Australasian Plant Pathol. (2013) 42:515–523 DOI 10.1007/s13313-013-0205-3 Identification and genetic diversity of Rosellinia spp. associated with root rot of coffee in Colombia Bertha L. Castro & Angela J. Carreño & Narmer F. Galeano & Jolanda Roux & Michael J. Wingfield & Álvaro L. Gaitán Received: 14 June 2012 / Accepted: 21 February 2013 / Published online: 3 July 2013 # Australasian Plant Pathology Society Inc. 2013 Abstract The genus Rosellinia includes species that cause root rot on a wide range of herbaceous and woody hosts. In Colombia, these fungi cause serious diseases of potato, forest and fruit trees, as well as coffee plants. The aim of this study was to identify isolates of Rosellinia collected from coffee and other hosts using DNA sequence comparisons of the internal transcribed spacer (ITS) region. Pathogenicity tests were conducted on coffee seedlings to confirm the role of the collected species in coffee root disease. Twenty six isolates were obtained and these were grouped into two clades representing R. bunodes and R. pepo. Isolates from Coffea arabica, Hevea brasiliensis, Macadamia integrifolia, Psidium guajava and Theobroma cacao were identified as R. pepo, while R. bunodes was obtained only from coffee plants. Low levels of genetic variability were observed among isolates of the two species. Pathogenicity tests on coffee with R. bunodes resulted in 98 % seedling death in an average of 10 days, while R. pepo killed 54 % of inoculated seedlings in an average of 16 days B. L. Castro (*) : J. Roux : M. J. Wingfield Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Hatfield, Pretoria 0028, South Africa e-mail: Berthalucia.castro@up.ac.za B. L. Castro e-mail: Berthal.castro@cafedecolombia.com A. J. Carreño : N. F. Galeano : Á. L. Gaitán National Coffee Research Center (CENICAFE), Manizales, Colombia A.A. 2427 URL: www.cenicafe.org confirming the compatibility of both species with this host. Pathogen characterization will be useful for further research in disease diagnosis, soil recovery and breeding for resistance. Keywords Coffea arabica . ITS . Phylogeny . Rosellinia bunodes . Rosellinia pepo . Soil-borne pathogens Introduction Based on symptoms and morphological characteristics, two species of Rosellinia are known in Colombian coffee growing areas, Rosellinia bunodes (Berk & Brome) Sacc., which causes a disease known as black root rot, and R. pepo Pat. causing stellate root rot (Fernández and López 1964; Castro and Esquivel 1991). Other than in Colombia, these pathogens are known to affect coffee in Africa (Saccas 1956), Brazil (Ponte 1996), Costa Rica (Bautista and Salazar 2000), Cuba (Herrera 1989), El Salvador (Procafé 1996), Guatemala (Hernández 1967) and Puerto Rico (Garcia 1945), while R. arcuata Petch, and/or R. bunodes are mentioned infecting coffee in India (Sivanesan and Holliday 1972; Muthappa 1977; Kannan 1995). Many Rosellinia spp. are saprophytes, some live endophytically and occasionally become pathogenic, and some species are well-known root pathogens on commercially grown plants such as potato (Guerrero 1990) and woody perennial trees in tropical and sub-tropical areas globally (Petrini and Petrini 2005; Ten Hoopen and Krauss 2006). Among the best known root pathogens are R. necatrix Berl.: Prill and R. desmazieresii (Berk. & Br.) Sacc. (= R. quercina 516 Hart), mostly known from temperate climates causing diseases on pear, apple and grape in Japan (Eguchi et al. 2009; Takemoto et al. 2009a, b), on avocado in Spain (LópezHerrera 1998; Pliego et al. 2012) and in Argentina on peach, plum, apple, pear, grapevine and other hosts (Sarasola and de Sarasola 1975). Rosellinia bunodes, R. pepo and R. arcuata, are known only from the tropics (Kannan 1995; Ten Hoopen and Krauss 2006). Various other Rosellinia spp. that infect coffee were mentioned by Saccas (1956), e.g. R. coffeae Sacc., R. didolotii Sacc, R. echinocarpa Sacc, R. lobayensis Sacc., R. mastoidiformis Sacc. and R. megalospora Sacc., but very little is known about these species. Rosellinia bunodes and R. pepo occur in the soil as saprophytes (Aranzazu 1996). After infection of suitable living hosts, patches of dying plants extend in a circular pattern due to the pathogen’s spread through root contact or via mycelial aggregations (Fernández and López 1964; Merchán 1988; Aranzazu 1996; Bautista and Salazar 2000). Many shade and fruit trees grown in association with coffee (e.g. Inga sp., Leucaena sp., Erytrina sp., Cordia alliodora (Ruiz & Pav.) Oken, Tabebuia rosea DC, Cedrela odorata L., Alnus acuminata Kunth) are susceptible to infection by Rosellinia spp. and are thought to provide initial sources of inoculum for coffee tree infection (Bermúdez and Carranza 1990; Aranzazu 1996; Castro and Serna 2009). In addition, debris of cassava (Manihot esculenta Crantz ) left in the soil after co-cultivation with coffee has been mentioned as increasing the survival of the pathogen and thus damage due to subsequent Rosellinia infection in Colombian coffee growing areas (Castro and Serna 2009). Infection of coffee plants by Rosellinia spp. results in chlorosis, wilt, die-back and death of plants. This may occur within a few weeks in the case of seedlings or young plants in the field or take up to three or four years after infection in the case of adult plants (Fernández and López 1964; Castro and Esquivel 1991; Ibarra et al. 1999). Important economic losses have been recorded by Castro and Serna (2009) in Colombian coffee growing areas. The diseases caused by Rosellinia spp. are also known to be difficult to control and numerous integrated measures have been investigated, with variable results (Merchán 1988; Ten Hoopen and Krauss 2006; Gutiérrez et al. 2006). Barceló-Muñoz et al. (2007), implemented a program aimed at selecting avocado rootstocks tolerant to white rot caused by R. necatrix in Spain. However, little research on resistance to tropical Rosellinia spp. has been published (Ten Hoopen and Krauss 2006). The genus Rosellinia belongs to the family Xylariaceae (Class Euascomycetes, subclass Pyrenomycetes, order Sphaeriales, syn. Xylariales) and includes more than one hundred species (Pliego et al. 2012). Teleomorphic structures, such as ascospore morphology, are considered valuable taxonomic characters for the identification of Rosellinia spp. (Pérez-Jiménez et al. 2003; Petrini and Petrini 2005; Takemoto et al. 2009a, b; Pliego et al. 2012). However, in B.L. Castro et al. tropical areas, stromata bearing fruiting bodies are rarely found in nature, making the identification of Rosellinia spp. reliant on characteristics of the anamorph (Dematophora) (Fernández and López 1964; Bermúdez and Carranza 1992; Ibarra et al. 1999; Realpe et al. 2006). A major diagnostic character at the generic level has been the presence of pearshaped swellings at the septa of the hyphae (Saccas 1956; Sarasola and de Sarasola 1975; Pérez-Jiménez 2006; Pliego et al. 2012). At the species level, R. pepo and R. bunodes have been distinguished based on the mycelial aggregates formed on the roots. R. pepo produces grayish cobweb-like strands, which become black and coalesce into a woolly mass. Beneath the bark, white, star-like fans can be observed macroscopically. Rosellinia bunodes shows black branching strands firmly attached to the roots, forming black dots and lines embedded in the tissues (Waterston 1941; Fernández and López 1964; Ibarra et al. 1999; Realpe et al. 2006). Rosellinia species have mostly been characterized based only on morphology (Petrini and Petrini 2005), with only limited DNA sequence data available for species in the genus. However, in the last decade, molecular tools have provided important means to elucidate genetic variation and phylogenetic relationships among global members of the Family Xylariaceae (Bahl et al. 2005; Peláez et al. 2008; Hsieh et al. 2010; Pliego et al. 2012). Sequencing of the internal transcribed spacer regions (ITS), fragments of the β-tubulin (BT), adenosine triphosphatase (ATP) and translation elongation factor 1α (TEF) gene regions and random amplified polymorphic DNA (RAPD) amplifications have mostly been used for identification of R. necatrix (López et al. 2008; Takemoto et al. 2009a, b). ITS Scorpion primer pairs have been successfully developed for large-scale detection of R. necatrix by realtime Scorpion- polymerase chain reaction (PCR) in soils and in plant materials (Schena and Ippolito 2003; Ruano-Rosa et al. 2007), and recently Takemoto et al. (2011) developed a species-specific PCR diagnostic for R. necatrix and R. compacta Takemoto in Japan. At the population level, inter-simple sequence repeat (ISSR) markers have been used to study R. necatrix diversity in Cyperus esculentus L. (Armengol et al. 2010). However, there is still a lack of information on tropical Rosellinia spp. that cause damage to commercially propagated plants such as coffee. López (2004), made a first attempt to study the genetic variability of R. bunodes and R. pepo from coffee, cocoa and potato in Colombia, using ITS and RAPD sequences and mentions high variability in these species. The primary aim of this study was to identify the species of Rosellinia damaging coffee and other associated plants in the Central Colombian coffee growing area. A pathogenicity test through artificial inoculation was carried out to confirm compatibility of the species with coffee. Identification and genetic diversity of Rosellinia spp. Materials and methods Sample collection and fungal isolation During 2008 and 2009, samples were collected from plants showing macroscopic signs of root rot caused by Rosellinia spp. Plant hosts sampled included coffee (Coffea arabica L.), macadamia (Macadamia integrifolia Maiden & Betche), rubber (Hevea brasiliensis Müll. Arg.), cocoa (Theobroma cacao L.) and guava (Psidium guajava L.). The areas sampled were located in the central coffee growing area of Colombia and included the Caldas, Risaralda and Quindío Provinces. Samples were selected and preliminary identifications were made based on in situ macroscopic observation of symptoms and signs as described by Fernández and López (1964) and Realpe et al. (2006). Plants thought to have root rot were identified based on external symptoms, including wilting, yellowing or dead trees. For fungal isolations, small segments (4–5 cm) were removed from fresh roots of symptomatic plants and placed in 2 % NaClO for 15 min, rinsed in sterile water and dried as described by López (2004) and Realpe et al. (2006). Small pieces of tissue, including fungal mycelium, were removed from the root sections and transferred to 2 % Malt Extract Agar (MEA), (Oxoid), pH 5.7, containing thiamine (100 μg/l) and antibiotic (100 mg/l rifampicin). Six to seven pieces were transferred to each Petri dish and the plates incubated at 24 °C for 3 to 4 days in the dark. Resultant colonies were transferred to fresh medium to obtain pure isolates, which were distinguished by the pear-shaped swellings at the septa, characteristic of Rosellinia spp. (Saccas 1956; Realpe et al. 2006; Pérez-Jiménez 2006; Pliego et al. 2012). Isolates were stored in liquid nitrogen (−196 °C) using the technique described by Ten Hoopen et al. (2004). DNA extraction, amplification and sequencing Twenty six isolates, identified as possible Rosellinia spp. based on morphology, and representing each of the hosts and areas sampled, were selected for characterization using DNA sequence comparisons. For each isolate, small pieces of mycelium from 15-day-old cultures were transferred to 100 ml Erlenmeyer flasks containing 100 ml Sabouraud medium (peptone-glucose-yeast extract). Flasks were incubated at 27 °C, for 8 days in darkness, with continuous shaking at 150 rpm. The resultant mycelium was harvested by filtration through Whatman No.1 filter paper and DNA was extracted using the method of Lee and Taylor (1990). Resultant DNA was diluted 20-fold with distilled water and stored at −20 °C until further use. Amplification of the ITS1, 5.8S and ITS2 nuclear gene regions of the ribosomal RNA operon was performed for 26 isolates as described by Hillis et al. (1996) using the primers 517 ITS1 (5′TCC GTA GGT GAA CCT GCG G3′) and ITS4 (5′ TCC TCC GCT TAT TGA TAT GC 3′) (White et al. 1990). PCR reactions consisted of 1.25 U of Taq polymerase (Promega, Southhampton, UK), 2 mM MgCl2; 0.2 mM dNTPs; 1X PCR Buffer; 0.2 μM of each Primer (ITS1, ITS4) and 100 ng of template DNA. Reactions were conducted with an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 55 °C and 2 min at 72 °C. A final elongation step was at 72 °C for 5 min. PCR products were separated on a 1.5 % agarose gel and stained with 1 μl ethidium bromide. Amplified products were visualized under UV light and their molecular mass estimated by comparison with Lambda DNA/HindIII Marker. PCR products were purified using PCR purification kit (QIAGEN). Sequencing reactions were conducted using BigDye terminator cycling conditions on an Applied Biosystems Automatic Sequencer 3730XL (Macrogen Inc, Seoul, Korea). Sequences were aligned with MAFFT 6 (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/) and a tree was generated using PAUP 4b10 (Swofford 2002). Analyses were done using the heuristic search option with 100 random addition sequence replications (Efron 1986). Sequences of known Rosellinia spp. were retrieved from Genbank National Center for Biotechnology Information (NCBI) and incorporated into the analyses (Table 1). Hypoxylon intermedium (Achwein.) Y.M. Ju & J.D. Rogers (Sánchez-Ballesteros et al. 2000) and Amphisphaeria umbrina (Fr.) de Not., both members of Xylariales, were used as outgroup taxa. Pathogenicity tests In order to preliminary evaluate the ability of the species of Rosellinia collected in the surveyed area to infect coffee, two pathogenicity tests (one in November and another in December 2009) were conducted under greenhouse conditions at Planalto-Cenicafé, in Chinchiná, Colombia, using seedlings of C. arabica variety Caturra. Isolates encoded as RCQ 60 (CBS134099), obtained from coffee with black root rot in a farm of Quimbaya (Quindio), and RCACC 67 (CBS 134106) obtained from cocoa roots with star rot, in Palestina (Caldas), were grown on twice-autoclaved parboiled rice (Doña Pepa ®) placed in plastic bags. One mycelial disc (6 mm diameter) taken from cultures of each isolate growing on MEA plates was added to each bag, then incubated at 25 °C in darkness for twenty five days, to allow the mycelium to completely colonize the rice. Coffee seedlings (65-days-old) previously grown in sterilized sand, were planted in plastic pots (one plant/pot) containing 150 g of sterilized soil (sandy loam, pH=4.9, organic matter=10 %). Eight days after planting, the seedlings were inoculated with 0.18 g of rice/plant, placing the inoculum in contact with the roots. For the controls, 518 B.L. Castro et al. Table 1 Sequences of isolates retrieved from GenBank included in this study Taxon Host and geographic origin Culture number Gen bank accession number Amphisphaeria umbrina (Fr.) de Not. Hypoxylon intermedium (Achwein.) Y.M. Ju & J.D. Rogers Rosellinia bambusae Henn. Unknown – AF009805 Unknown H4A AJ390396 Dendrocalamus latiflorus Munro (Taiwan) Calamus sp. (Australia) Unknown (England) Calamus sp. (Australia) ATCC 66430 AY908998 Sánchez-Ballesteros et al. (2000) Peláez et al. (2008) – ATCC 32869 – AY862573 AY909000 AY862570 Bahl et al. (2005) Peláez et al. (2008) Bahl et al. (2005) Unknown Unknown (Japan) Unknown (Japan) Calamus sp. (Australia) F-160.845 – 89112602 – AY908999 AB430457 AB430456 AY862572 Peláez et al. (2008) Takemoto et al. (2009b) Takemoto et al. (2009b) Bahl et al. (2005) Ehretia microphylla Lam (Taiwan) Camellia sinensis (L.) Kuntze (Taiwan) Serissa japonica (Thunb.) Thunb (Taiwan) Unknown (Japan) Acer morrisonense Pax (Taiwan) Unknown Unknown Wood (Illinois, USA) Hibiscus mutabilis L. (Bahamas) R210 R301 EF592569 EF592564 Sun et al. (2008) Sun et al. (2008) R203 EF592563 Sun et al. (2008) W536 R101 CBS350.36 ATCC36702 ATCC 58850 CBS. 347.36 AB430450 EF592568 AB017659 AB017661 AY909002 AB609598.1 Takemoto et al. (2009b) Sun et al. (2008) Rosellinia bambusae Henn Rosellinia buxi Fabre Rosellinia capetribulensis J. Bahl, Jeewon & K.D. Hyde Rosellinia corticium (Achwein.) Sacc. Rosellinia compacta Takemoto Rosellinia compacta Takemoto Rosellinia mirabilis (Berk & Broome) Y.M. Ju & J.D. Rogers Rosellinia necatrix Berl. Ex Prill Rosellinia necatrix Berl. Ex Prill Rosellinia necatrix Berl. Ex Prill Rosellinia Rosellinia Rosellinia Rosellinia Rosellinia Rosellinia Sacc. necatrix Berl. Ex Prill necatrix Berl. Ex Prill pepo Pat. quercina R. Hartig subiculata (Achwein.)Sacc. bunodes (Berk & Broome) uninoculated soil was used. The experimental unit was made up of ten seedlings placed individually in a row of plastic pots; the treatments corresponded to the isolates and five replicates were used per treatment as well as for the controls. Inoculated and control experimental units were placed in a fully randomized design in a greenhouse at an approximately 28 °C day and 20 °C night temperature regime. In both trials, plants were checked daily for symptoms for 35 days post-inoculation. Symptomatic and dead plants were inspected for the presence of mycelium on their stems or roots to confirm infection by Rosellinia spp. Experimental data, including the number of dead plants and days to mortality, were statistically analyzed using ANOVA and Tukey’s mean test (p=0.05) (SAS Statistical Software 2010). Results Sample collection and fungal isolations Twenty coffee farms located at an altitude of between 1,200 and 1,500 (m) where patches with symptoms of root rot Reference Peláez et al. (2008) were present were sampled. In these plantations, areas including infected coffee plants ranged from three to 3,000 trees. Cocoa plantations (two) and macadamia (three) had smaller numbers of infected trees ranging from three to 50 plants affected. Other hosts growing on coffee farms had fewer than 10 plants with symptoms per stand. Trees from which samples were collected showed typical symptoms of root rot caused by Rosellinia spp. including foliage yellowing or wilting as well as dead plants. In the root collar area, the bark was cracked and small black lines and dots could be seen macroscopically, embedded in the wood (Fig. 1a). These symptoms were most common on coffee and are similar to those recorded for R. bunodes (Fernández and López 1964; Ibarra et al. 1999; López 2004). Other plants, including cocoa, had white fan-shaped or stellate mycelial growth under the bark of roots (Fig. 1b), which is typical for R. pepo (Waterston 1941; Sivanesan and Holliday 1972; Merchán 1988; Realpe et al. 2006). Rosellinia spp. were successfully isolated from 90 % of the root samples collected, resulting in a total of 26 putative isolates of the fungus (Table 2). Cultures were grouped based on the signs of the pathogen observed on the roots Identification and genetic diversity of Rosellinia spp. Fig. 1 Signs of Rosellinia infection observed on coffee trees. a Black streaks and spots caused by Rosellinia bunodes, b white mycelial stars under the bark caused by Rosellinia pepo 519 a) at the time of sampling. Colonies of morphological group A, representing 18 isolates obtained from roots with star rot, were initially white and then turned dark gray and olive green or dark brown. Colonies of group B isolates, consisting of eight cultures originated from black root rot, produced a fluffy mycelium that was also initially white and later turned gray, brown or black, or with some areas white and other black. However there was no consistency with regard to the color of the colonies in each group after ten days of cultivation. No other structures were observed in addition to the mycelium, either on infected roots or in culture after 25 days of cultivation. The most important morphological characteristic observed using light microscopy for these isolates was the pyriform-swellings at the junctions of the septa in the hyphae (Fig. 2), as has been previously described for these fungi (Fernández and López 1964; Ibarra et al. 1999; López 2004; Realpe et al. 2006). The size range of the swellings was between 6.0 and 12.5 μm wide for both species, increasing as the mycelium aged. Strains of three (3) isolates of R. bunodes and eight (8) of R. pepo were deposited in the Centraalbureau voor Schimmelcultures (CBS, Netherlands). The deposit numbers are to be found in Table 2. ITS sequence comparisons PCR amplification of the ITS regions for 26 isolates putatively representing Rosellinia spp. yielded fragments of ~633 bp in length. All sequences generated for the phylogenetic analyses in this study were deposited in Genbank (Table 2). Parsimony analysis produced a data set with 363 constant characters, 101 parsimonyuninformative and 257 parsimony informative characters. Four hundred and sixty four uninformative characters were excluded and 30 trees were obtained, from which one was chosen for presentation (Fig. 3). This b) tree consisted of two major clades, the largest of these included 18 isolates from coffee and other hosts (colony type A), exhibiting little diversity, and strongly supported by a 100 % bootstrap value. The reported sequence in this clade corresponds to R. pepo (AB 017659) from the CBS (Netherlands). The second clade, including 8 isolates from Colombia (colony type B), all obtained from coffee plants, was strongly supported by a 100 % bootstrap value and was related to an R. bunodes sequence (AB 609598). The tree had a consistency index (CI) of 0.45, homoplasy index (HI) of 0.54, retention index (RI) of 0.76, and rescaled consistency index (RC) of 0.34 (TreeBase number TB2: S12799). Pathogenicity tests Isolates of both R. bunodes (RCQ 60) and R. pepo (RCACC 67) were pathogenic to C. arabica seedlings. In both trials, wilting symptoms caused by R. bunodes (RCQ 60) were seen as early as 9 days post-inoculation on most of the seedlings. All seedlings were killed in the first trial and 98 % in the second trial, within 10 to 11 days postinoculation. Wet rot, as well as brown, sunken discoloration were noticed in the tissues at the bases of the seedlings, with fine white mycelium invading the roots infected by R. bunodes. Wilt symptoms caused by R. pepo (RCACC 67) were evident 14 days post-inoculation on most of the seedlings in both trials. For the first trial, 62 % of the seedlings were killed and 46 % died in the second trial after 16 and 24 days post-inoculation respectively. Dry rot and brown tissue discoloration, but no sunken tissue, were observed at the bases of the seedlings, with gray mycelium invading the roots. Differences in mortality and days to death were statistically significant (P<0. 0001) in both tests. No mortality was found in any of the control plants for either of the tests. 520 B.L. Castro et al. Table 2 Isolates of Rosellinia spp. from coffee and other hosts in Colombia used in this study and for which internal transcribed spacer (ITS) regions sequence data were generated Taxon Culture number Host Origin Gen bank accession number Rosellinia bunodes “ “ “ “ “ “ “ Rosellinia pepo “ “ “ “ “ “ “ “ “ RCQ 48.2 (CBS 134097)b RCQ 68 (CBS 134098)b RCQ 67.2 RCQ 67 RCQ 66 RCQ 65 RCQ 60a (CBS 134099)b RCQ 48 RCACC 65 (CBS 134100)b RMACC 45 (CBS 134101)b RGUC 46 (CBS 134102)b RGUC 45 RGUC 28 RCR 14.2 (CBS 134103)b RCC 67 (CBS 134104)b RCC 64.2 RCC 64 RCC 60 Coffea arabica L. “ “ “ “ “ “ “ Theobroma cacao L. Macadamia integrifolia Maiden & Betche Psidium guajava L. “ “ Coffea arabica L. “ “ “ “ Circasia (Quindío) “ “ “ “ “ Quimbaya (Quindío) Circasia (Quindío) Palestina (Caldas) Chinchiná (Caldas) “ “ “ Pereira (Risaralda) Chinchiná (Caldas) Palestina (Caldas) “ Chinchiná (Caldas) JF263537 JF263538 JF263539 JF263540 JF263541 JF263542 JF263543 JF263544 JF263545 JF263546 JF263547 JF263548 JF263549 JF263550 JF263551 JF263552 JF263553 JF263554 “ “ “ “ “ “ “ “ RCAUR 18 (CBS 134105)b RCAUR 17 RCACC 67a (CBS 134106) b RCACC 66 RCACC 36 RMACQ 46 (CBS 134107)b RCR 24 RCR14 Hevea brasiliensis Müll. Arg. “ Theobroma cacao L. “ “ Macadamia integrifolia Maiden & Betche Coffea arabica L. “ Pereira (Risaralda) “ Palestina (Caldas) “ “ Buena Vista (Quindío) Pereira (Risaralda) “ JF263555 JF263556 JF263557 JF263558 JF263559 JF263560 JF263561 JF263562 Codes: RCC Rosellinia-Coffee-Caldas, RCQ Rosellinia-Coffee-Quindío, RCR Rosellinia-Coffee-Risaralda, RCACC Rosellinia- Cocoa-Caldas, RMACC Rosellinia-Macadamia-Caldas, RGUC Rosellinia-Guava -Caldas, RCAUR Rosellinia-Caucho (Hevea)-Risaralda, RMACQ RoselliniaMacadamia-Quindío a Isolates included in pathogenicity tests. b CBS deposit number Discussion This study provides the first detailed identification of Rosellinia species in Colombian coffee growing areas using DNA sequence data. Previous studies in the country relied on identification based only on morphology. We identified R. bunodes and R. pepo affecting several hosts, including coffee plants in three provinces of Colombia. Symptoms, signs and molecular characterization of R. bunodes and R. pepo in this study are consistent with the etiology of the diseases known as black root rot (R. bunodes) and stellate root rot (R. pepo). In this study, ITS sequence data confirmed that both R. bunodes and R. pepo are present in Colombia, confirming previous reports based on disease symptoms. Isolates obtained grouped into two distinct clades with 100 % bootstrap support and separate from any other Rosellinia sp. for which sequence data are available in GenBank. Currently, there are more than 100 reported Rosellinia species known (Kirk et al. 2001), but molecular data for these species is minimal or non-existent for most. Most of the sequence data available for the genus in GenBank are for R. necatrix. Our data for R. bunodes broadens the single sequence report, previously available for this species. The sequence data emerging from this study supports the reliability of observed differences in macroscopic characters on infected plant roots as diagnostic for discriminating between R. pepo and R. bunodes. Neither culture morphology nor microscopic features were sufficient to differentiate between species. Differences in mycelium color in culture were not consistent among isolates of the same species, or between species. Pyriform hyphal swellings at the junctions Identification and genetic diversity of Rosellinia spp. 521 of septa were also observed for both species, as previously reported by Fernández and López (1964), López (2004) and Realpe et al. (2006). These swellings have also been reported for R. necatrix (Saccas 1956; Pérez-Jiménez 2006; Pliego et al. 2012). Our observations showed that these swellings develop in mature hyphae (more than 8days-old) rather that in young mycelium. Eventually, synnemata and conidia were observed in some old cultures (more than 30 days), but these structures had dimensions very similar in R. pepo and R. bunodes, as reported by Saccas (1956) and Petrini and Petrini (2005). Thus, they did not add any taxonomic information useful for diagnostics. Fruiting bodies, such as stromata bearing perithecia were not observed, as frequently reported in other studies (Bermúdez and Carranza 1992; Ibarra et al. 1999). This might be related to the condition of the samples and the Fig. 2 Morphological characteristics of Rosellinia bunodes and Rosellinia pepo growing in culture. Typical pear-shaped swelling in the septa union of mycelia Fig. 3 The most parsimonious tree generated from DNA sequence data of the ITS regions for isolates of Rosellinia spp. Branch lengths are shown above and boostrap values below the branches. CI=0.4535; RI=0.7659; HI=0.5465; RC=0.3474 Tree Length=452 48 100 34 100 51 95 RCQ 48 JF263544 RCQ 48.2 JF263537 RCQ 60 JF263543 RCQ 65 JF263542 RCQ 66 JF263541 RCQ 67 JF263540 RCQ 67.2 JF263539 RCQ 68 JF263538 CBS347. 36 AB609598.1 RCACC 36 JF263559 RCACC 66 JF263558 RCACC 67 JF263557 RCR 14 JF263562 RCR 14 2 JF263550 RCR 24 JF263561 RGUC 28 JF263549 RMACC 45 JF263546 RMACQ 46 JF263560 RCAUR 17 JF263556 RCACC 65 JF263545 RCC 64 1 JF263553 RGUC 45 JF263548 RCAUR 18 JF263555 RCC 67 JF263551 RCC 60 JF263554 RGUC 46 JF263547 RCC 64.2 JF263552 AB017659 R. pepo AB430450 R necatrix 10 EF592563 R necatrix 62 EF592564 R necatrix EF592568 R necatrix EF592569 R necatrix 21 AY908998 R bambusae 21 100 AY862573 R bambusae 79 AY862572 R mirabilis AY909000 R buxi AY862570 R capetribulensis 35 AB430457 R compacta AB430456 R 100 22 AY908999 R corticium 23 25 AY909002 R subiculata 82 29 EF026118 R lamprostoma 17 AB017661 R quercina 49 AF009805 Amphisphaeria umbrina AJ390396 Hypoxylon intermedium 5 changes Rosellinia bunodes Rosellinia pepo 522 time required for these structures to appear after infection (Sarasola and de Sarasola 1975; de Texeira et al. 1995; Nakamura et al. 2000; Pliego et al. 2012). It could also mean that the pathogen does not require those parts of its life cycle under Colombian conditions where the temperature and humidity is high all year round. An effort should be made, nonetheless, to locate such structures and thus complement the molecular taxonomic understanding of Rosellinia spp. in Colombia. Acknowledgments This study was made possible through the financial support from the Colombian Institute for the Development of Science and Technology “Francisco José de Caldas” COLCIENCIAS; the Colombian National Center of Coffee Research (Cenicafé) and members of the Tree Protection Co-operative Program (TPCP), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. We thank James Mehl, Prof. Brenda Wingfield, Michael Mbenoum and Tuan Duong for their advice, assistance in the laboratory and with DNA sequence comparisons. Furthermore, we thank Dr. Juan Carlos Herrera (Cenicafé), Professor Jack Rogers (Washington State University) and Dr. Martijn ten Hoopen (CIRADFRANCE) for useful discussions and advice. References Aranzazu HF (1996) Comportamiento de la llaga estrellada Rosellinia pepo Pat. sobre raíces vivas y muertas. Agrocambio 2:10–15 Armengol J, Vicent A, León M, Berbegal M, Abad-Campos P, GarciaJiménez J (2010) Analysis of population structure of Rosellinia necatrix on Cyperus esculentus by mycelial compatibility and inter-simple sequence repeats (ISSR). Plant Pathol 59:179–185. doi:10.1111./j.1365-3059 Bahl J, Jeewon R, Hyde KD (2005) Phylogeny of Rosellinia capetribulensis sp. nov. and its allies (Xylariaceae). Mycologia 97:1102–1110 Barceló-Muñoz A, Zea-Bonilla T, Jurado-Valle I, Imbroda-Solano I, Vidoy-Mercado I, Pliego-Alfaro F, López-Herrera CJ (2007) Programa de selección de portainjertos de aguacate tolerantes a la podredumbre blanca causada por Rosellinia necatrix en el Sur de España (1999–2007). Proceedings VI World Avocado Congress. Viña del Mar Bautista PF, Salazar M (2000) Evaluación de daños económicos causados por Rosellinia spp. en un área afectada por el patógeno. In: Simposio Latinoamericano de Caficultura, 19. San José pp 459–464. ICAFE-PROMECAFE. Octubre 2–6 Bermúdez M, Carranza J (1990) Patogenicidad de Rosellinia bunodes en el jaúl (Alnus acuminata). Agron Costarric 14:181–188 Bermúdez M, Carranza J (1992) Estado anamórfico de Rosellinia bunodes (Berk&Br.) Sacc y Rosellinia pepo Pat. (Ascomycotina: Xylariaceae). Rev Biol Trop 40:43–46 Castro BL, Esquivel H (1991) Las llagas radicales del cafeto. Av Técnicos (Cenicafé) 163:1–4 Castro BL, Serna CA (2009) Incidencia de llagas radicales (Rosellinia spp.) en el sistema café-yuca en el Departamento del Quindío. Fitopatología Colomb 33:43–48 de Texeira SAJ, Guillaumin JJ, Harples GP, Whalley AJS (1995) Rosellinia necatrix and white root rot of fruit trees and other plants in Portugal and nearby regions. Mycologist 9:31–33 B.L. Castro et al. Efron B (1986) Bootstrapping methods: Another look at jacknife. Annals of Statistics. In: Hillis D, Moritz D, Mable B (eds) Molecular systematics. Snauer Associates Publishers, pp 1–6 Eguchi N, Kondo KI, Yamagishi N (2009) Bait twig method for soil detection of Rosellinia necatrix, causal agent of white root rot of Japanese pear and apple, at an early stage of tree infection. J Gen Plant Pathol 75:325–330. doi:10.17/s10327-009-0179-8 Fernández O, López S (1964) Las llagas radiculares negra (Rosellinia bunodes) y estrellada (Rosellinia pepo) del cafeto. I. Patogenicidad e influencia de la clase de inóculo en la infección. Cenicafé 15:126–144 Garcia LAA (1945) Studies on coffee root diseases in Puerto Rico. J Agric Univ P R 29:1–29 Guerrero O (1990) Mortaja blanca, enfermedad de la papa causada por el hongo Rosellinia sp. Rev ICA 25:243–249 Gutiérrez RA, Castro BL, Rivillas CA (2006) Manejo de la llaga negra del cafeto. Cenicafé 57:299–311 Hernández PMR (1967) El café: sus enfermedades. Rev Cafetalera 143:9–20 Herrera L (1989) La pudrición negra de las raíces del cafeto en la región del Escambray. Cent Agrícola 16:53–59 Hillis D, Moritz C, Mable B (1996) Molecular systematics. Associates Publishers Hsieh HM, Lin CR, Fang MJ, Rogers JD, Fournier J, Lechat C, Ju YM (2010) Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily. Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily. Mol Phylogenet Evol 54:957–969 Ibarra NL, Castro BL, Ponce CA (1999) Estudio del proceso infectivo de Rosellinia bunodes Berk y Br. Sacc. en café. Fitopatología Colomb 23:59–64 Kannan N (1995) Technical report on diseases affecting coffee in India— a review. Indian Coffee 59:11–17 Kirk PM, Cannon PF, David JC, Stalpers JA (2001) Dictionary of the fungi, 9th edn. CAB International, Wallingford, 456p Lee SB, Taylor JW (1990) Isolation of DNA from fungal mycelia and single spores. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press López JA (2004) Determinación de la variabilidad genética entre aislamientos de Rosellinia sp.; Rosellinia bunodes y Rosellinia pepo mediante la técnica de amplificación aleatoria de polimorfismos de DNA (RAPD) y análisis de los espaciadores de transcritos internos (ITS). Trabajo de grado. Biología. Universidad Nacional de Colombia. Departamento de Biología. Sede Bogotá 152p López M, Ruano-Rosa D, López CJ, Monte E, Hermosa R (2008) Intraspecific diversity within avocado field isolates of Rosellinia necatrix from south-east Spain. Eur J Plant Pathol 121:201–205. doi:10.1007/s10658-007-9253-2 López-Herrera CJ (1998) Hongos del suelo en el cultivo del aguacate (Persea americana Mill.) del litoral Andaluz. V Jornadas Andaluzas de frutos tropicales. Congresos y Jornadas 98:139–152, Sevilla Merchán VM (1988) La rosellinia del cacao. Rev Agron Univ Caldas 2:27–29 Muthappa BN (1977) Rosellinia bunodes on Coffee spp. J Coffee Res 7:109–110 Nakamura H, Uetake Y, Arawaka M, Okabe I, Matsumoto M (2000) Observation on the teleomorph of the with root rot fungus Rosellinia necatrix and a related fungus Rosellinia aquila. Mycoscience 41:503–507 Peláez F, González V, Platas G, Sánchez-Ballesteros J, Rubio V (2008) Molecular phylogenetic studies within the Xylariaceae based on ribosomal DNA sequences. Fungal Divers 31:111–134 Pérez-Jiménez RM (2006) A review of the biology and pathogenicity of Rosellinia necatrix- the cause of white root rot disease of fruit trees and other plants. J Phytopathol 154:257–266 Identification and genetic diversity of Rosellinia spp. Pérez-Jiménez RM, Zea-Bonilla T, López-Herrera CJ (2003) Studies of Rosellinia necatrix perithecia found in nature on avocado roots. J Phytopathol 151:660–664 Petrini LE, Petrini O (2005) Morphological studies in Rosellinia (Xylareaceae): the first step towards a polyphasic taxonomy. Mycol Res 109:569–580. doi:10.1017/S0953756 205002510 Pliego C, López-Herrera C, Ramos C, Cazorla F (2012) Developing tools to unravel the biological secrets of Rosellinia necatrix, an emergent threat to woody crops. Mol Plant Pathol 13:226–239. doi:10.1111/J.1364-3703.2011.00753.x Ponte J (1996) Clinica de doenςas de plantas. Universidade Federal do Ceará, Fortaleza, p 872 Procafé, Fundación Salvadoreña para investigaciones del Café (1996) Manejo integrado de la podredumbre negra de la raíz del cafeto Rosellinia sp. Boletin Técnico 2:1–7 Realpe CE, Villegas C, Riaño NM (2006) Aislamiento y caracterización morfológica de Rosellinia pepo Pat. en plantas de macadamia. Revista Facultad de Agronomía. Medellín 59:3509–3526 Ruano-Rosa D, Schena L, Ippolito A, López-Herrera J (2007) Comparison of conventional and molecular methods for the detection of Rosellinia necatrix in avocado orchards in south Spain. Plant Pathol 56:251–256. doi:10.1111/j.1365-3059 Saccas AM (1956) Les rosellinias des cafeiers en Oubangui-Chari. Agron Trop 11:687–706 Sánchez-Ballesteros J, González V, Salazar O, Acero J, Portal MA, Julián M, Rubio V, Bills GF, Polishook JD, Platas G, Mochales S, Peláez F (2000) Phylogenetic study of Hypoxylon and related genera based on ribosomal ITS sequences. Mycologia 92:964– 977 Sarasola A, de Sarasola MR (1975) Fitopatología, curso modernoTomo II-Micosis. Editorial Hemisferio Sur, Buenos Aires, 374p SAS Statistical software (2010) SAS/STAT users’s guide, version 9.2. SAS Institute Inc, Cary 523 Schena L, Ippolito A (2003) Rapid and sensitive detection of Rosellinia necatrix in roots and soils by real time ScorpionPCR. J Plant Pathol 85:15–25 Sivanesan A, Holliday P (1972) Rosellinia bunodes. CMI Description of pathogenic fungi and bacteria. 35:1–2 Sun EJ, Lin HS, Hsieh HJ (2008) Study on Rosellinia necatrix isolates causing white root rot disease in Taiwan. J Phytopathol 156:104– 111. doi:10.1111/j14390434 Swofford DL (2002) PAUP*. Phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland Takemoto S, Nakamura H, Degawa Y (2009a) The first record of Rosellinia aquila in Kanagawa Prefecture and the analysis of morphological variation among the collections. Bull Kanagawa Prefectural Mus Jpn Nat Sci 38:21–29 Takemoto S, Nakumura H, Sasaki A, Shimane T (2009b) Rosellinia compacta, a new species similar to the white root rot fungus Rosellinia necatrix. Mycologia 101:84–94. doi:10.3852/08-100 Takemoto S, Nakumura H, Sasaki A, Shimane T (2011) Speciesspecific PCRs differentiate Rosellinia necatrix from Rosellinia compacta as the prevalent cause of white root rot in Japan. J Gen Plant Pathol 77:107–111. doi:10.1007/s10327-011-0297-y Ten Hoopen GM, Krauss U (2006) Biology and control of Rosellinia bunodes, Rosellinia necatrix and Rosellinia pepo. Crop Prot 25:89–107. doi:10.1016/j.cropo Ten Hoopen GM, Ortiz JL, Aguilar ME, Krauss U (2004) Preservation methodology for cocoa pathogenic Rosellinia species. Mycol Res 108:274–282. doi:10.1017/S0953756204009712 Waterston JM (1941) Observations on the parasitism of Rosellinia pepo Pat. Trop Agric 18:174–186 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, pp 315–322