Morphological and molecular characterization of Cytospora species involved in apple decline in Iran

INTRODUCTION

Apple Cytospora canker which is commonly caused by different species of Cytospora Ehrenb., is one of the most important and destructive diseases of apples (Malus spp.) worldwide (Ashkan & Hedjaroude 1993, Lee et al. 2006, Abe et al. 2007, Suzaki 2008, Wang et al. 2011, Ma et al. 2018, Liu et al. 2020). The pathogen extensively invades host phloem and xylem tissues, causing formation of cankers on the main stems, twigs and branches, affect the cultivation, productivity and longevity of trees and severely limits the development of apple industry (Tamura & Saito 1982, Fotouhifar et al. 2010, Meng et al. 2019). The genus Cytospora (Cytosporaceae, Diaporthales, Sordariomycetes, Ascomycota) was introduced by Ehrenberg (1818) with C. chrysosperma (Pers.) Fr. as the type species. It is considered as asexual morph of several genera such as Leucocytospora, Leucostoma, Valsa, Valsella and Valseutypella (Fries 1823, Saccardo 1884, Spielman 1985, Adams et al. 2002, 2005, Castlebury et al. 2002), all of which were considered as synonyms of Valsa (Adams et al. 2005). According to one fungus one name (1F=1N), the generic name Cytospora has been recommended for use over Leucocytospora, Leucostoma, Valsa, Valsella and Valseutypella (Rossman et al. 2015). A search of MycoBank (October 2020, www.mycobank.org) revealed 634 legitimate names while a search of Index Fungorum (October 2020, www.indexfungorum.org) lists 662 names. At present, 22 Cytospora species have been reported from apple trees as causal agents of apple canker and dieback disease worldwide (Anonymous 1960, Ershad 2009, Mehrabi et al. 2011, Wang et al. 2011, 2020, Azizi et al. 2018, Ma et al. 2018, Farr & Rossman 2021). Thus far, eleven species viz. C. ambiens Sacc., C. chrysosperma (Pers.) Fr., C. cincta Sacc., C. leucostoma (Pers.) Sacc., C. paratranslucens Norph., Bulgakov, T.C. Wen & K.D. Hyde, C. rhodophila Sacc., C. rubescens Fr., C. schulzeri Sacc. & P. Syd., C. sorbicola Norph., Bulgakov, T.C. Wen & K.D. Hyde, Valsa malicola Z. Urb. and Leucostoma cinctum (Fr.) Höhn. have been recorded from Iran (Ershad 2009, Fotouhifar et al. 2010, Mehrabi et al. 2011, Azizi et al. 2018, Hanifeh et al. 2018).

West Azerbayjan Province is the main apple growing area in Iran with 60470 ha and total production of 970714 tonnes (Ahmadi et al. 2018). Two apple cultivars, ‘Golden Delicious’ and ‘Red Delicious’ are the predominant commercial apple cultivars grown widely in this area. Recently, various types of disease symptoms were observed in apple orchards leading to tree decline and dying. The aim of the present study was to identify Cytospora species involved in apple canker and dieback disease in West Azerbayjan province, based on morphological characteristics and phylogenetic analysis using ITS, LSU, RPB2 and ACT sequences data.

MATERIALS AND METHODS

Sample collection and fungal isolation

Apple orchards in different locations of West Azerbayjan Province, Iran, were visited for suspected decline symptoms. Samples were collected from twigs and trunks showing cankered, bark discoloration and scaling and wood discoloration, placed in new paper bags and brought to the laboratory. The samples were washed thoroughly under running tap water, then small pieces (5 × 5 mm) from the interfaces of healthy and diseased tissue were cut, surface disinfested with 70% ethanol for 1 min, rinsed three times in sterile distilled water and blotted dry on sterilized paper towel. The plant segments were placed on potato dextrose agar (PDA, Merck, 39 g/L) amended with streptomycin sulfate and penicillin G (100 ppm each) in 90 mm diam. Petri dishes were incubated at 25 ± 1 °C in the dark. Hyphae growing out from the samples were transferred into fresh PDA and were purified using hyphal tip or single spore methods. All purified isolates were maintained on PDA slants and stored at 4 °C for further examinations. In the cases that characteristic asexual conidiomata (pycnidia) were seen on the examined plant samples, the samples were washed gently with sterile distilled water, surface disinfested with 70% ethanol for 1 min, and kept under humid conditions for ooze formation. Then dilute spore suspension was prepared by suspending the oozing conidia in sterile distilled water and a drop of dilute spore suspension (≈25 µl) was spread out over PDA medium. Germinated solitary conidia were transferred into fresh PDA after 24–48 h. The growing colonies were purified again as described above. The isolates were deposited in the Fungal Culture Collection of the Iranian Research Institute of Plant Protection (“IRAN”) and Fungal Culture Collections of Urmia University (FCCUU). Sequence data were deposited in GenBank dataset and their accession numbers are provided in the text (Table 1).

Morphological examination

Colony characteristics of the isolates were recorded from cultures grown on PDA in triplicate 90 mm diam. glass Petri dishes incubated in the dark at 25 ± 1°C after 3, 7 and 30 days. Radial growth was measured by taking two measurements perpendicular to each other in triplicates (Fan et al. 2015, Lawrence et al. 2018, Ma et al. 2018, Zhang et al. 2019). Colony colors were determined based on Rayner’s color chart (Rayner 1970). Pycnidia formation were induced on pine needles embedded in 2% water agar (WA) medium or on one-year-old apple shoots embedded in PDA medium under near ultraviolet (NUV) light (12 h photoperiod). Both pine needles and apple shoots were autoclaved at 121 °C for 20 min. thrice, 24 h apart. Pycnidia formation was checked weekly for 30 days. Transverse and longitudinal sections of conidiomata were prepared by hand with a razor blade and microscopic slides were prepared in water or lactic acid as mounting solutions. Morphological characteristics including color, diameter, presence/absence of conceptacle; the color, shape and size of discs, number and size of ostioles per disc (n=20), arrangement of pycnidia locules and dimensions of conidia (n=40) and conidiogenous cells (n=20) were examined and measured at 1000 X magnification with an Olympus X61 microscope with differential interference contrast (DIC) illumination. Based on morphological grouping, 12 isolates were selected for molecular studies.

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from fungal mycelium scraped with a sterile scalpel from the surface of 14–day–old PDA cultures using Exgene^TM Cell SV mini kit (GeneAll Biotechnology Co., Korea) following the manufacturer’s instructions. The extracted DNA was checked for quality on a 1% agarose gel. The internal transcribed spacer (ITS), the nuclear ribosomal large subunit (LSU), the partial actin (ACT) and the partial RNA polymerase II subunit (RPB2) regions were amplified using the primer sets ITS1/ITS4 (White et al. 1990), NL1/NL4 (O'Donnel 1993), ACT512F/ACT783R (Carbone & Kohn 1999) and RPB2–5F2/fRPB2–7cR (Liu et al. 1999, Sung et al. 2007), respectively. The PCR mixtures for all reactions consisted of about 10 ng/µl of genomic DNA, 0.4 µM of each primer and 12.5 µl of 2X ready-to-use reaction mix (Taq DNA polymerase 2X Master Mix Red, 1.5 mM MgCl₂, Ampliqon, Denmark) in a total volume of 25 µl. Conditions for PCR of ITS, LSU and ACT consisted of an initial denaturation step of 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 57°C and 1 min at 72°C, and a final extension step of 10 min at 72°C. The RPB2 gene was amplified using a touchdown PCR consisted of an initial denaturation step of 5 min at 95°C, followed by 40 cycles of 45 s at 95°C, 45 s at 60–54°C (annealing temperature decreased 0.5°C per cycle in the first 10 cycles), 45 s at 72°C and a final extension step of 10 min at 72°C. The presence of expected amplicons was checked on a 1% agarose gel. PCR products were cleaned up and sequenced by Microsynth DNA Company (Balgach, Switzerland).

Table 1. Cytospora isolates used in molecular phylogenetic analysis in this study

Phylogenetic analysis

The newly generated sequences were edited in BioEdit v.7.2.6 (Hall 1999), checked manually and deposited in GenBank (Table 1). Sequences of the homologous regions with high similarity from the type or representative Cytospora species were obtained from GenBank database according to Fan et al. 2020 and were included in phylogenetic analysis. Multiple alignments were generated with MAFFT online service (https://mafft.cbrc.jp/alignment/server/index.html) for each locus separately. The combined sequence dataset (ITS + LSU + RPB2 + ACT) was produced and proofed in Mesquite v. 2.74 (Maddison and Maddison 2010). Maximum likelihood analysis (ML) was performed on concatenated dataset in RAxML-HPC BlackBox v. 8.2.12 (Stamatakis 2014) through the CIPRES Science Gateway v 3.3 (Miller et al. 2010) using the GTRGAMMA + I as substitution model. The resultant phylogenetic tree was observed in FigTree v. 1.4.4 (Rambaut 2019). Diaporthe vaccinii strain CBS 160.32 was used as an outgroup taxon.

Pathogenicity tests and statistical analysis

In order to assess the pathogenicity of fungal isolates, in vitro inoculation test was performed (Ma et al. 2018). One– to two–year-old healthy shoots of both ‘Red Delicious’ and ‘Golden Delicious’ cultivars were taken from an apple orchard, cut into 20 cm fragments in length, washed thoroughly under running tap water, then surface disinfested in 75% ethyl alcohol for 2 minutes and were kept under aseptic conditions for 30 minutes. The bark was removed with the aid of a sterilized 5 mm cork borer and immediately inoculated with a mycelium plug (5 mm in diameter) cut from the margin of a 7–day–old actively growing fungal culture. The inoculated site was covered with a moist cotton ball and wrapped with Parafilm to maintain high humidity (Azizi et al., 2020, Tang et al., 2012). In the control treatments, PDA plugs without fungal mycelium were used. One to two isolates from each species were used in the pathogenicity tests, three shoots were inoculated with each isolate and all the tests were replicated twice. The inoculated shoots were kept in clean plastic boxes lined with moistened sterile paper towels and kept at 25 ºC in darkness for 21 days. Shoots were examined for the presence or absence of necrotic lesions around the inoculated sites, and while present, the bark was removed carefully and the length of necrotic lesion was measured. To complete Koch’s postulates, fungi re-isolation was conducted from the diseased parts of inoculated shoots in the same way as described earlier. The identity of the isolates was confirmed again based on morphological characteristics. The experiments were conducted based on completely randomized design (CRD) with three replications. After analysis of variance (ANOVA), lesion length means were compared with Duncan’s test (P = 0.05) using SAS (9.1) software.

RESULTS

Disease symptoms

In total, 68 isolates with the typical characteristics of the genus Cytospora were obtained from 45 samples collected in different parts of West Azerbaijan province. Symptoms associated with Cytospora cankers include depressed lesions with bark splitting, cankers, discoloration of xylem and wood necrosis and dieback (Fig. 1).

Morphological identification

Morphological comparisons of the examined isolates with the species described in the literatures grouped our isolates with the isolates of four previously described Cytospora species. Two species, C. parasitica and C. paratranslucens have been reported previously from apple trees, but C. germanica and C. salicina are described as apple pathogens for the first time in the world and are reported here for the first time to mycobiota of Iran.

Molecular phylogenetic analysis

PCR amplifications of the selected 12 Cytospora isolates produced DNA fragments approximately 563 bp for ITS, 516 bp for LSU, 893 bp for RPB2 and 282 for ACT. Sequence combination of four loci for a total of 75 fungal strains including ingroup and outgroup taxa contained 1943 characters. The best scoring RaxML tree with the final ML optimization likelihood value of -10074.977337 (ln) is selected to demonstrate and discuss the phylogenetic relationships among the studies strains (Fig. 2). Maximum likelihood analysis of the combined dataset revealed that our strains clustered well with representative strains of four previously described species viz. C. chrysosperma, C. germanica, C. paratranslucens and C. salicina with 88–100% ML bootstrap values (Fig. 2) and confirmed morphological identification. All the identified species are illustrated and described.

Fig. 1. Disease symptoms associated with Cytospora spp. on apple trees. a, b. Apple trees with dieback symptoms. c, d. Bark discoloration, splitting, and canker formation on main branches and twigs. e. Asexual fruiting bodies formed on diseased bark of apple tree. f, g, h. Wood discoloration. 

Fig. 2. Phylogenetic tree of Cytospora spp. inferred from Maximum Likelihood (ML) analysis of combined dataset of ITS, LSU,RPB2 and ACT sequences. The Maximum Likelihood bootstrap support (BS) values >50% are given at the nodes. The tree was rooted to Diaporthe vaccinii strainCBS 160.32and newly identified strains are in red. The scale bar indicates the number of nucleotide substitutions.

Taxonomy

Cytospora chrysosperma (Pers.) Fr., Systema Mycologicum (Lundae) 2: 542 (1823) Fig. 3

Conidiomata pycnidial, scattered, semi-immersed in host tissue, erumpent through the bark surface, without conceptacle, with multiple locules, circular, (1066–)1128–2000(–2325) µm diam (n=20). Disk white to dilute brown, with one ostiole. Ostioles circular to ellipsoid and 145–500 µm diam. Conidiophores borne along the locules, hyaline, unbranched, thin-walled, reduced to conidiogenous cells. Conidiogenous cells entroblastic, phialidic, hyaline, smooth-walled, (6.5–)6.9–9.6(–10.7) × (1.2–) 1.3–1.8 µm (x̄= 8.7 × 1.6 µm, n= 20), tapering toward the apex. Conidia unicellular, hyaline, allantoid, thin-walled, eguttulate, (3.4–)3.6–4.8(–5) × 1–1.3 µm (x̄= 4.1 × 1.1 µm, n= 40) (Fig. 3). Sexual morph was not observed.

Culture characteristics. Colonies on PDA at 25±1ºC and in the dark reaches 40 mm diameter after 3 days and entirely covering the 9 cm Petri dishes after 7 days. Colonies at first white, becoming off-white to cream after 30 d, with short aerial hyphae giving a cottony appearance to colony surface, margin irregular. Pycnidia irregularly distributed on the PDA surface.

Specimens examined. Iran, West Azerbayjan province: Miyandoab, from apple trees with canker, decline and dieback symptoms, 5 September 2017, R. Azizi, IRAN 4240C, IRAN 4241C, FCCUU 333, FCCUU 334.

Hosts and distribution. Cytospora chrysosperma has been reported from more than 260 hosts in Africa, Asia, Australia, New Zealand, Europe and North and South America. In Iran, it has been reported from Armeniaca vulgaris, Crataegus azarolus, Ficus carica, Fraxinus excelsior, Fraxinus sp., Juglans regia, Ligustrum latifolium, Malus pumila, Morus alba, Olea sativa, Persica vulgaris, Platanus orientalis, Populus alba, Populus deltoides, Populus nigra, Prunus domestica, Prunus persicae, Robinia pseudoacacia, Salix aegyptiaca, Salix excelsa, Salix sp., Tamarix sp., Thuja orientalis and Vitis vinifera (Fotouhifar et al. 2010, Arzanlou & Narmani 2015,Bagherabadi et al. 2017).

Fig. 3. Cytospora chrysosperma (IRAN 4240C) on Malus domestica. a–b. Habit of conidiomata on branch with pale luteous to luteous tendril, c–d. Longitudinal section through conidiomata, e–f. Cross section through conidiomata, g. Conidiogenous cells with attached conidia, h. Conidia, i. Colony on PDA after 3 days (half part, left) and after 30 days (half part, right). Scale bars: b, c, e =250 µm; f, d=100 µm and g, h= 10 µm.

Cytospora germanica Sacc., Sylloge Fungorum 3:262 (1884) Fig. 4

Conidiomata pycnidial, scattered, semi-immersed in host tissue, erumpent through the bark surface, with multiple locules, circular, (800–) 850–1100 µm diameter (n=20). Disk white, circular, grey at center and with one ostiole. Ostioles circular to ellipsoid and 100–250 µm diam. Conidiophores borne along the locules, hyaline, branched at base or unbranched, thin walled. Conidiogenous cells entroblastic, phialidic, hyaline, smooth walled, (5.7–)6.3–14.3(–15.1) × (1.8–)1.9–3.4(–3.5) µm (x̄= 10 × 2.4 µm, n= 20), tapering toward the apex. Conidia unicellular, hyaline, allantoid, thin walled, eguttulate, (5.9–)6.2–9.4(–10.3) × (1.2–)1.5–1.9(–2) µm (x̄= 7.2 × 1.7 µm, n= 40) (Fig. 4). Sexual morph was not observed.

Culture characteristics. Colonies on PDA at 25±1 ºC and in the dark reaches 20 mm diameter after 3 days and 53 mm diameter after 7 days. Colonies at first white, becoming amber after 7 days and buff after 30 d, with short aerial hyphae giving a cottony to felt like appearance to colony surface, margin irregular.

Specimens examined. Iran, West Azerbayjan province: Oshnavieh, from apple trees with canker, decline and dieback symptoms, 24 October 2017, R. Azizi, IRAN 4242C, IRAN 4243C, FCCUU 333.

Hosts and distribution. Cytospora germanica has been reported from 15 hosts, mostly plants in the Salicaceae family in Asia, Europe and South Africa (Farr & Rossman 2021).

Fig. 4. Cytospora germanica (IRAN 4243C) on Malus domestica. a–b. Habit of conidiomata on branch, c–d. Longitudinal section through conidiomata, e–f. Cross section through conidiomata, g. Conidiogenous cells with attached conidia, h. Conidia, i. Colony on PDA after 3 days (half part, left) and after 30 days (half part, right). Scale bars: b, c, e = 250 µm; f, d = 100 µm and g, h = 10 µm.

Cytospora paratranslucens Norph., Bulgakov, T.C. Wen & K.D. Hyde, in Norphanphoun, Doilom, Daranagama, Phookamsak, Wen, Bulgakov & Hyde, Mycosphere 8(1): 75 (2017) Fig. 5

Conidiomata pycnidial, scattered, semi-immersed in host tissue, erumpent through the bark surface, with multiple locules, circular, 1000–1375 µm diameter (n=20). Disk olivaceous dark green with 1-2 ostioles. Ostioles circular to ellipsoid and 105–205 µm diam. Conidiophores borne along the locules, hyaline, unbranched, reduced to conidiogenous cells, thin walled. Conidiogenous cells entroblastic, phialidic, hyaline, smooth walled, (9.3–)9.5–14.2(–17.4) × (0.9–)1.3–2.1(–2.8) µm (x̄= 11.65 × 1.75 µm, n= 20), tapering toward the apex. Conidia unicellular, hyaline, allantoid, thin walled, eguttulate, (5.6–)5.8–8.4(–8.7) × (1.2–)1.3–1.9 µm (x̄= 6.9 × 1.6 µm, n= 40) (Fig. 5).

Sexual morph was not observed.

Culture characteristics. Colonies on PDA at 25 ± 1ºC and in the dark reaches 41 mm diameter after 3 days and entirely covering the 8-cm Petri dishes after 7 days. Colonies at first white, then becoming grey at margin and olivaceous grey at center after 30 d, margin regular.

Specimens examined. Iran, West Azerbayjan province: Mahabad, from apple trees with canker, decline and dieback symptoms, 31 October 2017, R. Azizi, FCCUU 339, IRAN 4245C; Iran, West Azerbayjan province: Salmas, from apple trees with canker, decline and dieback symptoms, 30 August 2017, R. Azizi, IRAN 4244C.

Fig. 5. Cytospora paratranslucens (IRAN 4245C) on Malus domestica. a–b. Habit of conidiomata on branch with short, bay to rust color tendril, c–d. Longitudinal section through conidiomata, e–f. Cross section through conidiomata, g. Conidiogenous cells with attached conidia, h. Conidia, i. Colony on PDA after 3 days (half part, left) and after 30 days (half part, right). Scale bars: b, c, e = 250 µm; f, d = 200 µm and g, h = 10 µm.

Cytospora salicina Norph., Bulgakov, T.C. Wen & K.D. Hyde, in Norphanphoun, Doilom,Daranagama, Phookamsak, Wen, Bulgakov & Hyde, Mycosphere 8(1): 80 (2017) Fig. 6

Conidiomata pycnidial, scattered, semi-immersed in host tissue, erumpent through the bark surface, with multiple locules, circular to ovoid, (1000–)1076–1650(–1775) µm diameter (n=20). Disk creamy-white, grey at center and with one ostiole. Ostioles circular to ellipsoid and 125–185 µm diam. Conidiophores borne along the locules, hyaline, unbranched, very rarely branched at base, thin walled, reduced to conidiogenous cells. Conidiogenous cells entroblastic, phialidic, hyaline, smooth walled, (8.1–)8.6–13.5(–15) × 1.5–2.3(–2.5) µm (x̄= 10.5 × 1.8 µm, n= 20), tapering toward the apex. Conidia unicellular, hyaline, allantoid, thin walled, eguttulate, (3.8–)4–5.6(–5.8) × (1–)1.1–1.5(–1.6) µm (x̄= 4.8 × 1.8 µm, n= 40) (Fig. 6). Sexual morph was not observed.

Culture characteristics. Colonies on PDA, at 25 ± 1ºC and in the dark reaches 12 mm diameter after 3 days and 29 mm diameter after 7 days, at first white, then change to olivaceous buff at margin, greenish olivaceous at middle and off-white to cream at center, margin irregular, without aerial mycelium and colony appearance felt like.

Specimens examined. Iran, West Azerbayjan province: Miyandoab, from apple trees with canker, decline and dieback symptoms, 5 September 2017, R. Azizi, IRAN 4246C; Iran, West Azerbayjan province: Nagadeh, from apple trees with canker, decline and dieback symptoms, 24 October 2017, R. Azizi, IRAN 4247C.

Hosts and distribution. Cytospora salicina has been reported from Salix alba, Salix fragilis and Salix × rubens (Salicaceae) and Cornus sanguinea (Cornaceae) in Europe (Russia) (Farr & Rossman 2021).

Pathogenicity tests

Seven days after inoculation of shoots with the fungal isolates, bark tissue around the inoculated site showed brown discoloration and shrinkage. The lesions were expanded to girdle the shoots, and after 14 days, small raised, black spots reminiscent of pycnidial initials were observed on discolored bark. Two tested isolates (IRAN 4246C and IRAN 4247C) of C. salicina caused characteristic lesions on detached branches of both ‘Golden Delicious’ and ‘Red Delicious’ cultivars (Fig. 7), but the tested isolates of C. chrysosperma, C. germanica and C.paratranslucens were only pathogenic on ‘Red Delicious’ cultivar and caused characteristic symptoms on inoculated shoots, while no symptoms were produced on ‘Golden Delicious’ cultivar as well as control treatments (Fig. 7). The fungal isolates were re-isolated from inoculated shoots and confirmed Koch’s postulates. The mean lesion length showed significant differences (P<0.05) among four species, five isolates and the two cultivars, ‘Red Delicious’ and ‘Golden Delicious’ (Fig. 8). The highest and lowest pathogenicity were observed in the IRAN 4245C (C. paratranslucens) and IRAN 4243C (C. germanica) isolates on ‘Red Delicious’ cultivar, respectively.