First Report of Colletotrichum gloeosporioides Causing Anthracnose of Tejocote (Crataegus gracilior) Fruits in Mexico

Crataegus, is a genus classified in family Rosaceae and includes several tree species commonly called Tejocote that are widely cultivated for their pome fruits in Mexico. During fall of 2014, 2015, and 2016, severe symptoms of anthracnose were observed on approx. 60% of tejocote (Crataegus gracilior) fruits in an orchard located in Tulancingo, Oaxaca, Mexico. Affected fruits showed sunken, prominent, dark brown to black necrotic lesions, and were exuding salmon spore masses. To isolate the fungus, small pieces from tissue adjacent to the lesions of 10 symptomatic fruits were excised and surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed three times in sterile distilled water, placed in Petri plates containing potato dextrose agar (PDA), and incubated at 25ºC for 5 to 7 days in darkness. Mycelial plugs were excised from the edge of the actively growing fungal colony and aseptically transferred to fresh PDA medium and incubated at 25°C for 6 days. Five monoconidial cultures were obtained by transferring germinated spores to Petri plates with fresh PDA. One isolate was selected as representative for morphological and molecular identification. Colonies of pure cultures exhibited greyish-white aerial mycelium and abundant salmon-pink conidial masses. Conidia (n= 100) were subcylindrical, hyaline, straight, one-celled, with rounded ends, measuring 13.6 to 17.7 × 4.4 to 5.9 μm. Conidial appressoria were ovoid and brown to dark brown. Based on morphological characteristics, the fungus was identified within the Colletotrichum gloeosporioides species complex (Weir et al. 2012). The isolate was designated UACH-177 and deposited in the Culture Collection of Phytopathogenic Fungi at the Chapingo Autonomous University. For molecular identification, the ITS region (White et al. 1990), and fragments of (Apn2) (Rojas et al. 2010), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin 2 (TUB2) genes (Weir et al. 2012) were amplified by PCR, and sequenced. The sequences were deposited in GenBank (Accessions numbers ITS:MG821312; Apn2:MG821310; GAPDH:MG821311; and TUB2:MG821313). A phylogenetic analysis using Bayesian inference and including published ITS, Apn2, GAPDH, and TUB2 data for C. gloeosporioides and other Colletotrichum species was performed. The phylogenetic analysis showed the sequences were grouped into the clade of C. gloeosporioides. To confirm the pathogenicity of the fungus, 20 tejocote fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 1 min, washed three times with sterile distilled water and dried on sterilized filter paper. Inoculations were performed by deposition of 10 μl of a conidial suspension (106 spores ml-1) on the fruit surface. Ten fruit were mock inoculated with distilled water as a control. All fruits were kept in a moist chamber at 25°C for 10 days. Pathogenicity test was repeated twice. Disease symptoms were observed on all inoculated fruit after 7 days, whereas control fruit did not develop symptoms. Fungal colonies were re-isolated from all symptomatic fruits and were found to be morphologically identical to the original isolate inoculated on tejocote fruits, thus fulfilling Koch´s postulates. In Mexico, García-Alvarez (1976) reported Colletotrichum sp. on fruits of Crataegus mexicana, however, that report was not supported by morphological characterization nor pathogenicity tests. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of Crataegus gracilior in Mexico and worldwide.Fil: Nieto López, Edgar Humberto. University of Nebraska; Estados UnidosFil: Everhart, Sydney. University of Nebraska; Estados UnidosFil: Ayala Escobar, Victoria. Colegio de Postgraduados; MéxicoFil: Camacho Tapia, Moises. Universidad Autónoma Chapingo; MéxicoFil: Bernardi Lima, Nelson. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Nieto Angel, Raúl. Universidad Autónoma Chapingo; MéxicoFil: Tovar Pedraza, Juan Manuel. Universidad Autónoma Chapingo; Méxic

First report of Cladosporium colocasiae causing leaf spot on taro (Colocasia esculenta) in Mexico

In September 2017, symptoms of leaf spot were observed on approx. 80% of taro (Colocasia esculenta var. antiquorum) plants at a commercial field located in San Juan Bautista Tuxtepec, Oaxaca, Mexico. Lesions on diseased leaves were dark brown and oval to irregular, sometimes coalesced to cover large areas of the leaf surface. To isolate the fungus, small fragments from adjacent tissue to lesions of symptomatic leaves were excised and surface disinfested by immersion in a 1% NaOCl for 2 min, rinsed three times in sterile distilled water, dried in blotter paper, and placed in Petri plates containing potato dextrose agar (PDA) amended with ampicillin (20 mg/l) and rifampicin (20 mg/l). The plates were incubated at 25ºC under constant white light for 8 days and then mycelial plugs were aseptically transferred to fresh PDA medium. Pure cultures were obtained by monoconidial isolation technique. Colonies on PDA exhibited aerial mycelium sparse, grey-olivaceous to darkolivaceous, and with sporulation profuse. Conidiophores were solitary, erect, straight or slightly flexuous, cylindrical-oblong, olivaceous-brown, nodulose, usually unbranched, and measuring up to 650 μm long. Conidia catenate in branched chains, ellipsoidal to cylindrical, smooth, aseptate or 1?2-septate, olivaceous-brown, 9 to 18 × 5 to 8 μm. Based on morphological characteristics, the fungus was identified as Cladosporium colocasiae (Bensch et al. 2012; García and Moya 2015). A representative isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Chapingo Autonomous University as UACH 291. For molecular identification, the internal transcribed spacer (ITS) region, and part of the translation elongation factor 1-α (EF1-α) gene were amplified by PCR, and sequenced using the primer sets ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The sequences were deposited in GenBank (Accession numbers ITS:MH191366 and EF1-α:MH191367). A phylogenetic analysis using Bayesian inference and including published ITS and EF1-α sequence dataset for C. colocasiae and other Cladosporium species was performed. The phylogenetic analysis resulted in a well-supported clade grouped with the type species of C. colocasiae. The pathogenicity of the fungus was verified on taro plants growing in a greenhouse. Two leaves from each of five 5-month-old taro plants were sprayed with a conidial suspension (104 spores/ml). Five leaves were mock inoculated with distilled water as a control. All plants were covered for 48 h with a plastic bag to keep moisture and then were maintained in a greenhouse at temperatures ranging from 25 to 32°C for 15 days. The pathogenicity test was performed twice. Symptoms of leaf spots were observed after 10 days on all leaves inoculated with conidial suspensions, while control leaves remained symptomless. Koch´s postulates were fulfilled when the pathogen was reisolated 100% from the diseased leaves. Cladosporium colocasiae has been reported on Colocasia spp. in North America (USA), Caribbean islands, South America, Europe, as well as widely distributed in Africa, Asia, and Australasia (Bensch et al. 2012; Farr and Rossman 2018). To our knowledge, this is the first report of C. colocasiae causing leaf spot on taro in Mexico. The occurrence of this pathogen presents a threat to the production of taro in this area of southeastern Mexico, and therefore, effective management strategies should be implemented.Fil: Vásquez-López, Alfonso. Instituto Politécnico Nacional; MéxicoFil: Palacios-Torres, Rogelio Enrique. Universidad del Papaloapa; MéxicoFil: Montiel-Frausto, Laura Belem. Instituto Politécnico Nacional; MéxicoFil: Medero Vega, Víctor Reinaldo. Instituto de Investigaciones de Viandas Tropicales; CubaFil: Bernardi Lima, Nelson. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Camacho-Tapia, Moises. Universidad Autonoma Chapingo; MéxicoFil: Tovar-Pedraza, Juan Manuel. Centro de Investigación En Alimentación y Desarrollo; Méxic

Colletotrichum brevisporum and C. musicola Causing Leaf Anthracnose of Taro (Colocasia esculenta) in Mexico

Taro (Colocasia esculenta var. antiquorum), family Araceae, is an important tropical crop cultivated throughout the world for its edible corms. During the summer of 2017, irregular brown lesions, typical of anthracnose were observed on approx. 40% of taro plants in a commercial orchard located in San Juan Bautista Tuxtepec, Oaxaca, Mexico. Small pieces of the lesions of five symptomatic leaves were surface disinfected with 1% NaOCl for 2 min followed by rinsing with sterile distilled water and plated on potato dextrose agar plates which were then incubated at 25°C for 5 days in darkness. Colletotrichum-like colonies were consistently isolated and 10 monoconidial isolates were obtained. Two isolates were selected as representatives for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were designated as UACH289 and UACH290 and were deposited in the Culture Collection of Phytopathogenic Fungi at the Chapingo Autonomous University. Conidia (n = 100) of isolate UACH289 were cylindrical, hyaline, aseptate, 15.1 to 18.4 × 4.4 to 5.2 μm, with the apex rounded and the base, rounded to truncate; appressoria (n = 20) were single, dark brown, and with undulate or lobate margin. Whereas conidia (n = 100) of isolate UACH290 were cylindrical to ellipsoidal, hyaline, aseptate, 12.3 to 17.0 × 3.8 to 5.0 μm; appressoria (n = 20) were irregular, bullet-shaped to elliptical, dark brown, with undulate or lobate margin. For molecular identification, the internal transcribed spacer (ITS) region (White et al. 1990), and fragments of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (TUB2) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic analysis based on Bayesian inference and including published ITS, ACT, GAPDH, and TUB2 data for Colletotrichum species was performed. After phylogenetic analysis, isolates UACH289 and UACH290 clustered with C. brevisporum (Accessions nos. ITS: MK862121; ACT: MK862124; GAPDH: MK862122; and TUB2: MK862123) and C. musicola (Accessions nos. ITS: MK882586; ACT: MK882589; GAPDH: MK882587; and TUB2: MK882588), respectively. Pathogenicity of the fungi was verified by spraying conidial suspensions (1 × 106 spores ml-1) on the upper surface of 10 taro leaves. Ten control leaves were sprayed using sterilized water. All plants were kept under greenhouse conditions at 25°C for 10 days. Anthracnose symptoms were observed on all inoculated leaves after 12 days, whereas control leaves remained symptomless. Koch´s postulates were fulfilled when the fungi were re-isolated 100% from the diseased leaves. The experiment was performed twice. Colletotrichum brevisporum has been previously reported as a pathogen on several crops including Annona muricata, Carica papaya, Sechium edule, Capsicum annuum, Passiflora edulis, Citrus medica, P. edulis, Cucurbita moschata, and C. pepo (Farr and Rossman 2019), whereas Colletotrichum musicola only has been associated with Musa sp. in Mexico (Damm et al. 2019). To our knowledge, this is the first report of C. brevisporum and C. musicola causing leaf anthracnose of taro in Mexico and worldwide.Fil: Vásquez-López, Alfonso. Instituto Politécnico Nacional. Unidad Oaxaca; MéxicoFil: Palacios-Torres, Rogelio Enrique. Universidad del Papaloapan, Instituto de Agroingeniería; MéxicoFil: Camacho-Tapia, Moises. Universidad Autónoma de Chapingo; MéxicoFil: Granados-Echegoyen, Carlos. Universidad Autónoma de Campeche; MéxicoFil: Bernardi Lima, Nelson. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Fitopatologia y Modelizacion Agricola. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Unidad de Fitopatologia y Modelizacion Agricola.; ArgentinaFil: Vera-Reyes, Ileana. Universidad Autonoma Chapingo; MéxicoFil: Tovar Pedraza, Juan Manuel. Universidad Autonoma Chapingo; MéxicoFil: Leyva-Mir, Santos Gerardo. Universidad Autonoma Chapingo; Méxic