Molecular and morphological characterization of Moniliophthora roreri isolates from cacao in Ecuador

Figures & data

Table 1. Morphological variables of M. roreri isolates observed after 15 days growth on malt extract agar at 27°C.

Variable	Category	Group
Colour^1	Cream-salmon only	1
	Intercalated dark brown and cream-salmon	2
	Cream-salmon to light brown to white	3
	Dark brown to light brown to white	4
	Dark brown to cream-salmon to white	5
	Dark brown to cream-salmon	6
	Dark brown only	7
	Light brown to white	8
	Cream-salmon to white	9
	Cream-salmon to light brown	10
	Cream-salmon to dark brown	11
	Light brown to cream-salmon	12
	Dark brown to light brown	13
	Light brown only	14
Edge	Regular	1
	Irregular	2
Texture	Powdery	1
	Plush-like	2
	Fluted	3
	Cotton-like	4
Meiospore shape	Globose	1
	Subglobose	2
	Elliptical	3

¹Colour described from centre to edge of the fungal colony.

Table 2. Percentage of isolates of M. roreri classified into each group described in Table 1.

Colour group^1,^2	Amazon	El Oro	Esmeraldas	Los Rios	Guayas	Manabi	Group
1a	14.7	21.4	30.0	0	0	0	12.2
2d	5.9	7.1	30.0	13.3	33.3	14.3	12.2
3a	5.9	7.1	20.0	0	0	7.1	6.7
4e	8.8	0	0	0	0	0	3.3
5f	2.9	0	10.0	6.7	33.3	35.7	10.0
6a	2.9	0	0	26.7	33.3	14.3	8.9
7g	11.8	0	0	6.7	0	14.3	7.8
8b	2.9	0	0	6.7	0	0	2.2
9b	5.9	0	0	6.7	0	0	3.3
10h	0	0	0	13.3	0	7.1	3.3
11b	5.9	0	0	6.7	0	0	3.3
12c	5.9	21.4	0	0	0	0	5.6
13i	14.7	14.3	0	0	0	0	7.8
14c	11.8	28.6	10.0	13.3	0	7.1	13.3

¹Colour groups with the same letter represent significant (P < 0.05) Pearson’s correlation coefficients with absolute values of 0.834 or higher.

²Values represent percentage of the 90 isolates analysed.

Fig. 1 (Colour online) Representative images of isolates belonging to the colour groups from 1 to 14 described in Table 1. The number in each figure represents the colour group from Table 1.

Fig. 2 (Colour online) Representative meiospore of M. roreri strains isolated from six regions of Ecuador.

Table 3. Parameter estimates based on the Gompertz model fitted to the growth of M. roreri isolates classified according to the colour groupings from Table 1.

Group^1	α			β			γ
1	3.746	±	0.006a,c	0.194	±	0.029a,b,c	0.290	±	0.006b,c
2	3.819	±	0.004d	0.279	±	0.017b	0.230	±	0.002e
3	3.724	±	0.004f,b	0.265	±	0.021a,b	0.305	±	0.008a,b
4	3.803	±	0.005d,e	0.199	±	0.023a,b,c	0.245	±	0.004d
5	3.792	±	0.008e	0.296	±	0.025b	0.252	±	0.006d
6	3.830	±	0.010d	0.209	±	0.021a,b,c	0.228	±	0.004e
7	3.783	±	0.009e	0.258	±	0.008b	0.258	±	0.004d
8	3.735	±	0.004a,f	0.173	±	0.015c	0.293	±	0.006b
10	3.822	±	0.023d,e	0.243	±	0.015a,b	0.239	±	0.011d,e
11	3.794	±	0.013d,e	0.232	±	0.007a,b	0.249	±	0.006d
12	3.712	±	0.002b,g	0.178	±	0.018a,c	0.322	±	0.003a
13	3.709	±	0.003 g	0.144	±	0.026c	0.320	±	0.003a
14	3.753	±	0.002c	0.174	±	0.028a,c	0.274	±	0.003c

¹ Parameters α, β and γ are estimates of the growth data fitted to a Gompertz model. Different letters represent significant differences (P < 0.05) between the parameter estimates of the colour groups.

Fig. 3 (Colour online) Growth curve of M. roreri isolates from colour groups that showed significant growth differences (P < 0.05) fitted to a Gompertz model.

Table 4. Genetic diversity of the six groups of M. roreri strains.

Region	PL^1	He^2
Amazon	37.5	0.16
El Oro	18.8	0.12
Esmeraldas	31.3	0.15
Guayas	12.5	0.11
Los Ríos	100	0.25
Manabí	93.8	0.26
Mean	48.98	0.18

¹Percentage of polymorphic loci at the 5% level.

²Expected heterozygosity estimated as unbiased Nei’s gene diversity.

Fig. 4 Representative gel photograph of PCR-RFLP of the ITS1-ITS2 regions of M. roreri using restriction enzymes. The lane numbers on the gels correspond to the sample codes in Supplementary Table 1. M indicates the DNA ladder.

Fig. 5 (Colour online) Factors 1 and 2 (F1 and F2) of the multiple discriminant analyses of ITS-RFLP patterns of M. roreri isolates grouped by colour (a) and provinces (b).

Table 5. Polymorphic nucleotides detected in the ITS1, 5.8 s, and ITS2 regions of representative isolates of M. roreri obtained in this study and compared with isolates from Ecuador previously deposited in GenBank.

Isolate^3	Polymorphic nucleotide^1,2
	111	196	204	397	446	522	579
A3.1B Amazon	G	C	A	A	-	C	-
A7.1C Amazon	G	C	A	A	-	C	-
G43E Guayas	-	C	A	-	T	T	G
M7C Manabi	-	C	A	-	T	T	G
LR22B Los Rios	-	C	A	-	T	T	G
LR22A Los Rios	-	-	-	-	T	T	G
LR24B Los Rios	-	A	G	-	T	T	G
LR30E Los Rios	G	C	A	A	-	C	-
A57E Amazon	-	-	-	-	T	T	G
JX315287.1	-	C	A	-	T	T	G
JX315288.1	-	C	A	-	T	T	G
JX315286.1	-	C	A	-	T	T	G
JX315285.1	G	C	A	A	-	T	-
AY194150.1	-	C	A	-	T	T	G

¹Numbers represent nucleotide position within the sequence.

²The absence of a nucleotide is indicated by (-).

³Accession numbers are provided for NCBI isolates.

Fig. 6 Neighbor-joining tree of the ITS1, 5.8 s and ITS2 region of the representative isolates obtained in this study and the most similar isolates from GenBank. The bootstrap values of 2000 interaction are also shown.