Sample collection

Walks in the rubber clonal gardens were made in October, 2016 in San Juan Bautista municipality, Tuxtepec, Oaxaca State. Twenty foliar samples and 10 stems with anthracnose symptoms were collected, which were processed at the Cenid Comef Forest Health Laboratory.

Isolation and purification of fungi

Plant material (leaves and stems) with anthracnose symptoms were cut into squares of approximately 1 cm²; they were disinfested with 1 % sodium hypochlorite for 5 minutes, then rinsed three times with sterile distilled water and excess moisture was removed on sterile filter paper. Finally, they were placed in a humid chamber and in Potato Dextrose Agar (PDA) culture medium.

Seeds of both methods were incubated at 28 ± 1 °C for five days with a 12: 12h controlled photoperiod. After the period, fungus growth was observed and purification was performed in PDA to obtain monosporic cultures. Those strains were preserved in inclined tubes with PDA culture medium with mineral oil.

Morphological characterization

From monosporic cultures in PDA, mycelium coloration and fungus growth were scored. Temporary and permanent preparations of the isolates were made, as well as mounting of cuts of the structures obtained in a humid chamber for visualization in optical microscopy with phase contrast (AxiolabdrbKT, Zeiss) and scanning electron microscopy (EVO MA15, Zeiss) by using the cryofracture technique, in order to obtain a higher resolution image that would allow to notice a more detailed approach to the structures present in the preparations. To determine the genus, general keys were used (^{Humber, 1997}; ^{Barnett and Hunter, 1999}).

Molecular characterization

For the molecular study, two isolated strains of stem (M51 and M95) and three of leaves (M39, M46 and M48) were selected. DNA extraction was carried out by the modified AP (Alkaline Phosphatase) method (^{Sambrook and Russell, 2001}) with four-day monosporic cultures of PDA growth. The ITS region was amplified with primers ITS5 (5´GGAAGTAAAAGTCGTAACAAGG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) (^{White et al., 1990}), found in the conserved regions of the 18S and 28S genes respectively.

The PCR amplifications were performed in a Biorad T100 thermocycler with the following program: an initial denaturation cycle of 3 min at 94 °C, followed by 34 cycles, each consisted of three steps: denaturation of 30 s at 94 °C, annealing of 30 s to 58 °C and an extension of 1 min at 72 °C, finally an extension of 1 min at 72 °C. The purification of the PCR products was made with the Wizard^TM SV Gel and PCR Clean-Up System Kit (^{Promega Corporation, 1999}). Sequencing was performed at Macrogen Inc. in Seoul, Korea.

The sequences obtained were cleaned, assembled in Mega X and a search of reference sequences of the related species of the Colletotrichum complex deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/) was downloaded.

All sequences were aligned with the muscle method (^{Edgar, 2004}) included in Mega X software (^{Kumar et al., 2018}). Phylogenetic reconstruction was performed by Bayesian inference using Markov Chains Monte Carlo (MCMC), implemented in the Mr Bayes v.3.2.1 program (^{Ronquist and Huelsenbeck, 2003}) with 1 000 000 generations. The 25 % of the trees produced were discarded with the ‘burn-in phase’ option and the subsequent probability was determined with the remaining trees. Colletotrichum boninense Moriwaki, Toy. Sato & Tsukib. accession number JQ005162 was used as an outgroup.

Symptom description

The samples collected from rubber plants of clone IAN-710 showed symptoms of anthracnose (Figure 1A): in the leaves, small, circular, irregular, cream-colored necrotic water spots were observed, which later turned dark with yellow edges (Figure 1B) ; in addition, split black lesions and descending petiole death were observed in the stems (Figure 1C).

A) Symptoms observed in the field; B) Leaves schowing necrotic spots; C) Stems with die back of petioles.

Figure 1 Anthracnose symptom in rubber.  

Morphological description

Seventeen monosporic isolates were obtained, seven of stems and 10 of leaves, which presented morphological characteristics of the Colletotrichum genus. White to light gray mycelial growth was observed, with small black dots and with salmon conidial masses in PDA (Figure 2A), cylindrical conidia, with rounded, hyaline, unicellular and fusiform ends (Figure 2B and 2C) that are located in a reproductive structure called acervulus. The septate and branched conidiophores originate in the upper part of the pseudoparenchyma.

A) Colonial development in PDA medium; B) Conidia observed in compound microscope (40x); C) Conidia seen in scanning electron microscope (mag 358X).

Figure 2 Colletotrichum morphology, isolated from rubber tree leaves. 

^{Li et al. (2012)} described Colletotrichum gloeosporioides with cylindrical conidia, obtuse at the ends, hyaline, smooth. The colonies in PDA with white, gray, dark gray or olive-gray mycelia and sporulation of orange and brown or olive green on the reverse, which is similar to that observed in the isolates obtained. Anthracnose in rubber cultivation is caused by species of the Colletotrichum genus, in particular by C. gloesporoides (^{Jaimes and Rojas, 2011}).

The disease caused by this pathogen, usually occurs in the production sites of some plant species in the tropics. Colletotrichum gloeosporioides Penz. is the causative agent of a variety of damages and symptoms in different crops in Mexico, and affects several plant organs at different phenological stages (^{Gutiérrez et al., 2001}). It shows in nurseries, clonal gardens and adult plants and limits production by causing the death of affected young tissues (^{Grupo Técnico Procaucho, 2012}).

Molecular description

The amplification of the PCR products with the ITS4 and ITS5 primers of the rDNA of the 17 isolates yield a fragment of approximately 550 bp (Figure 3).

M = Molecular marker 1kb, lane 1 to 7 strains of fungi obtained from stems, 8 to 17 strains of fungi obtained from leaves; (+) = Positive control (Colletotrichum sp isolated from mango); (-) = Negative control (Water free of nucleases).

Figure 3 PCR product amplification of Colletotrichum sp. associated with anthracnose in leaves and rubber stems. 

^{Silva and Ávila (2011)} obtained fragments of approximately 580 bp for Colletotrichum species isolated from avocado (Persea americana Mill.). On the other hand, ^{Dominguez-Guerrero et al. (2012)} reported a fragment of approximately 600 bp for Colletotrichum gloeosporioides of African oil palm (Elaeis guineensis Jacq.) and ^{Martínez et al. (2015)}, 580 bp for isolates of C. gloeosporioides in litchi (Litchi chinensis Sonn.).

Phylogenetic analysis

The resulting tree was obtained with 1 000 000 generations and a final standard deviation of 0.008780. The formation of two well-defined clades was observed, in the first clade, the species under study were included and in the second clade, six species corresponding to the C. acutatum J. H. Simmonds complex, the statistical support of both clades was equal to 1 (Figure 4).

Figure 4 Phylogenetic consensus tree based on Bayesian inference that illustrates the relationship of Colletotrichum isolates associated with anthracnose in rubber cultivation within the complexes of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. species. 

The sequences of the M39, M46 and M48 isolates were grouped with sequences corresponding to C. siamense Prihast, L. Cai & K. D. Hyde and C. aenigma B. S. Weir & P. R. Johnst., as well as M51 and M95 with C. fructicola Pihasti, L. Cai & H. D. Hyde, these species have recently been described within the C. gloeosporioides species complex (^{Weir et al., 2012}). The phylogenetic reconstruction showed that the isolates under study were different from the species included within the C. acutatum species complex (^{Damm et al., 2012a}) and outside the C. boninense group (^{Damm et al., 2012b}).

Based on the phylogenetic reconstruction of the ITS region of the rDNA, the M39, M46 and M48 isolates are found in the clade consisting of C. siamense and C. aenigma. Both species are phylogenetically related, although none of them have been declared as causing the effects of rubber in the country, so they represent ‘novel’ sequences for further studies.

With regard to the M51 and M95 isolates, they were identified as C. fructicola, together with the accession number AY376538 designated as C. gloeosporioides, this sequence has been reassigned in the C. fructicola subclade.