First Report of Apple Decline Caused by Botryosphaeria sinensis in Korea

Research Note
Seung-Yeol Lee12Leonid N. Ten1Chang-Gi Back3Hee-Young Jung12


Apple decline symptoms were frequently observed on cv. Fuji apple orchards located in Gyeonggi, Gyeongbuk, and Gangwon provinces during surveys conducted from May until the end of September 2020. Three fungal strains were isolated from the margins of internal lesions of diseased apple trees, and their morphological characteristics were considered similar to Botryosphaeria sinensis. Phylogenetic analysis using internal transcribed spacer (ITS) regions, translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), and the second largest subunit of RNA polymerase II (rpb2) gene sequences confirmed the closest relationship of isolates with B. sinensis at the species level. According to a pathogenicity test, the appearance of dark-brown discolorations and vascular necrosis on apple branches inoculated with the isolated strain KNUF-20-014 was observed. To the best of our knowledge, this is the first report of B. sinensis as the causal agent of apple disease in Korea.


Apple (Malus domestica) is one of the most economically important fruits, with more than 31,000 ha cultivation area in South Korea [1]. However, numerous destructive pathogens infect apple trees and significantly reduce the commercial apple production worldwide. Among the fungal diseases, canker, twig dieback, and plant decline frequently occurs in apple orchards [2]. During the apple cultivation period, apple decline symptoms were observed from May until the end of September 2020 in apple (cv. Fuji) orchards located in Gyeonggi, Gyeongbuk, and Gangwon provinces. Mostly, declined trees were young (less than ten years), showing warts on the surface, darkening from the trunk, detachment of epidermis of the stem, embedded pycnidia on the bark, withering of branch, and eventually leading to decline (Fig. 1A-1G, and I). In this present study, isolated fungal strains were described and illustrated as a causal agent of apple decline.

Fig. 1. Natural apple decline symptoms caused by Botryosphaeria sinensis and description of the KNUF-20-014. A, declined apple tree; B, warts on a diseased tree; C and D, blackened trunk and observed symptoms inside; E and F, black dots on a diseased trunk; G, observed necrosis on the internal of a diseased tree; H, pathogenicity test result shows canker and necrosis on the inoculated branch of cv. Fuji and its internal symptom (red arrows indicate inoculated zones); I and J, pycnidia on a diseased tree and on pine needle agar (PNA) medium; K, cross-sections through pycnidium; L and M, colony on malt extract agar after incubation at 28℃ for five days (L, front; M, reverse); N, conidia (scale bar=10 μm).

To isolate the causal fungus, the fragment (approximately 2 mm) was taken from the margin of internal lesions, transferred onto potato dextrose agar (PDA; Difco, Detroit, MI, USA), and incubated at 25℃. As a result, the three strains, KNUF-20-014, KNUF-20-072, and KNUF-20-074, were isolated from the diseased apple tree. For molecular identification of strains at the genus and species levels, the total genomic DNA was extracted from the isolates using a HiGene Genomic DNA prep kit (BIOFACT, Daejeon, Korea) according to the manufacturer’s instructions. Molecular identification of the strains was conducted using the nucleotide sequences of internal transcribed spacer (ITS) regions and translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), and second largest subunit of RNA polymerase II (rpb2) genes. The ITS regions, tef1, tub2, and rpb2 genes were amplified using the primer sets ITS1/ITS4 [3], EF1-668F/EF1-1251R [4], Bt2a/Bt2b [5], and RPB2-5F2/fRPB2-7cR [6], respectively. Amplified products of the polymerase chain reaction (PCR) were purified with EXOSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced by Macrogen Co. Ltd. (Daejeon, Korea). Amplification of the ITS, tef1, tub2, and rpb2 loci of strains KNUF-20-014, KNUF-20-072, and KNUF-20-074 yielded fragments of 526, 512, 413, and 1,020 bp; 520, 500, 422, and 1,005 bp; and 497, 496, 423, and 1,029 bp, respectively. The comparative sequence analyses of the molecular markers of the three strains revealed their similarity of 100%, indicating their affiliation to the same species. A BLAST search of the NCBI database showed that the sequences obtained from ITS, tef1, tub2, and rpb2 loci of the three strains exhibited highest similarities of 99.8% (517 bp out of 518 bp), 100% (267 bp out of 267 bp), 100% (363 bp out of 363 bp), and 100% (577 bp out of 577) with that of Botryosphaeria sinensis MUCC 2533 (accession numbers LC585268, LC585140, LC585164, LC585188, respectively). To confirm the closest relationship between the three strains and B. sinensis at the species level, a phylogenetic analysis was conducted using concatenated sequences of the ITS regions, tef1, tub2, and rpb2 genes. The sequences of allied species were retrieved from the NCBI database (Table 1). A phylogenetic tree was constructed with maximum-likelihood method using the program MEGA7 [7]. The evolutionary distances were calculated using the Kimura two-parameter model [8] and the topology of the tree was evaluated using bootstrap analysis based on 1,000 replicates. In the phylogenetic tree, the isolated strains occupied a position within the genus Botryosphaeria and clustered together with B. sinensis, indicating their closest relationship at the species level (Fig. 2).

Table 1. List of species used in phylogenetic analyses with the GenBank accession numbers.

The fungal strains isolated in this study and their accession numbers are indicated in bold.

ITS, internal transcribed spacer; tef1, translation elongation factor-1; tub2, beta-tubulin; rpb2, RNA polymerase II gene.

The representative isolate, KNUF-20-014, was cultured on pine needle agar (PNA) and malt extract agar (MEA) to check the cultural and morphological characteristics. On MEA the colonies grew up to 80 mm in three days at 28℃, showing gray aerial mycelium, white and gray surface with an olivaceous black reverse (Fig. 1L and 1M), the pycnidia were produced on PNA after 21 days (Fig. 1J and 1K), conidia were hyaline, aseptate, obtuse apex, fusiform, smooth with granular contents, and 19.7-28.9×4.9-7.3 μm (av. 23.4×6.0 μm, l/w 4.1, n=50) (Fig. 1N). These cultural and morphological characteristics were similar to those of the previously described B. sinensis [9], but strain KNUF-20-014 was readily distinguishable from B. dothidea by its conidia size. The average conidia length of the strain (23.4 μm) was distinctly shorter than that of B. dothidea (26.2 μm), while the width of its conidia (6.0 μm) was longer than that of B. dothidea (5.4 μm) [10]. The conidial length:width ratio of strain KNUF-20-014 (4.1) was the same as the reported value for B. sinensis [9], but clearly different from that of B. dothidea (4.9) [10].

Fig. 2. Maximum-likelihood phylogenetic tree, based on internal transcribed spacer (ITS) regions, translation elongation factor-1 (tef1) , beta-tubulin (tub 2) , and RNA polymerase II (rpb 2) gene sequences, showing the phylogenetic position of strains KNUF-20-076, KNUF-20-014, and KNUF-20-072, among related strains of the genus Botryosphaeria . Bootstrap values greater than 60% (percentage of 1,000 replications) are shown at branching points. The tree was rooted using Neofusicoccum parvum ATCC 58191 as an outgroup. Bar, 0.01 substitutions per nucleotide position.

Currently, there are controversies regarding the classification of a few species of the genus Botryosphaeria [11,12]. Based on phylogenetic analysis using the three combined ITS, tef1, and tub2 sequences, B. sinensis [9], B. minutispermatia [13], B. quercus [14], B. qinlingensis [15], and B. wangensis [16] were reclassified as a later synonym of B. dothidea by Zhang et al. [12]. At the same time, Hattori et al. [11] reexamined the genus Botryosphaeria based on phylogenetic analyses using the four molecular markers, namely ITS, tef1, tub2, and rpb2, and showed that B. sinensis and B. dothidea represent separate species. Simultaneously, the conidia size was highlighted as a key morphological characteristic for the differentiating species in the genus Botryosphaeria. Typically, an increase in the number of genes used in multi locus sequence analysis (MLSA) resulted in more accurate identification of isolated strains, therefore, we conducted MLSA using the four molecular markers in our study. According to the phylogenetic analysis, cultural and morphological characteristics, KNUF-20-014 was identified to be the same with B. sinensis at the species level but differed from B. dothidea. These data are in good agreement with the results of the reexamination of the genus Botryosphaeria conducted by Hattori et al. [11].

A pathogenicity test of strain KNUF-20-014 was conducted with healthy branches cv. Fuji. The branches were inoculated by placing a mycelial plug (4-5 mm) from the five-day-old colony on fresh wound sites made with a sterilized needle, while PDA plugs were placed into similar wounds on the three branches as mock inoculation. The inoculation points were wrapped in parafilm to maintain the moisture for three days at 25℃ in a growth chamber. After four weeks, all inoculated branches showed dark-brown discolorations and vascular necrosis, whereas no symptoms were observed in the mock inoculated branches (Fig. 1H). Pathogenicity test was conducted three times, and the pathogen was re-isolated from the inoculated branches.

Botryosphaeria species are known as plant saprobes, pathogens, and endophytes, with global distribution, on a wide variety of mainly woody hosts [13,17]. Diseases caused by pathogens belonging to the genus Botryosphaeria have resulted in significant losses in various economically important agricultural crops. Apple black rot caused by B. obtusa has resulted in fruit loss of 25-50% in the USA [18] and 20% annual losses of vineyard productivity from B. stevensii infection were reported in France [19]. Among them, B. dothidea is known to be a serious pathogen mainly of woody plants that were reported in 66 countries and confirmed in more than 24 host genera [20]. In Korea, B. dothidea has been previously reported to cause canker and warts on infected apple branches, mainly occurring on the cv. Hongro and rarely observed on the cv. Fuji [21,22]. However, in this study, B. sinensis was mainly isolated from the cv. Fuji, which could be related to host specificity.

B. sinensis was recorded on the twigs of Populus sp., Morus alba, Juglans regia in China [9], Paulownia tomentosa and Prunus sp. in Japan [11], and recently identified on Mangifera indica in Australia [23]. Our results increase the awareness of Botryosphaeria distribution, thereby improving our understanding of apple decline associated with B. sinensis and can be used for developing the control methods to prevent economic losses.


This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ014998022021), funded by the Rural Development Administration, Republic of Korea.


1 1. Voluntary report: 2020 Apple update. KS2020-0045 [Internet]. Bethesda (MD): United States Department of Agriculture Foreign Agricultural Service; 2021 [cited 2021 Aug 12] Available from:  

2 2. Sutton T, Aldwinckle H, Agnello A, Walgenbach J. Compendium of apple and pear diseases and pests. 2nd ed. Saint Paul: American Phytopathological Society Press; 2014.  

3 3. White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: A guide to methods and applications. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. San Diego: Academic Press; 1990. p. 315–22.  

4 4. Alves A, Crous PW, Correia A, Phillips AJL. Morphological and molecular data reveal cryptic speciation in Lasiodiplodia theobromae. Fungal Divers 2008;28:1–13.  

5 5. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 1995;61:1323–30.  

6 6. Liu YJ, Whelen S, Hall BD. Phylogenetic relationships among ascomycetes: Evidence from an RNA polymerase II subunit. Mol Biol Evol 1999;16:1799–808.  

7 7. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870–4.  

8 8. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111–20.  

9 9. Zhou Y, Dou Z, He W, Zhang X, Zhang Y. Botryosphaeria sinensia sp. nov., a new species from China. Phytotaxa 2016;245:43–50.  

10 10. Slippers B, Crous PW, Denman S, Coutinho TA, Wingfield BD, Wingfield MJ. Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea. Mycologia 2004;96:83–101.  

11 11. Hattori Y, Ando Y, Sasaki A, Uechi N, Nakashima C. Taxonomical study of noteworthy species of Botryosphaeria in Japan. Mycobiology 2021;49:122–32.  

12 12. Zhang W, Groenewald JZ, Lombard L, Schumacher RK, Phillips AJL, Crous PW. Evaluating species in Botryosphaeriales. Persoonia 2021;46:63–115.  

13 13. Ariyawansa HA, Hyde KD, Liu JK, Wu SP, Liu ZY. Additions to karst fungi 1: Botryosphaeria minutispermatia sp. nov., from Guizhou Province, China. Phytotaxa 2016;275:35–44.  

14 14. Wijayawardene NN, Hyde KD, Wanasinghe DN, Papizadeh M, Goonasekara ID, Camporesi E, Bhat DJ, McKenzie EHC, Phillips AJL, Diederich P, et al. Taxonomy and phylogeny of dematiaceous coelomycetes. Fungal Divers 2016;77:1–316.  

15 15. Liang LY, Jiang N, Chen WY, Liang YM, Tian CM. Botryosphaeria qinlingensis sp. nov. causing oak frogeye leaf spot in China. Mycotaxon 2019;134:463–73.  

16 16. Li GQ, Chen SF, Liu QL, Li JQ, Liu FF. Botryosphaeriaceae from Eucalyptus plantations and adjacent plants in China. Persoonia 2018;40:63–95.  

17 17. Marsberg A, Kemler M, Jami F, Nagel JH, Postma-Smidt A, Naidoo S, Wingfield MJ, Crous PW, Spatafora JW, Hesse CN, et al. Botryosphaeria dothidea: A latent pathogen of global importance to woody plant health. Mol Plant Pathol 2017;18:477–88.  

18 18. Brown EA, Britton KO. Botryosphaeria diseases of apple and peach in the southeastern United States. Plant Dis 1986;70:480–84.  

19 19. Larignon P, Fulchic R, Cere L, Dubos D. Observation on black dead arm in French vineyards. Phytopathol Mediterr 2001;40:S336–42.  

20 20. Batista E, Lopes A, Alves A. What do we know about Botryosphaeriaceae? An overview of a worldwide cured dataset. Forests 2021;12:313.  

21 21. Uhm JY. Reduced fungicide spray program for major apple diseases Korea. Anyang: Agriculture and Horticulture Press; 2010.  

22 22. Lee DH, Lee SW, Choi KH, Kim DA, Uhm JY. Survey on the occurrence of apple disease in Korea from 1992 to 2000. Plant Pathol J 2006;22:375–80.  

23 23. Tan YP, Shivas RG, Marney TS, Edwards J, Dearnaley J, Jami F, Burgess TI. Australian cultures of Botryosphaeriaceae held in Queensland and Victoria plant pathology herbaria revisited. Australas Plant Pathol 2018;48:25–34.