First Report of Colletotrichum tropicale Causing Anthracnose on Common Fig in Taiwan

Ficus carica L. known as common fig is one of the most profitable fruit crops in Taiwan. Their fruit are harvested for high-priced market. Common fig can be eaten fresh or dried and processed to make different food products. In September 2015, an anthracnose-like disease was widely observed on common fig fruit planted in an orchard in Lukang township (24°04'36" N, 120°27'15" E) in Changhua County, central Taiwan. Symptoms were sunken, water-soaked lesions covered with salmon-colored spore masses and were observed on all stages of fruit, especially when fruit was ripe. Four fungal isolates were collected from four diseased fruit of different plants in the same orchard. Conidia were spread on 2% water agar, and a single conidium was separated by a handmade glass needle. Fungal isolates were grown on potato dextrose agar (PDA) at 24 to 28°C with diffused light. All four strains produced white, aerial, and cottony mycelia covered with abundant salmon-colored conidial masses on PDA. The conidia were hyaline, single celled, round cylindrical on both ends, thin walled, and the contents guttulate. The sizes of conidia were 15.4 (18.5 to 13.1) × 4.73 (5.8 to 3.6) μm [average (max. to min.); length/width ratio = 3.25, n = 40]. DNA was isolated from the representative isolate FC1 and used for amplification of partial sequences of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin 2 (TUB2), manganese-superoxide dismutase (SOD2), calmodulin (CAL), chitin synthase 1 (CHS-1) (Weir et al. 2012) and the intergenic region of apn2 and MAT1-2-1 gene (ApMat) genes (Sharma et al. 2013). A BLAST search against the NCBI database revealed that FC1 gene sequences [GenBank accession nos. MT192648 (ITS), MT155819 (GAPDH), MT199873 (ACT), MT199874 (TUB2), MT815916 (SOD2), MT815917 (CAL), MW684717 (CHS-1) and MT221652 (ApMat)] displayed 99.1, 98.2, 99.3, 99.6, 99.5, 100.0, 92.8 and 100.0% nucleotide identity to the respective gene sequences of Colletotrichum tropicale CBS 124949 (ICMP18653) (JX010264, JX010007, JX009489, JX010407, JX010329, JX009719, JX009870 and KC790728). Multilocus phylogenetic analysis performed with reference sequences showed that the isolate FC1 clustered with C. tropicale in accordance with BLAST results. A conidial suspension (1 × 106 conidia/mL) prepared from FC1 isolate was inoculated by spraying onto detached, ripe, healthy, non-wounded and surface-disinfected common fig fruit (cv. China, n = 4). Fruit sprayed with sterile water were used as control. Fruit were kept in a moist chamber (greater than 90% relative humidity, 24 to 28°C) for 24 h and then maintained in the lab for additional 5 days. The inoculated fruit developed lesions similar to the disease symptoms in the orchard. No symptom was observed on fruit treated with water. C. tropicale was re-isolated from symptomatic fruits and had similar morphological characteristics to FC1 isolate, thus fulfilling Koch’s postulates. The experiment was repeated once showing similar results. The FC1 isolate of C. tropicale with the identification number BCRC FU31436 has been deposited at Taiwan Bioresource Collection and Research Center. This fungus had previously been found on lotus and mango in Taiwan (Chen and Kirschner 2018; Wu et al. 2020), while the pathogenicity among the isolates from different origins is not yet known. To our knowledge, this is the first report of C. tropicale causing anthracnose on common fig fruit in Taiwan.

First report of Colletotrichum tropicale causing anthracnose on Passiflora edulis in Brazil

Brazil is the world's largest producer and consumer of yellow passion fruit (Passiflora edulis f. flavicarpa), mainly for the manufacture of concentrate and frozen juice as well as for fresh consumption (Faleiro et al. 2005). Between June and July 2018, passion fruit plants with symptoms of anthracnose were observed in commercial planting in the municipality of Coruripe (20 ha), northeastern state of Alagoas, Brazil. Approximately 70% of the plants showed leaves with relatively large, watery, circular spots that affected 30% of the leaf surface. Small fragments taken from the transition region of symptomatic tissue were superficially disinfected in 70% ethanol for 30 s and in 1% NaClO for 1 min, rinsed in sterile distilled water (SDW), dried on filter paper, plated on potato dextrose agar (PDA-Kasvi) incubated at 25°C under white light and 12 h photoperiod, for 3 days. Two isolates were obtained and deposited in the Collection of Phytopathogens at the Universidade Federal de Alagoas (COUFAL0281 and COUFAL0282). To identify the isolates, partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (TUB2) genes and of the rDNA-ITS (ITS) region were amplified by PCR. The partial sequences were deposited at GenBank (MT299339, MT334694, MT310553, MT299340, MT334695 and MT310554). Based on the BLASTn analysis, sequences of the PCR products showed high nucleotide similarity with sequences of the species C. tropicale (CBS 124949/ex-type and ICMP 18672), for GAPDH (98.94% and 100%), TUB2 (99 and 100%) genes and ITS (100%). This result was also confirmed in the phylogenetic tree of Bayesian Inference assembled with concatenated data (GAPDH, TUB2 and ITS). The colonies of the isolates were white with a white reverse, with dense mycelium, and mean growth rate of 7.54 mm/day, after 7 days on PDA medium at 25° C. Conidia were subcylindrical with rounded ends, hyaline, smooth walls and measured 13.63-20.59μm (= 17.54μm; n= 50) in length and 4.40-7.93 μm (= 5.88 μm; n= 50) in width. Appressoria were melanized, subglobose, irregular and measured 7.44 - 18.57 μm (= 10.04 μm; n= 50) in length and 5.49-10.16 μm (= 7.66 μm; n= 50) in width. These morphological characteristics were consistent with those described for Colletotrichum tropicale E.I. Rojas, S.A. Rehner & Samuels (Rojas et al. 2010). To confirm pathogenicity, 30 µL of a 106 conidia/mL sterile distilled water (SDW) conidia suspension, together with a drop of 20% Tween were deposited on the adaxial surface of passion fruit leaves wounded with a sterile needle, with four repetitions. The control consisted of leaves inoculated only with SDW. The leaves were placed in a plastic Gerbox box with sterilized filter paper moistened with SDW and maintained in a Biochemistry Oxygen Demand (BOD) incubator stove at 25 ºC and photoperiod of 12 h. After 7 days, typical anthracnose symptoms were observed on inoculated leaves. The pathogen was re-isolated and confirmed by morphological characterization, according to Koch's postulates. No symptoms were observed in the negative control. The occurrence of this species has been frequently reported in several other crops grown in northeastern Brazil (Silva et al. 2017; Veloso et al. 2018; Vieira et al. 2018; Costa et al. 2019). Additionally, many of these crops are grown in close proximity to the passion fruit orchards, thus favoring pathogen movement between hosts, probably, due to the anthropic influence, circulation of animals and insects, as well as wind driven rain splashes. However, this is first report of C. tropicale in Passiflora edulis in the world.

Tryptophan-derived metabolites and BAK1 separately contribute to Arabidopsis postinvasive immunity against Alternaria brassicicola

Nonhost resistance of Arabidopsis thaliana against the hemibiotrophic fungus Colletotrichum tropicale requires PEN2-dependent preinvasive resistance and CYP71A12 and CYP71A13-dependent postinvasive resistance, which both rely on tryptophan (Trp) metabolism. We here revealed that CYP71A12, CYP71A13 and PAD3 are critical for Arabidopsis’ postinvasive basal resistance toward the necrotrophic Alternaria brassicicola. Consistent with this, gene expression and metabolite analyses suggested that the invasion by A. brassicicola triggered the CYP71A12-dependent production of indole-3-carboxylic acid derivatives and the PAD3 and CYP71A13-dependent production of camalexin. We next addressed the activation of the CYP71A12 and PAD3-dependent postinvasive resistance. We found that bak1-5 mutation significantly reduced postinvasive resistance against A. brassicicola, indicating that pattern recognition contributes to activation of this second defense-layer. However, the bak1-5 mutation had no detectable effects on the Trp-metabolism triggered by the fungal penetration. Together with this, further comparative gene expression analyses suggested that pathogen invasion in Arabidopsis activates (1) CYP71A12 and PAD3-related antifungal metabolism that is not hampered by bak1-5, and (2) a bak1-5 sensitive immune pathway that activates the expression of antimicrobial proteins.

bak1-5 mutation uncouples tryptophan-dependent and independent postinvasive immune pathways triggered in Arabidopsis by multiple fungal pathogens

ABSTRACTRobust nonhost resistance of Arabidopsis thaliana against the nonadapted hemibiotrophic fungus Colletotrichum tropicale requires PEN2-dependent preinvasive and CYP71A12/CYP71A13-dependent postinvasive resistance, which both rely on tryptophan (Trp) metabolism. Here we report that CYP71A12 and CYP71A13 are critical for Arabidopsis’ postinvasive resistance toward both the necrotrophic Alternaria brassicicola and the adapted hemibiotrophic C. higginsianum fungi. Metabolite analyses suggest that the production of indole-3-carboxylic acid derivatives (ICAs) and camalexin is induced upon pathogen invasion, while phenotypic comparison of cyp79B2 cyp79B3 and pen2 cyp71A12 cyp71A13 plants indicates that the contribution of ICAs to postinvasive resistance is dose-dependent. We also found that the disruption of intact pattern recognition receptor complex caused by bak1–5 mutation significantly reduced postinvasive resistance against C. tropicale and A. brassicicola, indicating that pattern recognition commonly contributes to this second defense-layer against pathogens with distinct infection strategies. However, the bak1–5 mutation had no detectable effects on Trp-metabolite accumulation triggered by pathogen invasion. Together with this, further comparative gene expression analyses suggested that pathogen invasion in Arabidopsis activates (i) bak1–5 insensitive Trp-metabolism that leads to antimicrobial secondary metabolites, and (ii) a bak1–5 sensitive immune pathway that activates the expression of antimicrobial proteins.