Detection of Colletotrichum pyricola on urban trees of Embothrium coccineum in Chile
Detección de Colletotrichum pyricola asociado a árboles urbanos de Embothrium coccineum en Chile
* Corresponding author: a Servicio Agrícola y Ganadero, Laboratorio Regional Chillan, Unidad de Fitopatología, Claudio Arrau 738, Chillan, Chile, tel.: 56 42 2221235, firstname.lastname@example.org
The genus Colletotrichum comprises several species in cryptic complexes that cannot be easily recognizable using morphological and cultural characteristics. As a consequence of the lack of morphological characters suitable for identification, DNA sequence analyses are now typically used as the primary basis in diagnosis and description of new species of Colletotrichum. In this study, based on a multi-locus phylogeny analysis, C. pyricola was identified on leaves of Embothrium coccineum in Chile, corresponding to the first report of this fungus in the country. Colletotrichum pyricola is a member of the C. acutatum complex, morphologically sharing features with several species of this group, being necessary the combination of morphological and molecular data for its identification. Species of Colletotrichum cause diseases in a wide range of hosts, making it important to establish an accurate diagnosis of the species for plant pathology or quarantine purposes.
Key words: Colletotrichum pyricola, Embothrium coccineum, anthracnose, morphological characteristics, multi-locus analysis.
El género Colletotrichum comprende varias especies crípticas dentro de complejos que no pueden ser fácilmente identificables usando características morfológicas y culturales. Como consecuencia de la falta de caracteres morfológicos adecuados para la identificación, los análisis de secuencias de ADN son comúnmente usados, en la actualidad, en el diagnóstico e identificación de nuevas especies. En este estudio, mediante análisis filogenético multi-locus se identificó a C. pyricola en hojas de Embothrium coccineum, correspondiente a primer reporte de este hongo en Chile. Colletotrichum pyricola pertenece al complex de C. acutatum, morfológicamente comparte características con varias especies de este grupo, siendo necesario la combinación de datos morfológicos y moleculares para su identificación. Especies de Colletotrichum causan enfermedades en un amplio rango de hospederos, haciendo importante establecer con precisión el diagnóstico de la especie para fines fitopatológicos o de cuarentena.
Palabras clave: Colletotrichum pyricola, Embothrium coccineum, antracnosis, características morfológicas, filogenia multi-locus.
Embothrium coccineum J.R. Forst. et G. Forst, known by the common names of notro, ciruelillo or fosforito, is a member of the Proteaceae family, endemic of the southern forests of South America whose latitude distribution in Chile extends from Curicó - Linares (35° S) to Tierra del Fuego (55° S) (Rovere et al. 2010). Due to its ornamental value, this species has been used in landscaping, park and garden designs, especially in public spaces (Teiller 2008). As a native species and part of the urban trees, it has been object of phytosanitary surveys by the Servicio Agrícola y Ganadero (SAG). In surveillance activities carried out in May 2015, a Colletotrichum species associated with leaf spots was detected on E. coccineum, in the county of Chillán Viejo, region of Biobio, Chile. The affected tree was an isolated individual showing chlorosis symptoms and partial defoliation of branches. Preliminary results, using specific polymerase chain reaction (PCR) primers, identified the specie as C. acutatum sensu lato (Sreenivasaprasad et al. 1996, EPPO 2003). Currently it is known that C. acutatum sensu lato is a complex comprising about 30 species closely related, sharing hosts and morphological and cultural characteristics that require a multi-locus DNA analysis for its correct identification (Damm et al. 2012). Due to that C. acutatum sensu stricto is part of this complex, and as a pathogen is considered a quarantine pest on several agriculture crops in Chile (SAG 2015), it is necessary to establish with precision the diagnostic of the detected species. Therefore, the main objective of this study was to determine the species of Colletotrichum found on E. coccineum, through the morphological and multi-locus molecular analysis.
Samples and isolates. The sample analyzed corresponds to leaves of Embothrium coccineum collected in May of 2015, from an isolated tree, located in the urban park Bernardo O'Higgins of Chillan Viejo County (36°51'45" S, 72°23'46" W). The leaves with symptoms of anthracnose were left in a wet chamber at room temperature for seven days (18 ± 5 °C). After this period, abundant orange masses of conidia characteristic of the Colletotrichum genus was observed, from which monosporic cultures were established in potato dextrose agar (PDA, Merck 110130). One of the monosporic isolates was conserved for morphological and molecular studies, which was stored in the mycology collection of the Regional SAG Chillan Laboratory, with the number SAG:60192-2015.
Morphological analyses. The colony growth, color and texture were evaluated on the PDA culture medium, oatmeal agar (OA) (Smith et al. 2002) and synthetic nutrient agar (SNA) (Nirenberg 1976), incubated at 20 °C under alternating cycles of 12 h near UV light and darkness for 10 days. The measuring of the size of conidia and appressoria (n = 30), as well as their morphological description, was completed only in SNA (Damm et al. 2012). Additionally, the size measurements of the conidia were completed in vivo, collected directly from leaves on E. coccineum maintained in a wet chamber. The microscopic preparations of the structures were done with 60 % lactic acid and afterwards were observed with a 100x objective using immersion oil. For the measuring of the structures, the software Piximetre version 5.6 was used.
Molecular analyses. From aerial mycelium growing on PDA, genomic DNA was extracted using the modified protocol of Cooke and Duncan 1997. The internal transcribed spacer (ITS), the intron 2 of the glyceraldehyde 3-phospha-te dehydrogenase (GAPDH), part of the actin (ACT) and beta-tubulin 2 (TUB2) were amplified with the primer pairs ITS-5 / ITS-4 (White et al. 1990), GDF1 / GDR1 (Guerber et al. 2003), ACT-512F / ACT-783R (Carbone and Kohn 1999) and TUB2Fd / TUB4Rd (Aveskamp et al. 2009), respectively, according to the protocol described by Damm et al. (2012). The amplicons obtained were sent to MACROGEN Korea for sequencing in both directions with the primers mentioned before. The sequences ITS, GAPDH, ACT and TUB2 resulting from the sequencing were aligned with another C. acutatum species complex belonging to the clade 5 according to Damm et al. 2012 (table 1), including C. orchidophilum as outgroup, using the algorithm Clustal-W of the software BioEdit 7.2.0. The phylogenetic reconstruction of the concatenated genes was done using criteria of maximum parsimony (MP) with a heuristic search and TBR (tree-bisection-reconnection) with the software PAUP 4.0b10 (Swofford 2003). The support of the internal topology of the phylogenetic tree was completed through the bootstrap analysis with 1,000 iterations.
Table 1. Species and strain information used in the phylogenetic analyses with NCBI
*CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity
The Colletotrichum isolate SAG:60192 showed different cultural characteristics according to the growth medium. Colonies on PDA were flat with entire margin, moderate grey aerial mycelium and orange mass conidia in the agar surface, with a radial growth of 32 mm in 10 days. Colonies on OA developed more aerial mycelium than that developed by colonies on PDA, but of lighter gray color with orange conidial masses mainly in the center of the dish, exhibiting a radial growth of 30 mm after 10 days. Colonies produced on OA were hyaline, with very low aerial mycelium and radial growth reaching only 26 mm in 10 days. The sizes of the conidia measured on E. coccineum leaves were 14,7 [15,8 - 16,2] 17,3 x 4,2 [4,6 - 4,8] 5,3 um with a relationship L:W= 2,9 [3,3 - 3,5] 3,9 and in the SNA medium 14,9 [15,9 - 16,3] 17,3 x 4,2 [4,5 - 4,6] 4,8 um with a relationship L:A = 3,1 [3,5 - 3,6] 4 (95 % of confidence). The conidia that formed both on the leaves and in the culture medium were hyaline, smooth-walled, aseptate, straight to cylindrical with one rounded and one acute end. Appressoria, measured on the back side of a plate with SNA medium, registered a size of 6,6 [8,8 - 9,8] 12 x 4,6 [5,6 - 6,1] 7,1 um with a relationship L:W = 1,1 [1,5 - 1,7] 2,1 (95 % of confidence), with smooth edges and wavy, brown color, mostly oval, rarely irregular or elongated (figure 2). The morphological and cultural characteristics here studied were common to several members of C. acutatum species complex, making it not possible to establish a clear differentiation on the basis of these observations.
In the multi-locus phylogenetic analysis (gene boundary in the alignment: ITS: 1-540, GAPDH: 541-800, ACT: 801-1.048, TUB2: 1.049-1539), 1539 characters were processed, including the gaps, of which 94 were parsimony-informative. After a heuristic search using PAUP, six trees were retained. One of them is shown in figure 1 (Length of tree = 130 steps, consistency index (CI) = 0.82, homoplasy index (HI) = 0.18, retention index (RI) = 0.96, rescaled consistency index (RC) = 0.79). In this phylogenetic tree the isolate SAG:60192 can be observed forming a clade with C. pyricola, with 99 % bootstrap support for the node (figure 1). The pairwise distance between our isolate SAG:60192 and the CBS reference of C. pyricola showed 99.7 % of similarity in the sequenced regions (four nucleotides of difference in the data set). In contrast, pairwise comparisons among the eight other Colletotrichum species included in the analysis identified similarities of 90-99 %. The DNA sequences for the ITS, GAPDH, ACT, TUB2 obtained in this study were deposited in GenBank with the accession numbers KU963516, KU963517, KU963518 and KU963519, respectively.
Figure 1. One of the most parsimonious trees obtained from heuristic search of the
Figure 2. Morphology of Colletotrichum pyricola observed for the isolate SAG:60192:
In this study, a combination of morphological characteristics and a multi-locus phylogenetic analysis enabled the identification of Colletotrichum pyricola on leaves of Embothrium coccineum, being this determination the first report of this fungus in Chile. Even though the morphological and cultural characteristics observed in the Colletotrichum isolate SAG:60192 correspond to the C. pyricola description, their identification can be difficult because of overlapping ranges of conidial size and variation in colony characteristics with other members of the C. acutatum species complex. In fact, C. pyricola may not be clearly distinguishable from C. johnstonii using any morphological or cultural characteristics (Damm et al. 2012). To overcome taxonomic problems associated with these traditional identification methods, DNA sequence analyses are now typically used as the primary basis in diagnosis and description of new species of Colletotrichum (Bailey 1997, Cannon et al. 2012, Liu et al. 2016). Based on the multi-locus analysis using ITS, GAPDH, ACT and TUB2 sequences, we have identified clearly C. pyricola, one of the 29 species known in the complex acutatum. Furthermore, this mycological determination has the additional value of discarding the presence of C. acutatum sensu stricto, a quarantine pest for Chile, which is an important pathogen on strawberry (SAG 2015, CABI 2016).
Colletotrichum pyricola was recently described from fruits rot of Pyrus communis L., appearing to be endemic to New Zealand and no other information exists about hosts, distribution and pathological significance (Damm et al. 2012). Many species belonging to the genus Colletotrichum are causal agents of plant diseases, generally referred to as anthracnose, on a wide range of economically important agricultural crops. For this reason, a future line of research should consider if C. pyricola is associated with the genus Pyrus or other hosts in Chile, and determine it pathological relevance.
The authors would like to thank Rodolfo Yévenes of the SAG Chillan for sample collection, the technical team of the Unidad de Fitopatología del Laboratorio Regional Chillan for the anonymous work that they performed in this identification, Gloria Cuevas, Head of the Unidad de Fitopatología and María Teresa Illesca, Head of the Laboratorio Regional Chillan, both for granting the necessary facilities to complete this work, and finally, Marcos Béeche of the Division de Protección Agrícola y Forestal of SAG Central, for supporting this technical-scientific dissemination.
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