First record of Dactylonectria macrodidyma causing black root rot on strawberry

INTRODUCTION

Strawberry (Fragaria ananassa Duch.) is a major fruit crop in Iran. Strawberry was imported to Iran about 100 years ago from France (Eshghi et al. 2007). It is cultivated in fields and greenhouses. According to agriculturestatistic book ofIran (2019), the greenhouse strawberry production area has been approx. 522 ha in 2019, from which 266 ha were located in Kerman province (including southern parts of Kerman). Root and foot diseases caused by fungi as well as post-harvest diseases are major constraints to strawberry production worldwide, including Iran (Embaby 2007, Ayoubi et al. 2016, Fang et al. 2011,Petrasch et al. 2019). Botrytis gray mold (Petrasch et al. 2019), wilt disease caused by Verticillium dahliae Kleb. (Harris & Yang 1996), red stele, caused by Phytophthora fragariae Hickman (Newton et al. 2010), crown rot (vascular collapse) and leather fruit rot caused by Phytophthora cactorum (Leb. and Cohn) Schröeter (Stensvand et al. 1999) are major fungal diseases on this crop. Botrytis cinerea Pers., Colletotrichum spp. and Rhizopus stolonifer, are of great importance among fungal diseases of strawberry in Iran. Black root rot disease on strawberry has been reported to be associated with many fungal species, including Ceratobasidium fragariae (Wilhelm et al. 1972), Pythium spp., (Watanabe et al. 1977), Gnomoniopsis fructicola (Moročko-Bičevska et al. 2019), Fusarium spp. (Koike & Gordon 2015) and Dactylonectria torresensis (Weber & Entrop 2017). The disease symptoms appear as deterioration and black necrosis of the root system and decline in productivity. The aim of this study was to identify the causal agent of strawberry black root rot, which was observed during visits to greenhouse strawberry productions in Kerman.

MATERIALS AND METHODS

Sample collection and fungal isolation

During visiting strawberry cultivation greenhouses in different locations in Kerman County, several plants with typical symptoms of black root rot were observed. Symptomatic plants showing stunted growth, leaf margins necrosis and black root rot were collected and transferred to the laboratory. Plant materials were washed under running tap water for 30 minutes to remove excess soil particles and spores of fast-growing contaminant fungi on the tissue's surface.  Small segments of symptomatic tissues were surface disinfected in 70% EtOH for 10 s, 1% NaOCl for 1 min, and rinsing in sterile deionized H₂O for 1 min. Plant materials were dried on sterile filter papers. Tissue pieces (3–5mm long) were placed on potato dextrose agar (PDA), amended with streptomycin sulfate at 100 mg/L and incubated at 25°C for seven days in darkness. Single conidial cultures were prepared and stores on PDA slants at 10°C. The isolates were deposited in (Kerman Graduate University of Advanced Technology, Kerman, Iran) fungal culture collection and stored in 15% glycerol at -80°C.

Cylindrocarpon-like isolates were consistently isolated from infected tissues. The species identification was based on morphological and molecular criteria. A total of 12 isolates of D. macrodidyma were obtained from roots of strawberries showing black root rot symptoms from Kerman strawberries cultivation greenhouses. Colonies of D. macrodidyma on PDA were brown with yellow (honey) pigmentation at the margins. Conidiophores arising laterally from the aerial mycelium unbranched or sparsely branched and 1–4-septate. Phialides cylindrical tapering towards the tip. Macroconidia on SNA medium were 1–3 (–4) septate, straight, cylindrical (sometimes widening toward the tip), apical cell slightly bent to one side, 40 (±11) × 6.3 (±1.8) µm with free-standing, slender, unbranched conidiophores. Microconidia were 0–1 septate, ellipsoid and ovoid 10.5 (±3.2) × 4.1 (±1.6) µm. Chlamydospores in short, intercalary chains (Fig. 1). Morphological characteristics are corresponding with published descriptions of D. macrodidyma (Haleen et al. 2004). The results of BLAST search against sequences in GenBank and phylogenetic analysis confirmed the species identification. In the phylogenetic tree (Fig. 2), the isolates obtained from strawberries grouped with D. macrodidyma in a well-supported clade.

The results of pathogenicity tests showed that the tested isolates were pathogenic to strawberry and the first symptoms were observed 15 days after inoculation while control plants remained healthy and asymptomatic. Dactylonectria macrodidyma was consistently re-isolated from symptomatic tissues. The inoculated plants showed identical symptoms to those observed in strawberry cultivation greenhouses. The inoculated plants showed stunted growth, necrosis in leaf margins and black root rot symptoms with necrotic lesions on roots (Fig. 3).

Species of Cylindrocarpon / Dactylonectria are plant pathogens causing black foot and root diseases (Vitale et al. 2012, Dos Santos et al. 2014, Adesemoye et al. 2016). This study represents the first report of D. macrodidyma on strawberry in Iran. However, the distribution of this disease in other strawberry production areas remains to be investigated. The impact of this pathogen on strawberry production in Kerman greenhouses is not clear yet. Black foot rot resulting from Dactylonectria has been wildly studied in nurseries and vineyards of grapevines (Halleen et al. 2003, 2004, Cabral et al. 2012). According to Halleen et al. (2003), the main practice for disease management is using a clean potting medium collected from unaffected areas where the disease has not been observed. Greenhouse staff should pay attention to root decay symptoms and discard seedlings that exhibit symptoms.

Table 1. Strains used in the phylogenetic analysis.

Fig. 1. Dactylonectria macrodidyma. a. Colony after 14 days at 24°C; b-c. macroconidia; d-e. macroconidia and microconidia; f. chlamydospores. — Scale bars = 10 μm.

Fig. 2. Parsimony tree based on aligned sequences of ITS region. Bootstrap values (1000 replicates) indicated at the nodes. The scale bar indicates the number of nucleotide changes. The tree was rooted in Nectria balansae.

Fig. 3. Disease symptoms on strawberry including necrosis in leaf margins (upper) and black root rot symptoms with necrotic lesions on roots (beneath). 

ACKNOWLEDGEMENTS

This research was supported by Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.