Diversity and evolutionary relationships of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the Central Andean region of Colombia

Nematology, 2007, Vol. 9(3), 333-341

Diversity and evolutionary relationships of entomopathogenicnematodes (Steinernematidae and Heterorhabditidae)

from the Central Andean region of Colombia

Juan Carlos LÓPEZ-NÚÑEZ 1, Liliana CANO 1,Carmenza E. GÓNGORA-B. 1 and S. Patricia STOCK 2,∗

1 Disciplina de Entomología, Centro Nacional de Investigaciones de Café (Cenicafé), Cenicafé Planalto, Kilómetro 4,Vía Antigua a Manizales, Chinchiná, Caldas, Colombia

2 Department of Entomology, The University of Arizona, Forbes 410, 1140 E. South Campus Drive, Tucson,AZ 85721-0036, USA

Received: 12 December 2006; revised: 8 February 2007Accepted for publication: 9 February 2007

Summary – A survey of entomopathogenic nematodes (EPN) was conducted in the Central Andean region of Colombia. Out of a totalof 945 soil samples, 28 (3%) were positive for entomopathogenic nematodes. Of them, 26 samples (92.8%) contained Steinernema andtwo (7.2%) harboured Heterorhabditis isolates. Characterisation of the recovered isolates was done by analysis of rDNA sequences.The large subunit of rDNA (28S) was considered for diagnosis of Steinernema species whereas the internal transcribed spacer region(ITS) of rDNA was used to identify Heterorhabditis isolates recovered in this study. Five Steinernema spp. were isolated and identifiedas S. websteri and four new, undescribed species. Steinernema websteri was isolated from two sites: Naranjal and Paraguaicito stations.At Naranjal, this species was recovered from a coffee plantation and a forest habitat surrounding these plantations. At Paraguaicito, thesame species was recovered from a coffee-guamo habitat. Steinernema sp. 1 was collected from nogal cafetero (Cordia allaidora) grovesand forest habitats at Paraguaicito station. Steinernema sp. 2 was found in coffee-guamo habitats both at Naranjal and Paraguaicitostations. Steinernema sp. 3 was the only EPN species recovered at Santa Barbara station in association with plantain. This specieswas also found at La Catalina station in association with forest habitats and also from soil samples taken from a landscaped area withHeliconia sp. and grass cover (Arachis pintoi) at Cenicafé La Granja station and a coffee-guamo habitat at Naranjal station. Steinernemasp. 4 was found at La Catalina in a forest habitat, also associated with Steinernema sp. 3 (isolate JCL002) and Heterorhabditis sp. 1(isolate JCL 003). The Heterorhabditis isolates were identified as two undescribed Heterorhabditis spp. Heterorhabditis sp. 1 (isolateJCL003) was recovered at La Catalina station associated with a forest habitat. Heterorhabditis sp. 2 (isolate JCL040) was collectedfrom a coffee-guamo habitat at Naranjal station. Although limited to one geographic region, this study suggests entomopathogenicnematodes in Colombia are quite diverse and are perhaps widely distributed in this country. A more intensive survey covering allgeographic regions is currently underway.

Keywords – biological diversity, Colombian Andes, Heterorhabditis, phylogeny, Steinernema.

Entomopathogenic nematodes (EPN) of the generaSteinernema Travassos and Heterorhabditis Poinar areobligate pathogens that infect a wide range of soil insects(Kaya & Gaugler, 1993). EPN provide an environmen-tally-safe and economical alternative for the control of awide range of arthropod pests (Grewal & Georgis, 1998;Grewal et al., 2005). Progress in the development of EPNas biological control agents has been outstanding. Effi-cient in vitro mass production techniques involving liq-uid media (Ehlers, 2001) and solid substrates (Bedding,

∗ Corresponding author, e-mail: [email protected]

1984), and formulation (Grewal, 2002) have been devel-oped. Advances in production and application technologyhave led to the use of these nematodes in citrus groves,ornamentals (nurseries and glasshouses), turf, strawberryplantations and cranberry bogs (Grewal & Georgis, 1998).Because of these reasons, interest in EPN has increasedrapidly in recent years and research with these benefi-cial organisms is being conducted in approximately 100laboratories representing 60 countries worldwide (Stock,2005). Numerous surveys provide evidence of the om-

© Koninklijke Brill NV, Leiden, 2007 333Also available online - www.brill.nl/nemy

J.C. López-Núñez et al.

nipresence of these entomopathogens in natural and agri-cultural soils (Hominick, 2002), and new species and iso-lates are described every year. More importantly, many ofthese indigenous species appear to possess unique traitsthat enable their survival in unique environments, andthese traits can be exploited in novel pest managementstrategies.

At present, only a few studies have focused on docu-menting the diversity of EPN in South America (Stock,1995; Caicedo & Belloti, 1996; Aguilera, 2002; Caicedoet al., 2004; Leite, 2005). In Colombia, information onthe presence of indigenous EPN and their symbiotic bac-teria is scarce. Caicedo and Belloti (1996) reported thepresence of Heterorhabditis bacteriophora Poinar associ-ated with the cassava bug, Cyrtomenus bergi (Hemiptera:Cydnidae). In another study several Steinernema and He-terorhabditis species were reported from in the Cundina-marca sabana (Parada, unpubl.). However, identity of thespecies encountered in this study is questionable and re-quires a thorough taxonomic examination. More recently,Melo et al. (2004) surveyed for entomopathogenic nema-todes in Colombia and Panama, reporting the presence ofuncharacterised Steinernema and Heterorhabditis spp.

Colombia encompasses an area of more than 1.1 mil-lion km2. It is the only country in South America withboth Caribbean and Pacific coastlines (Fig. 1). Four ge-ographic regions characterise this country: the Andeanhighlands, consisting of the three Andean ranges (East,Central and West Andes) and intervening valley lowlands;the Caribbean lowlands coastal region; the Pacific low-lands coastal region, separated from the Caribbean low-lands by swamps at the base of the Isthmus of Panama;and eastern Colombia, the great plain that lies to theeast of the Andes Mountains (Zambrano-Pantoja, 1998).These geographic regions are quite diverse in climateand ecosystems, placing Colombia among the world’s topfive countries for biodiversity (Zambrano-Pantoja, 1998).Among these geographic regions, the Andean highlandsare considered a hotspot for biodiversity and endemismof global significance (Myers, 2000). Moreover, in thisregion, agricultural practices are quite diverse and blendin with natural habitats such as coniferous and decidu-ous forests. Coffee is the most important commercial crop,with approximately 560 000 coffee-growing families cul-tivating 2.0 million acres (ca 800 000 ha) and producing720 000 tons of coffee, equivalent to approximately 12%of the worldwide coffee market (Ramirez et al., 2002;Federación Nacional de Cafeteros, 2006). Next in impor-tance are bananas, flowers, sugarcane, oil palm and cit-

Fig. 1. Map of Colombia showing Central Andean region andsampling sites departments. 1. Risaralda, 2. Caldas, 3. Cund-inamarca, 4. Quindío. Steinernema websteri: ; Steinernema

sp. 1: ; Steinernema sp. 2: ; Steinernema sp. 3: ; Steiner-

nema sp. 4 ; Heterorhabditis sp. 1 ; Heterorhabditis sp. 2:.

rus. Other crops for domestic consumption are corn, rice,sorghum, potatoes, manioc, tobacco, cacao and vegeta-bles.

As in most Latin American and Caribbean countries,chemical pesticides in Colombia have been, and still are,of importance for control of agricultural and forest pests.However, in recent years, public awareness of the prob-lems developed from the use of chemical pesticides (i.e.,adverse impact on human health, wildlife, environmen-tal pollution, pesticide resistance and pest resurgence) hasmade farmers and growers become more receptive to con-sidering natural enemies and entomopathogens for con-trol of pests of agricultural and forestry significance. Withthese considerations in mind, a survey for native EPNin the Central Andean highlands was conducted. In thisstudy, and for the first time, the diversity, distribution and

334 Nematology

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evolutionary relationships of Colombia-native EPN are re-ported.

Materials and methods

SAMPLING SITES AND SAMPLING STRATEGY

A selection of crops and natural habitats in the west-ern slopes of the central Andean highlands was consid-ered for this survey and five experimental stations (sam-pling sites) of the National Federation of Coffee Growersof Colombia (Cenicafé) were used. Sampling was donebetween August 2003 and December 2004. The experi-mental stations are located in four different departments:Caldas, Quindío, Risaralda and Cundinamarca (Table 1).Each of the sampling sites represented a particular ‘cof-fee ecotype’ with known topography, soil characteristicsand climate (Table 1) (Gomez et al., 1991). The fol-lowing crops were considered: coffee (Coffea arabicavar. Castillo® and var. Caturra), plantain (Musa spp.),citrus (Citrus sinensis), corn (Zea mays), macadamia(Macadamia integrifolia and M. tetraphyla) and guamo(Inga edulis, I. spectabilis and I. densiflora). Patches ofpremontane humid forests surrounding these crops werealso sampled. Typical vegetation of these natural habitatsincluded: nogal cafetero (Cordia alliodora), quiebabar-rigo (Trichanthera gigantean), zurrumbo (Trema micran-tha), bore (Alocasia macrorrhiza), chusque (Chusqueaspp.), pine groves (Pinus oocarpa), pizamo (Erythrinafusca) groves, and a secondary growth guadua (Guaduaangustifolia) woodland (at least 10 years old). Addition-

ally, a landscaped area with Heliconia spp. and grass cover(Arachis pintoi) was also sampled at Cenicafe/La Granja).

At each site, a total of 20-50 samples, at least 10 mapart, were taken. Each soil sample (approximately 2 kg)consisted of a composite of five random sub-samplestaken in a 2 m2 area at a depth of 0-20 cm. Samples wereplaced in polyethylene bags to prevent water loss and keptin coolers (at ca 15◦C) during transit to the laboratory.A total of 945 samples (Naranjal 250, La Catalina 250,Paraguaicito 260, Santa Barbara 165, La Granja Cenicafe20) were taken. For each EPN-positive sample, a portionof the soil (ca 250 g) was analysed for physical (texture)and chemical characteristics (i.e., pH, organic matter andnitrogen content). Soil analysis was done at the MultilabAgroanalitica Soil Laboratory, Cenicafé.

NEMATODE RECOVERY AND PROPAGATION

EPN were recovered from soil samples by the modi-fied insect baiting technique described by Bedding andAkhurst (1975). Samples were thoroughly mixed andtap water (amount depending on the dryness of thesample) was added to moisten the soil. Ten last-instarGalleria mellonella (L.) larvae (Lepidoptera: Pyralidae)were placed in 300 ml plastic containers (five contain-ers/sample) with soil obtained from each sample. Contain-ers were covered with a lid, turned upside down and keptat room temperature (23 ± 2◦C). After 7 days G. mel-lonella larvae were recovered, and parasitised cadavers,recognised by a change in colour (usually red/purplefor heterorhabditids, and ochre/brown/black for steiner-nematids), were placed in modified White traps (Kaya& Stock, 1997) to allow the infective-stage juveniles to

Table 1. Sampling sites: geographic location, climate characteristics (annual precipitation and temperature) and coffee ecotypes.

Department Latitude Longitude Altitude Annual Mean annual Relative Coffeeprecipitation temperature humidity ecotype**

(mm) (◦C)* (%)*

Risaralda1 04◦45′ 75◦44′ 1321 1800-2046 21.7 73.8-74.7 209ACaldas2 05◦00′ 75◦36′ 1310 2293-2800 21.6 78.2-81.2 206ACaldas3 04◦58′ 75◦39′ 1400 2400-2887 21.4 72.8-74.3 206AQuindío4 04◦24′ 75◦44′ 1203 1700-2200 21.1 78.9-81.0 211A

Cundinamarca5 04◦56′ 74◦25′ 1478 1800-2427 20.1 76.6-78.3 311A

* Values of temperature and relative humidity are annual averages. Data from Federación Nacional de Cafeteros de Colombia (Cenicafé2004, 2006a, b).** From Gomez et al. (1991).County – station: 1Pereira – La Catalina; 2Chinchiná – Cenicafé/La Granja; 3Chinchiná – Naranjal; 4Buenavista – Paraguaicito;5Sasaima – Santa Barbara.

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J.C. López-Núñez et al.

emerge. Emerging nematodes were pooled from the modi-fied White traps and were thoroughly rinsed to remove anyinsect debris. They were used to infect fresh G. mellonellalarvae to confirm Koch’s postulates for pathogenicity andfor future identification and establishment of cultures.A second baiting round was done by placing fresh G. mel-lonella into the same soil. All nematode cultures werestored in 250 ml tissue culture flasks at 10-15◦C followingprocedures described by Kaya and Stock (1997).

NEMATODE IDENTIFICATION

Morphological characterisation

A preliminary morphological diagnosis (i.e., IJ bodysize and male spicule/gubernaculum morphology) of therecovered isolates was done to help sort them, accord-ing to their morphological characteristics, into simi-lar species-groups. Morphological observations followedtaxonomic criteria suggested by Stock and Kaya (1996)and Hominick et al. (1997). Briefly, ten first generationmales and ten IJ were randomly selected from differentG. mellonella cadavers. Nematodes were examined live orheat-killed in 60◦C Ringer’s solution. The heat-killed ne-matodes were placed in triethanolamine-formalin (TAF)fixative (Kaya & Stock, 1997) and processed to anhy-drous glycerin for mounting (Seinhorst, 1959). Observa-tions were made on live and mounted specimens usingan Olympus BX51 microscope equipped with differen-tial interference contrast optics and digital image software(AnalySIS Image software, Soft Imaging System, Lake-wood, CO, USA).

Molecular characterisation and phylogeneticassociations

All isolates were molecularly characterised by analysisof rDNA sequences. Two nuclear genes, the large subunit(LSU) and internal transcribed spacer region (ITS) wereconsidered for steinernematids and heterorhabditids, re-spectively. DNA extraction, PCR conditions and sequenc-ing followed methods described by Stock et al. (2001).The resulting sequences were compared to a library ofmore than 40 EPN species (Stock Lab Database, Univer-sity of Arizona, Tucson, AZ, USA). Phylogenetic analyses(maximum parsimony analysis) of LSU and ITS sequencedata was done using PAUP* v 4.0b (Swofford, 2001) fol-lowing criteria described by Stock et al. (2001) and Stockand Gress (2005).

Results

SAMPLING RESULTS AND SPECIES DIVERSITY

From a total of samples (945), 28 (3%) were EPNpositive. Of these, 26 samples contained steinernematids(92.86%) and two (7.14%) held heterorhabditids. EPN-positive samples were distributed at each sampling site asfollows: Naranjal with 28.6%, La Catalina with 10.7%,Paraguaicito with 50%, Santa Barbara with 3.6%, Ceni-cafe La Granja 7.1%. EPN recovery varied also amongthe different crops, agro-forest and forest groves (Table 2).Soil characteristics of each EPN positive are showed inTable 2. The heterorhabditid isolates were identified astwo undescribed species of Heterorhabditis. Overall mor-phology of these isolates resembles that of species in theindica-group, with an average IJ body length of less than550 µm (Stock & Hunt, 2005). Heterorhabditis sp. 1 wasrecovered at La Catalina station in a forest habitat at over1300 m elevation. The soil at this site was acidic withlow organic matter content (Table 2). Heterorhabditis sp.2 was isolated at Naranjal station in a coffee plantationsurrounded by guamo trees at 1400 m elevation. At thissite the soil was more acidic than at La Catalina stationand was richer in organic matter content (Table 2).

Preliminary morphological observations (IJ body sizeand male spicule/gubernaculum morphology) help placedisolates into different species groups. Isolates JCL001,JCL002, JCL004, JCL008 and JCL027 presented mor-phological similarities in their IJ body length and malespicule morphology. However, isolate JCL001 was de-picted as a unique taxon based on sequence data (seebelow). Isolates JCL007, JCL009, JCL010, JCL013 andJCL014 were very similar to each other and shared sim-ilar IJ body length to that of species in the bicornutum-group. Isolates JCL006, JCL015, JCL030 and JCL032showed morphological similarities in the IJ body lengthand male spicule and gubernaculum morphology to that ofS. websteri. Isolates JCL 011, JCL012, JCL025, JCL016,JCL018, JCL019, JCL020, JCL021, JCL022, JCL024 andJCL025 showed similarities in the examined morpholo-gical traits (IJ body length and male spicule morphology)with each other.

Interpretation of 28S rDNA sequences indicated thesteinernematid isolates represent five species, includingS. websteri Stock & Cutler and four new undescribedtaxa, referred to as Steinernema sp. 1, Steinernema sp. 2,Steinernema sp. 3 and Steinernema sp. 4.

Isolates JCL006, JCL015, JCL030 and JCL032 wereidentified as S. websteri. Isolates JCL006 and JCL030 and

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Table 2. EPN positive sites: soil types, habitat characteristics and EPN species diversity.

Station Soil type pH Organic N Clay:silt: Vegetation EPN-positive Isolate EPN GenBankmatter sand samples/ code Accession

content total no. Number(%) of samples

La Catalina Chinchiná Unit(Melanudands),Volcanic ashes

5.0-5.4 12.2-14.8 0.47-055 16-18:38-40:44-52

Forest 3/50 JCL001 Steinernemasp. 4

EF187019

JCL002 Steinernemasp. 3

EF187020

JCL003 Heterorhabditissp. 1

EF217327

CenicafeLa Granja

Chinchiná Unit(Melanudands),Volcanic ashes

4.5-4.7 14.9-17.1 0.55-0.61 13-18:26-30:57-61

Landscaped 2/20 JCL004, Steinernemasp. 3

EF187021,Heliconiasp. andArachispintoi

JCL005 EF187022

Naranjal Chinchiná Unit(Melanudands),Volcanic ashes

3.9-5.2 13.5-19 0.51-0.65 10-17:23-33:54-66

Forest 1/50 JCL006 Steinernemawebsteri

EF217323

Coffee 2/50 JCL030,JCL032

S. websteri EF217325,EF217326

Coffee-Guamo

5/50 JCL007, Steinernemasp. 2

EF187023

JCL008, Steinernemasp. 3

EF187024

JCL009,JCL010

Steinernemasp. 2

EF187025,EF187026

JCL040 Heterorhabditissp. 2

EF217328

Paraguaicito Tablazo, Cas-carero, GenovaUnits (Tro-porthents andDystropepts)Chinchiná andMontenegroUnits(Melanudands),Volcanic ashes

4.4-5.9 5.3-10.8 0.24-0.43 10-21:14-24:63-72

Forest 2/50 JCL011,JCL012

Steinernemasp. 1

EF187027,EF187028

Coffee-Guamo

3/50 JCL013,JCL014,JCL015

Steinernemasp. 2

EF187029,EF187030

S. websteri EF217324

Coffee-NogalCafetero

9/50 JCL016,JCL017,

Steinernemasp. 1

EF194278,EF194279,

JCL018,JCL019,

EF194280,EF194281,

JCL020,JCL021,JCL022,JCL024,JCL025

EF194282,EF194283,EF194284,EF194285,EF194286

SantaBarbara(Sasaima)

Villeta Unit 4.5 15.8 0.57 35:25:40 Plantain 1/20 JCL027 Steinernemasp. 3

EF217322

(Troporthents,Dystropeptsand Eutropepts)Volcanic ashes

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J.C. López-Núñez et al.

JCL032 were recovered at Naranjal station from a forestand coffee habitats, respectively. Isolate JCL015 was alsorecovered from a coffee plantation surrounded by a guamogrove at Paraguaicito station. At both sites the pH of thesoil was acidic and with moderate organic matter content(Table 2).

Steinernema sp. 1 was isolated at Paraguaicito station.A total of nine isolates representing this species wererecovered from a nogal cafetero. Two additional isolateswere collected in a forest habitat. Soil characteristics forthese positive samples are listed in Table 2.

Steinernema sp. 2 was found at Naranjal and Paraguai-cito stations. All isolates representing this species werefound in coffee-guamo habitats at both stations.

Steinernema sp. 3 was the most ubiquitous EPN en-countered, being recovered at four out of the five Ceni-cafe stations: Catalina, Cenicafe/La Granja, Naranjal andSanta Barbara. The habitats where this species was col-lected were also quite diverse and included forest (LaCatalina), a landscaped area with Heliconia sp. and grasscover (Arachis pintoi), coffee-guamo (Naranjal) and plan-tain (Santa Barbara).

Steinernema sp. 4 (isolate JCL001) was found at LaCatalina station in a forest habitat and in association withSteinernema sp. 3 (isolate JCL002) and Heterorhabditissp. 1 (isolate JCL003).

NEMATODE DIAGNOSIS AND PHYLOGENETIC

RELATIONSHIPS

Heterorhabditis sp. 1 has morphological similarities tospecies in the bacteriophora-group in the IJ body size (av-erage 580 µm) and male tail (number and arrangement ofbursal rays) and spicule morphology. Analysis of ITS se-quences also placed this taxon close to H. bacteriophora.

Isolate JCL040 was depicted as a new Heterorhabdi-tis sp. (Heterorhabditis sp. 2). IJ of this species resemblemost taxa in the indica-group in their body length (av-erage: <550 µm). Comparison of ITS sequences of thisnematode also placed it as more closely related to H. bau-jardi, itself a member of the indica-group (Fig. 2).

Morphological examination of isolates JCL006,JCL015, JCL030 and JCL032 indicated that these isolateshave morphological similarities to S. websteri. Moreover,analysis of 28S rDNA sequences for these isolates gener-ated identical sequences to that of the type isolate of S.websteri.

Steinernema sp. 1 was represented by 11 isolates(JCL011, JCL012, JCL016, JCL017, JCL018, JCL019,JCL020, JCL021, JCL022, JCL024, JCL025). This

Fig. 2. Strict consensus of ITS-1 rDNA sequences tree showingphylogenetic relationships of Heterorhabditis spp. Tree length =783, Consistency index (excluding uninformative characters) =0.75. Numbers above the node indicate bootstrap values.

species most resembles species in the feltiae-group, al-though morphological differences exist in both adult andjuvenile stages and warrant further study.

Isolates JCL007, JCL009, JCL010, JCL013 andJCL014 generated identical 28S rDNA sequences to eachother, and were grouped together as a new species,Steinernema sp. 2. This species seems to be related to thebicornutum-clade although this association was not wellsupported by bootstrap resampling (Fig. 3).

Steinernema sp. 3 encompasses five isolates (JCL002,JCL004, JCL005, JCL008, JCL027) with morphologicaltraits (i.e., IJ body size) that place them as closelyrelated to the carpocapsae-group. Analysis of 28s rDNAsequences also supports these observations.

Isolate JCL001 was depicted a Steinernema sp. 4.This species also resembles the carpocapsae-group andanalysis of sequence data placed this isolate as a closerelative of Steinernema sp. 3.

A further and detailed morphological analysis, in-cluding DIC and SEM microscopy, together with cross-hybridisation tests is currently underway to formally de-scribe all new EPN species recovered in this study.

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Fig. 3. Strict consensus of 28S rDNA sequences tree showing evolutionary relationships of Steinernema spp. Tree length = 1200,Consistency index (excluding uninformative characters) = 0.57. Numbers above the node indicate bootstrap values.

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Discussion

The present study recorded for the first time the occur-rence of indigenous EPN species in Colombia. Nematodeswere recovered from all five Cenicafe stations sampled.These stations are quite distinct in their coffee ecotypesand habitat diversity. Although EPN were recovered fromonly 3% (28 out of 945) of all samples taken, the recov-ery of two Heterorhabditis and five Steinernema speciesclearly shows the incredible richness of EPN species inthis region. Furthermore, this study highlights the im-portance of conducting a more intensive EPN survey inother natural areas and geographic regions of this coun-try. Based on these results we think it likely that moreindigenous new species will be discovered in future sur-veys, thereby adding significant information to the diver-sity and biogeography of this group of nematodes. Thesenative isolates may prove more effective in controlling lo-cal pests than exotic EPN isolates. Important agriculturalpests such as the coffee-berry borer (Hypothenemus ham-pei (Ferrari)), the corn earworm (Spodoptera frugiperdaSmith), palm weevils (Cyparissius daedalus Craemer;Sagalassa valida Walker), banana weevil (Cosmopolitessordidus Germ.), white grubs (Euetheola spp., Anomalaspp., Phyllophaga sp., Ancognatha spp.), cassava bug(Cyrtomenus bergi Froeschner) and other pests of eco-nomically important crops in this region may be more sus-ceptible to these native EPN, providing the opportunity todevelop and implement these entomopathogens in biolog-ical control or integrated pest management programmes inthis country and/or other countries of the Andean region.Moreover, implementation of EPN as bioinsecticides mayhelp protect biodiversity of natural ecosystems adjoiningcrops and help to preserve the environment of these fragileecosystems.

Acknowledgements

This study was funded in part by a Colombia Ministryof the Environment grant (Project ENT1801), and TheNational Federation of Coffee Growers of Colombia, Cof-fee Research Centre (Cenicafe). We acknowledge Ceni-cafe research leaders G. Cadena-Gómez, A.E. Bustillo, P.D. Rodríguez and C. Quintero for their support on theEPN project. We also thank Cenicafé station managersJ.C. García, C. Arboleda and J.D. Arias, and advisors M.Cristancho and A. Gaitan, for their technical assistanceand collaboration during the survey.

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