Carbendazim sensitivity in populations of C

1. Introduction

Strawberry (Fragaria×ananassa) is an important fruit crop in the world. The cultivated area of strawberry in China has increased rapidly in recent years. In 2015, China became the largest producer of strawberry, with a total planting area of 130 000 hectares and a production of 3.5 million tons per year (MOA 2017). Anthracnose can occur throughout the entire production cycle, particularly during the seedling and transplanting stages of strawberry plants. Strawberry infected with Colletotrichum species may develop lesions on all parts of plants, resulting in the collapse and death of the plant in serious cases (Freeman et al. 1998). The etiology of strawberry anthracnose is complex, and 12 Colletotrichum species have been regarded as causal agents (Weir et al. 2012; Han et al.2016). In a preliminary study, Colletotrichum siamense (in synonymy with Colletotrichum murrayae) (Liu et al. 2016),Colletotrichum fructicola, Colletotrichum gloeosporioides,and Colletotrichum aenigma in C. gloeosporioides complex were identified to cause anthracnose on strawberry in Hubei Province of China. Isolates of C. siamense and C. fructicola showed strong virulence to petioles of gloeosporioides and C. aenigma showed weak virulence to petioles. C. siamense was the most frequently isolated species, accounting for 81% of all isolates (Han et al. 2016).

Yams (Dioscorea spp.) are widely cultivated in area used for yams production has increased rapidly in recent years. Yam infected with Colletotrichum species may develop lesions on leaves and stems, resulting in reduction of the effective photosynthetic surface area of the plants and seriously affecting tuber production (Abang et al. 2002).Anthracnose has become an important disease in most of the yam-growing regions, particularly in Hubei Province.

Anthracnose caused by Colletotrichum species affects a wide variety of crops and fruit trees, particularly in tropical and subtropical areas (Hyde et al. 2009; Wikee et al. 2011;Cannon et al. 2012). The genus has been denoted the world’s eighth most important group of plant-pathogenic fungi based on perceived economic and scientific importance (Dean et al. 2012). Colletotrichum species may exhibit significant differences in their response to example, most wild-type isolates of C. gloeosporioides are sensitive to methyl-benzimidazole carbamate (MBC)fungicides. Thus MBC fungicides are still recommended for the control of anthracnose (Brannen and Smith 2015).However, C. acutatum is tolerant to MBC fungicides (Peres et al. 2014). Chung et al. (2006) and Maymon et al. (2006)discussed the MBC resistance of C. gloeosporioides, the phylogenetic tree obtained from the internal transcribed spacer (ITS) sequences indicated that the sensitive and resistant genotypes are two separate and independent populations. Single-gene phylogenetic analyses have not been successful in delineating Colletotrichum gloeosporioides is a species complex that consists of 34 taxa (Weir et al. 2012; Hyde et al. 2014). The separate populations in the phylogenetic tree based on ITS sequences in Maymon et al. (2006) may belong to different species.

Carbendazim is one of the most commonly used fungicides to control strawberry and yam anthracnose in Hubei Province. Lin et al. (2016) have evaluated the benzimidazole resistance of Colletotrichum gloeosporioides complex populations from strawberry in Zhejiang Province of China and found 99.1% isolates were resistant to benzimidazoles. But, little was known about the carbendazim resistance of species in the C. gloeosporioides complex from strawberry and yam in Hubei , the objectives of this study were (i) to detect the occurrence of resistance to carbendazim within C. gloeosporioides complex isolates obtained from strawberry and yam and (ii) to further understand the genetic evolution of fungicide resistance in the plant pathogen.

2. Materials and methods

2.1. Isolate collection

Fig. 1 Field symptoms of anthracnose caused by Colletotrichum spp. in strawberry (A) and yam (B).

From 2012 to 2016, symptomatic leaves and stems with diagnostic symptoms (Fig. 1-A and B) were collected from 17 fields in eight cities across Hubei Province of China. A 5 mm×5 mm block of tissue was cut from the edge of the lesion and surface disinfested by rinsing in 0.5% sodium hypochlorite for 3 min, and then washed three times with sterilized distilled water. The disinfested tissue was placed in the center of potato dextrose agar (PDA) plate acidified with lactic acid at a final concentration of 0.05% to inhibit bacterial growth. The plates were incubated for 3 to 7 days at 28°C in the dark. After incubation, a 5 mm×5 mm block of agar taken from the growing edges of any fungal hyphae developing on the PDA plates was then transferred aseptically to new PDA plates amended with lactic acid at 0.05%. Monoconidial cultures were subsequently obtained through serial dilution, and isolates without conidia on the PDA plates were purified by transferring samples from the advancing mycelial edge of each colony to new PDA plates three to five times. All Colletotrichum isolates were identified to the species using a multilocus phylogenetic analysis identification method (Han et al. 2016).

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