Res. Plant Dis > Volume 22(2); 2016 > Article
Park, Kim, Back, Lee, Kang, and Jung: First Report of Botryosphaeria parva Causing Stem Blight on Rubus crataegifolius in Korea

ABSTRACT

In 2015, stem blight of Rubus crataegifolius was observed in Pohang, Korea. The symptoms began as dark red spots in the stem, which led to stem blight, then leaf blight, and eventually resulted in death. A fungal isolate was obtained from a symptomatic stem and incubated on a potato dextrose agar plate. The isolated fungus produced white, cloudy mycelia turned black in 3 days. Based on the morphological characteristics, the causal fungus was assumed to be Botryosphaeria sp. A pathogenicity test was conducted according to Koch's postulates. To identify the causal agent, the combined sequence of the internal transcribed spacer, β-tubulin, and translation elongation factor 1α genes were used for phylogenetic analysis. Approximately 1,200 bp of the combined sequence clearly suggested that the isolated pathogen was Botryosphaeria parva. This is the first report on stem blight in R. crataegifolius caused by B. parva in Korea.

Body

Rubus crataegifolius Bunge belongs to the family Rosaceae, which is commonly found in the mountainous areas of Korea, Japan, and northeast China. R. crataegifolius has been used as food and traditional medicine for many years in this region by its various bioactivities such as immunological and antioxidant activities (Moon et al., 2011; Ni et al., 2009; Zhang et al., 2010). According to the usefulness of R. crataegifolius, the importance of disease management of R. crataegifolius has increased in Korea (Korea Forest Service, 2015). Several pathogens that infect R. crataegifolius have been reported in Korea, including Septoria rubi, Rhizopus stolonifer, Phragmidium griseum, and Phytoplasma (Kim et al., 2009). In July 2015, stem blight symptoms were observed in a field of R. crataegifolius in Pohang, Gyeongbuk province, Korea. Early in the course of the disease, plants showed dark red spots on stems; these spots expanded to black lesions associated with stem blight (Fig. 1A). As the disease progressed, pycnidia appeared on the stem lesions (Fig. 1B). The blight eventually spread to the leaves and resulted in plant death.
Fig. 1
(A) Symptoms of stem blight on Rubus crataegifolius. (B) Conidia discharged from pycnidia on the lesion (yellow arrows). (C) Seven-day-old fungal isolate on potato dextrose agar medium. (D) Coni­dia. Scale bar=10 μm. (E) Symptoms following experimental inoculation.
RPD_22_116_fig_1.jpg
A fungus, the presumed causal agent, was isolated from the diseased plant and designated as PPL02. The infected part of the stem was cut into small pieces and the surface of pieces was sterilized with 1% NaOCl for 1 minute and washed three times with 70% ethanol. The surface-sterilized pieces were placed onto potato dextrose agar (PDA) medium and incubated at 25°C. For isolation of a pure culture, hyphal tips of mycelia on the PDA medium were cut and transferred to fresh PDA plates. The isolated pathogen PPL02 produced white, cloudy mycelia that reached the edge of the PDA plate within 7 days (Fig. 1C). The center of the colony turned black after 3 days of incubation, with several dark spots appearing on the back side of the plate, and the black part extended all over the plate within 21 days of incubation. When the fungal colony was exposed to visible light with a 12-hour photoperiod for 21 days, the fungus produced conidia, which were aseptate, hyaline, and ellipsoid, ranging from 13-18 μm×4-6 μm (Fig. 1D). The morphology of the colonies and conidia showed similar characteristics to Botryosphaeria species. Although the culture characteristics and morphologies of B. parva and B. dothidea are similar, guidelines to distinguish these species have been previously suggested (Phillips, 2002). According to the previous report, conidia of B. parva have 14 to 18 μm length with length to width (L/W) ratio 3.2 to 3.9 while conidia of B. dothidea have 20 to 28 μm length with L/W ratio 4.3 to 5.2 (Phillips, 2002). Previously reported B. parva isolates from various hosts have satisfied these criteria (Haleem et al., 2012; Linaldeddu et al., 2007; Phillips, 2002; Slippers et al., 2005). Thus, morphological and cultural observation suggested that PPL02 was presumed to be B. parva, based on its morphological and culture characteristics (Table 1).
Table 1
Morphological comparision of the isolate obtained from Rubus crataegifolius Bunge and of Botryosphaeria species described previously
Present isolate B. parva* B. dothidea*
Colony color White to black White to black White to black
Conidia  Size (μm)  13-18×4-6 14-19×5-7 19-27×4-6
 Setae Absent Absent Absent
 Shape Ellipsoid Ellipsoid Ellipsoid, fusiform
 L/W 3.4 3.3 4.1

L/W, the ratio of length to width of conidia.

* Described by Pennycook and Samuels (1985) (Mycotaxon 24: 445-458).

To verify pathogenicity of PPL02, a fungal disc was obtained from the PDA plate at 7 days of incubation. The fungal disc was attached to an artificially wounded stem of a healthy plant of mature R. crataegifolius and then was wrapped with parafilm to maintain humidity. Ten days after inoculation, the same symptoms that were observed in the original plant appeared on the inoculated stem (Fig. 1E). Re-isolated pathogen from the disease lesion on the inoculated stem exhibited the same morphological characteristics of the original isolate. Therefore, PPL02 fulfilled Koch's postulates for establishing the causative agent of blight on R. crataegifolius.
A phylogenetic analysis was performed to clarify the species of this causal agent of stem blight in R. crataegifolius. Genomic DNA was extracted from PPL02, grown on the PDA plate, using a HigeneTM Genomic DNA Prep Kit (Biofact, Daejeon, Korea). PCR was performed with genomic DNA to amplify the internal transcribed spacer (ITS) region, β-tubulin, and translation elongation factor 1α (EF1-α) genes. Since some species belonging to Botryosphaeria genus are indistinguishable in the phylogenetic analysis using ITS only (Slippers et al., 2004), in this study, we used three genes and its combined sequences for further analysis. Each gene was amplified with the following, respective primer sets: ITS1F/ITS4 for ITS region (White et al., 1990), Bt2a/Bt2B for β-tubulin (Glass and Donaldson, 1995), and EF1-728F/EF1-986R for EF1-α (Carbone and Kohn, 1999). The sequence of each gene was compared with sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST) by BLAST search. Nucleotide sequences of each gene showed >99% similarity with sequences of B. parva (accession Nos. EU938334, FJ238525, DQ487158). Analyzed sequence data were deposited in GenBank (accession Nos. LC120826 for ITS region, LC120827 for β-tubulin, and LC120828 for EF1-α). For the phylogenetic analysis, reference genes of various species belonging to the genus Botryosphaeria were obtained from GenBank (Table 2). Sequences of three genes for each species, including the fungal isolate characterized in this study, combined as follows: ITS region, β-tubulin, and EF1-α. Combined sequences were aligned and analyzed using analysis tool provided in the DDBJ (DNA databank of Japan) homepage (http://www.ddbj.nig.ac.jp/). A phylogenetic tree was constructed using TreeView program with the combined sequences (Page, 1996). A results showed that our combined sequence was clustered with B. parva sequences (Fig. 2). Taken togather, the pathogen causing stem blight on R. crataegifolius was identified as B. parva, based on morphological and phylogenetic analyses.
Table 2
Isolates of Botryosphaeria and Guignardia species considered in this study
Isolates Identity Host Accession No.

ITS β-Tubulin EF1-α
 CMW991 Botryosphaeria dothidea Populus nigra AF241175 AY236924 AY236895
 CMW7780 B. dothidea Fraxinus excelsior AY236947 AY236925 AY236896
 CMW7999 B. dothidea Ostrya sp. AY236948 AY236926 AY236897
 CMW8000 B. dothidea Prunus sp. AY236949 AY236927 AY236898
 CMW9075 B. dothidea P. nigra AY236950 AY236928 AY236896
 CMW9078 B. parva Actinidia deliciosa AY236940 AY236914 AY236885
 CMW9079 B. parva A. deliciosa AY236941 AY236915 AY236886
 CMW9080 B. parva P. nigra AY236942 AY236916 AY236887
 CMW9081 B. parva P. nigra AY236943 AY236917 AY236888
 CMW1130 B. parva Sequoia gigantea AY236945 AY236919 AY236890
 CMW7054 B. ribis Ribes rubrum AF241177 AY236908 AY236879
 CMW7772 B. ribis Ribes sp. AY236935 AY236906 AY236877
 CMW7773 B. ribis Ribes sp. AY236936 AY236907 AY236878
 CMW992 B. lutea A. deliciosa AF027745 AY236923 AY236894
 CMW9076 B. lutea Malus domestica AY236946 AY236922 AY236893
 CMW10309 B. lutea Vitis sp. AY339258 AY339266 AY339250
 CMW6837 B. australis Acacia sp. AY339262 AY339254 AY339270
 CMW9072 B. australis Acacia sp. AY339260 AY339252 AY339268
 CMW9073 B. australis Acacia sp. AY339261 AY339253 AY339269
 CMW10126 B. eucalyptorum Eucalyptus grandis AF283687 AY236921 AY236892
 CMW6233 B. eucalyptorum Eucalyptus nitens AY615138 AY615122 AY615130
 CMW10125 B. eucalyptorum E. grandis AF283686 AY236920 AY236891
 CMW7774 B. obtusa Ribes sp. AY236953 AY236931 AY236902
 CMW7775 B. obtusa Ribes sp. AY236954 AY236932 AY236903
 CMW7060 B. stevensii F. excelsior AY236955 AY236933 AY236904
 UCP130 B. stevensii Citrus sp. JF271751 JF271769 JF271787
 CMW9074 B. rhodina Pinus sp. AY236952 AY236936 AY236901
 CMW10130  B. rhodina Vitex donniana AY236951 AY236929 AY236900
 CMW7063 Guignardia philoprina Taxus baccata AY236956 AY236934 AY236905
 PPL02* B. parva Rubus cratargifolius LC120826 LC120827 LC120828

ITS, internal transcribed spacer; EF1-α, elongation factor 1α.

* The sequence obtained in this study.

Fig. 2
Phylogenetic analysis of Botryosphaeria species using a combined sequence of the internal transcribed spacer, β-tubulin, and elongation factor 1α genes. The tree was constructed using the neighbor-joining method with 1,000 replicates.
*The fungus isolated in this study.
RPD_22_116_fig_2.jpg
B. parva was first reported to cause soft rot on kiwifruit in New Zealand (Pennycook and Samuels, 1985). Since this first report, presence of B. parva has been reported in 71 host species across six continents and 21 countries (Sakalidis et al., 2013). This pathogen causes serious symptoms, including dieback, canker, stem blight, and fruit rot in various hosts, such as grapevines, kiwi vines, cherry trees, raspberry bushes, and mango (Abdollahzadeh et al., 2013; Haleem et al., 2012; Linaldeddu et al., 2007; Pennycook and Samuels, 1985; Phillips, 2002; Sakalidis et al., 2013; Slippers et al., 2005; van Niekerk et al., 2004). Thus, the characterization and management of this pathogen is very important. In Korea, B. parva was not reported until 2009 (Kim et al., 2009). In 2012 and 2013, dieback symptoms caused by Neofusicoccum parvum, an anamorphic synonym of B. parva, on blueberries and walnuts has been reported (Cheon et al., 2013; Choi et al., 2012), but there has been no report hitherto that B. parva (N. parvum) was isolated from R. crataegifolius. Thus, this is the first report of stem blight on R. crataegifolius caused by B. parva in Korea. Considering economic value of R. crataegifolius, methods to manage this pathogen are urgently needed to prevent low production of R. crataegifolius.

Acknowledgements

This research was supported by a grant (NIBR 2015-01205) from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea for projects on the survey and discovery of indigenous Korean fungal species.

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