Short Communication pISSN: 2288-9744, eISSN: 2288-9752 J F E S Journal of Forest and Environmental Science Journal of Forest and Vol. 31, No. 4, pp. 312-316, November, 2015 Environmental Science http://dx.doi.org/10.7747/JFES.2015.31.4.312 Effect of Sodium Chloride (NaCl) on the Mycelial Growth and Ascospore Germination of Rhizina undulata, the Root Rot Fungus of Coniferous Trees Sun Keun Lee1, Dong Hyeon Lee2, Kyoung-Tae Lee3, Yong Bae Park3, Sang-Tae Seo1 and Jong Kyu Lee4,* 1Division of Forest Insect Pest and Diseases, National Institute of Forest Science, Seoul 02455, Republic of Korea 2Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Hatfield, Pretoria 0028, South Africa 3Southern Forest Resources Research Center, National Institute of Forest Science, Jinju 52817, Republic of Korea 4Department of Forest Environment Protection, Kangwon National University, Chuncheon 24341, Republic of Korea Abstract Rhizina undulata, the causal agent of Rhizina root rot, is a soil-borne fungus occurring on coniferous trees. The destruction of coastal forests caused by R. undulata infection has been mainly associated with bonfires at camping sites. However, Rhizina root rot was observed in the western coastal forests without fire. It was hypothesized that Rhizina root rot in this area might be closely related to the soil salinity, which can facilitate the growth and survival of R. undulata. So, the variation in sodium chloride (NaCl) resistance among isolates of Rhizina undulata was compared using liquid media containing different concentrations of NaCl ranging from 0 mM to 300 mM. Our results showed that, albeit of no growth at a higher concentration of NaCl (300 mM), most of R. undulata isolates were capable of germinating and grew at up to 100 mM, indicating that NaCl resistance varies among R. undulata isolates. It was further found that isolates from coastal areas seemed to be more tolerant to NaCl than those further away the coast. We demonstrated that R. undulata could be possible to survive in coastal areas, but was lower NaCl tolerance than other fungi. Key Words: Rhizina undulata, NaCl resistance, mycelial growth, ascospore germination Introduction and the incineration of plant material in proximity to coastal forests, implying that fires are a main source of the disease Rhizina undulata Fr ex Fr. (Syn. Rhizina inflata (Schaeff) (Ginns 1968; Gibson 1970). Specifically, spore germination Karst), the causal agent of Rhizina root rot, is a soil-borne is dependent on high temperatures and the duration of ex- fungus occurring on coniferous trees (Germmen 1971). R. posure to heat; conditions ranging from 35oC for 24 hr to undulata has been reported to cause severe damage to costal 45oC for 17 hr, are generally referred to as the ‘heat shock’ ef- pine forests in Korea (Lee and Kim 1990; Lee et al. 2005). fect (Jalaluddin 1967; Germmen 1971; Lee and Kim 1990). The destruction of coastal forests caused by R. undulata in- In June 2002, Rhizina root rot was identified in a 20 hec- fection has been associated with bonfires at camping sites tare Japanese black pine (Pinus thunbergii) forest located in Received: October 27, 2015. Revised: November 6, 2015. Accepted: November 6, 2015. Corresponding author: Jong Kyu Lee Department of Forest Environment Protection, Kangwon National University, Chuncheon 24341, Republic of Korea Tel: 82-33-250-8364, Fax: 82-33-259-5620, E-mail: [email protected] 312 Journal of Forest and Environmental Science http://jofs.or.kr Lee et al. Taean on the western coast of South Korea (Lee et al. and 2009 (Fig. 1, Table 1). Fungal materials collected in the 2005). Subsequently, R. undulata infected P. t h u n b e r g i i were field were placed separately for each tree in paper bags, and observed in the national park of Seocheon in 2005 (Korea then transported to the laboratory for further isolations. Forest Service: unpublished data). However, disease in- cidence at both sites was not found to be associated with fire; it was hypothesized that those cases of Rhizina root rot might be closely related to other environmental factors, fa- cilitating the growth and survival of R. undulata. Salinity is one of the main features of coastal environ- ments. Although sodium chloride (NaCl) may have sig- nificant impacts on coastal environments, little is known re- garding the effect that the salinity could have on the growth and survival of microorganisms in the coastal environment. It was thus hypothesized that levels of salinity might have an ef- fect on the growth, spore germination, and survival of R. un- dulata, and thus, its potential impact was determined in vitro. Materials and Methods Collections of R. undulata isolates were made from P. thunbergii and P. densiflora across the country between 2002 Fig. 1. Schematic representation of R. undulata collection site. Ta ble 1 . Mycelial dry weights of R. undulata isolates incubated in PDB with different NaCl concentrations NaCl concentration NaCl concentrations in PDB (mM) Collected Distance from Host Descriptionsa of sampling location locations coast (km) (mM)b 0 50 100 300 Buan 0.3 Pinus Coast, Sand, 0.342 18.84±5.83c 7.34±2.99* 6.06±2.78* 0* Gangneung 1.4thunbergii Camping site 0.855 13.62±6.58 9.00±2.30* 7.18±2.50* Incheon 0.7 0.513 14.68±3.93 5.50±0.73* 4.00±1.98* Uljin 2.2 0.342 18.84±5.83 7.34±2.99* 6.06±2.78* Taean 1.1 0.684 10.10±6.56 7.42±4.01* 5.80±4.59* Yangyang 0.6 0.513 8.38±0.79 4.04±1.10* 4.16±1.62* Andong 60.0 Pinus Mountain, - 9.18±4.23 8.06±1.48* 5.48±1.52* 0* Gimhae 20.9densiflora Loam, 11.62±5.07 5.02±3.52* 4.16±2.17* Gumi 95.7Burned area 14.06±2.74 8.78±2.59* 5.30±5.24* Gyeongju 21.6 8.02±3.83 3.56±2.15* 3.68±2.63* Haenam 3.8 15.08±2.33 8.52±3.84* 4.78±2.52* Hongcheon 76.4 14.26±2.73 9.70±1.29* 2.60±1.89* Namwon 60.0 14.82±4.64 8.76±2.03* 4.84±2.30* Okcheon 85.6 16.40±6.64 11.50±5.51* 7.68±2.10* Suncheon 8.5 13.24±4.48 6.28±2.67* 7.54±3.55* Ulsan 15.2 12.08±2.36 5.34±2.98* 1.22±1.70* ageographical condition, soil texture and descriptions of collection site; bwere referred from the research report of National Institute of Forest Science (2012); cDry weight (mg): Mean values±SD(Standard deviation) of 5 replicates. *indicates significant differences between 0 mM and different NaCl concentrations based on one-way ANOVA tests at p=0.05 (Tukey's test). J For Env Sci 31(4), 312-316 313 Effect of NaCl on R. undulata Single ascospore isolations were made according to the dia were sealed and incubated under the dark condition for method described by Jalaluddin (1967), and subsequently 7 days at 25oC. The rate at which R. undulata ascospores the isolates were cultured on PDA (DifcoTM potato dex- were germinated was measured using the optical micro- trose agar; Becton, Dickinson Co., USA) at 25oC for one scope (Carl Zeiss Axio Imager A1; Zeiss Co., Göttingen, week under the dark condition prior to use. In total, sixteen Germany). Five replicates were prepared for each treat- R. undulata isolates were obtained and all isolates used in ment. Each measured value was used in the following for- this study were deposited with the Tree Pathology and mula to calculate the germination rate: Germination rate Mycology laboratory (TPML), Kangwon National Uni- (%)=(Total number of the germinated ascospores)/(Total versity, South Korea. Genomic DNA for all isolates in- number of ascospores)x100. cluded in this study was extracted using DNeasy Mini One-way analysis of variance (ANOVA) and Tukey's Plant Kit (Qiagen, Valencia, CA) according to the manu- method were used to determine the significance of the dif- facturer's instructions. To ensure the correct identity of iso- ferences in NaCl resistance measured for concentrations lates obtained in this study, internal transcribed spacer ranging from 0 mM to 300 mM. All statistical tests were (ITS) regions, including the 5.8S rDNA region were se- performed using the SPSS program (IBM SPSS Statistics quenced, following the technique described by Lee et al. Ve rsion 19, USA). (2007) (White et al. 1990). The resulting sequences were then blasted against the nucleotide database of the National Results and Discussion Center for Biotechnology Information (NCBI; http://www. ncbi.nlm.nih.gov). All sequences obtained in this study Soil salinity varies spatiotemporally in coastal areas. were deposited with GenBank under accession numbers, NaCl concentrations at a site within 40 m of the shoreline in EU346951 to EU346953. Kadoori, Japan exceeded 400 mM under typhoon con- To determine the effect of different concentrations of ditions, but were lower than 30 mM post-typhoon (Ishikawa NaCl on the growth of R. undulata isolates, five-millimeter et al. 1995). P. t h u n b e r g i i appears to be highly resistant to of agar disks containing fungal mycelium from the pre-cul- high levels of NaCl, since it is capable of colonizing sand tured fungal colonies were taken from the edges of actively dunes and rocky cliffs where seawater (with a NaCl concen- growing isolates.