Short Communication


Dieback of rose caused by Acremonium sclerotigenum as a new causal agent of rose dieback in Iran


Maryam Mirtalebi

Shiraz University, College of Agriculture, Department of Plant Protection. Shiraz, Iran

Zia Banihashemi

Shiraz University, College of Agriculture, Department of Plant Protection. Shiraz, Iran

Fatemeh Sabahi

Shiraz University, College of Agriculture, Department of Plant Protection. Shiraz, Iran

Hamze Mafakheri

Shiraz University, College of Agriculture, Department of Plant Protection. Shiraz, Iran



Severe dieback of rose has been recently observed in several rose greenhouses in Fars province of Iran. During 2014 and 2015, stems of rose plants showing yellow to brown discoloration and dieback were collected from rose greenhouses. Coniothyrium fuckelii, Botrytis cinerea and Acremonium were subsequently isolated from the margin between healthy and symptomatic tissue. B. cinerea and C. fuckelii isolates were similar to those previously reported for dieback of rose worldwide. Morphological and cultural characters along with molecular analysis based on partial sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA genome allowed confirming the affiliation of the Acremonium isolates, corresponding to A. sclerotigenum as a new causal agent of rose dieback. To determine its pathogenicity on rose, Koch’s postulates were fulfilled by stem inoculation of nine rose cultivars under greenhouse conditions. While A. sclerotigenum is considered as a soil-born pathogen, and produces sclerotia that are resistant to adverse conditions enables the fungus to survive extended period in soil, propagule trapping in our study revealed that conidia can become airborn, imply that an aerial phase, forms an important component of the disease cycle.

Additional key words: pathogenicity; spore trapping; airborn; growth rate; spore germination.

Abbreviations used: ITS (internal transcribed spacer region of the ribosomal RNA genome); MEA (malt extract agar); OA (oatmeal agar); PDA (potato dextrose agar); TMN-PDA (trimethylnonanol-potato dextrose agar); WA (water agar).

Authors’ contributions: Conceived and designed the experiments, and wrote the paper: MM and ZB. Performed the experiments, and contributed reagents/materials/analysis tools: MM, FS and HM. Analyzed the data: MM. Critical revision of the manuscript for important intellectual content: ZB.

Citation: Mirtalebi, M.; Banihashemi, Z.; Sabahi, F.; Mafakheri, H. (2016). Short communication: Dieback of rose caused by Acremonium sclerotigenum as a new causal agent of rose dieback in Iran. Spanish Journal of Agricultural Research, Volume 14, Issue 4, e10SC03.

Received: 04 Apr 2016. Accepted: 14 Nov 2016

Copyright © 2016 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Funding: Shiraz University (Iran).

Competing interests: The authors have declared that no competing interests exist.

Correspondence should be addressed to Maryam Mirtalebi:





Material and methods

Results and discussion



Stem canker and dieback are very serious diseases of rose (Sweets et al., 1982). This disease is usually more prevalent on plants that are under some type of stress due to poor cultural practices. The stem canker and dieback phase of the disease appears as a yellow to tan or dark brown to black lesion that enlarges until the stem is girdled. Once the stem is girdled, the foliage above the canker wilts and dies that causes brown discoloration and death of the terminal areas of the cane (Waterman, 1982).

Several fungi are capable of causing stem canker and dieback of roses. The most commonly reported fungi worldwide are Coniothyrium fuckelii Sacc., Botryodiplodia theobromae pat., Botrytis cinerea Pers. ex Fr., Trichothecium roseum (Pers.) Link ex. S.F. Gray, Phomopsis sp. and Pestalotia spp. (Guba, 1961; Pitta & Teranishi, 1973; Sweets, 1982). B. cinerea, Phomopsis oblonga, Pestalotiopsis sp. and C. fuckelii have been reported on roses in Alborz and Esfahan provinces of Iran (Mirabolfathi & Ershad, 2004; Mahdizadehnaraghi & Bakhtiari, 2014). Recently, a relatively high incidence of rose dieback disease has been observed in Shiraz and Noorabad areas rose greenhouses in the Fars province of Iran. There is a lack of information concerning pathogens associated with cane dieback of greenhouse roses in Fars province. During 2014 and 2015, isolations were made from roses with stem dieback obtained from Fars province greenhouses. Fungi isolated from these stems included C. fuckelii, B. cinerea and Acremonium sp. (Domsch et al., 2007). C. fuckelii and B. cinerea are both well recognized pathogens as causal agents of stem dieback of roses worldwide (Sweets, 1982). To date there is no available information related to Acremonium spp. as causal agent of rose dieback, worldwide. This study was therefore carried out to investigate the identity of Acremonium species in terms of morphological and molecular characteristics and pathogenicity test on different rose cultivars.

Material and methodsTop

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.

Results and discussionTop

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).

Figure 1. Rose dieback symptoms observed in greenhouses (a-c). Necrotic lesion development in pathogenicity trials (d); Morphological characteristics of A. sclerotigenum; a whitish tomentose (e) and floccose-cottony (f) colony of the fungus on potato dextrose agar; mycelial strand (g); simple, long, stiff phialids with conidial heads (h); hyaline, cylindrical conidia (i); firm, globose, hyaline sclerotia consisting of isodiametrical cells of equal size formed on the nettle stem (j-k). Bars= 20 mm (h, i, k); 100 mm (g, j).

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

Figure 2. Effect of incubation temperature on radial growth rate (a) and percentage of spore germination (b) of Acremonium sclerotigenum isolates on potato dextrose agar.

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).

Figure 3. Phylogram of neighbor-joining analysis of two Acremonium sclerotigenum isolates from this study (indicated in bold), together with 10 isolates of Acremonium taxa and two isolates of Plectosphaerella cucumerina (indicated using accession number retrived from GenBank within parentheses) based on the internal transcriberd spacer region of rDNA (ITS). Bionectria ochroleuca isolate KF055399 is included as outgroup. Bootstrap values (> 70%) are shown as percentages of 1,000 replicates.

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.


Armengol J, Sanz E, Martinez-Ferrer G, Sales R, Bruton BD, Garcia-Jimenez J, 1998. Host range of Acremonium cucurbitacearum, causal agent of Acremonium collapse of muskmelon. Plant Pathol 47: 29-35.https:/
Asgari B, Zare R, Payghami E, 2004. Hyphomycetous fungal community of barley phylloplane in East Azerbaijan province with emphasis on new taxa for Iranian fungal flora. Rostaniha 5(2): 67-70.
Banihashemi Z, de Zeeuw DJ, 1969. Two improved methods for selectively isolating Fusarium oxysporum from soil and plant roots. Plant Dis Rep 53: 589-591.
Chase AR, Munnecke DE, 1980. Shasta daisy vascular wilt incited by Acremonium strictum. Phytopathology 70: 834-838.https:/
Choi GJ, Jin-Cheol K, Kyoung SJ, Kwang YC, Heung TK, 2008. Mycoparasitism of Acremonium strictum BCP on Botrytis cinerea, the gray mold pathogen. J Microbiol Biotechnol 18: 167-170.
Dhingra OD, Sinclair JB, 1995. Basic Plant Pathology Methods, 2nd ed. USA, Boca Raton, FL, CRC Press, 355 pp.
Domsch KH, Gams W, Anderson TH, 2007. Compendium of soil fungi. IHW-Verlag and Verlagsbuchhandlung, Eching, Germany, 627 pp.
El-Shafey HA, Abdel-Rahim MF, Rafatt MM, 1979. A new Cephalosporium wilt of grain sorghum in Egypt. Proc 3rd Egypt Phytopath Soc, Cairo, Egypt, pp: 514-532.
Eskalen A, Gubler, W, 2001. Association of spores of Phaeomoniella chlamydospora, Phaeoacremonium inflatipes, and Pm. aleophilumwith grapevine cordons in California. Phytopath Mediterr 40: 429-432.
Garcia-Jimenez J, Velazquez MT, Jorda C, Alfaro-Garcia A, 1994. Acremonium species as the causal agent of muskmelon collapse in Spain. Plant Dis 78: 416-419.https:/
Glenn AE, Bacon CW, Price R, Hanlin RT, 1996. Molecular phylogeny of Acremonium and its taxonomic implications. Mycologia 88: 369-383.https:/
Guba EF, 1961. Monograph of Monochaetia and Pestalotia. Harvard Univ Press, Cambridge, MA, USA. 342 pp.
Harrington TC, Steimel JP, Workneh F, Yang XB, 2000. Molecular identification of fungi associated with vascular discoloration of soybean in the North Central United States. Plant Dis 84 (1): 83-89.https:/
Kamlesh-Mathur Shekhawat KS, Siradhana BS, Mathur K, 1987. Acremonium wilt of pea, a new record. Veget Sci 14: 206-208.
King SB, 1981. Time of infection of maize kernels by Fusarium moniliforme and Cephalosporium acremonium. Phytopathology 71: 796-799.https:/
Larkin MA, Blackshields G, Brown NP, Chenna R, Mc Gettigan PA, William Mc H, Valentin F, Wallace IM, Wilm A, Lopez R, et al., 2000. Clustal W and X version 2.0. Bioinformatics 23: 2947-2948.https:/
Li BH, Wang, CC, Dong XL, Zhang ZF, Wang CX, 2014. Acremonium brown spot, a new disease caused by Acremonium sclerotigenum on bagged apple fruit in China. Plant Dis 98: 1012.https:/
Mahdizadehnaraghi R, Bakhtiari M, 2014. Identification of Coniothyrium fuckelii as the causal agent of rosa stem canker disease in the greenhouses of Hamedan. Proc 21th Iran Plant Prot Congress, August. Oromieh, Iran, pp: 61.
Martinez-Culebras PV, Abad-Campos P, Garcia-Jimenez J, 2004. Molecular characterization and PCR detection of the melon pathogen Acremonium cucurbitacearum. Eur J Plant Pathol 110: 801-809.https:/
Mirabolfathi M, Ershad D, 2004. Twig and cane canker of rose in the greenhouses of central area of Iran. Iran J Plant Pathol 40: 84 [In Persian with English summary].
Mirtalebi M, Banihashemi Z, Linde CC, 2013. Phylogenetic relationships of Fusarium oxysporum f. sp. melonis in Iran. Eur J Plant Pathol 136 (4): 749-762.https:/
Morgan-Jones G, Gams W, 1982. Notes on Hyphomycetes. XLI. An endophyte of Festuca arundinacea and the anamorph of Epichloe tvphina, new taxa in one of two new sections of Acremonium. Mycotaxon 15: 311-318.
O’Donnell K, Gray LE, 1995. Phylogenetic relationships of the soybean sudden death syndrome pathogen Fusarium solani f. sp. phaseoli inferred from sequence data and PCR primers for its identification. Mol Plant Microb Int 8 (5): 709-716.https:/
Pitta GPB, Teranishi J, 1973. Phomopsis e Botriodiplodia associados a seca de roseiras. O Biologico 39: 21-23.
Racedo J, Salazar SM, Castagnaro AP, Diaz Ricci JC, 2013. A strawberry disease caused by Acremonium strictum. Eur J Plant Pathol 137: 649-654.https:/
Rekah Y, Shtienberg D, Katan J, 2000. Disease development following infection of tomato and basil foliage by airborne conidia of the soilborne pathogens Fusarium oxysporum f. sp. radicis-lycopersici and F. oxysporum f. sp. basilici. Phytopathology 90: 1322-1329.https:/
Rowe RC, Farley DF, Coplin DL, 1977. Airborne spore dispersal and recolonization of steamed soil by Fusarium oxysporum in tomato greenhouses. Phytopathology 67: 1513-1517.https:/
Saitou N, Nei M, 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425.
Scarlett K, Tesoriero L, Daniel R, Maffi D, Faoro F, Guest DI, 2015. Airborne inoculum of Fusarium oxysporum f. sp. Cucumerinum. Eur J Plant Pathol 141: 779-787.https:/
Sweets LE, 1982. Control of fungi associated with cankers of greenhouse roses. Plant Dis 66: 491-494.https:/
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28 (10): 2731-2739.https:/
Uchida JY, Kadooka CY, Hamasaki RT, 1996. First report of Fusarium wilt of basil in Hawaii and foliar disease initiation. Plant Dis 80: 105.https:/
Waterman, 1982. Rose diseases: Their causes and control. US Dep Agric Farm Bull 1547: 19 pp.
White TJ, Bruns TD, Lee SB, Taylor JW, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: A guide to methods and applications; Innis MA et al. (eds). pp: 315-322. Academic Press, San Diego, CA, USA.https:/