Plant Pathol. J. 34(2) : 143-149 (2018) https://doi.org/10.5423/PPJ.NT.12.2017.0281 The Plant Pathology Journal pISSN 1598-2254 eISSN 2093-9280 ©The Korean Society of Plant Pathology Note Open Access Comparison of Microbial Community Structure in Kiwifruit Pollens Min-Jung Kim1†, Chang-Wook Jeon2†, Gyongjun Cho2, Da-Ran Kim1, Yong-Bum Kwack3, and Youn-Sig Kwak1,2* 1Department of Plant Medicine, Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Korea 2Dvision of Applied Life Science (BK21plus), Gyeongsang National University, Jinju 52828, Korea 3Namhae Sub-Station, NIHHS, RDA, Namhae 52430, Korea (Received on December 26, 2017; Revised on January 30, 2018; Accepted on January 31, 2018) Flowers of kiwifruit are morphologically hermaph- Plant-associated microbes are fundamental determinant of roditic and survivable binucleate pollen is produced plant health and productivity (Berendsen et al., 2012). Mi- by the male flowers. In this study, we investigated crobes living on surfaces of plant are called epiphytes and microbial diversity in kiwifruit pollens by analyzing living in inner tissues are endophytes. Habitat of plant-asso- amplicon sequences of 16S rRNA. Four pollen samples ciated microbes are mainly divided into phyllosphere which were collected: ‘NZ’ was imported from New Zealand, is the aerial surfaces of fresh plant on the ground and rhizo- ‘CN’ from China in year of 2014, respectively. ‘KR13’ sphere which is the attached soil of root surfaces (Johnston- and ‘KR14’ were collected in 2013’ and 2014’ in South Monje et al., 2016). These plant microbiome makes the Korea. Most of the identified bacterial phyla in the four host to enhance nutrient accumulation from the soil, toler- different pollens were Proteobacteria, Actinobacteria ate against abiotic stresses, produce phytohormones and and Firmicutes. However, the imported and the domes- protect from pathogens (Bulgarelli et al., 2013). Accord- tic pollen samples showed different aspects of micro- ingly, it is crucial to understand the plant microbiome that bial community structures. The domestic pollens had how they coexist and what they do ecosystem. In the labo- more diverse in diversity than the imported samples. ratory, microbes are separated on culture media and identi- Among top 20 OTUs, Pseudomonas spp. was the most fied with biochemical and morphological methods (Yang dominant specie. Interestingly, a bacterial pathogen of and Crowley, 2000). However, the total environmental kiwifruit canker, Pseudomonas syringae pv. actinidiae DNA sequence data suggested that less than 1% in a gram was detected in ‘NZ’ by the specific PCR. This study of soil microbes can be cultured. As the ‘Pyrosequencing’ provides insights microbial distribution and community or ‘Metagenomics’ emerges, it can explore microbial com- structure information in kiwifruit pollen. munity and diversity research without culturing microbes (Gilbert et al., 2010). Therefore, the microbial studies using Keywords : kiwifruit, microbial community, pollen the culture-independent methods have recently provided much more ecological information of phyllosphere mi- Handling Associate Editor : Oh, Chang-Sik crobiology than previously studied (Lopez-Velasco et al., 2013). The plant rhizosphere contains the vast microbial diversity that colonize on the roots. One gram of soils con- tains about 2 × 109 CFU (Daniel, 2005). The majority of †These authors contributed equally to this work as first authors. bacteria in the rhizosphere habitats belonged to Proteobac- *Corresponding author. teria, Acidobacteria, Actinobacteria and Bacteroidetes. Mi- Phone) +82-55-772-1922, FAX) +82-55-772-1929 crobial community composition differs depending on the E-mail) [email protected] This is an Open Access article distributed under the terms of the plant species, the soil type and the cultural practices. Plant Creative Commons Attribution Non-Commercial License (http:// organic matter such as root exudates, epidermal cells and creativecommons.org/licenses/by-nc/4.0) which permits unrestricted mucilage also influence the microbial community in rhizo- noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. sphere (Coats et al., 2014). Compared with bulk soil, the microbial biomass and activity in rhizosphere are enhanced Articles can be freely viewed online at www.ppjonline.org. as a result of compounds exudation by the root (Sørensen, 144 Kim et al. 1997). Likewise, such studies focused on rhizosphere have were transferred to 1.5 ml tube, centrifuged at 13,000 rpm been widely reported. However, phyllosphere microbiome for 1 min then the supernatant was discarded. The process has been less studied then rhizosphere one (Vorholt, 2012). was repeated 3 times. Total DNA were extracted using Bacteria have often distributed in numbers averaging 106 DNA prep kit (Solgent Co., Daejeon, Korea) divided into to 107 cfu/cm2 on leaves. Proteobacteria containing α-, β- gram-negative and gram-positive. In the case of gram- and γ-proteobacteria are known as the dominant bacterial positive bacteria, the pellets were resuspended in 300 μl group of the phyllosphere with Firmicutes, Acidobacteria, of R1 solution, added 2 μl of Lysozyme (100 mg/ml). The Actinobacteria and Cyanobacteria (Whipps et al., 2008). tubes were incubated at 37oC for 1 h, centrifuged at 10,000 Most studies about phyllosphere microbial community has rpm for 1 min and the supernatant was discarded. From focused on leaves but pollen-associated microbes are poor- here, experimental procedures were performed as same ly investigated. Pollen-associated microbial communities both gram-positive and gram-negative bacteria. SGD1 are different by pollination types such as insect-pollinated solution (350 μl) and 5 μl of protenase K (20 mg/ml) were plants and wind-pollinated ones or species-specific in same added, mixed using vortex for 1 min and incubated at 65oC environmental conditions (Ambika Manirajan et al., 2016). for 10 min. After cooling the mixtures thoroughly at room Actinidia spp. (Kiwifruit) is an economically important temperature for 5 min, 400 μl of SGD2 solution was added, crop widely cultivated in many countries. It is dioecious mixed then centrifuged at 10,000 rpm for 5 min. During fruit tree divided into opposite sex: male and female centrifuged, spin column was installed to 2-ml collection flower. These flowers are morphologically hermaphroditic, tube. SuperBinderTM Solution (100 μl) was added to the however, survivable binucleate pollen is produced by the spin column, centrifuged at 10,000 rpm for 30 sec and the male flowers only (Fraser et al., 2009). The kiwifruit bacte- pass-through was discarded. Spin column was transferred rial canker caused by Pseudomonas syringae pv. actinidiae to a new 2-ml collection tube. WB (500 μl, 80% Ethanol) (PSA) is the severe problem in the global kiwifruit indus- was added to spin column and centrifuged at 10,000 rpm try (Kim et al., 2017). The symptoms of bacterial canker for 30 sec. The pass-through was discarded, in the same appear brown spots surrounded by a yellow halo on the way, DNA was washed with 500 μl of WB (80% Ethanol) leaves, milky bacterial exudates from the many organs once again. The empty spin column was centrifuged at (twigs, vines, leaves and flower) and typical canker on the 10,000 rpm for 2 min to eliminate the WB completely and trunks (Kim et al., 2017). Pollen contaminated by the PSA transferred to a new 1.5 ml tube. DNA Hydration Solution is a serious vector to spread the canker pathogen (Chapman (50 μl) was added, incubated at room temperature for 1 min et al., 2011). Overwintering PSA can disperse to host via and centrifuged at 10,000 rpm for 2 min. Spin column was pollen in the disease cycle (Donati et al., 2014). Artificial eliminated and final DNA were stored in a new 1.5 ml tube pollination with the PSA contamination pollen obviously at -80oC. spread and transfer the pathogen in orchard and transfer Bacterial 16S rRNA regions were amplified by forward the pathogen from infected orchard to non-infected orchard primer 27F (10 pmol): 5’-GAGTTTGATCMTGGCT- (Tontou et al., 2014). Therefore, understanding of the mi- CAG-3’ and the reverse primer 518R (10 pmol): 5’ WT- crobial community structures of the pollen is necessary to TACCGCGGCTGCTGG 3’ (Kwon et al., 2010). The implement the management strategies of kiwifruit bacterial volume of PCR products was adjusted to total volume 20 canker. μl with 13.8 μl of distilled water, 100 ng of template DNA, In this study, four kiwifruit pollen samples were collect- 2 μl of 10X reaction buffer, 1 μl of 10 mM dNTP, 1 μl of ed from several orchards in New Zealand, China and South respective primers and 5 unit of Taq DNA polymerase Korea. Two pollen samples, New Zealand and China were (Bioneer Co., Daejeon, Korea). Reactions were performed imported in year of 2014 and two domestic pollen samples using 5 min of denaturation step at 95oC, 30 cycles consist- were collected from a kiwifruit orchard in Sacheon, Ko- ing of 30 sec at 95oC, 30 sec at 55oC and 30 sec at 72oC, rea in year of 2013 and 2014, respectively. One gram of followed by 5 min of final extension at 72oC (Rondon et the pollen was added to a test-tube containing 9 ml of 1X al., 2000). PCR products containing the amplified DNA Phosphate buffered saline (PBS; 8 g NaCl, 0.2 g KCl, fragments were checked their size by electrophoresis on 1% 1.44 g Na2HPO4, 0.24 g KH2PO4, bring total volume to 1l agarose gel containing ethidium bromide in 1% Tris-ace- with ddH2O, pH 7.4). To lyse the cellular organelles and tate EDTA (TAE) buffer. The PCR products were purified extract DNA, the samples were sonicated with frequencies using the ExpinTM gel SV kit (Geneall biotech Co., Seoul, of 35 kHz for ten minutes using ultrasonic bath (Bandelin Korea). Pyrosequencing was performed using the Roche electronic GmbH and Co., Berlin, Germany).