Sample collection and fungal isolation

During 2014 and 2015, rose plants with yellow to brown discoloration and dieback of the tips of shoots, were collected randomly from the major rose-producing greenhouses of the Fars province. Wood segments were cut from the affected stems, washed under running tap water, surface disinfested in a 1% sodium hypochlorite solution for 5 min, and washed twice with sterile distilled water. Small pieces from the margin between healthy and discolored stem tissue were plated on acidified potato dextrose agar (PDA; potato extract 300 g/L, dextrose 20 g/L, agar 15 g/L). Plates were incubated at 25 °C for 5 to 10 days, and growing colonies were transferred to PDA. Single spore colonies were derived prior to morphological and molecular identification using the serial dilution method (Dhingra & Sinclair, 1995). Among isolated fungi, Acremonium species was found to be the most abundant one. Using compound microscope, morphological characters were used to distinguish the species of isolated fungi included conidiophore morphology, phialides type and shape, and conidial size and shape. The length and width of 50 conidia per isolate were measured. Colony characters and pigment production on PDA, 2% malt extract agar (MEA; 20 g/L malt extract, 16 g/L agar) and oatmeal agar (OA; 60 g/L oatmeal, 16 g/L agar) incubated at 25 °C were evaluated after 10 days (Domsch et al., 2007). Presence/absence of chlamydospores and sclerotia was evaluated using OA and 2% water agar (WA; agar 20 g/L) cultures with sterilized nettle (Urtica sp.) stems respectively and incubating them at 25 °C (Domsch et al., 2007). Isolates were examined weekly for formation of chlamydospores and sclerotia.

Representative isolates were deposited in the culture collection of Plant Protection Department, College of Agriculture, Shiraz University.

Growth rates and percentage of spore germination

Plugs of agar, 2 mm in diameter, were cut from the edge of 7 day-old colonies and placed in the center of PDA plates which were then incubated at temperatures ranging from 15±2 to 35±2 °C in 5 °C increments using three replicate plates per isolate. Growth rates of colonies were recorded after 10 days.

Concentration of about 10⁴ spore/mL of each isolate was made and dispersed to 2% WA plates using a sterilized glass rod. Percent germination of spores was determined under a compound microscope Zeiss at × 100 magnification following 16 h at temperatures ranging from 15 to 35 °C in 5 °C increments.

Spore trapping in rose greenhouses

The spore trapping method described by Eskalen & Gubler (2001) was used for this study with the following modification. Spore traps including TMN-PDA (PDA amended with 0.5 mL/L trimethylnonanol and 25 mg/mL pentachloronitrobenzene) (Banihashemi & de Zeeuw, 1969) plates placed in selected greenhouses where rose dieback were known to occur. The plate traps were collected after 24 h, incubated at 25 °C and observed for pathogen presence after 7 days. The colonies of each fungus were recorded and a representative of each isolate of fungus was subculture onto PDA.

Pathogenicity tests

Nine rose cultivars (ˈRed Oneˈ, ˈOtopiaˈ, ˈTintoˈ, ˈAvalancheˈ, ˈAttractaˈ, ˈFiestaˈ, ˈSamuraiˈ, ˈDolce Vitaˈ, ˈShirazˈ) were used for pathogenicity tests in the greenhouse test. Six shoots of each cultivar were used for each isolate. Wounds were made on the internode of the shoots with a disinfested pruning shear. Agar plug from 10-day-old cultures were placed in the wounds and parafilm was wrapped over the wounds to prevent desiccation. Fresh PDA plugs instead of mycelium plugs were used as control. Four weeks after inoculation, the canes were inspected for lesion development and disease symptoms. Small pieces of necrotic tissue from the edge of each lesion were cut and placed on PDA and the pathogen was re-isolated from the inoculated plants to confirm Koch’s postulates.

Molecular identification

Morphological identification of Acremonium species as dominant fungi in this study was confirmed by sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA genome. DNA extraction was carried out following the procedures described by Mirtalebi et al. (2013). The ITS region was amplified by polymerase chain reaction (PCR) using the universal primers ITS1 and ITS4 (White et al., 1990). Each 20 µL amplification reaction mixture contained 5-10 ng of total DNA, 2 µL of 10× reaction buffer, 0.2 mM dNTP, 2.5 mM MgCl2, 1 U of Taq polymerase (CinnaGen) and 0.8 µM of each primer. Amplification conditions consisted of 34 cycles of denaturation at 94 °C for 40 s, annealing at 60 °C for 90 s, and extension at 72°C for 2 min. Each PCR reaction included an initial denaturation step at 95 °C for 2 min and final extension step at 72 °C for 5 min. The amplification products were purified with GeneJet PCR Purification Kit (Fermentas) to remove excess of primers and nucleotides. Subsequently, purified amplification products were sequenced in both directions with an ABI PRISMBigDye Terminator v3.1 sequencing kit (Applied Biosystems) on an ABI-3100 automated sequencer. Phylogenetic analyses were performed using DNA sequences of the ITS region that were either retrieved from published ITS sequences in the GenBank or determined in this study (Table 1). DNA sequences were edited with DNASTAR (Seq Man II) and aligned with ClustalX 1.8 (Larkin et al., 2000). Manual adjustment of sequence alignments was performed to accommodate insertions/deletions. Phylogenetic analyses were conducted in MEGA 5 (Tamura et al., 2011) using the Neighbor-Joining method (Saitou & Nei, 1987). The sequence of Bionectria ochroleuca (KF055399) was used as outgroup.

Table 1. GenBank accession numbers of internal transcriberd spacer sequences of rDNA (ITS) of isolates used in this study. Isolate’s name is indicated where known.

Isolation and identity of causal agent of rose dieback

Disease symptoms on roses in greenhouses including yellow, brown to black areas on a cane or stem, canker and dieback were observed (Fig. 1). In this study, 10 isolates of Botrytis cinerea and 34 isolates of Coniothyrium fuckelii were isolated. These fungi have been previously reported for dieback of rose worldwide (Sweets, 1982; Mirabolfathi & Ershad, 2004). Based on morphological characteristics (Domsch et al., 2007), 51 isolated fungi were preliminarily identified as Acremonium spp. The species of these isolates obtained from single conidia were characterized as follows: Colonies deeply floccose-cottony to tomentosus, whitish to pale pinkish; conidiation abundant, mainly plectonematogenous; phialides very numerous on hyphal strands, mostly simple and long (25-55 mm, mean 43 ± 1.6), distinctly chromophilic near the base; conidia were aggregated in heads, cylindrical or tapering towards the tips and slightly fusiform, mostly homopolar, smooth- walled, hyaline 4.2-5.5 (mean 4.9 ± 0.3) × 1.2-1.7 (mean 1.5 ± 0.2) mm; sclerotia formed on nettle stems, scattered in the mycelium, firm, globose, smooth walled, hyaline, 15-50 (mean 34 ± 1.8) mm, consisting of isodiametrical cells of equal size; chlamydospores were absent (Fig. 1). These characteristics were typical of A. sclerotigenum (Moreau & R. Moreau ex Valenta) W. Gams (Domsch et al., 2007).

Optimum temperatures for growth and spore germination of A. sclerotigenum were 25-30°C (Fig. 2).

Molecular identification

To confirm the identification based on morphology, two isolates of Acremonium (Acr1 and Acr2) previously identified based on morphological and culture characters, were amplified using the primers ITS1 and ITS4. An amplicon of about 600 bp was obtained for both isolates. BLASTn searches in GenBank showed that ITS sequences of Acremonium isolates had 99-100% identity with isolates of A. sclerotigenum (GenBank KT878352, KT878350 and KJ194115). ITS sequences of the two isolates of Acremonium have been submitted to the GenBank (Table 1). According to DNA sequence analyses and morphological characteristics, Acremonium isolates recovered from stem of roses showing dieback symptoms could be assigned to A. sclerotigenum (Fig. 3). This study revealed Acremonium sclerotigenum as a new causal agent of rose dieback. Results of inoculation of healthy rose plants with A. sclerotigenum isolates confirmed its pathogenic feature, a fact which is particulary important because there is no information in the literature about the capacity of A. sclerotigenum to affect roses. Fungi of the genus Acremonium have been found as plant endophytes (Morgan-Jones & Gams, 1982), mycoparasites of plant pathogenic fungi (Choi et al., 2008) and pathogens (Garcia-Jimenez et al., 1994). Acremonium cucurbitacearum is the causal agent of Acremonium collapse, a soil-borne disease affecting muskmelon and watermelon worldwide (Armengol et al., 1998; Martinez-Culebras et al., 2004). Acremonium strictum has proved to be pathogenic to sorghum, cotton, okra, shasta daisy, maize, pea and strawberry (El-Shafey et al., 1979; Chase & Munnecke, 1980; King, 1981; Kamlesh-Mathur et al., 1987; Racedo et al., 2013). A. sclerotigenum is obviously a widely distributed saprophyte (Domsch et al., 2007). The species has also been reported as epiphytic fungus on barley and causal agent of bagged apple brown spot (Asgari et al., 2004; Li et al., 2014).

Spore trapping and pathogenicity tests

According to records obtained from spore trapping in rose greenhouses, several fungi including Penicillium spp., Aspergillus spp., Cladosporium spp., Alternaria spp., Botrytis cinerea and Acremonium sclerotigenum were detected. Trapping frequency was 29-30%, 25-27%, 12-13%, 20-22% and 14-16%, respectively.

Three isolates of Acremonium sclerotigenum obtained from roses with dieback symptoms and two isolates obtained from spore trapping were used for pathogenicity tests. Four weeks after inoculation, disease symptoms including brown discoloration lesions, necrosis and dieback of stems were observed for each five isolates on each nine rose cultivars. Brown necrotic lesions extended downwards from the inoculation site (Fig. 1d). A. sclerotigenum was re-isolated from all inoculated stems on nine cultivars. Control plants remained symptomless and no fungi were re-isolated. Based on spore trapping results, the possibility of airborne inoculum of A. sclerotigenum infecting rose wounds such as pruning wounds is not negligible in our study. Different formae speciales of Fusarium oxysporum have been shown to enter into greenhouses by airborne inoculum (Scarlett et al., 2015). Infection through leaf wounds by airborne propagules of F. oxysporum f. sp. radicis-lycopersici in tomatoes (Rekah et al., 2000) and F. oxysporum f. sp. basilici in basil (Uchida et al., 1996) have also been reported. A. sclerotigenum probably survive on disease canes or plant debris in greenhouses and spores of the causal fungus are dispersed by air currents and splashing water. The disease may also be spread by fungus-contaminated pruning tools. Rowe et al. (1977) demonstrated that while organic waste can provide a source for the survival of F. oxysporum f. sp. lycopersici, entry of the pathogen into tomato greenhouse that utilize soilless growth substrates can be due to the aerial inoculum of the pathogen. Understanding the threat associated with airborne conidial germination on wound sites will establish a basis for practices in relation to management the risk of disease associated with aerial inoculum of the fungus (Scarlett et al., 2015).

Although the occurrence of the causal agents of rose dieback such as Coniothyrium fuckelli, Botryodiplodia theobromae, Botrytis cinerea, etc. is much more frequent worldwide (Sweets, 1982), the disease caused by Acremonium sclerotigenum is only at the early stage of development and measures should be taken to prevent or reduce its spread into new areas. Currently we are investigating the use of fungicidal pruning wound protectants to reduce the incidence of rose dieback disease development.