In order to assess plant protection of endophytes against seed-borne pathogens, soybean seeds (cultivar Daewon) were treated with microbial suspension from individual endophytic isolates, followed by treatment with seed-borne pathogen suspension. Then, vegetative growth and resistance to pathogenicity were assessed through pot experiment (Fig. 1). Soybean seeds were washed with 70% ethanol and dipped into 1% sodium hypochlorite solution for 5 min to disinfect the surface and rinsed with sterilized water before microbial treatment. Effectiveness of surface sterilization was confirmed by no colony formation on the agar media plated with distilled water used to rinse the surface-sterilized seeds.

The seed-borne pathogens C. sojina (KACC No. 49847), S. glycines (KACC no. 43091), X. axonopodis pv. glycines (KACC no. 10491) and P. syringae pv. tabaci (KACC no. 17820) were acquired from the Korean Agriculture Culture Collection (KACC). All the pathogens obtained from the KACC were described in the website as being isolated from the seeds. Diaporthe sp. and Cercospora sp. were isolated from diseased soybean seeds and identified by sequencing of PCR product using the primers EF1-728F (5′-CATCGAGAAGTTCGAGAAGG-3′) and EF1-986R (5′-TACTTGAAGGAACCCTTACC-3′) (Gomes et al., 2013) and the primers cfp_F1 (5′-GCCGATCGATTGCAGGAGTTGGC-3′) and cfp_R1 (5′-TTGCTGATCCAAGTAGTCGGACG-3′) (Soares et al., 2015) for identification of Diaporthe sp. and Cercospora sp., respectively. Then they were identified as D. eres and C. kikuchii corresponding to accession no. KJ490498 and no. KP752257 each with 100% similiarity. Seed endophytes were selected from our previous study, based on their in-vitro antagonistic activities against the seed-borne pathogens (Kim et al., 2022). We tested three species of bacteria (Bacillus sp. DWB21155, Pseudomonas koreensis DWB21122, and Stenotrophomonas maltophilia DWB21134) and three species of fungi (Alternaria hungarica DWF21130, Cladosporium pseudocladosporioidies DWF21127, and Penicillium steckii DWF21107).

Three soybean seeds were planted in one experimental pot with six replications per one treatment (pot size: 11.3 cm upper diameter, 6.5 cm bottom diameter, 9.7 cm high). The pot trials were conducted in a phyto-chamber at 25°C and 70% relative humidity with a 12-h light and dark alternation from a fluorescent lamp. Before planting, seeds were treated with endophyte isolates for 2 h by immersing them in bacterial suspension or spore suspension, followed by another immersion in bacterial suspension or spore suspension of seed-borne pathogens for 2 h. Then, the treated seeds were moved on the to the petri dishes with wet paper disc on the bottom and incubated for 3 days to allow seed germination. The endophytic treatments consisted of the bacterial isolates DWB21155, DWB21235, and DWB21122 and the fungal isolates DWF21130, DWF21232 and DWF21107 obtained from the previous study (Kim et al., 2022). The soil used to grow the plants consisted of 70% cocopeat, 8.5% peatmoss, 9.5% pearlite, 6% vermiculite, 3.748% zeolite, 0.01% humic acid, 0.01% wooden charcoal and 0.18% fertilizer. Bacterial suspensions for seed treatment were prepared with tryptic soy broth (Difco, Detroit, MI, USA) incubated for approximately 24 h at 30°C, 120 rpm from cultures grown for 24 h in trypsin soy agar at 30°C, until the suspension concentration of each of the bacteria reached an OD₆₀₀ = 0.5-0.6 in spectrophotometer. Then, the cultures were centrifuged at 1,500 ×g for 5 min at 4°C, the bacterial pellet was resuspended in distilled water. To obtain fungal suspension, each of the fungal isolates (5 mm in diameter of a cylindrical agar plug) was inoculated on the center of potato dextrose agar medium and incubated at 25°C until mycelia reached the edge of the plate (9 cm in diameter). Next, the fungi were scraped with sterile water from the medium before being transferred to a sterile falcon tube, and filtered through a Miracloth layer (Millipore, Billerica, MA, USA). Then, the number of spores in the spore suspension was counted using a hemocytometer. Concentration of spore suspensions were adjusted to a final concentration of 4-5 × 10⁴ spores/ml before seed treatment. The soybean seeds were immersed in the suspension for 2 h under 50 rpm at 30°C and 25°C for bacterial and fungal treatment, respectively. The control group with no inoculation was treated with distilled water instead of bacterial or fungal suspensions.

We measured shoot length (cm), root length (cm), number of leaves, fresh leave weight (g), fresh stem weight (g), fresh root weight (g), dry leave weight (g), dry stem weight (g), and dry root weight (g), as the indicator of plant development 14 days after planting the soybean seeds. To compare each measured value, we used the following formula; (V_c − V_exp)/V_c, where V_c represents each measured value of control group and V_exp represents each measured value of experimental group treated with microbes. Then, the averages of resulting values were taken and shown in heatmaps generated using the R program (version 4.2.1). The control was referred to healthy control (HC), and the control with pathogen inoculation was referred to infected control (IC).

The control seeds treated with distilled water swelled up a few hours after the treatment without any color change, while seeds treated with some pathogens showed morphological changes after the treatment in addition to discoloration. In the D. eres-treated seeds, for example, seeds looked quite different from the controls at six hours after treatment. D. eres-treated seeds were brown in whole or in part on the surface of the seed, compared to control (‘De-trt’ in Fig. 2A). The width and length of the seeds was comparable to the control, but D. eres-treated seeds exhibited difference in thickness (Fig. 2B, Supplementary Table 1). We also often observed brown spots or cracks in the hilum up of the D. eres-treated seeds.

When comparing C. kikuchii-treated seeds with control seeds, there was little difference in length, width, and thickness. However, the difference in color was clearly visible at six hours after treatment. We found pink to purple stain in every seed coat treated with C. kikuchii. Some of C. kikuchii-treated seeds displayed relatively small range of stain, while other seeds showed stain over the entire area of seed coat (‘Ck-trt’ in Fig. 2A). The seeds treated with Pst did not differ significantly from the control seeds, except that the seed surface tended to become slightly darker (‘Pst-trt’ in Fig. 2A). The seeds treated with C. sojina, S. glycines, and Xag showed no clear difference from the control at the seed stage.

To gauge the effects of seed treatment with endophytes, we first observed the effect of seed inoculation with pathogens on seed germination and seedling growth. After C. kikuchii inoculation, only 3 out of 6 soybean seeds succeeded in germination, and 2 of those did not reach the leaf development stage (Fig. 3 Trt (P) row and Supplementary Table 2). We also observed dark discoloration in the leaves of C. kikuchii-treated group (‘Ck-trt’ in Supplementary Fig. 1B).

When seeds were inoculated with C. sojina, five out of six seeds germinated, which was the highest germination rate among pathogen-only treated groups. Three out of six seeds (50%) were able to germinate after inoculation with S. glycines. Of the six D. eres-treated seeds, three showed severe decay symptoms in consistent with the observed abnormalities in D. eres-treated seeds, and three others also did not develop normally (Supplementary Fig. 1A). Only 1 out of 6 seeds inoculated with D. eres succeeded in germination, and even 14 days after planting, the germinated seed did not succeed in leaf release.

Only half of the Xag-treated seeds germinated, and the growth of soybean plant from the seed was very poor. Notably, the leaves of Xag-treated group exhibited yellow to brown lesions with pustules (‘Xag-trt’ in Supplementary Fig. 1B). Similarly, three out of six Pst-treated seeds germinated and showed relatively poor growth. In the leaves of plants, yellow halos were also observed at the edges (‘Pst-trt’ in Supplementary Fig. 1B).

Given the responses of plants to seed-borne pathogens, we set out to test whether the seed endophytes, which we had isolated in the previous work (Kim et al., 2022), would improve or remedy such responses of plants to the pathogen treatment. To do the test, seeds were treated first with the endophytes before being challenged with the pathogens and planted in the pot. The endophytes we tested in this study include three bacteria (P. koreensis DWB21122, Bacillus sp. DWB21155, and S. maltophilia DWB21134) and three fungi (A. hungarica DWF21130, C. pseudocladosporioidies DWF21127, and P. steckii DWF21107), which will be referred to as their isolation number (for example, DWB21122 for P. koreensis) (Fig. 3, Supplementary Table 2).

For C. kikuchii, treatment with DWF21107 and DWB21155 did not show noticeable difference, compared to the C. kikuchii-treated plant (Trt(P)). DWB21155 treatment even reduced the stem weight lightly more (0.275 g and 0.113 g of fresh stem weight and 0.175 g and 0.738 g of dry stem weight at Trt(P) and Bacillus sp. DWB21155, respectively). In contrast, the treatment with DWF21130, DWF21127, DWB21122 and DWB21134 improved plant growth, especially in shoot length, number of leaves, leaves weight, and root weight (Figs. 3 and 4A, Supplementary Table 2).

For C. sojina, DWF21127 and DWB21155 had a positive effect on seed germination and plant growth. Especially, DWF21127 increased shoot length, fresh leaves weight, and dry leaves weight (52%, 80%, and 81%, respectively). In contrast, DWF21107 appeared to have a negative effect, resulting in a decrease of fresh and dry stem weight (86% in both) (Figs. 3 and 4B, Supplementary Table 2).

For S. glycine, the germination of soybean seeds was improved by treatment with DWF21130, DWB21155, DWB21122 and DWB21134 (100%, 83%, 100%, and 67%), but not in seeds treated with DWF21127 and DWF21107. The most significant effect was observed in plants grown out of seeds inoculated with DWF21130 (77%, 83%, 76%, 94%, 73%, 83%, 94%, 77%, and 85% increase in shoot length, root length, number of leaves, fresh leave weight, fresh stem weight, fresh root weight, dry leave weight, dry stem weight, and dry root weight, respectively). DWF21127 and DWF21107 had little effect against S. glycines in the pot experiments (Figs. 3 and 4C, Supplementary Table 2).

Among all the pathogens we tested in our work, D. eres had the most devastating effects on soybean plants, causing seed decay and suppressing the plant growth and development (Fig. 3). Seed treatment with endophytes except DWB21134 dramatically recovered the germination rate and plant growth (Figs. 3 and 4D, Supplementary Table 2). Leaf weight in both of fresh and dry condition was stimulated positively by five strains. In particular, DWB21155 displayed plant growth promoting effect even in comparison with the non-treatment control.

For Xag, treatment with endophytes was able to improve shoot length, root length, number of leaves, fresh and dry stem weight, and fresh and dry root weight (Figs. 3 and 4E, Supplementary Table 2). In addition, the germination rate was 100% in plant groups inoculated with endophytes, while it was 50% in infected control. However, none of the endophytes could recover the stem length and leaf weight (both fresh and dry).

For Pst, the seed treatment with endophytes increased seed germination rate in all cases (Figs. 3 and 4F, Supplementary Table 2). DWF21130, DWF21134 and DWF21127 was most effective in promoting plant growth, compared to the Trt(P). Especially, when seeds were treated with DWB21134, all measurements showed increase in value with fresh root weight even higher than non-treatment control (31%).