Plant Pathology (2003) 52, 476 485

Phylogeny of the frosty pod rot pathogen of cocoa

Blackwell Publishing Ltd.

H. C. Evansa*, K. A. Holmesa and A. P. Reidb

a
CABI Bioscience, Ascot Centre, Silwood Park, Ascot, Berkshire SL5 7TA; and bCABI Bioscience, Egham Centre, Bakeham Lane, Egham,
Surrey TW20 9TY, UK

Morphological, cytological and molecular evidence is presented which confirms that the frosty pod rot pathogen
of cocoa, formerly classified as the mitosporic fungus Moniliophthora roreri (Deuteromycota), belongs to the hymeno-
mycetous genus Crinipellis (Basidiomycota) and that two varieties should now be recognized: Crinipellis roreri var.
roreri and the new variety C. roreri var. gileri. The latter was collected on Theobroma gileri, an endemic tree of
submontane forests in north-west Ecuador, and can be distinguished from Ecuadorian and Peruvian isolates from cocoa
(T. cacao) on the basis of spore morphology, incompatibility and nucleotide sequence data. As with var. roreri, meiosis
is shown to occur within the dispersive and infective spore stage of var. gileri and these meiospores are interpreted
to represent a much modified probasidium. In addition, in a field inoculation experiment, an isolate from T. gileri
proved to be noninfective to cocoa pods when compared with positive control strains isolated from T. cacao in western
Ecuador and T. bicolor in eastern Ecuador. It is concluded that var. gileri is the vestigial progenitor of the frosty pod
rot pathogen of cocoa, with a host range and distribution restricted to T. gileri in the mesic forests of north-west South
America.

Keywords: characterization, Crinipellis, evolution, frosty pod rot, Moniliophthora, Theobroma

Leach et al., 2002), the disease has now been confirmed

Introduction
Frosty pod rot of cocoa (Theobroma cacao) is a relatively
minor disease in global terms, accounting for an estimated
30 000 tonnes in loss of cocoa production, or less than
4% of total crop losses (Bowers et al., 2001). Thus, the
disease has been of limited interest and, until recently,
largely confined to its purported centre of origin in western
Ecuador and /or Colombia (Holliday, 1971; Evans,
1981). It has been further speculated that the pathogen
evolved on the endemic forest host, Theobroma gileri
(Holliday, 1971; Evans, 2002). The disease, however, is
currently in an invasive phase, having arrived in and
spread throughout Peru within the last decade; posing a
direct threat to the neighbouring cocoa-growing countries
of Bolivia and Brazil (Evans et al., 1998; Evans, 2002).
Similarly, following its arrival in Costa Rica in the late
1970s (Enriquez & Suarez, 1978), and its subsequent and
continuing dramatic impact on cocoa yields (Evans, 1986;
*To whom correspondence should be addressed.
E-mail: h.evans@cabi.org
Present address: Scottish Agricultural Science Agency,
Diagnostics and Molecular Biology, 82 Craigs Road, East
Craigs, Edinburgh, EH12 8NJ, UK
Accepted 4 March 2003
in Nicaragua, Honduras and Guatemala (Evans, 2002),
and it must be only a matter of time before it reaches
the more extensive and ancient cocoa plantations in
Mexico. Wherever the pathogen has invaded, cocoa pro-
duction has been severely affected, with, for example, an
estimated 4050% fall in production in Peru (Evans et al.,
1998). The socioeconomic and ecological implications
could be profound. In Peru and Bolivia, cocoa constitutes
an important component in the campaign against illicit
drug crops. If cocoa farming were to become uneconomic
due to disease pressure, then such commodity crops
would be abandoned in favour of the more lucrative coca
(Erythroxylum coca). In both Central America and Brazil,
the loss of a sustainable, forest-shaded tree crop, such as
cocoa, and the predicted switch to annual, subsistence or
cash crops could spell ecological disaster as the forest
corridors for bird migration in Meso-America, and the
forest refuges for endemic mammals in Brazil, especially
in Bahia state, disappear (Rice & Greenberg, 2000;
Saatchi et al., 2001).
The main objective of the current study was to test the
theory that the pathogen which devastated Ecuadorian
cocoa plantations during the early part of the 20th
century (Rorer, 1926) originated on T. gileri in the sub-
montane forests of north-west Ecuador (Evans, 1981;
Evans, 2002).

476 2003 BSPP

 Frosty pod rot of cocoa 477

Table 1 Geographical and host origins of frosty pod rot isolates used in the study

Isolate code Locality Country Date Host Habitat

DIS 67 Nueva Primavera, Rio Napo, Ecuador March 1999 Theobroma bicolor Secondary forest
Napo Province
DIS 116 Guadual, Esmeraldas Ecuador September 1999 T. gileri Primary forest
Province
DIS 122 Anangu, Rio Napo, Ecuador September 1999 T. cacao Plantation
Napo Province
DIS 160 Juanjui, Rio Huallaga, Peru November 1999 T. cacao Plantation
San Martin Department
DIS 329a Guadual, Esmeraldas Ecuador November 2001 T. gileri Primary forest
Province
DIS 331 Guadual, Esmeraldas Ecuador November 2001 T. gileri Primary forest
Province

a
Isolated as an endophyte from an immature, symptomless pod; all other isolates from diseased pods with symptoms of frosty pod rot.

mutual repulsion (barrage reaction) occurred between the

Materials and methods
Fungal isolates
The origins of the isolates used in the present study are
listed in Table 1. Those from T. gileri, the suspected wild
host, were collected from remnant, submontane forest in
north-west Ecuador, using published (Cuatrecasas, 1964)
as well as unpublished (herbarium) data to locate the
holotype site. All isolates were made by plating pod tissues
directly on potato carrot agar (PCA) supplemented
with 10 mg mL1 penicillinstreptomycin solution (Sigma-
Aldrich, Poole, UK) or malt extract agar (MEA) contain-
ing benomyl (5 mg mL1) and incubating at 25C in the
dark.
Spore morphology
Mature spores were shaken from 10-day-old colonies
grown on MEA at 25C in the dark, and stained in
aceto-carmine. Measurements of the length and breadth
were made from 200 spores for each isolate and their
mean size and range calculated. The length:breadth ratios
were compared by analysis of variance (anova) after
angular transformation using Genstat 5 (Payne et al.,
1993).
Spore cytology
A modified Giemsa method (Punithalingam, 1983) was
used to stain the nuclei of spores in various stages of devel-
opment, including mature spores pregerminated on PCA
for 2448 h. In order to ensure that sufficient material
adhered to the staining slides, spores were spread over the
slides in a thin film of sterile distilled water and allowed
to air dry slowly at 2025C for a minimum of 6 h.
Compatibility reactions
In order to detect genotype differences, compatibility or
noncompatibility was assessed by observing if aversion or
various isolates. These were evaluated by taking 5-mm-
diameter plugs from 10-day-old colonies and transferring
them to MEA in 5-cm-diameter plates in paired or quad-
rupled combinations of the isolates from T. cacao and
T. gileri listed in Table 1, followed by incubation at 25C
for 30 days in the dark.
Field inoculation
The experiment was carried out in April 2002 at the
Estacin Experimental Tropical (EET), Pichilingue, Los
Rios Province, Ecuador, in a 40- to 50-year-old, mixed
hybrid cocoa plantation. Approximately 1-month-old
pods (cherelles), 35 cm in length, were inoculated by
soaking a pad (2 cm in diameter) of absorbent cotton
wool in an aqueous spore suspension (1 108 spores
mL1) of the selected isolate and wrapping this centrally
around the pod. All inoculated pods, including controls
treated with sterile distilled water only, were immediately
enclosed in clear plastic bags (30 20 cm) and fastened
to the peduncle with a plant tie, in order to maintain
humidity and prevent contamination during the 4- to 5-
month growth and maturation period. Healthy mature
and infected pods were harvested and assessed macro-
scopically for frosty pod rot symptoms. For those diseased
pods with no evident external sporulation, samples were
plated onto potato dextrose agar (PDA) to determine the
identity of the causal agent.
PCR amplification of the ITS region
Amplification of the ITS region of the rDNA operon was
carried out by direct PCR from mycelia growing on agar
plates. A small section of mycelium was scraped off each
plate and placed into sterile 05 mL thin-walled PCR
tubes. An equal volume (20 L) of lysis buffer (LB;
10 mm Tris, pH 83, 50 mm KCl, 25 mm MgCl2, 045%
NP40, 045% Tween 20, 001% gelatin, 60 g mL1
Proteinase K) was added and the tubes frozen at 80C
for 10 min. These were then incubated at 65C for 60 min

2003 BSPP Plant Pathology (2003) 52, 476 485

478 H. C. Evans et al.

followed by 10 min at 95C and 5 L added to a fresh
thin-walled tube containing 45 L PCR reaction mix
(10 mm Tris, pH 83, 50 mm KCl, 25 mm MgCl2, 2 mm
dNTP (Gibco Life Sciences, Paisley, UK), 50 pmol of each
primer and two units of Taq polymerase (Sigma)). The
primers used were ITS 1F (White et al., 1990) and ITS 4
(Gardes & Bruns, 1993) (Amersham Biosciences UK Ltd,
Little Chalfont, UK). Cycling conditions were 95C for
5 min followed by 35 cycles of 95C for 1 min, 50C for
45 s and 72C for 2 min; a final extensions step of 72C
for 10 min was included and the reactions held at 10C
until purification. Amplification was carried out in a
Hybaid PCR Express Thermal Cycler (Hybaid Ltd,
Teddington, UK).
Cloning and sequencing of the PCR products
The ITS PCR products were purified using a Wizard PCR
Preps Purification System (Promega UK Ltd, Southampton,
UK) and cloned using a pGEM-T Easy Vector System
II (Promega) according to the manufacturers instruc-
tions. A number of colonies were picked from each plate
and grown overnight in 1 mL LB containing 50 g mL1
ampicillin at 37C in an orbital incubator at 200 rpm.
The presence of the correct insert was checked by heating
10 L from each culture at 95C in a thin-walled tube
for 10 min and adding 15 L PCR mix and amplifying
as detailed earlier.
Clones containing inserts were then grown overnight
in 5 mL LB containing 50 g mL1 ampicillin at 37C
in an orbital incubator at 200 rpm and the plasmids
purified using a Wizard Plus Minipreps DNA Purifica-
tion System (Promega) according to the manufacturers
instructions. The purified plasmids were sequenced
using a SequiTherm Excel II DNA Sequencing Kit
(Cambio Ltd, Cambridge, UK) with labelled M13 primers
obtained from MWG-Biotech (Ebersberg, Germany).
Electrophoresis was carried out on a Li-Cor DNA Gene
Reader 4200 (Li-Cor Biosciences UK Ltd, Cambridge,
UK).
Data analysis
Forward and reverse sequences of clones from each strain
were assembled using Sequencer v4.0.5 (Gene Codes
Corporation, Ann Arbor, MI, USA). Alignment of the
consensus sequences was carried out using the ClustalW
function in MacVector v70r2 (Accelrys Ltd, Cambridge,
UK) using the slow alignment speed. Pairwise matrices
were obtained by use of the Pustell DNA matrix function
within MacVector with a window size of 30 base pairs
(bp) and a minimum percentage score of 60%. Phyloge-
netic analysis was done by upgma analysis with 100 boot-
strap replicates. Details of the isolates used in the analysis,
with sequence data from GenBank, are presented in
Table 2.

Table 2 Details of the fungal isolates used in

Fungus Isolate code GenBank accession number
the ITS sequence phylogenetic analysis. All
Frosty pod rot* DIS 160 AY230254 obtained from GenBank, apart from those
Frosty pod rot* DIS 331 AY230255 marked with an asterisk (*)
Alternaria alternata AA1 AF314569
A. infectoriaa BMP 21-11-15 AF229458
A. sesamicola AZM AF314588
A. smyrnii EGS 37-093 AF229456
A. tenuissima ATCC 16423 AF229476
Cladosporium macrocarpum CBS 17562 AJ244229
C. sphaerospermum CBS 12247 AJ244228
Colletotrichum acutatum BBA 71427 AJ301987
C. gloeosporioides b BBA 65797 AJ301925
C. gloeosporioides BBA 71407 AJ301986
C. lindemuthianum BBA 65483 AJ301958
Crinipellis perniciosa AF335590
Gaeumannomyces sp. sp. 153 AJ010038
Magnaporthe poae C4 AF0774403
M. poae 1832 AJ010041
M. poae 2562 AJ010042
M. grisea 2690 U17328
M. grisea 2692 U17329
Phialophora graminicola P4 U17217
Pyricularia rabaulensis IMI 388508
Trichoderma reesii ATCC 56765 X93938
T. saturnisporum CBS 33592 X93973
T. virgatum ATCC 24961 Z48949
T. viride Tr 22 AJ230678

a
As teleomorph, Lewia infectoria.
b
As teleomorph, Glomerella cingulata.

2003 BSPP Plant Pathology (2003) 52, 476 485

 Frosty pod rot of cocoa 479

Figure 1 Frosty pod rot pathogen on hosts and in culture: (a) on mature pod of Theobroma gileri, in understorey submontane forest, Ecuador (isolate
DIS 116), healthy green pod indicated by arrow in canopy background; (b) same locality, healthy pod of T. gileri from which DIS 329 was isolated as
an endophyte; (c) same locality, isolate DIS 331 on immature pod showing mature brown spore mass developing on white pseudostroma; (d) healthy
and diseased pods in cocoa plantation, Huallaga Valley, Peru, showing white pseudostroma covering pod on which immature (pink) spores are
developing; (e) same locality, external and internal pod symptoms, showing more advanced stage in sporogenesis with brown, flaking spore masses
beginning to obscure white pseudostroma, and compacted beans surrounded by white mycelium; (f ) dual plate culture (MEA) of two cocoa isolates,
DIS 122 ex Ecuador and DIS 160 ex Peru; (g) cocoa isolate (DIS 160) challenged with T. gileri isolate (DIS 331); (h) T. gileri isolate (DIS 116)
intermingling centrally from lateral inoculum plugs and inhibiting the growth of cocoa isolate (DIS 160) from proximal and distal plugs.

2003 BSPP Plant Pathology (2003) 52, 476 485

480 H. C. Evans et al.

Table 3 Microscopic analysis of frosty pod rot spores from Theobroma gileri and T. cacao

Isolate code Host Spore lengtha (m) Spore breadth (m) Overall mean (m)

DIS 116 T. gileri (75)95 160 (180) 55 90(100) 1113 701

DIS 329 T. gileri (75)110 160(220) 55 80(105) 1186 696
DIS 331 T. gileri (70)80 155(175) (50 )60 110 1071 844
DIS 122 T. cacao (55)65 105(120) 55 90(100) 812 720
DIS 160 T. cacao 55 105(125) 50 80(100) 822 735

a
Minimum of 200 spores.

Table 4 Comparison of spore sizes of frosty pod rot isolates from Table 5 Field evaluation of pathogenicity to cocoa (T. cacao) pods of
Theobroma gileri and T. cacao frosty pod rot isolates from Theobroma spp.

Mean spore Angular Isolate code Host Pods infectedb

Isolate length:breadth ratioa transformation
DIS 67 T. bicolor 9 (9)
T.gileri (DIS 329) 1730 7435ab DIS 116 T. gileri 0 (10)
T.gileri (DIS 116) 1651 7581a EET 1a T. cacao 16 (20)c
T.gileri (DIS 331) 1551 7288a Control 1d (10)
T.cacao (DIS 160) 1164 6063b
a
T.cacao (DIS122) 1143 6117b Source was an infected pod, collected on day of inoculation, within an
experimental plot.
a b
Means of 200 spores. Number in parentheses shows total pods inoculated.
b c
Values followed by the same letter are not significantly different Remainder infected by Crinipellis perniciosa.
d
according to the least significant difference test (P = 005). Natural infection from field inoculum.

Results
Spore form and function
Macroscopic morphology of the frosty pod rot pathogen
on T. cacao and T. gileri is illustrated in Fig. 1(a,ce).
Microscopic analysis showed that spores of the isolates
from T. gileri differed significantly from the cocoa isolates
(Tables 3 and 4), a high proportion of the T. gileri spores
being ellipsoidal to cylindrical and relatively thin-walled
(Fig. 2b), compared with the predominantly globose,
thick-walled spores from T. cacao (Fig. 2a).
Nuclear staining of the sporophores and the mature
and germinating spores from T. gileri revealed evidence of
a meiotic cycle (Fig. 2ch), as recently reported for cocoa
isolates (Evans et al., 2002), and therefore that they func-
tion as meiospores rather than mitospores as previously
interpreted.
Isolate compatibility
Paired cocoa isolates produced a compatible reaction
as did T. gileri isolates (Fig. 1f), whereas incompatibility,
or a barrage reaction, was consistently demonstrated in
mixed combinations, with well-defined demarcation lines
between isolates from the two different hosts (Fig. 1g h).
Pathogenicity
The results of the field inoculation experiment are shown
in Table 5. Due to confusion over isolate numbering, only
one isolate from T. gileri was tested. The second isolate
turned out to be from another Theobroma host, T.
bicolor, but the mistake was only detected when the seem-
ingly ambiguous results were being analysed. Thus, there
were insufficient data to assess pathogenicity critically,
but provisional evidence indicated that T. gileri isolates
were nonpathogenic to cocoa, whilst cocoa isolates were
cross-infective within the genus Theobroma (Evans,
1981).
Phylogenetic analysis
The consensus sequence for the T. cacao strain (DIS 160)
was 795 bp in length and 796 bp for the T. gileri strain
(DIS 331). The alignment of the two sequences (Fig. 3)
showed that the two strains were 988% homologous
with only nine polymorphic sites; all of these sites were
consistent within the various clones sequenced. Most of
these took the form of single nucleotide polymorphisms,
although there was one region containing four polymor-
phisms in a 14-bp region. In the cocoa isolate, at bases
460 474, the sequence was 5-AAAAAAGCTTTTTC-3,
but in T. gileri it was 5-AAAAAGCTTTTTTT-3. It is
possible that these two regions form a hairpin structure
within the 58S rRNA gene. However, upon aligning
both sequences with the ITS regions of numerous fungal
sequences from GenBank, this region was just outside
the gene. The closest match was with Crinipellis perniciosa
(Stahel) Singer, the causal agent of witches broom disease
of cocoa, which displayed 89% homology.
Pairwise comparisons of the frosty pod isolates (Fig. 4a),
showed a high degree of similarity, whilst those between
the cocoa isolate (DIS 160) and Crinipellis perniciosa
(Fig. 4b) revealed the polymorphisms, the majority of
which occurred in the ITS 2 region.

2003 BSPP Plant Pathology (2003) 52, 476 485

 Frosty pod rot of cocoa 481

Figure 2 Spore morphology and sporogenesis of the frosty pod rot pathogen: (a) cocoa isolate (DIS 122) with predominantly globose, thick-walled
spores; (b) T. gileri isolate (DIS 116) with both globose and ellipsoidal, thinner-walled spores. (ch) Sporogenesis in T. gileri isolate DIS 116: (c) early
stage (prophase), showing erect sporophores surrounded by an amorphous layer (arrow) with newly formed diploid nuclei, the recent narrow hyphae
of the pseudostroma are binucleate; (d) swelling of sporophore with first meiotic division (anaphase); (e) binucleate, globose to ellipsoidal mature
spores with abnormally large cylindrical spore in foreground in late telophase stage of meiosis; (f) meiospore germinating after 48 h on PCA, showing
a four-celled metabasidium, with distal and apical cells reverting to a binucleate condition; (g, h) disrupted meiospore chains exhibiting various
stages of nuclear division; the majority of meiospores mature in the binucleate condition with the second meiotic divisions occurring during
germination. (All scale bars = 10 m.)

A phylogenetic analysis with 24 other fungal sequences

yielded a tree (Fig. 5) which clustered the frosty pod rot
Discussion
isolates with Crinipellis perniciosa, a relationship with a Until very recently, the phylogeny of the frosty pod rot
100% bootstrap support. pathogen of cocoa was based on a few facts, supported by

2003 BSPP Plant Pathology (2003) 52, 476 485

482 H. C. Evans et al.
several assumptions and some speculation. The facts
were established by Evans et al. (1978), who reported that
the fungus had basidiomycetous origins rather than the
ascomycetous (Leotiales) origin that the name Monilia
roreri Ciferri suggested. The evidence was obtained from
scanning and transmission electron microscopic studies
which revealed the unique sporogenesis, basipetal rather
than acropetal as in Monilia, and the presence of dolipore
septa in the mycelium. The new genus Moniliophthora was
erected to accommodate the pathogen. It was assumed
that this represented the mitotic state (anamorph) of an
Figure 3 Aligned nucleotide sequences of frosty pod
rot isolates DIS 160 (ex T. cacao, GenBank accession
no. AY230254) and DIS 331 (ex T. gileri, GenBank
accession no. AY230255).
unknown basidiomycete. Later, Evans (1981) speculated
that the teleomorph could be a Crinipellis species since
the unusual pleomorphic, hemibiotrophic life-cycle is
mirrored by that of the other major South American
cocoa pathogen, Crinipellis perniciosa (Evans, 1980): the
former evolved on an endemic Theobroma host, or its
near relative Herrania, on the western or Pacific side of
the Andean Cordillera (Rorer, 1926; Holliday, 1971;
Evans, 1981), and the latter on T. cacao, in its native range
on the eastern or Amazonian side (Baker & Holliday,
1957).
2003 BSPP Plant Pathology (2003) 52, 476 485
 Frosty pod rot of cocoa
Figure 4 Pustell DNA matrices for pairwise comparisons of frosty pod
rot isolates DIS 160 (ex T. cacao) and DIS 331 (ex T. gileri) (a) and frosty
pod rot isolate DIS 160 and Crinipellis pernicosa (b).
The increasing invasiveness of the frosty pod rot
pathogen and its actual impact on and potential threat
to cocoa cultivation in Latin America have offered the
opportunity to test these assumptions and hypotheses
using modern molecular techniques. The work reported
here, as well as in recent studies on the ITS region of ribos-
omal RNA (G.W. Griffith, Institute of Biological Sciences,
Figure 5 UPGMA tree derived from fungal ITS
sequence data. Bootstrap values displayed at
the nodes. *DIS 160, isolated from T. cacao;
**DIS 331, isolated from T. gileri.
2003 BSPP Plant Pathology (2003) 52, 476 485
483
University of Wales Aberystwyth, Dyfed, UK, personal
communication, 2001), and also of mitochondrial DNA
(W. Phillips, University of Reading, UK, personal commu-
nication, 2001), has revealed that the fungus does indeed
lie very close to C. perniciosa in the respective phyloge-
netic trees. However, the assumption that the sporophores
and spores represent the anamorph has now been found
to be wanting. Using more traditional cytological methods,
it has been discovered that the purported conidia or mit-
ospores are, in fact, meiospores and that the frosty pod rot
pathogen of cocoa is a much modified agaric teleomorph.
The new combination Crinipellis roreri (Ciferri) H.C.
Evans has now been proposed, the genus Moniliophthora
being reduced to synonymy with Crinipellis (Evans et al.,
2002). The cytological results presented here (Fig. 2ch)
serve to confirm this behaviour, and also that a similar
meiotic cycle occurs during sporogenesis and germination
in the forest isolates from T. gileri.
The discovery of C. roreri on pods of T. gileri in its type
locality (Fig. 1a and c) gave initial support to the hypoth-
esis that the pathogen which ravaged cocoa plantations
in western Ecuador in the early 1900s originated on this
forest host.
Subsequent morphological examination, however, casts
doubt on this and significant differences in spore form
were found between the isolates from T. gileri and T.
cacao (Tables 3 and 4, Fig. 1a,b). Those from the former
produce a much higher proportion (30 40%) of thin-
walled, ellipsoidal to cylindrical spores compared with the
cocoa isolates (12%). Moreover, the molecular studies
also showed that the Ecuadorian T. gileri strain differs
in a number of base pairs from a selected T. cacao strain.
484 H. C. Evans et al.
This morphological and molecular evidence, as well as
that from ongoing work on the genetic diversity of cocoa
isolates of frosty pod rot (W. Phillips, University of Read-
ing, UK, personal communication, 2002), suggests that
the T. gileri strain in Ecuador has never moved from its
forest habitat.
The results of the field study lend support to this isola-
tion theory since there was no cross-infection. However, a
statistical analysis was not undertaken because of back-
ground contamination, mainly by C. perniciosa, and
insufficient replicates in the T. gileri treatment. Initially,
the experimental design included two T. gileri isolates, but
due to confusion over code identification, an isolate from
T. bicolor was substituted erroneously. The latter (DIS 67)
was obtained from a forest species endemic in eastern
Ecuador, where the pathogen is thought to have arrived in
the late 1970s (Evans, 1986; Evans et al., 1998). A much
larger field experiment, involving 100 pods per treatment
and additional isolates from T. gileri, is being established
to confirm these results.
The reason why the cocoa strain or isolate can attack
not only most of the cocoa clones and varieties against
which it has been screened, but also all of the species of
Theobroma and Herrania in germplasm collections where
the disease occurs (Evans, 1981), can now be explained by
the presence of a sexual stage in the life-cycle. What was
once thought to be a clonal pathogen, which cycled only
through mitospores, has now been proven to be geneti-
cally diverse within its native and exotic ranges, with the
current T. gileri isolates forming a separate clade from all
the cocoa isolates, including those from western Ecuador
(W. Phillips, University of Reading, UK, personal commu-
nication, 2002). Whether the sexual mechanism involves
outbreeding through heterothallism has yet to be estab-
lished; however, by virtue of the fact that many billions of
meiospores are produced per pod (Fig. 1e) estimated at
44 106 cm2 (Evans, 1981) suggests that sufficient gene
movement is possible through recombination and muta-
tion in order to respond to selection pressures. Indeed, this
enormous inoculum potential demonstrates why frosty
pod rot has so rapidly displaced witches broom disease as
the major constraint to cocoa production in Peru follow-
ing its relatively recent arrival (Evans et al., 1998).
The question remains: if not in Ecuador on T. gileri,
then where and on what host did the cocoa pathogen
evolve? The genetic diversity data (W. Phillips, University
of Reading, UK, personal communication, 2002) show that
by far the greatest variation occurs in northern Colombia
and, significantly, it was from this region that Baker et al.
(1954) first confirmed the disease on T. gileri in the forests
of north-west Antioquia Department. In an earlier report,
this host had been confused with T. microcarpum Mart.
(Cope, 1953) the Amazonian vicariant of T. gileri (Cuat-
recasas, 1964) since at that time the taxon T. gileri had
not yet been described (Cuatrecasas, 1953). This part of
Antioquia, together with north-west Ecuador, is included
by Gentry (1982) in the Choc phytogeographic region,
now recognized by Myers et al. (2000) as a biodiversity
hotspot, due to its exceptional concentrations of
endemic species. Thus, the Andean uplift which sepa-
rated the Choc and Amazonian regions in the mid-
Pleistocene (Gentry, 1982) resulted not only in the
evolutionary isolation of the genus Theobroma, but also
of its mycobiota: the frosty pod rot pathogen evolved to
the west and witches broom in the much more extensive
forests to the east, possibly from a common endophytic
Crinipellis ancestor. Indeed, isolate DIS 329 was isolated
as an endophyte from a healthy pod of T. gileri during the
present study (Fig. 1b). Both pathogens appear to have
extended their host colonization abilities by invading mer-
istematic tissues, causing auxin imbalances in the form of
hypertrophy and hyperplasia. Although the frosty pod rot
fungus can induce such symptoms in cocoa seedlings at
high spore loads (Evans, 1981), in the field situation it is
strictly a pod pathogen, whereas C. perniciosa can invade
all the meristems of cocoa leading to a multiplicity of
symptoms (Baker & Holliday, 1957).
The ice-age isolation of the Choc refuge (Gentry,
1982), resulting in the paucity of both Theobroma and
Herrania hosts compared with Amazonia (Schultes, 1958;
Cuatrecasas, 1964; Thorold, 1975), may have provided
the selection pressure whereby the agaric basidiomata
evolved to maximize the production of a multipurpose
meiospore, which, unlike the basidiospore, has a survival
as well as a dispersal function, adapted for efficient long-
distance movement. This would have been essential for a
pathogen of a low-density, understorey forest tree such as
T. gileri, with a disparate range from northern Colombia
along the western slopes of the Andes to northern Ecua-
dor. Certainly in the latter country, T. gileri is confined to
submontane forests, between 500 and 700 m above sea
level (asl), and this species appears to be unknown either
side of this narrow strip, as well as from the forests to the
south of Pichincha Province (unpublished observations).
The thick white pseudostroma covering the diseased
pod prior to sporogenesis, and which imparts the frosted
appearance (Fig. 1b and c), may represent the vestigial pileus.
Additional circumstantial evidence that the fungus on
T. gileri is the progenitor of the cocoa pathogen, and that
this evolved in northern Colombia, comes from historical
accounts of cocoa cultivation in Latin America. Baker
et al. (1954) suggest that frosty pod rot may have been
recorded on cultivated cocoa in northern Colombia as
long ago as 1850, and that in this area cocoa may have
been grown for many centuries or even millennia since
wild cocoa was discovered in the dense forests bordering
the Rio Len. The origin of T. cacao remains controver-
sial, but past and recent evidence strongly indicates that
cocoa evolved in the Upper Amazon and later became
naturalized in northern Colombia and Meso-America
(Cheesman, 1944; Baker et al., 1954; B.G.D. Bartley, Pao
de Arcos, Portugal, personal communication, 2002). Thus,
the ancient cultivations in northern Antioquia would have
been planted near to or even amongst T. gileri populations
and exposed to frosty pod inoculum, with inevitable
adaptation to the new host through sexual selection. The
added selection pressure of drier microclimates in the
cocoa plantations, compared with the buffered humid
2003 BSPP Plant Pathology (2003) 52, 476 485
 Frosty pod rot of cocoa
forests, may have contributed to the gradual loss of the
thinner-walled, ellipsoidal spores and the increased pro-
duction of thick-walled, globose spores which distinguish
cocoa isolates from those of T. gileri. It is considered that
these morphological characters, in addition to the molec-
ular and pathogenicity differences presented here, justify
taxonomic recognition at the varietal level as follows:
Crinipellis roreri (Ciferri) H. C. Evans var. gileri H.C.
Evans & K.A. Holmes var. nov. (Figs 1a and c, and 2bh)
Etym: derived from its host, Theobroma gileri.
Meiosporae catenulata, ellipsoidea vel globose, 80 160
(220) 55110 m, plerumque tenuitunicata. Evans HC, 2002. Invasive neotropical pathogens of tree crops.
Holotypus: DIS 116, in Theobroma gileri (Sterculiaceae)
in silvestri primiario, lectus in Guadual, Esmeraldas
Provincia, in Ecuador, 14 September 1999.
Material examined: holotype DIS 116 ex Theobroma gileri
(Sterculiaceae), in primary forest, Guadual, Esmeraldas
Province, Ecuador, 550 m asl, 14 September 1999;
paratypes DIS 329 and 331, same host and locality, 5
November 2001.
This variety is distinguished from C. roreri var. roreri
by the significantly greater proportion of thin-walled, ellip-
soidal spores, which raises the mean length from 8 m in
var. roreri, range 5512 m, to 1112 m in var. gileri, Gardes M, Bruns TD, 1993. ITS primers with enhanced
range 8 22 m. In addition, var gileri is nonpathogenic
to cocoa and differs in its genetic profile.
Acknowledgements
This project was carried out within USDA-ARS Alterna-
tive Crops Programme (project no. 0500-00008-001-21).
We would like to thank Dr Carmen Suarez and staff at
the Estacin Experimental Tropical Pichilingue, Ecuador,
for invaluable assistance with the field experiment.
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