Microbes Environ. Vol. 30, No. 4, 301-309, 2015 https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME15109 Community Analysis of Root- and Tuber-Associated Bacteria in Field-Grown Potato Plants Harboring Different Resistance Levels against Common Scab AKIRA KOBAYASHI1,2, YUKI OHDAIRA KOBAYASHI1,2, NOBUTAKA SOMEYA1, and SEISHI IKEDA1* 1National Agricultural Research Center for Hokkaido Region, National Agriculture and Food Research Organization, 9–4 Shinsei-minami, Memuro-cho, Kasai-gun, Hokkaido 082–0081, Japan; and 2Present affiliation: NARO Kyushu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization, 6651–2 Yokoichi, Miyakonojo, Miyazaki 885–0091, Japan (Received July 22, 2015—Accepted September 21, 2015—Published online December 9, 2015) Eight genotypes of potato plants with different resistance levels against common scab were grown in a field infested with Streptomyces turgidiscabies. DNA was extracted from the roots, tubers, and rhizosphere soils of each of the eight genotypes at the flowering stage, and the quantity ofS. turgidiscabies genomic DNA was assessed by real-time PCR using a TaqMan probe. The results obtained showed that the different potato genotypes had significant impacts on the population levels of S. turgidiscabies between resistant and susceptible genotypes in the tubers, but not in the roots or rhizosphere soils. Clone analyses of 16S rRNA gene libraries from the eight potato genotypes identified three phyla (Proteobacteria, Firmicutes, and Actinobacteria) as dominant taxa in root and tuber clone libraries, while a clustering analysis identified 391 operational taxo- nomic units (OTUs) at the species level. Eleven OTUs closely related to Aquicella siphonis, Arthrobacter nicotinovorans, Streptomyces rishiriensis, Rhodococcus baikonurensis, Rhizobium radiobacter, Rhizobium etli, Phyllobacterium myrsinacearum, Paenibacillus pabuli, Paenibacillus alginolyticus, and Bacillus halmapalus were detected in the root or tuber libraries of all the potato genotypes examined. Furthermore, an abundance of OTUs related to Aquicella and Rhodococcus was observed in the rhizospheres of resistant and susceptible potato genotypes, respectively. Based on this ecological information, an efficient survey may be conducted for biological agents from the potato rhizosphere. Key words: 16S rRNA gene, common scab, community analysis, potato, rhizosphere Common scab of potato (Solanum tuberosum L.) is preva- bacteria from the genera Bacillus (11, 28, 38); Pseudomonas, lent in most potato-growing areas around the world. It is caused Enterobacter, and Acinetobacter (46); and bacteriophages by soil- or seed-borne pathogenic Streptomyces, including (27) are known for their potential suitability as biocontrol Streptomyces scabies, S. acidiscabies, S. turgidiscabies, S. agents for potato common scab. However, only limited suc- europaeiscabiei, S. stelliscabiei, and other species (6, 24, 44, cess has been achieved in the development of a commercial 49). When scab-like lesions are formed on tubers, their market application to control common scab using these microorgan- value is significantly reduced. Several methods are currently isms because of the inconsistent performance of biocontrol used to control common scab, including the chemical treat- agents in field conditions, and, hence, this has become an ment of seed potatoes (43), crop rotation (23, 45), incorpora- important technical issue in the development and utilization tion of green manure (41, 45), reduction in soil pH (45), and of beneficial microorganisms in agronomic environments. irrigation during tuber formation (23, 29). However, the disease- The success of the smart utilization of beneficial microorgan- suppressive effects of these methods are not sufficient. Strong isms is now considered to largely depend on a comprehensive reductions in the incidence of common scab may be achieved knowledge of plant-microbe interactions at a community by soil fumigation with chemicals such as methyl bromide level in field conditions (42). Thus, a better understanding of and chloropicrin; however, this treatment impairs the integ- the diversity and functionality of the plant-associated micro- rity of the ecosystem. Furthermore, these chemicals reduce bial community in field conditions may facilitate the utilization not only the populations of pathogens, but also those of of beneficial microorganisms in order to promote plant growth non-targeted microorganisms including beneficial soil micro- and control plant pathogens in agricultural practices (3). organisms for disease control with a potential environmental Several studies using culture-independent methods have risk and high cost. Therefore, alternative methods to control been reported for the characterization of the rhizosphere- potato common scab are being widely sought. associated bacterial community structures of potato plants (3, Biological control is considered as a desirable approach for 9, 20, 35, 39, 40, 42). These culture-independent analyses not controlling soil-borne diseases because it is eco-friendly and only revealed the tissue-specific distribution of potato-associated expected to replace or supplement chemical pesticides (51). bacteria, but also contributed to the identification of candidate A number of antagonistic microorganisms including fungal microorganisms for biological control (3, 35, 40, 47). However, species from the genus Trichoderma (19, 30); actinomycetes the genetic background of host plants, even at the cultivar from the genus Streptomyces (2, 13, 18, 22, 50); general soil level, has been suggested to affect the diversity and function- ality of the community structure of plant-associated microor- * Corresponding author. E-mail [email protected]; ganisms (31). The potential impact of host plant genotypes on Tel:+81–155–62–9276; Fax:+81–155–61–2127. the plant-associated microbial community may be significant 302 KOBAYASHI et al. in the case of the potato because its intragenetic diversity is Materials and Methods generally considered to be large owing to polyploidy and self-incompatibility. Plant materials and sampling In order to achieve the successful biological control of Eight potato genotypes (seven cultivars: Yukirasha (17), Snow plant diseases, including potato common scab, it is also March (16), Star Ruby, Snowden, Irish Cobbler [Japanese name important to understand the ecology and epidemiology of Danshakuimo], Toyoshiro, and Piruka, and an experimental line pathogens through the specific detection and quantification of 02005-10) were used to quantify the populations of S. turgidiscabies in rhizospheres and assess the diversity of rhizosphere bacteria. pathogenic Streptomyces on potato tissue and in soil under field Disease severity in the eight potato genotypes against common conditions. A TaqMan real-time PCR assay using virulence scab was evaluated between 2004 and 2009 in an experimental field gene nec1 (5), SYBR Green quantitative real-time PCR assay artificially infested with S. turgidiscabies at the Memuro Research using the txtAB operon (33), and quantitative competitive Station, Hokkaido Agricultural Research Center (42°89.2' N, quenching probe PCR assay using the nec1 gene (26) have 143°07.7' E, 93 m a.s.l.) based on a previously reported method (17). been used for the accurate detection and quantification of After harvesting, the disease index of each tuber was determined by pathogenic Streptomyces in the potato. Although a good the percentage of the surface area with scab lesions: no lesions=0 points; 1–3%=1 point; 4–13%=2 points; 14–25%=3 points; more than correlation has been reported between the presence of nec1 26%=4 points. Disease severity was calculated using the following and pathogenicity, some nonpathogenic nec1-positive strains formula (Σ[score of the disease index×number of tubers]/[4×total and numerous pathogenic nec1-negative strains have been number of tubers]×100). Based on these field evaluations, the resis- described (33). The Streptomyces txtAB operon encodes the tance levels of the eight potato genotypes were categorized as highly non-ribosomal peptide synthetase required for phytotoxin resistant against (Yukirasha [R1] and 02005-10 [R2]), resistant thaxtomin A production and is essential for the pathogenicity against (Snow March [R3]), moderately resistant against (Star Ruby of all known pathogens of potato common scab (12). Thus, [M1] and Snowden [M2]), and susceptible to (Irish Cobbler [S1], Toyoshiro [S2], and Piruka [S3]) common scab (Table 1). These the txtAB operon is regarded as an optimal marker for patho- eight genotypes were planted on May 19, 2010 in an experimental genic Streptomyces for potato common scab, and, as such, the field artificially infested with S. turgidiscabies at the Memuro development of a TaqMan real-time PCR assay system using Research Station, with single rows of 3.6 m in length and a plant the txtAB operon is preferred for the highly accurate quantifi- spacing of 30 cm per genotype in triplicate. The field was dressed cation of pathogenic Streptomyces for potato common scab with a commercial fertilizer (60, 170, and 102 kg for N, P2O5, and –1 in soil. K2O ha , respectively) as a side dressing for basal fertilization. Nine healthy plants in each genotype were selected visually at In the present study, we utilized a TaqMan real-time PCR random and sampled on July 7, 2010. After the root system of each assay system using the txtAB operon in order to monitor potato plant was carefully sampled,