Trop. plant pathol. DOI 10.1007/s40858-017-0182-0

SHORT COMMUNICATION

Colletotrichum species causing anthracnose on lima bean in Brazil Enayra Silva Sousa 1 & Janaíne Rossane Araújo Silva 2 & Iraildes Pereira Assunção 2 & Maruzanete Pereira de Melo 1 & Frederico Monteiro Feijó 2 & Kedma da Silva Matos 3 & Gaus Silvestre de Andrade Lima 2 & José Evando Aguiar Beserra Jr 1

Received: 30 March 2017 / Accepted: 1 September 2017 # Sociedade Brasileira de Fitopatologia 2017

Abstract The taxonomy of the genus Colletotrichum has undergone various changes. These alterations arise from the use of modern molecular tools. Currently, Colletotrichum species are grouped into complexes. Species of Colletotrichum associated with diseases in lima bean have been investigated very little. In Brazil, anthracnose in lima bean has often been associated with Colletotrichum truncatum, but only morphological characteristics have been used for identification purposes. In this study, samples of lima bean with symptoms of spots on the leaves and pods have been collected in Piauí and Alagoas states. Comparisons of morphological characteristics revealed nine isolates with cylindrical or curved conidia. The ITS region and partial sequences of GAPDH, β-tubulin and Actin regions were amplified by PCR, sequenced and submitted to multilocus phylogenetic analysis. The isolates analyzed grouped with reference specimens from Colletotrichum truncatum, C. cliviae and C. fructicola. All isolates were pathogenic to 25-day-old lima bean seedlings. Therefore, this work confirms the pathogenicity of C. truncatum to lima bean and for the first time records the occurrence of C. cliviae and C. fructicola as pathogens of this host in Brazil.

Section Editor: Marciel J. Stadnik * José Evando Aguiar Beserra, Jr [email protected] 1

Centro de Ciências Agrárias, Departamento de Fitotecnia, Universidade Federal do Piauí, Ininga, Teresina, PI 64049-550, Brazil

2

Centro de Ciências Agrárias, Universidade Federal de Alagoas, Br 104, km 85 norte, Rio Largo, AL 57100-000, Brazil

3

Centro de Ciências Agrárias, Universidade Federal de Roraima, Boa Vista, RR 69310-000, Brazil

Keywords Colletotrichum cliviae . Colletotrichum fructicola . Colletotrichum truncatum . multilocus analysis . Phaseolus lunatus

Lima bean (Phaseolus lunatus L.) is the second most important agronomic species from the genus Phaseolus. It is a legume that is consumed worldwide, as it contains high level of proteins (Seidu et al. 2015). In Brazil, specifically in the Northeast, it is cultivated by small-scale growers (Vieira, 1992). Both low level of technology of cultural practices and phytosanitary problems contribute to low yields of lima bean in many regions in Brazil (Paula Junior et al. 1995; Silva et al. 2014). Anthracnose, caused by Colletotrichum truncatum (Schw.) Andrus & Moore, is the main disease of P. lunatus, and it is characterized by necrotic spots on leaves, petioles and pods (Carvalho et al. 2015). In severe attacks, young leaves and pods can appear rolled and wrinkled and, at advanced stages of the disease, masses of whitish spores are formed, with numerous setae (Paula Junior et al. 1995). Curiously, anthracnose is also an important disease of common bean in various parts of the world, causing significant losses in Brazil (Peloso 1992). Recently, two new lineages of Colletotrichum sp. were confirmed in Brazil, and these belong to the Colletotrichum gloeosporioides complex (Barcelos et al. 2014). Previously, the identification of species was based mostly on the evaluation of morphological characteristics, which is often subjective, because some species of Colletotrichum have variable morphological characteristics, due to the influence of environmental factors (Hyde et al. 2009). Currently, the species of Colletotrichum are grouped into complexes and the systematics is based on molecular markers (Hyde et al. 2009; Damm et al. 2012; Weir et al. 2012; Damm et al. 2014).

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Molecular tools have been used to identify isolates of Colletotrichum spp. with greater precision. By using multigene analysis, for example, what was known as C. gloeosporioides (Penz.) Penz. & Sacc. was divided into 22 distinct phylogenetic lineages (Weir et al. 2012). Since then, a number of diseases that had C. gloeosporioides as their etiological agent have been attributed to other species in the genus. As an example is the anthracnose in mango fruits, where the causal agent was denominated as C. gloeosporioides and, when the concept of phylogenetic species was applied, five distinct species were confirmed (Lima et al. 2013). In Brazil, there are records of C. truncatum and C. lindemuthianum associated with lima bean (Paula Junior et al., 1995; Farr and Rossman 2017); however, these records may not be accurate, because they are based only on morphological characteristics. Furthermore, cultures of these isolates were not deposited in collections, and tests were not carried out to prove their pathogenicity. Therefore, using the concept of morphological species, phylogenetics and the pathogenicity test, this study aimed to characterize isolates of Colletotrichum obtained from lima bean in the Northeast of Brazil. Samples of leaves and pods (Fig. 1A, E) with symptoms of spots were collected in the municipality of Teresina (experimental area of the Universidade Federal do Piauí), state of Piauí, and in the municipalities of Rio Largo, Messias, Murici and São José da Lage, state of Alagoas. Small fragments of colonized tissue were removed from the samples and disinfested in ethanol 70% for 1 min, and sodium hypochlorite 2% for 2 min; then transferred to sterilized distilled water, and dried on sterile paper. The isolates were cultivated for seven days in Potato-Dextrose-Agar culture medium (PDA). Monosporic cultures were deposited in the Maria Menezes Culture Collection of Phytopathogenic Fungi at the Universidade Federal Rural de Pernambuco, Recife, Brazil or in the Collection of Phytopathogenic Fungi at the Universidade Federal de Alagoas, Rio Largo, Brazil (Table 1). For molecular characterization, DNA was extracted using the AxyPrep Multisource Genomic DNA Miniprep kit (Axygen Biosciences, Union City, CA, USA). Biomass was produced for DNA extraction on plates containing PDA medium and transferred to a 1.5 mL microcentrifuge tube. The samples were centrifuged at 10,000×g for 1 min; the supernatants (culture medium) were discarded. PCR products were analyzed by 1% agarose electrophoresis gels stained with SYBR® Safe (Thermo Fisher Scientific, Waltham, MA, USA) and visualized under UV light to check for amplification size and purity. The PCR was performed using 12.5 μL 2× Taq PCR Max Mix, 2 μL of each primer (forward and reverse) and 9 μL of ultra-pure water in a final volume of 25.0 μL. The gene regions coding for Internal Transcribed Spacers (ITS) were

amplified using the primers ITS 1 (forward; 5′-GTAA CAAGGTTTCCGTAGGTG-3′) and ITS 4 (reverse; 5′TTCTTTTCCT CCGCTTATTGATATGC-3′) and specific amplification conditions (White et al. 1990). A fragment of the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was amplified using primers GDF1 (forward; 5′GCCGTCAACGACCCCTTCATTGA-3′) and GDR1 (reverse; 5′-GGGTGGAGTCGTACTTGAGCATGT-3′) (Templeton et al. 1992). A portion of the beta tubulin gene (TUB2) was amplified with primers TB5 (forward; 5′GGTAACCAGATTGGTGCTGCCTT-3′) and TB6 (reverse; 5′-GCAGTCGCAGCCCTC AGCCT-3′) (Panaccione and Hanau 1990). A fragment of the Actin gene (ACT) was amplified with primers ACT-512 (forward; 5′-ATGT GCAAGGCCGGTTTCGC-3′) and ACT-783R (reverse; 5′TACGAGTCCTTCTGGCCCAT-3′) (Carbone and Kohn 1999). The PCR products were analyzed on 2% agarose gels after staining with GelRed™ (Biotium Inc., CA, USA) in 1× TAE buffer (Sambrook et al. 1989) and visualization under UV light. The PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Sequencing was performed by the Biological Institute of São Paulo. The nucleotide sequences were edited using SeqAssem (Hepperle 2004). Additional sequences of Colletotrichum spp. isolates from different hosts were obtained from GenBank (Table 1). The sequences were aligned using the program ClustalW (Thompson et al. 1994) as implemented in the software MEGA v. 6 (Tamura et al. 2013). The resulting alignment was deposited into TreeBASE as accession number 21246. Bayesian inference analyses were performed using the Monte Carlo chain method (MCMCMC). Mr. Modeltest 2.3 (Posada and Buckley 2004) was used to determine the evolutionary model of the nucleotides that best fit the data; the models used in the phylogenetic analyses were: GTR + G for ITS and TUB2; HKY + G for GAPDH; and HKY + I for ACT. Phylogenetic analysis was performed at the CIPRES web portal (Miller et al. 2010) using MrBayes version v. 3.2 (Ronquist et al. 2011). Markov chains were run simultaneously from random trees to 10,000,000 generations. Trees were sampled every 1,000th generation for a total of 10,000 trees. The first 2500 trees were discarded as burn-in in each analysis, and the remaining 7500 trees were used to calculate the posterior probabilities of the branches that were determined based on the consensus of most sampled trees. Tree was visualized using Figtree (Rambaut 2009) and exported to a graphics program. The sequences obtained in this study were deposited in GenBank (Table 1). To evaluate the morphological characteristics the isolates were cultivated in PDA at 25 °C under constant fluorescent light. The color and aspect of the colony were evaluated (Cai

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Fig. 1 Colletotrichum spp. in lima bean. Symptoms on leaves (A and B), stems (C) and pods (E) caused by C. truncatum. (D) Stem with conidial masses (arrow). Symptom of leaf spot caused by C. fructicola and

C. cliviae (F). Setae (G). Conidiomata in SNA (H). Conidia of C. truncatum (I). Conidia of C. fructicola (J). Asci and ascospores of C. cliviae (K). Bar: G, H = 20 μm; I, J, K = 40 μm

et al. 2009). To evaluate the format of the conidia and formation of conidiomata, Spezieller nährstoffarmer agar (SNA) medium was used, and the cultures were incubated for 10 days at 20 °C with a photoperiod of 12 h. All isolates were tested for their ability to infect 25-day-old lima bean seedlings. The inoculum was prepared at the concentration of 1 × 105 spores mL−1 and sprayed on the upper and lower leaf surfaces of the plants 10 days after they emerged, until dripping. After inoculation, the seedlings were maintained in a moist chamber for 48 h, using plastic bags. These bags were then removed, and the seedlings were kept in an environment with controlled temperature at 25 ± 2 °C. Disease development was observed until 15 days after inoculation. Seven replicates (seedlings) were tested for each

isolate. The experiment was repeated twice. The control seedlings were sprayed with sterile distilled water. Nine isolates were obtained, of which two were from Piauí and seven from Alagoas. All isolates were pathogenic to lima bean. The symptoms began to appear on the third day after inoculation. For isolates CMM 3365, CMM 3366 and COUFAL 0122, the symptoms consisted of red points that were initially rounded on the adaxial leaf surface and then increased in size, becoming irregular as the disease progressed (Fig. 1F). On the veins dark lesions could be observed, containing acervuli and black setae. Seven days after inoculation, the leaves turned yellow, with leaflets falling later. Brown lesions were formed in stems, with subsequent formation of conidia masses (Fig. 1C and D). For the other isolates, the

Trop. plant pathol. Table 1

Isolates and sequences of Colletotrichum species used in phylogenetic analysis this study

Species name

Culture collection numbera

Host

Origin

GenBank accession numbersb

ITS

GAPDH

TUB

ACT

C. aenigma C. alienum

ICMP 18608* ICMP 12071*

Persea americana Persea americana

Israel Australia

JX010244 JX010251

JX010044 JX010028

JX010389 JX010411

JX009443 JX009572

JQ005240

JQ005588

JQ005501

C. boninense

CBS 123755*

Crinum asiaticum

Japan

JQ005153

C. cliviae C. cliviae

CBS 125375* CMM 3366

Clivia miniata Phaseolus lunatus

China Teresina, PI

GQ485607 GQ856756 GQ849440 GQ856777 KY498350 KY498351 KY498355 KY498353

C. cliviae

COUFAL 0122

Phaseolus lunatus

Murici, AL

KY654707 KY654705 KY677743 KY654706

C. conoides

CAUG17*

China

KP890168

C. curcumae

IMI 288937*

Capsicum annuum var. conoides Curcuma longa

India

GU227893 GU228285 GU228187 GU227991

C. dracaenophilum CBS 118199*

Dracaena sanderiana

Egypt

JX519222

C. fructicola C. fructicola

Coffea arabica Phaseolus lunatus

Thailand Teresina, PI

JX010165 JX010033 JX010405 FJ907426 KY488562 KY488563 KY498354 KY498352

– Citrus sp. Vitis vinifera

Thailand New Zealand China

KT290266 KT290255 KT290256 KT290251 JQ005152 JQ005239 JQ005587 JQ005500 KF156863 KF377495 KF288975 KF377532

ICMP 18581* CMM 3365

C. fusiforme MFLUCC 12-0437* C. gloeosporioides CBS 112999* C. hebeiense MFLUCC 13-0726*

KP890162

JX546707

KP890174

JX519247

KP890144

JX519238

C. jasminigenum

MFLUCC 10-0273*

Jasminium sambac

Vietnam

HM131513 HM131499 HM153770 HM131508

C. liaoningense C. musae

CAUOS2* ICMP 19119*

Capsicum spp. Musa sp.

China EUA

KP890104 JX010146

KP890135 JX010050

KP890111 KP890097 HQ596280 JX009433

C. nupharicola

ICMP 18187*

EUA

JX010187

JX009972

JX010398

C. truncatum C. truncatum

CBS 151.35* COUFAL 0117

Nuphar lutea subsp. polysepala Phaseolus lunatus Phaseolus lunatus

EUA Messias, AL

GU227862 GU228254 GU228156 GU227960 KY677746 KY677745 KY677744 –

C. truncatum C. truncatum

COUFAL 0118 COUFAL 0119

Phaseolus lunatus Phaseolus lunatus

KY677749 KY677748 KY677747 – KY829020 KY829019 KY819084 –

C. truncatum C. truncatum C. truncatum

COUFAL 0120 COUFAL 0121 COUFAL 0123

Phaseolus lunatus Phaseolus lunatus Phaseolus lunatus

Messias, AL S. J. da Laje, AL Murici, AL Murici, AL Rio Largo, AL

KY851325 KY851322 KY816209 – KY851326 KY851323 KY819083 – KY851327 KY851324 KY829018 –

C. viniferum C. yunnanense

GZAAS5.08601* CBS 132135*

Vitis vinifera Buxus sp.

China China

JN412804 JN412798 NR137098 JX546706

JN412813 JX519248

JX009437

JN412795 JX519239

a

CAUOS: Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS); CBS: Culture collection of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CMM: Coleção de Culturas de Fungos Fitopatogênicos “Prof. Maria Menezes”, Universidade Federal de Pernambuco, Recife, Brazil; COUFAL: Coleção de Culturas de Fungos Fitopatogênicos, Universidade Federal de Alagoas, Maceió, Brazil; IMI: International Mycological Institute, Kew, England; ICMP: International Collection of Microorganisms from Plants, Lincoln, New Zealand; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand. b ITS: internal transcribed spacer regions 1 and 2 including the 5.8S rRNA gene; GAPDH: glyceraldehyde 3-phosphate dehydrogenase gene; TUB: beta-tubulin 1-α gene; ACT: actin gene. *ex-type strains

symptoms consisted of reddening of the veins on the abaxial side of the leaves (Fig. 1B). Later, the adaxial side showed large reddish spots, on which acervuli formed, and with the presence of numerous setae and conidia. Control seedlings did not present symptoms. All the isolates were recovered from inoculated seedlings. Based on multigene phylogenetic analysis, the isolates were identified as Colletotrichum cliviae, C. fructicola and C. truncatum. Isolates CMM 3366 and COUFAL 0122 were grouped with the reference isolate of C. cliviae (CBS 125375), with high support (Bayesian posterior probability (Bpp) = 1)

(Fig. 2), while isolate CMM 3365 grouped with the reference isolate of C. fructicola (CBS 238.49), also with high support (Bpp = 0.99). The isolates (COUFAL 0117, COUFAL 0118, COUFAL 0119, COUFAL 0120, COUFAL 0121 and COUFAL 0123) were grouped with the epitype isolate of C. truncatum (CBS 151.35) with high support (Bpp = 1). The isolate cultures presented light or dark gray coloration, with a black underside, in PDA culture medium. In SNA the presence of orange conidiomata was seen on fragments of filter paper and carnation leaves (Fig. 1H). The conidia of C. fructicola were cylindrical, hyaline, aseptate, and with a

Trop. plant pathol. Fig. 2 Bayesian phylogenetic tree of Colletotrichum spp. isolates from Phaseolus lunatus. The tree was built using concatenated sequences of the ITS, GAPDH, TUB2 and ACT genes. Bayesian posterior probability values >0.7 are indicated above the nodes. The sequences generated in this study are highlighted in bold. The analysis was carried out with sequences from ex-type or authentic culture. The scale bar (0.04) represents nucleotide substitutions per site. This tree is rooted with Colletotrichum boninense

length and width ranging between 9.0 to 13.3 μm (~11.1) × 3.1 to 5.7 μm (~4.4), respectively (n = 50) (Fig. 1J). The isolate of C. cliviae did not produce the asexual phase, but perithecia were formed at 20 days of incubation in PDA culture medium. Asci were unitunicate with 8 ascospores each (Fig. 1K). The ascospores possess a curved and hyaline format, with a length and width ranging between 7.1 to 13.4 μm (~ 10.2) × 1.8 to 6.8 (~ 4.3) μm (n = 50). The isolates of C. truncatum possess curved conidia with pointed, hyaline and aseptate tips, measuring 16.87 to 22.6 (~19.7) × 2.2 to 2.5 (~2.3) μm (n = 50) (Fig. 1I). The setae produced were dark and sometimes septate, and measure 50.7 to 83.9 (~ 67.3) × 2.7 to 5.3 (~ 4.0) μm (n = 50) (Fig. 1G). This study represents the first characterization of isolates of Colletotrichum associated with lima bean using a morphological and phylogenetic approach. In Brazil, C. truncatum is known as the etiological agent of anthracnose in lima bean (Cavalcante et al. 2012). However, the identifications had previously been carried out using only morphological characteristics, which could result in incorrect identifications, because some species of Colletotrichum share morphological characteristics with C. truncatum (Damm et al. 2009; Yang et al. 2014). The isolates in this group that have curved conidia grouped with the epitype CBS 151.35 of C. truncatum obtained from lima bean plants in the United States (Damm et al. 2009). Although this species has often been associated with lima bean in some parts of the United States and Brazil, it is also commonly found causing disease in other plants from the

Fabaceae family (Gossen et al. 2009; Ramos et al. 2010). In Brazil, all the isolates obtained from soybean cultivated in different regions were identified as C. truncatum (Rogério et al. 2017) and C. cliviae (Barbieri et al. 2017). This species has also been recorded as the etiological agent of a new disease in brassicas (Damm et al. 2009, 2012; He et al. 2016). Colletotrichum fructicola and C. cliviae represent two new causal agents of anthracnose in P. lunatus in Brazil. Colletotrichum fructicola was originally described causing anthracnose in coffee in Thailand (Prihastuti et al. 2009). This species is associated with various plants, such as Pyrus pirifolia (Japan), Limonium (Israel), Malus domestica and Fragaria x ananassa (USA and Brazil), Persea americana (Australia), Mangifera indica (Brazil) (Farr and Rosmann 2017), and in the fruit of Capsicum sp. (China) (Diao et al. 2017). Colletotrichum cliviae has a more restricted host range and was originally described in plants of Clivia miniata (Yang et al. 2009), besides being associated with ornamental hosts such as Cymbidium dentulum and Cammelia sinensis and more recently in fruits of Capsicum sp. causing post-harvest rot (Jayawardena et al. 2016). The range of hosts has extended to other plants, such as Phaseolus, Saccharum and Calamus thwaitesii (Sharma and Shenoy 2013). Although C. fructicola is widely distributed in the tropics, there has so far been no report of this species or of C. cliviae in plants of the genus Phaseolus in Brazil (Farr and Rosmann 2017). The isolates of C. fructicola presented the typical morphology of members of C. gloeosporioides (Weir et al. 2012).

Trop. plant pathol.

Morphologically, it is difficult to distinguish the species from the C. gloeosporioides complex (Phoulivong et al. 2010). Isolate CMM 3366 of C. cliviae produced only the sexual phase, possibly having lost its ability to produce the asexual phase. However, isolate COUFAL 0122 produced only the asexual phase. In the original description of C. cliviae, the authors observed only structures of the asexual phase (Yang et al. 2009), but in China, isolates from orchids (Cymbidium sp.) produced perithecia and ascospores (Liu et al. 2015). Studies using isolates from Colletotrichum acutatum Simmonds ex Simmonds demonstrated that the loss of the capacity to produce conidia is a common event (Fernando et al. 2000; Du et al. 2005). Due to the production of perithecia in culture medium, this isolate may be homothallic, since the culture comes from monosporic cultivation. Species of C . a l i e n u m We i r & J o h n s t o n , C . f r u c t i c o l a , C. queenslandicum Weir & Johnston, and C. salsolae Weir & Johnston, as well as three species from the Kahawae clade, produce the sexual phase in culture medium. The characteristics of the sexual phase, such as shape and size of the ascospores, may be useful for identifying species of Colletotrichum (Weir et al. 2012). Some reports confirm the existence of homothallic isolates of C. lindemuthianum, but most isolates are usually heterothallic (García-Serrano et al. 2008). In Brazil, a study using phylogenetic analysis confirmed that isolates associated with anthracnose lesions in common bean is distinct from C. lindemuthianum (Barcelos et al. 2014). Besides C. truncatum, C. dematium (Pers.) Grove, C. lilii Plakidas ex Boerema & Hamers, C. circinans (Berk.) Voglino, and C. lineola Corda, among other important pathogens, present curved conidia that have pointed tips (Damm et al. 2012). It is therefore impossible to identify the species of this complex using only morphological characteristics (Damm et al. 2009). Many isolates with curved conidia were denominated as C. dematium, but later, using phylogenetic approaches, it has become clear that this morpho-species is represented by distinct phylogenetic species (Damm et al. 2009; Yang et al. 2014). In the complex Colletotrichum destructivum there are species with curved conidia, but the tips of the spores are not pointed (Damm et al. 2014). Correct identification of phytopathogenic fungi is important for the adoption of efficient control methods (Than et al. 2008). Various pathogens present different epidemiological strategies, and thus require specific control strategies, such as the development of resistant varieties or the application of fungicides. It is important to make a precise determination of the species that cause anthracnose in lima bean, with the aim of understanding epidemiological aspects of its control more thoroughly. This study confirms the occurrence of C. truncatum, C. cliviae and C. fructicola as causal agents of anthracnose in lima bean in the Northeast of Brazil. Molecular

characterization and identification are important for the adoption of future strategies in monitoring pathogens, in the chemical control of anthracnose and in breeding programs for P. lunatus with a view to selecting resistant accessions. Acknowledgments ESS thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for granting a scholarship and MPM thanks the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for granting PNPD fellowship.

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