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.
For morphological examination of colony characteristics and growth, isolates were grown on PDA, water agar (WA), synthetic poor nutrient agar (SNA) at 24°C in darkness (Schroers et al. 2008) and examined after 7–20 days of incubation. Morphology of conidia and chlamydospores were determined. An average of 30 conidia was measured. Microphotographs of fungal features were taken using a Dinoeye microscope camera USB lens (The Microscope Store, LLC., USA). Colony diameters were measured on three replicate plates on PDA after 7 days.
DNA extraction, PCR and Sequencing
For molecular identification at the species level, three representative isolates were selected for sequencing of ITS rDNA regions. Fresh fungal mycelia were scraped off from 7-day-old PDA plates of single spore cultures, homogenized using liquid nitrogen and Genomic DNA was extracted using a CTAB extraction procedure (Zhang et al. 2010). A standard polymerase chain reaction (PCR) protocol was used to amplify ITS rDNA regions with primers ITS1 and ITS4 (White et al. 1990). Amplifications were performed in a Biometra TAdvanced Thermal Cycler (Biometra, Göttingen, Germany) with an initial denaturation of 5 min at 95°C followed by 35 cycles of 30 s denaturation at 95°C, 30 s annealing at 59°C, 60 s extension at 72°C and a final extension of 5 min at 72°C. The quantity and quality of PCR products were evaluated on 1% agarose gels. The PCR products sequencing was performed by Bioneer (Bioneer Co., Korea). The sequences generated in this study were deposited in GenBank and the accession numbers were obtained (Table 1).
The obtained sequences were manually edited using Geneious v. 7 (Biomatters)s and compared with those in the GenBank database using a basic local alignment search tool (BLAST) (Altschul et al. 1990). Generated sequences were added to sequences retrieved from GenBank according to the reliable published papers and included in the phylogenetic analysis (Table 1). The sequences were aligned using Geneious version 7 (Biomatters, USA). Phylogenetic relationships and identification of the isolates in the species level were performed using PAUP* 4.0a133 (Swofford 2002) for parsimony. Gaps were treated as missing data. To assess the branch support, bootstrap analysis with 1000 replicates using a heuristic search was performed. Nectria balansae (GenBank accession no. HM484857) was used as an outgroup taxon.
Representative isolates were tested for pathogenicity confirmation on strawberry. Potted symptomless strawberry plants (Fragaria ananassa cv. Paros) were surface-sterilized by dipping the roots in 0.5% (w/v) NaOCl for 30 s and washed with sterile distilled water for 2 min. Ten-day-old fungal cultures growing on PDA were used to obtain conidial suspensions. Spore concentration was adjusted to 5×106 spores mL-1 using a hemocytometer. Plants were inoculated by immersing in a conidial suspension of the isolate (106 conidia mL–1) for 20 min. Control plants were inoculated with sterile distilled water. Plants were incubated at 20°C for 24 h, then transferred to a greenhouse and inspected daily for symptoms.
RESULTS AND DISCUSSION
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).
This research was supported by Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.