b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 269–278 ht tp://www.bjmicrobiol.com.br/ Environmental Microbiology Antagonistic endophytic bacteria associated with nodules of soybean (Glycine max L.) and plant growth-promoting properties a,∗ a b LongFei Zhao , YaJun Xu , XinHe Lai a Shangqiu Normal University, College of Life Sciences, Key Laboratory of Plant-Microbe Interactions of Henan, Shangqiu, Henan, PR China b The First Affiliated Hospital of Wenzhou Medical University, Institute of Inflammation & Diseases, Wenzhou, China a r t i c l e i n f o a b s t r a c t Article history: A total of 276 endophytic bacteria were isolated from the root nodules of soybean (Glycine Received 5 January 2017 max L.) grown in 14 sites in Henan Province, China. The inhibitory activity of these bacte- Accepted 19 June 2017 ria against pathogenic fungus Phytophthora sojae 01 was screened in vitro. Six strains with Available online 13 October 2017 more than 63% inhibitory activities were further characterized through optical epifluore- scence microscopic observation, sequencing, and phylogenetic analysis of 16S rRNA gene, Associate Editor: Fernando Andreote potential plant growth-promoting properties analysis, and plant inoculation assay. On the basis of the phylogeny of 16S rRNA genes, the six endophytic antagonists were identified Keywords: as belonging to five genera: Enterobacter, Acinetobacter, Pseudomonas, Ochrobactrum, and Bacil- Endophytes lus. The strain Acinetobacter calcoaceticus DD161 had the strongest inhibitory activity (71.14%) Soybean against the P. sojae 01, which caused morphological abnormal changes of fungal mycelia; Antagonisis such changes include fracture, lysis, formation of a protoplast ball at the end of hyphae, and Phytophthora sojae split ends. Except for Ochrobactrum haematophilum DD234, other antagonistic strains showed Plant growth-promoting potential the capacity to produce siderophore, indole acetic acid, and nitrogen fixation activity. Regres- sion analysis suggested a significant positive correlation between siderophore production and inhibition ratio against P. sojae 01. This study demonstrated that nodule endophytic bacteria are important resources for searching for inhibitors specific to the fungi and for promoting effects for soybean seedlings. © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). ∗ Corresponding author at: College of Life Sciences, Shangqiu Normal University, 298 Wenhua Middle Road, Shangqiu, Henan 476000, PR China. E-mail: [email protected] (L. Zhao). https://doi.org/10.1016/j.bjm.2017.06.007 1517-8382/© 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 270 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 269–278 obtained from fields of 14 sites subordinate to 9 districts of Introduction Henan Province, China (map available as Supplementary Fig. 21,22 1). Three healthy root nodules with similar sizes were The root nodules of legume plants are symbiotic organs excised from the lateral roots of each plant. Soil debris was induced by soil bacteria known as rhizobia. As part of brushed away from the nodules, and the nodules were stored the root system, root nodules harbor symbiotic bacte- ◦ in sterile plastic bags at 4 C until they were processed for ria and many endophytes, including Agrobacterium 1 2 isolation within 24 h. tumefacien, A. rhizogenes, Phyllobacterium, Stenotrophomonas, 3 4 In each site, soil cores were sampled at five locations with Enterobacteriaceae, Bacillus species, Bacillus, Bordetella, Cur- 5 a depth of 15–20 cm and 5 cm away from the taproots, which tobacterium, and Pantoea. Aside from their diversity, which were bulked and thoroughly mixed to form composite sam- has been studied extensively, the effect of nodule endo- ples. Soil samples were stored in loosely tied plastic bags at phytes on host legumes was revealed. The nodule endophytic ◦ 4 C. Soil texture was defined according to the international Agrobacterium strains specifically inhibit the nodulation of institution triangle coordinate graph, and soil pH was deter- Rhizobium gallicum on the common bean (Phaseolus vulgaris 31 6 mined as described in Zhao et al. L.) or facilitate the unspecific nodulation of Sinorhizobium 7 A phytopathogenic fungus, P. sojae 01, was provided by the meliloti on woody legumes. Some nodule endophytes that College of Life Sciences of Northwest A & F University in China belong to Bacillus, Bordetella, Curtobacterium, or A. rhizogenes 5 and was incubated on potato dextrose agar plate (PDA: extract could promote the growth or nodulation of red clover. These of 200 g potato, 20 g of glucose, 18 g of agar, 1 L of distilled phenomena are similar to that of endophytes isolated from ◦ ◦ water) at 30 C for 3 days and maintained at 4 C for temporary other parts of plants and could benefit host plants by pro- storage. ducing phytohormones, 1-aminocyclopropane-1-carboxylase The seeds of soybean (G. max L.) cultivar Zhonghuang 13, (ACC) deaminase, and antibiotic compounds, as well as by which is the principle cultivar used in the sampling region, fixing nitrogen, solubilizing phosphate, or suppressing phy- 8–11 were bred by the Institute of Crop Sciences of the Chinese topathogens through the competence of invasion sites. Academy of Agricultural Sciences. Owing to the above mentioned advantages, endophytes are considered novel resources in the biocontrol of plant diseases 12–14 and in the promotion of plant growth. Isolation and purification of soybean nodule endophytes As a major legume crop, soybean (Glycine max L.) plays an important role in sustainable agriculture and in the economy Bacteria were isolated from root nodules according to a 32 33 of many countries. Soybean has a great nitrogen-fixing abil- standard method as described by Ma et al. and Miller et al. ity due to its symbiosis with rhizobia in root nodules. The A single colony of the isolate was repeatedly streaked on the presence of Bradyrhizobium japonicum, B. liaoningense, B. yuan- same medium and examined with a microscope. Pure cul- ◦ 15,16 17 18 mingense, B. elkanii, B. huanghuaihaiense, B. daqingense, tures were preserved on plates at 4 C for temporary storage 19 B. pachyrhizi, B. iriomotense, B. canariense, Sinorhizobium fredii, or in sterile vials with 30% (v/v) glycerol for long-term storage − ◦ 20 and S. sojae has been reported in China, which is the at 80 C. To confirm if the surface sterilization process was 21,22 center of origin of soybean. Similar to other plants, endo- successful, several surface-sterilized nodules were rolled over phytic bacteria have been isolated from different parts of nutrient agar (NA) plates and aliquots of water from final rinse 34 19,23–26 soybean, and some of these parts showed antagonis- solutions and then plated onto NA plates. Plates without any 27–29 tic and growth-promoting potential. Diverse endophytic contaminants were considered effectively surface-sterilized, bacteria, including Pantoea, Serratia, Acinetobacter, Bacillus, and the corresponding plates were used for the isolation of Agrobacterium, and Burkholderia, have also been isolated from endophytes. 30 soybean nodules. However, antagonistic endophytic bacte- ria within nodules of soybean for P. sojae in Henan Province Screening of antagonistic endophytic bacteria have not been sufficiently studied. On the basis of the above mentioned background knowl- The antifungal activity of endophytes against pathogenic fun- edge and considering the nodule endophytes as a new bacteria gus P. sojae 01 was detected by using the point inoculation resource with potential in biotechnology, we conducted this 35 method. Spores of fungal cultures were inoculated on PDA study (1) to screen antagonistic endophytic bacteria from soy- plates, and a small block of agar with fungal mycelia cut with bean nodules against P. sojae; (2) to explore the potential a sterile puncher (Ø = 4 mm) was placed in the center of a fresh plant-beneficial traits of endophytic bacteria; and (3) to assay plate. Tested strains were spot inoculated on the edge of PDA the seedling growth response of soybean to the inoculation of plates approximately 25 mm from the center. After incuba- endophytic bacteria. ◦ tion at 28 C for 7 days, the inhibition zone was measured. Fungal mycelia that were cultivated without inoculation were 36 Materials and methods included as control. Experiments were performed in tripli- cate for each bacterial isolate. Collection of root nodules, soil samples, phytopathogenic Secondary screening of antifungal activity was performed fungus, and soybean seeds similar to the primary screening method, but bacteria were spot inoculated as bacterial suspension (OD600 ≈ 1). Antago- Nodules from cultivated soybean were collected from July to nistic activities were evaluated by measuring inhibition zones August 2012, when the plants were blooming. Samples were between pathogens and tested bacteria. b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 269–278 271 30,44 Microscopic observation of phytopathogenic fungi mycelia didates of nitrogen fixers. Experiments were performed in triplicate. The forward primer nifH40F (5 -GGN ATC GGC To determine the effect of endophytic bacteria on pathogenic AAG TCS ACS AC-3 ), reverse primernifH817R (5 -TCR AMC fungus, treated and untreated pathogenic fungi were cul- AGC ATG TCC TCS AGC TC-3 ),and the procedure described 45 tured for 2 days on PDA medium.