First Report of Colletotrichum tropicale Causing Anthracnose on Pitahaya Fruit in Mexico.

Pitahaya (Hylocereus spp.), also called dragon fruit, is a cultivated cactus that is native to Mexico as well as Central and South America. In October 2021, anthracnose symptoms were observed on fruit of pitahaya (Hylocereus costaricensis) in a commercial orchard located in Culiacán, Sinaloa, Mexico. Lesions on fruit were circular, sunken, dark brown and with halo. To fungal isolation, small pieces from adjacent tissue to lesions of symptomatic fruits were surface disinfested by immersion in a 2% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently observed on PDA and five monoconidial isolates were obtained. An isolate was selected as a representative for morphological identification, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited as CCLF186 in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa). On PDA, initially white colonies turned grey with abundant orange conidia masses at 8 days after incubation at 25 ºC. Conidia were cylindrical, with ends rounded, aseptate, hyaline, and measuring 15.2 to 18.9 × 4.3 to 6.4 µm (n= 100). Appressoria were terminal, subglobose to clavate, of 7.4 to 11.6 × 5.9 to 8.2 µm (n= 30). Setae were not observed. These morphological characters were consistent with those reported for the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To determine the phylogenetic identity of the isolate CCLF186, genomic DNA was extracted following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region, the ApMat intergenic region, as well as partial sequences of actin (act) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were amplified and sequenced using the primers pairs ITS5/ITS4 (White et al. 1990), AM-F/AM-R (Silva et al. 2012), GDF/GDR, and ACT-512F/ACT-783R (Weir et al. 2012), respectively. The sequences were deposited in GenBank under accession nos. OP269659 (ITS), OP302778 (gapdh), OP302777 (act), and OP302779 (ApMat). BLASTn searches revealed high identity with sequences of C. tropicale (CBS 124949) for ITS (100%), ApMat (100%), act (100%), and gapdh (100%). A phylogenetic tree based on Bayesian inference and Maximum Likelihood methods, including published ITS, ApMat, act, and gapdh sequence datasets for isolates in the Colletotrichum gloeosporioides species complex was generated. The phylogenetic analysis based on the concatenated sequences clustered the isolate CCLF186 with the C. tropicale reference isolates. Pathogenicity of the isolate CCLF186 was confirmed on 10 healthy pitahaya fruits without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on two locations on each fruit. Ten control fruits were treated with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated pitahaya fruits exhibited sunken and necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation. Recently, C. tropicale causing anthracnose in dragon fruit (Selenicereus monacanthus) was reported from Philippines (Evallo et al. 2022). Now, this is the first report of C. tropicale causing fruit anthracnose in H. costaricensis in Mexico and worldwide. These findings provide a basis for research about the distribution and effective disease-management strategies.

First Report of Colletotrichum siamense Causing Anthracnose of Guava (Psidium guajava) in Mexico.

Guava (Psidium guajava L.) is a small tree belonging to the Myrtaceae family and it is distributed worldwide in the tropical and subtropical areas. During the summer of 2019, symptoms of fruit anthracnose were observed on approx. 90% of 250 guava trees located in backyards in Juan Jose Rios, Sinaloa, Mexico. Lesions on guava fruit were irregular, necrotic, and sunken. On advanced infections, acervuli containing salmon-pink masses of spores were observed on the lesions. Twenty fruits were collected from 10 trees (2 fruits per tree). Colletotrichum-like colonies were consistently isolated on PDA medium and 20 monoconidial isolates were obtained. Four isolates were selected as representatives for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of El Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF205-FAVF208). Colonies on PDA medium were flat with an entire margin, with abundant felty and white aerial mycelium, with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with ends rounded, and measuring 14.8 to 18.1 × 4.4 to 5.3 µm. Based on morphological features, the isolates were tentatively allocated in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), as well as partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), chitin synthase (CHS-1) and glutamine synthetase (GS) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference and including published ITS, GAPDH, TUB2, ACT, CHS-1, and GS data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the four isolates FAVF205-FAVF208 as C. siamense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accessions nos. ITS: MW598512-MW598515; GAPDH: MW595216-MW595219; TUB2: MW618012-MW618015; ACT: MW595208-MW595211; CHS-1: MW595212-MW595215; and GS: MW618008-MW618011). Pathogenicity of the four isolates was verified on 40 healthy guava fruits. Twenty fruits were wounded with a sterile toothpick (2 mm in depth) and a mycelial plug (6 mm of diameter) was placed on each wound. Ten fruits inoculated with a PDA plug without mycelial growth served as controls. The fruit was kept in a moist plastic chamber at 25°C for 7 days. Pathogenicity of each isolate was tested with both non-wound and wound inoculation methods. The experiments were repeated twice with similar results. All inoculated fruits developed sunken necrotic lesions 4 days after inoculation, whereas no symptoms were observed on the control fruits. The fungi were consistently re-isolated only from the diseased fruits, fulfilling Koch´s postulates. Colletotrichum siamense has been previously reported on guava fruit in India (Sharma et al. 2015). However, to our best knowledge, this is the first report of C. siamense causing fruit anthracnose on guava in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species on guava in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.