Light grey	22.86	11.43	11.43	0.00	2.86	2.86
Light yellow	0.00	0.00	0.00	11.43	0.00	8.57
White	20.00	20.00	14.29	17.14	11.43	11.43

^aNumbers indicate the proportion of isolates showing a particular colony color.

PDA, Potato Dextrose agar; MEA, Malt Extract Agar; SDA, Sabouraud Dextrose Agar.

Figure 1. Colony characteristics of Colletotrichum species isolated from diseased coffee berry where (a) CBD007, (b) CBD33, (c) CBD014, (d) CBD119, (e) CBD005 and (f) CBD099 (PDA, Potato Dextrose agar; MEA, Malt Extract Agar; SDA, Sabouraud Dextrose Agar)

Table 3. Comparisons among Colletotrichum isolate on colony density, sector and acervuli

Isolate	Colony density^a			Sectoring or saltation^b			Acervuli^c
	PDA	MEA	SDA	PDA	MEA	SDA	PDA	MEA	SDA
CBD005	+	++	+	-	-	-	+	+	+
CBD007	+	+	+	+	+	+	-	-	-
CBD011	+	+	+	+	+	+	-	-	-
CBD013	+	+	+	-	-	-	-	-	-
CBD014	+++	+++	++	-	-	-	+	+	-
CBD021	+	++	+	+	-	-	-	-	-
CBD027	+++	++	+	-	-	-	+	-	+
CBD033	+	+	+	+	+	+	-	-	-
CBD039	+	+	++	+	+	-	-	-	-
CBD042	+	+	+	-	-	-	+	+	+
CBD043	+	+	+	-	+	-	-	-	-
CBD051	+	+	+	-	+	-	-	-	-
CBD059	++	+	+	-	-	-	-	-	-
CBD068	+	+	+	+	+	+	-	-	-
CBD094	++	+++	++	-	-	-	-	-	-
CBD099	+++	+++	++	-	-	-	+	+	+
CBD103	+	+	+	+	+	+	-	-	-
CBD110	+	+	+	+	-	-	-	-	-
CBD119	++	++	+	-	-	-	+	+	-
CBD121	++	+	+	+	+	-	-	-	-
CBD135	++	++	+	-	+	+	-	-	-
CBD140	++	+	+	+	+	-	-	-	-
CBD145	++	++	++	-	-	-	-	-	-
CBD153	+	+	+	+	+	-	-	-	-
CBD154	+	++	+	-	-	-	+	+	+
CBD156	++	+	+	+	-	-	-	-	-
CBD163	+	+	+	-	-	-	-	+	-
CBD164	++	+	+	+	+	-	-	-	-
CBD166	+	+	+	+	+	-	-	-	-
CBD182	++	++	+	-	-	-	+	-	-
CBD184	+	+	+	+	+	-	-	-	-
CBD186	++	++	+	-	-	-	+	+	-
CBD188	+	+	+	-	-	-	-	-	-
CBD196	+	+	+	-	-	-	-	-	-
CBD211	+	+	+	+	+	+	-	-	-

^aColony density: + = Dense, ++ = Irregular (scarce), +++ = Very scarce.

^bSalitaion: + = Present, - = Absent

^cConidia mass (acervulus) = + = Present, - = Absent

PDA, Potato Dextrose agar; MEA, Malt Extract Agar; SDA, Sabouraud Dextrose Agar.

Table 4. Radial mycelia growth of Colletotrichum species on PDA, MEA and SDA

Isolate	Culture media			Mean
	PDA	MEA	SDA
CBD01	4.41 ± 0.20^no	3.84 ± 0.02^r	5.16 ± 0.01^i-n	4.47 ± 0.11^pq
CBD05	6.88 ± 0.06^d	6.64 ± 0.17^e	7.22 ± 0.06^c	6.92 ± 0.04^e
CBD07	5.31 ± 0.11^g-i	4.22 ± 0.10^pq	4.88 ± 0.20^l-p	4.81 ± 0.08^no
CBD11	4.47 ± 0.24^m-o	5.63 ± 0.18^gh	6.15 ± 0.24^e-g	5.41 ± 0.22^ij
CBD13	5.13 ± 0.56^h-j	4.87 ± 0.21^j-m	4.83 ± 0.10^m-p	4.95 ± 0.22^l-n
CBD14	8.73 ± 0.18^a	8.99 ± 0.10^a	8.35 ± 0.12^b	8.69 ± 0.08^a
CBD21	4.78 ± 0.22^k-m	4.95 ± 0.20^i-k	4.52 ± 0.18^o^−q	4.75 ± 0.03^no
CBD27	8.72 ± 0.10^a	8.28 ± 0.25^b	7.03 ± 0.15^c	8.01 ± 0.13^b
CBD42	4.43 ± 0.30^m-o	4.55 ± 0.17 ^k-p	4.21 ± 0.05^q	4.39 ± 0.13^q
CBD43	5.45 ± 0.05^gh	4.70 ± 0.04^k-o	5.20 ± 0.30^i-n	5.12 ± 0.11^kl
CBD33	5.63 ± 0.17^g	5.86 ± 0.33^fg	6.36 ± 0.08^d-f	5.95 ± 0.17^g
CBD39	5.19 ± 0.27^h-j	4.58 ± 0.08^k-p	5.10 ± 0.16 ^j-o	4.95 ± 0.18^l-n
CBD51	4.53 ± 0.09^m-o	4.03 ± 0.29^qr	4.41 ± 0.63^pq	4.33 ± 0.28^q
CBD59	4.56 ± 0.07 ^l-o	4.75 ± 0.04 ^k-o	5.42 ± 0.07 ^h-m	4.91 ± 0.01 ^l-o
CBD68	4.59 ± 0.22^l-o	4.49 ± 0.43^l-p	4.92 ± 0.04^k-p	4.67 ± 0.16^o^p
CBD94	5.59 ± 0.02^g	5.26 ± 0.05^h-j	5.67 ± 0.12^g-j	5.51 ± 0.05^hi
CBD99	6.44 ± 0.05^e	6.69 ± 0.13^e	6.79 ± 0.11^cd	6.64 ± 0.06^f
CBD103	5.06 ± 0.10^i-k	4.62 ± 0.10^k-p	5.56 ± 0.07^g-j	5.08 ± 0.08^k-m
CBD110	3.90 ± 0.18^p	3.26 ± 0.01^s	4.76 ± 0.36^n-q	3.97 ± 0.12^r
CBD119	7.67 ± 0.01^b	7.56 ± 0.17^c	6.91 ± 0.18^cd	7.38 ± 014^c
CBD121	5.57 ± 0.12^g	5.28 ± 0.11^hi	6.15 ± 0.19^e-g	5.66 ± 0.03^h
CBD135	5.57 ± 0.25^g	5.51 ± 0.36^gh	5.61 ± 1.40^g-j	5.56 ± 0.27^hi
CBD140	5.28 ± 0.01^g-i	4.83 ± 0.12^k-n	5.52 ± 0.34^h-k	5.21 ± 0.14^jk
CBD145	7.45 ± 0.05^bc	6.94 ± 0.26^de	5.82 ± 0.10^f-h	6.74 ± 0.04^ef
CBD153	5.32 ± 0.10^g-i	4.91 ± 0.07^i-l	5.48 ± 0.16^h-l	5.24 ± 0.03^jk
CBD154	7.27 ± 0.05^c	7.31 ± 0.16^cd	6.86 ± 0.40^cd	7.15 ± 0.19^d
CBD156	4.74 ± 0.21^k-n	4.43 ± 0.10^n-q	5.18 ± 0.15^i-n	4.78 ± 0.09^no
CBD166	4.48 ± 0.13^m-o	4.27 ± 0.34^pq	5.25 ± 0.13^h-n	4.67 ± 0.08^o^p
CBD163	6.06 ± 0.08^f	7.17 ± 0.08^d	7.17 ± 0.16^c	6.80 ± 0.06^ef
CBD164	4.91 ± 0.16 ^j-l	4.36 ± 0.01^o^−q	5.27 ± 0.10^h-n	4.84 ± 0.05^m-o
CBD196	4.76 ± 0.2^k-n	4.48 ± 0.58^m-p	5.47 ± 0.16^h-l	4.90 ± 0.29^l-o
CBD182	8.42 ± 0.15^a	8.26 ± 0.28^b	8.95 ± 0.16^a	8.54 ± 0.02^a
CBD184	4.27 ± 0.26^o	3.46 ± 0.16^s	4.20 ± 0.06^q	3.98 ± 0.13^r
CBD186	5.17 ± 0.26^h-j	5.49 ± 0.27^gh	5.77 ± 0.29^g-i	5.48 ± 0.08^hi
CBD188	5.33 ± 0.22^g-i	6.10 ± 0.13^f	6.44 ± 0.05^de	5.96 ± 0.05^g
Mean	5.60	5.44	5.79	5.61
SE	0.22	0.24	0.19	0.21
CV(%)	3.43	4.05	5.49	2.44

*Values (mean of three replications) followed with the same latter(s) are not statistically different at p < 0.05

PDA, Potato Dextrose agar; MEA, Malt Extract Agar; SDA, Sabouraud Dextrose Agar.

Table 5. Variation among isolates of Colletotrichum species in sporulation capacity

Isolate	Conidia production (×10,000/ml)			Mean
	PDA	MEA	SDA
CBD05	216.00^h	256.00^g	220.00^e-h	230.67^h
CBD07	204.00^hi	196.00^hi	232.00^d-g	210.67^i
CBD11	93.33^n-p	110.67^o^−r	209.33^f-i	137.78^mn
CBD13	100.00^n-p	164.00^j-l	212.00^f-i	158.67^k
CBD14	1058.67^b	1006.67^a	36.00^p	700.44^b
CBD21	98.67^n-p	107.33^o^−s	270.67^c	158.89^k
CBD27	666.67^c	308.00^f	18.67^p	331.11^e
CBD33	129.33^l-n	76.00^s-v	198.67^h-j	134.67^mn
CBD39	73.33^pq	118.67^n-p	222.67^e-h	138.22^mn
CBD42	1268.00^a	1008.00^a	129.33^m	801.78^a
CBD43	118.67^l-o	128.67^m-o	208.00^g-i	151.78^k-m
CBD51	24.00^r	65.33^t-v	170.67^kl	86.67^r
CBD59	84.00^o^−q	85.33^q-u	100.00^n	89.78^qr
CBD68	84.00^o-q	81.67^q-u	152.00^lm	105.89^o-q
CBD94	128.00^l-n	113.33^n-q	128.00^m	123.11^no
CBD99	426.67^e	444.00^d	320.00^b	396.89^d
CBD103	88.00^op	142.00^l-n	244.00^de	158.00^kl
CBD110	104.00^n-p	90.67^p-t	136.00^m	110.22^°op
CBD119	349.33^f	372.00^e	28.00^p	249.78^g
CBD121	72.00^pq	154.67^k-m	254.67^cd	160.44^k
CBD135	30.67^r	28.67^w	132.00^m	63.78^s
CBD140	49.33^qr	48.00^vw	180.00 ^jk	92.44^qr
CBD145	117.33^m-o	80.00^r-u	93.33^n	96.89^p-r
CBD153	177.33^i-k	152.00^k-m	252.00^cd	193.78 ^j
CBD154	76.00^pq	612.00^c	754.67^a	480.89^c
CBD156	96.00^n-p	54.67^u-w	88.00^no	79.56^rs
CBD163	186.67^h-j	640.00^b	66.67^o	297.78^f
CBD164	149.33^k-m	96.00^p-t	200.00^h-j	148.44^k-m
CBD166	93.33^n-p	77.33^s-v	168.00^kl	112.89^op
CBD182	466.67^d	186.67^h-j	25.33^p	226.22^hi
CBD184	154.67 ^j-l	132.67^m-o	186.67^i-k	158.00^kl
CBD186	314.67^g	212.00^h	21.33^p	182.67^j
CBD188	120.00 ^l-o	58.67^uv	170.67^kl	116.44^o
CBD196	118.67 ^l-o	92.00^p-t	210.67^f-i	140.44^l-n
CBD211	152.00^k-m	176.00^i-k	236.00^d-f	188.00^j
Mean	219.69	219.30	179.31	206.10
SE	45.99	41.26	21.52	27.61
CV(%)	8.73	7.66	8.21	4.76

*Values (mean of three replications) followed with the same latter(s) are not statistically different at p < 0.05

PDA, Potato Dextrose agar; MEA, Malt Extract Agar; SDA, Sabouraud Dextrose Agar.

Table 6. Variation of conidia size of Colletotrichum isolates on potato dextrose agar medium

Isolate	Conidia size (μm)
	Length	Width
CBD005	12.97 ± 0.05^o	3.57 ± 0.09^r
CBD007	14.53 ± 0.21^j-l	4.64 ± 0.05^f-i
CBD011	15.03 ± 0.13^hi	4.70 ± 0.05^d-h
CBD013	15.25 ± 0.17^gh	4.60 ± 0.03^g-j
CBD014	16.03 ± 0.21^bc	3.97 ± 0.02^no
CBD021	15.44 ± 0.13^fg	4.63 ± 0.04^f-j
CBD027	15.97 ± 0.05^b-d	3.97 ± 0.09^no
CBD033	15.55 ± 0.21^d-g	4.44 ± 0.10 ^j-l
CBD039	15.50 ± 0.22^e-g	4.33 ± 0.04^l
CBD042	12.92 ± 0.08^n	5.10 ± 0.07^a
CBD043	15.69 ± 0.17^c-f	4.46 ± 0.07^i-l
CBD051	15.53 ± 0.09^e-g	4.71 ± 0.04^d-h
CBD059	15.89 ± 0.21^b-e	4.67 ± 0.08^e-h
CBD068	14.78 ± 0.12^ij	4.75 ± 0.07^d-g
CBD094	14.08 ± 0.16^lm	4.63 ± 0.12^f-j
CBD099	12.86 ± 0.09^n	3.75 ± 0.08^pq
CBD103	15.64 ± 0.20^c-g	4.58 ± 0.08^g-j
CBD110	14.42 ± 0.16 ^j-m	4.29 ± 0.04^lm
CBD119	14.17 ± 029^lm	4.04 ± 0.07^no
CBD121	14.33 ± 0.08^k-m	4.71 ± 0.15^d-h
CBD135	14.17 ± 0.65^lm	4.45 ± 0.05^j-l
CBD140	14.28 ± 0.21^k-m	4.14 ± 0.05^mn
CBD145	14.11 ± 0.09^lm	3.96 ± 0.17^o
CBD153	16.14 ± 0.25^b	4.93 ± 0.02^a-c
CBD154	12.72 ± 0.51^n	3.89 ± 0.03^o^p
CBD156	16.02 ± 0.11^bc	4.83 ± 0.04^b-e
CBD163	14.28 ± 0.13^k-m	4.39 ± 0.10^kl
CBD164	17.38 ± 0.08^a	4.99 ± 0.02^ab
CBD166	17.71 ± 0.34^a	4.88 ± 0.04^b-d
CBD182	13.97 ± 0.43^m	3.61 ± 0.05^qr
CBD184	16.06 ± 0.10^bc	4.46 ± 0.29^i-l
CBD186	12.33 ± 0.00^o	3.63 ± 0.00^qr
CBD188	14.67 ± 0.41^i-k	4.79 ± 0.16^c-f
CBD196	14.42 ± 0.25 ^j-m	4.77 ± 0.10^c-g
CBD211	14.78 ± 0.13^ij	4.52 ± 0.19^h-k
SE	0.21	0.07
CV(%)	1.59	2.20

*Values are mean of three replications Mean in the same column followed with the same latter are not statistically different at p < 0.05 DMRT.

Figure 2. Pathogenicity of isolates of Colletotrichum species on detached berry test on coffee cultivar 370 and (a-c) Disease symptom development on green berries inoculated with isolates of C. kahawae (CBD068) (d) Isolates of C. gloeosporioides (CBD182), (e) Isolates of C. acutatum (CBD154) and (f) Control

Figure 3. Pathogenicity of isolates of Colletotrichum species on seedling inoculation test on 370 coffee cultivar (a-c) Isolates of C. kahawae; (d) Isolates of C. gloeosporioides; (e) Isolates of C. acutatum and (f) Control

Table 7. Pathogenic variability among isolates of Colletotrichum species

Isolate	Zone	Identified as	DBT^a	Hypocotyls test
				Reaction	Disease index
CBD005	Gedeo	C. acutatum	-	-	0.0
CBD007	Gedeo	C. kahawae	+	+	100
CBD011	Gedeo	C. kahawae	+	+	93.8
CBD013	Gedeo	C. kahawae	+	+	93.8
CBD014	Gedeo	C. gloeosporioides	-	-	0.0
CBD021	Borena	C. kahawae	+	+	90.6
CBD027	Sidama	C. gloeosporioides	-	-	0.0
CBD033	Sidama	C. kahawae	+	+	96.8
CBD039	Sidama	C. kahawae	+	+	90.6
CBD042	Sidama	C. acutatum	-	-	0.0
CBD043	Sidama	C. kahawae	+	+	100
CBD051	Sidama	C. kahawae	+	+	100
CBD059	Bench Maji	C. kahawae	+	+	100
CBD068	Jimma	C. kahawae	+	+	100
CBD094	Jimma	C. kahawae	+	+	93.6
CBD099	Jimma	C. acutatum	-	-	0.0
CBD103	Jimma	C. kahawae	+	+	84.4
CBD110	Jimma	C. kahawae	+	+	84.4
CBD119	Jimma	C. gloeosporioides	-	-	0.0
CBD121	Illubabor	C. kahawae	+	+	93.8
CBD135	Illubabor	C. kahawae	+	+	84.4
CBD140	Illubabor	C. kahawae	+	+	87.5
CBD145	Illubabor	C. kahawae	+	+	89.3
CBD153	West Wollega	C. kahawae	+	+	87.5
CBD154	West Wollega	C. acutatum	-	-	0.0
CBD156	West Wollega	C. kahawae	+	+	75.0
CBD163	West Wollega	C. gloeosporioides	-	-	0.0
CBD164	West Wollega	C. kahawae	+	+	92.9
CBD166	West Wollega	C. kahawae	+	+	79.2
CBD182	East Hararghe	C. gloeosporioides	-	-	0.0
CBD184	East Hararghe	C. kahawae	+	+	100
CBD186	East Hararghe	C. acutatum	-	-	0.0
CBD188	East Hararghe	C. kahawae	+	+	100
CBD196	East Hararghe	C. kahawae	+	+	98.9
CBD211	Kaffa	C. kahawae	+	+	93.8

^aDBT = detached berry test; + = Pathogenic— = Not pathogenic.

Figure 4. Dendrogram showing relationships among Colletotrichum isolates associated with diseased coffee berries based on cultural, morphological and pathological characteristics