Variability of Colletotrichum Kahawae in Relation to Other Colletotrichum Species from Tropical Perennial Crops and the Development of Diagnostic Techniques

J. Phytopathology 156, 274–280 (2008) doi: 10.1111/j.1439-0434.2007.01354.x 2008 CAB International Journal compilation 2008 Blackwell Verlag, Berlin

CABI Europe-UK, Egham, Surrey, UK

Variability of Colletotrichum kahawae in Relation to Other Colletotrichum Species from Tropical Perennial Crops and the Development of Diagnostic Techniques

P. D.Bridge 1, J. M.Waller 2, D.Davies 2 and A.G.Buddie 2 AuthorsÕ addresses:1British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge, CB3 0ET, UK; 2CABI Europe-UK, Bakeham Lane, Egham, Surrey, TW20 9TY, UK (correspondence to J. M. Waller. E-mail: [email protected]) Received January 24, 2007; accepted August 3, 2007

Keywords: coﬀee, coﬀee berry disease, Colletotrichum gloeosporioides, molecular analysis, vegetative compatibility grouping

Abstract [teleomorph: Glomerella cingulata (Stonem.) Spauld & Twenty-six isolates of Colletotrichum kahawae, the cau- Schrenk], as evidenced from the internal transcribed sal agent of coffee berry disease, from coffee in Africa, spacer sequence data (Sreenivasaprasad et al., 1993), and 25 isolates, mostly of Colletotrichum gloeosporio- and presumably evolved from it in the recent past ides, from coffee and other tropical perennial crops, (Waller and Bridge, 2000). The pathogen was formerly were examined for the ability to metabolize citrate and referred to as a form of Colletotrichum coffeanum tartrate and their molecular genetic variability was Noack, a name applied to Colletotrichum isolated from assessed using restriction fragment length polymor- coffee in Brazil, where CBD does not exist, and this phisms (RFLP) and variable number tandem repeats name is probably synonymous with C. gloeosporioides. (VNTR). Twenty-four isolates of C. kahawae were The distinctiveness of the CBD pathogen in terms of also assessed using amplified fragment length polymor- colony morphology, physiological properties and ecol- phisms (AFLP). Vegetative compatibility within a ogy enabled it to be classified as a distinct species and collection of nine isolates, including two of to be clearly separated from other C. gloeosporioides C. gloeosporioides was also assessed. All isolates of and Colletotrichum acutatum, which are also members C. kahawae from across Africa failed to metabolize of the coffee mycobiota (Waller et al., 1993). CBD citrate or tartrate, but all other isolates metabolized one remains restricted to the African continent, but it or both. Colletotrichum kahawae isolates also showed poses a major threat to the Arabica coffee industries minimal variability using the molecular techniques with of other tropical countries; hence, the accurate and two isolates from Cameroon showing slightly different timely identification of the pathogen is of particular banding patterns in RFLP analysis. All other isolates concern to those involved in the coffee industry and had variable VNTR and RFLP banding patterns. AFLP the maintenance of plant health. Because of the close analysis failed to detect variability within 12 isolates relationship of the CBD pathogen to C. gloeosporioides from Kenya, but did detect differences between isolates and the lack of distinctive microscopic characters, from other countries. Five isolates from Kenya were there is a need to develop simple diagnostic techniques vegetatively compatible but differed from two from for the pathogen. The work described here uses bio- Cameroon and from two C. gloeosporioides isolates. chemical and molecular biological techniques to inves- Results demonstrate some geographic variability within tigate variability within C. kahawae and between it C. kahawae isolates, although this is small, probably and other closely related species in order to develop due to the relatively young age of C. kahawae popula- simple diagnostic techniques for the CBD pathogen. tions. The biochemical and molecular techniques used Despite the recent evolution of C. kahawae, patho- showed clear differences from other Colletotrichum iso- genic variability has been found in the Kenyan popula- lates, and can be used to distinguish the species. Lack of tion (Omondi et al., 2000), and strains resistant to citrate and tartrate metabolism provides a readily appli- fungicides had soon developed when these chemicals cable diagnostic method. were used for control (Ramos and Kamidid, 1982; MwangÕombe, 1994). Small differences between populations from East and West Africa have also been Introduction detected using vegetative compatibility techniques The fungal pathogen causing coffee berry disease (Bella-Manga et al., 2001; Varzea et al., 2002). Further (CBD), Colletotrichum kahawae Waller & Bridge, is knowledge of this variability is required before reliable closely related to Colletotrichum gloeosporioides Penz. diagnostic tests can be developed.

www.blackwell-synergy.com Variability of Colletotrichum kahawae 275

A collection of isolates of the pathogen from Kenya DNA solutions were stored at >1 mg⁄ml in Tris-EDTA and elsewhere, in addition to other Colletotrichum iso- (TE) buffer at )20C. DNA solutions (containing lates from coffee and other perennial crops maintained approximately 5 lg) were digested with 100 U HaeIII at CAB International UK Centre (Egham, UK) was (Gibco BRL, Paisley, UK) in the manufacturerÕs examined using molecular biological techniques and restriction buffer at 37C overnight. DNA fragments enzyme function to determine the levels of variability at were separated by electrophoresis at 5 V⁄cm in 1% (w⁄v) the sub-specific and population levels. In addition, SeaKem LE agarose (FMC Products, Rockland, ME, while examining the variability of the populations of USA) in Tris⁄acetate EDTA buffer (TAE; Sambrook the pathogen from Africa, the opportunity was taken to et al., 1989). DNA fragments were visualized under UV compare these with non-pathogenic isolates of Colleto- light after staining with ethidium bromide. Gel images trichum from coffee and from other perennial crops. were captured using the GDS5000, Paisley, UK gel doc- umentation system (UVP Ltd., Cambridge, UK) and Materials and Methods stored as tiff files on disk for later analysis. Source of material Colletotrichum isolates used in the study are listed in Amplified fragment length polymorphism (AFLP) analysis Table 1, together with information on host, location DNA solutions obtained as for the RFLP analysis, were and pathogenicity (where known). Some groups of cul- used to produce AFLP fingerprints following the tures were collected specifically to investigate their vari- method of Mueller et al. (1996) with some modifica- ability, and include numbers 17–20 and 57–62, tions. Restriction–ligation (using 1 lg of DNA in the showing a range of pathogenicity (Dr C. Omondi, reaction) and precipitation of the product was carried Coffee Research Station, Kenya), numbers 66–73 from out as published. Minor alterations were made to the oil spot lesions on coffee in Colombia (Dr F. Gil, PCR amplification. Three primer pairs (B, D and E; Cenecafe, Colombia) and numbers 23–32 from a study Table 2) with 2-bp extensions at the 3Õ end were used to of isolates from anthracnose lesions from different compare variation within different groups of isolates. perennial crops in the mixed perennial crop gardens of The adapted Pst1 fragments were diluted 10-fold, and Sri Lanka, where coffee grows in close juxtaposition 2.5 ll of this dilution was subjected to PCR under the with other crops (DfID Crop Protection Research Pro- following conditions: 15 pmol primer, 200 lm dNTP ject R 5586). The isolates were maintained during the mix, 0.625 units Tth enzyme, 10· Tth buffer, sterile work on potato carrot agar (PCA; Smith and Onions, HPLC-grade water to a volume of 25 ll and using a 1994) and preserved by lyophilization. Cultures were profile of 33 cycles at 94, 60 and 72C for 1, 1 and derived from single spores on receipt according to the 2.5 min, respectively. The DNA fragments were sepa- methods given in Waller et al. (1993). rated by electrophoresis at 100 V in 2% (w⁄v) agarose in Tris⁄borate⁄EDTA buffer (TBE; Sambrook et al., 1989). Citrate and tartrate metabolism The DNA fragments were visualized as for RFLP. The ability of the isolates to utilize sodium citrate or ammonium tartrate as a sole source of carbon was Variable number tandem repeats (VNTR)–PCR tested on agar plates, in a variation of the method Cultures were grown for 72 h in 500 ll of GYM med- described by Waller et al. (1993). Medium B (Lynch ium in micro-centrifuge tubes. DNA was extracted by et al., 1981) with 1.2% (w⁄v) agar (Oxoid no.3) was the rapid extraction procedure described by Cenis supplemented with 1% w⁄v citric acid or ammonium (1992). PCR reactions were set up with the single tartrate and 0.005% (w⁄v) bromocresol purple. The pH primers RY and MR (Table 2) according to Bridge of the media were adjusted to 4.5 with NaOH prior to et al. (1997), and the PCR products were separated by sterilization at 105C for 20 min. Utilization was electrophoresis at 5 V⁄cm in 3% NuSieve agarose assessed as growth and a subsequent rise in the pH of (FMC Products) in TAE buffer. The DNA fragments the medium sufficient to give a dark blue to purple col- were visualized as for RFLP. our from the bromocresol pH indicator. Vegetative compatibility groups (VCG) Restriction fragment length polymorphism (RFLP) analysis Nine strains isolated from coffee, seven of which Mycelial plugs from PCA cultures were sliced finely and caused CBD, were selected to test for vegetative com- inoculated into 10 ml of liquid glucose yeast medium patibility. Chlorate-resistant nitrate non-utilizing (GYM) (Mugnai et al., 1989) to give starter cultures. mutants were generated using the procedure described Starter cultures were incubated at 27C in an orbital by Brooker et al. (1991) with a few modifications. The shaking incubator for 72 h and then transferred into cultures were sub-cultured onto tap water agar, held 250 ml conical flasks containing a further 60 ml of for 4 days at 25C and then transferred to potato dex- GYM. These flasks were incubated for a further 72 h trose agar or a minimal medium containing 1.5% when mycelial growth was harvested by vacuum filtra- KClO3. Fast growing sectors, which appeared from 1 tion. The resulting mycelial mats were washed with ster- to 3 weeks later, were sub-cultured onto minimal med- ile distilled water and lyophilized. DNA was extracted ium supplemented with 0.3% (w⁄v) NaNO3 (Brooker from lyophilized mycelium according to the cetrimide et al., 1991). Those that grew as sparse colonies were procedure given in Paterson and Bridge (1994). The final considered nit mutants, and these colonies were 276 Bridge et al.

Table 1 Details of Colletotrichum isolates and results of VNTR, MtDNA and carbohydrate utilization tests

Our IMI MtDNA Number number VNTR MR VNTR RY RFLP Citrate Tartrate Host Location Comments

1 170660 I I I - - Coﬀea arabica Kenya CBD Colletotrichum kahawae 2 190857 I I I - - Co. arabica Ethiopia CBD C. kahawae 3 299393 I I Ia - - Co. arabica Cameroon CBD C. kahawae 5 319418 I I I - - Co. arabica Kenya CBD C. kahawae ÔtypeÕ isolate 8 337195 I I I - - Co. arabica Malawi CBD C. kahawae 9 344766 I I I - - Co. arabica Kenya CBD C. kahawae 10 348840 I I I - - Co. arabica Malawi CBD C. kahawae 12 356654 I I I - - Co. arabica Kenya CBD C. kahawae (yellow isolate ex CRS Ruiru) 13 357057 I I I - - Co. arabica Malawi CBD C. kahawae 15 361501 I I Ia - - Co. arabica Cameroon CBD C. kahawae 17 363577 I I I - - Co. arabica Kenya CBD C. kahawae 18 363578 I I I - - Co. arabica Kenya CBD C. kahawae 19 363579 I I I - - Co. arabica Kenya CBD C. kahawae 20 363580 I I I - - Co. arabica Kenya CBD C. kahawae 52 300964 I I I - - Co. arabica Zimbabwe CBD C. kahawae 53 301220 I I I - - Co. arabica Malawi CBD C. kahawae 54 311655 I I I - - Co. arabica Tanzania CBD C. kahawae 55 338730 I I I - - Co. arabica Zambia CBD C. kahawae 56 334963a I I I - - Co. arabica Malawi CBD C. kahawae 57 363576 I I I - - Co. arabica Kenya CBD C. kahawae 58 364303 I I I - - Co. arabica Kenya CBD C. kahawae 59 364304 I I I - - Co. arabica Kenya CBD C. kahawae 60 364305 I I I - - Co. arabica Kenya CBD C. kahawae 61 364306 I I I - - Co. arabica Kenya CBD C. kahawae 62 364307 I I I - - Co. arabica Kenya CBD C. kahawae 65 361500 I I I - - Co. arabica Burundi CBD C. kahawae 6 319423 X VIII XI + + Co. arabica Kenya Colletotrichum acutatum 16 361502 IV IV II + + Co. arabica Cameroon Ex-berry Colletotrichum gloeosporioides 7 325945 III III II + + Co. arabica Malawi Ex-berry -ve pathogenicity C. gloeosporioides 11 356 457 III III II + + Co. arabica Colombia Ex-leaf C. gloeosporioides 4 303106 VII VI II - + Co. arabica Colombia Ex-berry -ve pathogenicity C. gloeosporioides 23 W4055e IX VI III + + Piper nigrum Sri Lanka Leaf lesion C. gloeosporioides 24 W4056a IX VI III + + Co. arabica Sri Lanka Leaf lesion C. gloeosporioides 25 W4056e IX VI IV - + Co. arabica Sri Lanka Leaf lesion C. gloeosporioides 30 W4065c IX VI V + + Eugenia aromatica Sri Lanka Leaf lesion C. gloeosporioides 22 W4055a V V IV + + P. nigrum Sri Lanka Leaf lesion C. gloeosporioides 26 W4057f VI VI VI + + Myristcum fragrans Sri Lanka Leaf lesion C. gloeosporioides 32 W4065f VI VI VI + + E. aromatica Sri Lanka Leaf lesion C. gloeosporioides 27 W4061b VIII VI VII + + Mangifera indica Sri Lanka Shoot blight C. gloeosporioides 31 W4065d VIII VII nt - + E. aromatica Sri Lanka Leaf lesion C. gloeosporioides 50 226802 nt nt IX - + Citrus sinensis Belize Premature fruit drop 63 321881 XI IX VII - + M. indica Trinidad Anthracnose C. gloeosporioides 64 334134 XII X VIII + - Dioscorea alata Barbados Anthracnose C. gloeosporioides 66 nt nt X - + Co. arabica Colombia Oil spot leaf C. gloeosporioides 67 nt nt X - + Co. arabica Colombia Oil spot leaf C. gloeosporioides 68 nt nt X - + Co. arabica Colombia Oil spot leaf C. gloeosporioides 69 nt nt X - + Co. arabica Colombia Oil spot leaf C. gloeosporioides 70 nt nt XI - + (o) Co. arabica Colombia Oil spot fruit C. gloeosporioides 71 nt nt XI - + (o) Co. arabica Colombia Oil spot fruit C. gloeosporioides 72 nt nt XI - + (o) Co. arabica Colombia Oil spot fruit C. gloeosporioides 73 nt nt XI - + (o) Co. arabica Colombia Oil spot fruit C. gloeosporioides

Each Roman numeral describes a particular banding pattern for that primer or method. VNTR, variable number tandem repeats; IMI, authentic culture collection accession; MR and RY are primers; RFLP, restriction fragment length polymorphism; CBD, coffee berry disease; (o), production of orange pigment in culture; nt, not tested. maintained on a chlorate medium from which they tation every 3 or 4 days over a 3-week period. A line had been selected. Mutant phenotypes were identified of dense aerial growth at the junction of the two colo- using the five media containing different nitrogen nies was classed as a compatible reaction. sources (Brooker et al., 1991). To determine if the isolates used in this study belonged to the same VCG, up Results to six nit mutant from each isolate (two from each Table 1 shows details of isolates and the results from phenotypic class where possible) were paired in all the RFLP analysis of MtDNA, VNTR–PCR and combinations. The plates were scored for complemen- carbohydrate utilization tests. Variability of Colletotrichum kahawae 277

Table 2 Table 3 Oligonucleotide primers used in this study Kenyan isolate comparison

Primer name Primer sequence AFLP VNTR primer MR 5Õ-GAGGGTGGCGGTTCT-3Õ Our IMI RY 5Õ-CAGCAGCAGCAGCAG-3Õ number number MR RYMtDNARFLP B D E Citrate Tartrate AFLP-B 5Õ-GACTGCGTACATGCAGGA-3Õ AFLP-D 5Õ-GACTGCGTACATGCAGAC-3Õ 5 319418 I I I I I I - - AFLP-E 5Õ-GACTGCGTACATGCAGAG-3Õ 12 356654 I I I I I I - - 17 363577 I I I I I I - - 18 363578 I I I I I I - - 19 363579 I I I I I I - - 20 363580 I I I I I I - - 57 363576 I I I I I I - - 58 364303 I I I I I I - - 59 364304 I I I I I I - - 60 364305 I I I I I I - - 61 364306 I I I I I I - - 62 364307 I I I I I I - -

IMI, authentic culture collection accession; VNTR, variable number tandem repeats; MR and RY are primers; RFLP, restriction fragment length polymorphism; AFLP, ampliﬁed fragment length polymorphism.

AFLP analysis The heterogeneity of groups within the C. kahawae isolates was assessed from the banding patterns obtained from primers pairs B, D and E. Twelve isolates of C. kahawae from Kenya showed the same con- sistent banding pattern with all three primers (Table 3). Another group of isolates from elsewhere in Africa were tested with primers B and E (Table 4). Fig. 1 Restriction fragment length polymorphism patterns for repre- These gave similar patterns except for an isolate from sentative Colletotrichum isolates. Isolate 26, pattern VI; 25, pattern Malawi (no. 10) that gave a slightly different pattern IV; 24, pattern III; 4, pattern II; 15-5, pattern I; difference between with both primers (1 band absent), and isolates 15 and isolate 15 and pattern I shown by arrow 3 from Cameroon that gave a different pattern with primer E (Fig. 2).

Citrate and tartrate metabolism VNTR–PCR analysis None of the 26 isolates of C. kahawae from CBD were PCR amplifications with primers RY and MR gave able to grow on citrate or tartrate sufficiently to pro- patterns consisting between 2 and 10 bands. Twelve duce any colour change. All other isolates used in this different MR and 10 RY band patterns were obtained study grew and changed the indicator on either one or from 42 isolates (Table 1). All isolates received as or both substrates. Isolates from oil spot fruit lesions presumed to be C. kahawae gave a single band pattern from Colombia grew on the tartrate medium, where for each primer, and these patterns were not obtained they produced an orange pigmentation. from any other isolates. In contrast, the other patterns were obtained from small groups of, or single, isolates. RFLP analysis Eleven different banding patterns were obtained from Vegetative compatibility groups the 51 isolates used in the study (Table 1; Fig. 1). The The number of nit mutants generated from the 40 plates 26 isolates, all received as or presumed to be C. kaha- per isolate varied from 10 to 50, some plates yielding wae, gave a single pattern (I), although the pattern for more than one sector. Following growth on media that isolates 3 and 15 differed slightly in the position of one contained one of the five different nitrogen sources, of the higher molecular weight fragments (Fig. 1). One three nit mutant classes were identified as nit1, nit3and banding pattern (XI) was obtained from one isolate of nitM (Brooker et al., 1991). The largest number of C. acutatum from Coffea arabica, and four other iso- mutants were nit1 phenotype 1 (48–78%) followed by lates from Colombian coffee fruit with oil spot. The nit3 (10–43%), although one isolate generated none of other banding patterns were obtained from small the latter. The smallest number was nitM (4–26%). groups of isolates. In some cases, apparently unique Mutants derived from each of the test isolates were able banding patterns were obtained from particular hosts to complement mutants in other phenotypic classes that or locations, but the host⁄location range and the sam- had been derived from the same isolate. Complementa- ple sizes for these were too heterogeneous to identify tion generally occurred between nitM mutant and either any other direct correlations. nit1 and⁄or nit3. Occasionally, complementation 278 Bridge et al.

Table 4 Comparison of non-Kenyan coﬀee isolates

AFLP VNTR primer

Our Number IMI number MR RYMtDNA RFLP B E Citrate Tartrate Location

2 190857 I I I 1 1 - - Ethiopia 3 299393 I I Ia 1 2 - - Cameroon 8 337195 I I I 1 1 - - Malawi 10 348840 I I I 2 3 - - Malawi 13 357057 I I I 1 1 - - Malawi 15 361501 I I Ia 1 2 - - Cameroon 52 300964 I I I 1 1 - - Zimbabwe 53 301220 I I I 1 1 - - Malawi 54 311655 I I I 1 1 - - Tanzania 55 338730 I I I 1 1 - - Zambia 56 334963a I I I 1 1 - - Malawi 65 361500 I I I 1 1 - - Burundi

Table 5 Vegetative compatibility groupings (VCG)

VCG Number IMI Origin Comments

1 12 356654 Kenya CBD C. kahawae (yellow isolate ex CRS Ruiru) 1 17 363577 Kenya CBD C. kahawae 1 19 363579 Kenya CBD C. kahawae 1 20 363580 Kenya CBD C. kahawae 1 55 338730 Zambia CBD C. kahawae 2 3 299393 Cameroon CBD C. kahawae 2 15 361501 Cameroon CBD C. kahawae 3 7 325945 Malawi Ex-berry -ve pathogenicity C. gloeosporioides 4 24 4056a Sri Lanka Leaf lesion C. gloeosporioides

Four C. kahawae isolates from Kenya and one from Zambia had same VCG. Two from Cameroon were compatible with each other but different from the others. Both were different from two non- coffee isolates. IMI, authentic culture collection accession; CBD, coffee berry dis- Fig. 2 Amplified fragment length polymorphism patterns from ease; C., Colletotrichum. primer E for representative Colletotrichum kahawae isolates. Differ- ences for 15, 3 and 10 are shown by arrows tical argument for considering C. kahawae as a distinct taxon, especially for quarantine and disease-manage- occurred between the two nitM mutants. The results are ment purposes. The results presented here demonstrate shown in Table 5. Complementation between the that C. kahawae can be separated from C. gloeosporio- mutants of different isolates occurred between the four ides by a number of different relatively simple tech- CBD isolates from Kenya and one from Zambia. The niques. The RFLP method used was originally two CBD isolates from Cameroon complemented each described as a method for assessing mitochondrial var- other but no other isolate. The two non-CBD isolates iability (Spitzer et al., 1989), and has been used subse- did not complement any other isolate. quently to determine the mitochondrial lineages in a range of fungi (e.g. Whittaker et al., 1994; Typas et al., Discussion 1998). This method has been undertaken in several Although Sreenivasaprasad et al. (1993) showed that Colletotrichum studies (e.g. Freeman and Rodriguez, C. kahawae is very close to C. gloeosporioides on the 1995; Bridge et al., 1997; Buddie et al., 1999; Marti- basis of rDNA sequences, Martinez-Culebras et al. nez-Culebras et al., 2003). Such studies have shown (2003) were able to generate an RFLP that separated the utility of the method for defining functional popu- C. kahawae from C. gloeosporioides. However, this was lations below species level (e.g. Thomas et al., 1994). based on a single base difference in rDNA, hence the Similarly, typing procedures, such as VNTR and underlying difference is very small. Nevertheless, the AFLP, have been used to define populations of host fungus causes a very specific disease that is both host or geographically restricted isolates within single spe- and geographically limited, and exists as an ecologi- cies (e.g. Nicholson and Rezanoor, 1994; Edel et al., cally distinct population. There remains a strong prac- 1995; Arora et al., 1996). Variability of Colletotrichum kahawae 279

The results of the molecular typing carried out here pathogenic strains are known to occur (Omondi et al., indicate that the population of C. kahawae within 2000). Again, the isolates from Cameroon differed in Kenya (and, by inference, younger populations else- their AFLP patterns. Populations resistant to methyl where derived from this) are genetically very uniform benzimidazole carbamate-based fungicides have devel- with only slight separation at the population level as oped (Ramos and Kamidid, 1982), and VCG differ- yet. The fact that differences in aggressiveness and fun- ences between geographically separated populations gicide tolerance have not become associated with other from East and West Africa have been confirmed by indicators of genetic heterogeneity reflect the relative this work. Isolate 10 from Malawi also showed slight young age of C. kahawae as a species and that there differences. This separation of East and West African has been little selection pressure for other population isolates as two distinct, but very closely related popu- changes. lations has been reported with other fungal patho- A previous research on Colletotrichum diseases of gens, one example being Armillaria heimii on tea perennial crops has shown some specialization of path- (Pe´rez-Sierra et al., 2004), although in that case, dif- ogenicity associated with the host species (Alakahoon ferences were maintained within a single meiotic mat- et al., 1994). An examination of DNA profiles using ing group. There is no known perfect state of ribosomal and mitochondrial DNA RFLP from a col- C. kahawae; hence, the opportunities to develop lection of C. gloeosporioides, isolated from fruit lesions genetic heterogeniety are limited. There has also been of banana, avocado, mango and papaya originating limited selection pressure for emergence of new worldwide (Hodson et al., 1993), showed that among genetic traits on this successful pathogen. Under these the considerable variation that was found, isolates conditions, the rapid spread of a seemingly clonal could be distinguished in relation to their host source population occurred but with some evidence now that within geographic localities. This may indicate adapta- variability at the functional level within the population from the local C. gloeosporioides gene pool. How- tion is developing. ever, isolates from different hosts never had the same The CBD pathogen did not evolve in the centre of banding patterns and those from mango were specific diversity of Co. arabica (Ethiopia) which the disease did to that host suggesting a unique form pathogenic to not reach until 1970 when much of the wild Co. arabica mango. was found to be susceptible to the disease. When The results show considerable heterogeneity in terms Co. arabica was newly planted in western Kenya in the of mitochondrial DNA among Colletotrichum isolates 1920s, the crop encountered this pathogenic fungal from perennial crops, but with evidence that clonal population which was able to spread relatively rapidly populations are associated with particular hosts or dis- across the new areas of Co. arabica in Africa (Masaba eases. For example, isolates from oil spot disease and Waller, 1992). The pathogen most likely originated (mancha mantecosa) of coffee obtained from leaves from the general C. gloeosporioides gene pool in the and fruit in South America show different mtDNA forests of the great lakes region of central Africa on the RFLP patterns, but within these mtDNA populations, diploid progenitors of the tetraploid Co. arabica, which there is little evidence of further heterogeneity, and have their centres of diversity in that area. each group produces homogenous patterns with PCR- The inability of C. kahawae to utilize either citrate fingerprinting techniques. Within this group are the or tartrate as a sole carbon source provides a useful isolates obtained from fruit from oil spot infected test to distinguish the pathogen from other Colletotri- plants. These isolates were homogenous in that they chum isolates from perennial crops. Increased pathoge- all produced a type XI RFLP banding pattern. Apart nicity of fungi has frequently been associated with a from this group of isolates, this RFLP pattern was reduction of saprophytic ability and the loss of only obtained from a single isolate identified as so-called avirulence genes, and the loss of enzyme C. acutatum. The oil spot fruit isolates also produced a functions associated with citrate and tartrate metabo- strong orange pigment in some of the agar plate-based lism is likely to be a manifestation of this in C. kaha- tests, and this may indicate that these strains actually wae. The molecular biochemical tests also show that are isolates of C. acutatum. these may be used to distinguish C. kahawae from Examination of a range of C. kahawae isolates other closely related taxa. from across Africa show a single main mtDNA geno- type with slight variation with the two isolates from Acknowledgements Cameroon. These two isolates also differed in their Funding for a part of this work was provided by the UK Depart- VCG reactions (Beynon et al., 1995). Gichuru et al. ment for International Development Crop Protection Research Pro- (2000) found that 44 East African isolates of C. kaha- ject no R 5586. This study is dedicated to the memory of Diana wae Davies, who sadly passed away before seeing the final interpretation (42 from Kenya, one from Ethiopia and one of the work she had carried out over a number of years. from Malawi) all belonged to the same VCG, except for one white sector mutant. Further analysis of References C. kahawae isolates with VNTR primers, simple repet- Alakahoon PW, Brown AE, Sreenivasaprasad S. (1994) Cross-infec- itive sequence fingerprinting primers and AFLP shows tion potential of genetic groups of Colletotrichun gloeosporioides almost no variability within the main group, indicat- on tropical fruits. Physiol Mol Plant Pathol 44:93–103. ing a virtually clonal population, although different 280 Bridge et al.

Arora DK, Hirsch PR, Kerry BR. (1996) PCR-based molecular dis- Nicholson P, Rezanoor HN. (1994) The use of random amplified crimination of Verticillium chlamydosporium isolates. Mycol Res polymorphic DNA to identify pathotype and detect variation in 100:801–809. Pseudocercosporella herpotrichoides. Mycol Res 98:13–21. Bella-Manga , Bieyss D, Bedimo M, Nyass S, Berry D. (2001) Omondi CO, Ayecho PO, MwangÕombe AW, Hindorf H. (2000) Elaboration dÕune strate´gie de lutte durable et efficace contra Reaction of some Coffea arabica genotypes to strains of Colletotri- lÕanthracnose des baies du cafeíer Arabica dans les hautes terres de chum kahawae, the cause of coffee berry disease. J Phytopathol lÕOuest Cameroun; Bilan des connaissances acquises et perspec- 148:61–63. tives. 19eme Coll Sci Int Cafe´ Trieste 1–9. Paterson RRM, Bridge PD. Biochemical Techniques for Filamentous Beynon SM, Coddington A, Lewis BG, Varzea V. (1995) Genetic Fungi. IMI Technical Handbooks Vol. 1. Wallingford, UK, CAB variation in the coffee berry disease pathogen, Colletotrichum kah- International, 1994. awae. Physiol Mol Plant Pathol 46:457–470. Pe´rez-Sierra A, Guillaumin J-J, Spooner BM, Bridge PD. (2004) Bridge PD, Pearce D, Rutherford M, Rivero A. (1997) VNTR Characterization of Armillaria heimii from Africa. Plant Pathol derived oligonucleotides as PCR primers for population studies in 53:220–230. filamentous fungi. Lett Appl Microbiol 24:426–430. Ramos AH, Kamidid RE. (1982) Determination and significance of Brooker NL, Leslie JF, Dickman MB. (1991) Nitrate non-utilizing mutation rates for Colletotrichum coffeanum from benomyl sensi- mutants of Colletotrichum and their use in studies of vegetative tivity to benomyl tolerance. Phytopathology 72:181–185. compatibility and genetic relatedness. Phytopathology 81:672–677. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Labora- Buddie AG, Martinez-Culebras P, Bridge PD, Garcıá MD, Querol tory Manual, 2nd edn. New York, USA, Cold Spring Harbor Lab- A, Cannon PF, Monte E. (1999) Molecular characterization of oratory Press, 1989. Colletotrichum strains derived from strawberry. Mycol Res Smith D, Onions AHS. The Preservation and Maintenance of Living 103:385–394. Fungi. 2nd ed. CAB International, Wallingford, UK, 1994. Cenis JL. (1992) Rapid extraction of fungal DNA for PCR amplifi- Spitzer ED, Lasker BA, Travis SJ, Kobayashi GS, Medoff G. (1989) cation. Nucleic Acids Res 20:2380. Use of mitochondrial and ribosomal DNA polymorphisms to clas- Edel V, Steinberg C, Avelange I, Laguerre G, Alabouvette C. (1995) sify clinical and soil isolates of Histoplasma capsulatum. Infect Comparison of three molecular methods for the characterization Immun 57:1409–1412. of Fusarium oxysporum strains. Phytopathology 85:579–585. Sreenivasaprasad S, Brown AE, Mills PR. (1993) Coffee berry dis- Freeman S, Rodriguez RJ. (1995) Differentiation of Colletotrichum ease pathogen in Africa: genetic structure and relationship to the species responsible for anthracnose of strawberry by arbitrarily group species Colletotrichum gloeosporioides. Mycol Res 97:995– primed PCR. Mycol Res 99:501–504. 1000. Gichuru E, Varzea VMP, Rodrigues CJ, Masaba DM. (2000) Vege- Thomas V, Rutherford MA, Bridge PD. (1994) Molecular differenti- tative compatibility grouping of Colletotrichum kahawae in Kenya. ation of two races of Fusarium oxysporum special form cubense. J Phytopathol 148:233–237. Lett Appl Microbiol 18:193–196. Hodson A, Mills PR, Brown AE. (1993) Ribosomal and mitochon- Typas MA, Mavridou A, Kouvelis VN. Mitochondrial DNA differ- drial DNA polymorphisms in Colletotrichum gloeosporioides iso- ences provide maximum intraspecific polymorphism in the ento- lated from tropical fruits. Mycol Res 97:329–335. mopathogenic fungi Verticillium lecanii and Metarhizium Lynch JM, Slater JH, Bennett JA, Harper SHT. (1981) Cellulase anisopliae, and allow isolate detection⁄identification. In: Bridge P, activities of some aerobic micro-organisms isolated from soil. Couteaudier Y, Clarkson J (eds),Molecular Variability of Fungal J Gen Microbiol 127:231–236. Pathogens. Wallingford, UK, CAB International, 1998, pp. 227– Martinez-Culebras PV, Querol A, Suarez-Fernandez MB, Garcia- 237. Lopez MD, Barrio E. (2003) Phylogenetic relationships among Varzea VMP, Rodriguez CJ, Lewis BG. (2002) Distinguishing char- Colletotrichum pathogens of strawberry and design of PCR acteristics and vegetative compatibility of Colletotrichum kahawae primers for their identification. J Phytopathol 151:135–143. in comparison with other related species from coffee. Plant Pathol Masaba DM, Waller JM. Coffee berry disease – the current status. 51:202–207. In: Bailey JA, Jeger MJ (eds),Colletotrichum; Biology, Pathology Waller JM, Bridge PD. Recent advances in understanding Colletotri- and Control, Wallingford, UK, CAB International, 1992, pp. 237– chum diseases of some tropical perennial crops. In: Prusky D, 249. Freeman S, Dickman MB (eds),Colletotrichum; Host Specificity, Mueller UG, Lipari SE, Milgroom MG. (1996) Amplified fragment Pathology and Host-Pathogen Interactions. St Paul, MN, USA, length polymorphism (AFLP) fingerprinting of symbiotic fungi APS Press, 2000, pp. 337–345. cultured by the fungus-growing ant Cyphomyrmex minutus. Mol Waller JM, Bridge PD, Black R, Hakiza G. (1993) Characterisation Ecology 5:119–122. of the coffee berry disease pathogen Colletotrichum kahawae Mugnai L, Bridge PD, Evans HC. (1989) A chemotaxonomic evalua- sp. nov. Mycol Res 97:989–994. tion of the genus Beauveria. Mycol Res 92:199–209. Whittaker SL, Assinder SJ, Shaw DS. (1994) Inheritance of mito- MwangÕombe AW. (1994) Tolerance in isolates of Colletotrichum cof- chondrial DNA in Phytophthora infestans. Mycol Res 98:569–575. feanum to prochloraz-Mn in Kenya. J Phytopathol 14:114–122.