The base soil was prepared by mixing vermiculite (Kanuma Kosan, Tochigi, Japan), decomposed granite soil (Kohnan Shoj, Osaka, Japan), black soil (Tachikawa Heiwan Nouen, Tochigi, Japan), and peat moss (Kanuma Kosan) at a ratio of 5:3:1:1 (v/v) [13] . Cow manure, chicken manure, rice bran, oil cake, soybean meal, and bone meal were used as organic fertilizers, and the amounts added were 38.8 g, 3.9 g, 7.8 g, 1.9 g, 1.9 g, and 1.2 g, respectively, per kg base soil followed by previously developed fertilization method [13] . Chemical fertilizers (Kuki-hiryou, Mie, Japan) was added at a rate of 1.33 g per kg base soil. A specimen of Chlorella residue was purchased from Sun Chlorella Co., Ltd. (Kyoto, Japan), and was composed of four species of Chlorella (Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidea, and Chlorella regularis). The Chlorella residue was applied to both soil types at a concentration of 0%, 1%, 5 and 10%. The two types of prepared soil were adjusted to 30% (w/w) water content. The o soils were stored for 1 week at 23˚C prior soil bacterial biomass analysis. The soil added to cylindrical pots (10.5 cm in diameter, 22 cm in height) to a soil depth of 20 cm for plant cultivation.

In this study, the plant species Brassica rapa var. periviridis and Spinacia oleracea L. were used to observe the effect of Chlorella residue on plant growth. The seeds were purchased from Takii & Co., Ltd. (Kyoto, Japan) and were sown in red clay soil. Seven days of 2 seedlings were transplanted in each pot at different application rate (0%, 0.5%, and 1%, v/v) of Chlorella residue and cultivated for 4 weeks in a plant growth chamber (23˚C, light: 12 hours, dark: 12 hours) [14] . The water content of soil was maintained at 30% during cultivation period. After 4 weeks of cultivation, plant growth was measured in terms of fresh shoot weight. The experiment was conducted at triplicates.

To prepare pathogenic soil, resting spores of Plasmodiophora brassicae were obtained from the infected roots of B. rapa, and a spore suspension was prepared to create pathogenic soil. Infected B. rapa roots were carefully extracted and washed, and the galls were collected. The galls were then crushed at a ratio of 1 g galls:1.5 mL water using a mixer. The resulting suspension was filtered through 500- and 100-mesh sieves, and then the filtrate was centrifuged (500 rpm for 5 min) to separate the debris. The supernatant was subjected to a second centrifugation step (1000 rpm for 10 min.) and the pellet containing resting spores was suspended in distilled water. This purification process was repeated three times to ensure spore purity. Storage buffer (Hoagland’s solution) was added to the final pellet to inhibit spore germination. The number of resting spores in the suspension was determined using a hemocytometer under a microscope (BX-50, Olympus, Tokyo). The spore suspension (4.0 × 10⁸ spores/mL) was stored at 4˚C.

Pathogenic soils containing chemical and organic fertilizers were created by adding the resting spore suspension. The pathogenic soils were prepared with a spore concentration of 5 × 10⁵ spores/g-soil. Chlorella residue was added a concentration of 0.5% (v/v) in both chemical and organic pathogenic soil. Seven days of 2 seedlings of B. rapa were transplanted in each pot and cultivated for 4 weeks in a plant growth chamber (23˚C, light: 12 hours, dark: 12 hours). The water content of soil was maintained at 30% during cultivation period.

Disease indexes (DI) were evaluated based on the infection status of root (gall formation) to assess the severity of clubroot disease. The disease status of the roots was categorized into five classes (Class 0: no symptoms, Class 1: small galls only on the lateral roots, Class 2: small galls on the taproot, Class 3: large galls on the taproot, but lateral roots unaffected, Class 4: galls on the entire root).

The DI was calculated for each soil treatment using the following equation:

$DI=\frac{\left(1n_1+2n_2+3n_3+4n_4\right)}{4N_t}\times 100$

where $n_1-n_4$ is the number of plants in each class, and N_t is the total number of plants in the treatment.

The TC (total carbon) concentration was determined using the total organic carbon analyzer (TOC-VCPH, Shimadzu, Kyoto, Japan). To determine TN (total nitrogen), TP (total phosphorus), and TK (total potassium) contents, soil samples were extracted using CuSO₄·5H₂O, H₂SO₄, and H₂O₂, respectively, at 420˚C [15] . Subsequently, the TN and TP concentrations were assessed followed by our previous method [8] . The TK, TNa (total sodium), TCa (total calcium), TMg, (total magnesium), TFe (total iron), TMn (total manganese), TZn (total zinc), and TCu (total copper) concentrations were measured by atomic absorption spectrophotometry using a Z2300 instrument (Hitachi, Tokyo, Japan). The contents of ammonia nitrogen (NH₄-N) and nitrate nitrogen ( $N{O}_3^{-}-N$) were analyzed according to our previous study [16] after extracting soil samples with 1 M KCl. Total bacterial biomass was determined by quantifying environmental DNA (eDNA) extracted using the slow-stirring method [17] [18] . Following eDNA separation by agarose gel electrophoresis, the eDNA band was quantified using Kodak 1D Image Analysis Software (Kodak, Rochester, NY, USA).

PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) was conducted to investigate the effect of chlorella residue on bacterial diversity in the pathogenic chemical and organic soil. The 16S rRNA bacterial gene was amplified using the primer pair DGGE-F (5’-CGCCC GCCGC GCCCC GCGCC CGTCC CGCCG CCCCC GCCCG CCTAC GGGAG GCAGC AG-3’) and DGGE-R (5’-CCGTC AATTC CTTTG AGTTT-3’) [19] [20] . The PCR reaction mixture (50 μL) consisted of 0.01 ng/μL of DNA template, 1.5 U rTaq DNA polymerase, 5.0 μL 10× buffer, 5.0 μL 2 mM dNTPs, 3.0 μL MgCl₂, and 2.0 μL of each 10 mmol/L primer. The DNA polymerase, dNTPs, and PCR buffer were obtained from TOYOBO (Osaka, Japan), and all primers were synthesized by Sigma-Aldrich (Tokyo, Japan). The thermal cycling program for PCR consisted of initial denaturation at 95˚C for 1 min, followed by 35 cycles of denaturation at 95˚C for 1 min, primer annealing at 55˚C for 30 s, and extension at 72˚C for 1 min, with a final extension at 72˚C for 5 min. Subsequently, the amplified 16S rRNA bacterial genes were subjected to denaturing gradient gel electrophoresis (DGGE) analysis using the D Code System (BioRad Laboratories Inc., Hercules, CA, USA). A total of 20 μL PCR product was loaded onto an 8% (w/v) polyacrylamide gel with a denaturing gradient ranging from 27.5% to 67.5%. The gel was run in 1× Tris-acetate EDTA buffer at a constant voltage of 70 V at 60˚C for 15 hours. After electrophoresis, the gel was stained with ethidium bromide for 30 min and then rinsed with distilled water.

To investigate the effects of Chlorella residue as a bio-stimulant, its composition was analyzed ( Table 1 ). The Chlorella residue contained a range of nutrients. The concentrations of the primary macronutrients such as TN, TP, and TK were high at 97,910 mg/kg, 11,760 mg/kg, and 5500 mg/kg, respectively. However, the concentrations of inorganic nitrogen, such as $N{O}_3^{-}-N$ (92 mg/kg) and NH₄-N and (160 mg/kg), were relatively low. These results indicate that Chlorella residue contains a large amount of organic nitrogen.

The concentrations of TMg and TCa were 4300 mg/kg and 9700 mg/kg, respectively. The Chlorella residue was also rich in micronutrients; the TFe concentration was 1850 mg/L and the TMn concentration was 359 mg/kg. The low C/N ratio (5.1) confirmed the suitability of Chlorella residue as an organic nitrogen fertilizer. Furthermore, the Chlorella residue contained substantial amounts of secondary macronutrients and micronutrients.

The addition of organic materials to soil affects the microbial community. We analyzed the bacterial biomass in the organic soil after adding Chlorella residue ( Table 2 ), and found that the bacterial biomass was increased by 2.44 times after addition of Chlorella residue at 1% w/w, and by 2.13 times after addition of Chlorella residue at 5% w/w. However, the addition of Chlorella residue at a higher concentration (10% w/w) did not lead to an increase in bacterial biomass. Thus, the lower concentrations of Chlorella residue had positive effects on the bacterial biomass in the soil.

The effects of Chlorella residue on plant growth were investigated using B. rapa and S. oleracea ( Figure 1 and Figure 2 ). When Chlorella residue was added to the soil at 0.5% or 1.0% (w/w), the fresh weight of B. rapa was increased by 1.64 times and 1.75 times, respectively ( Table 3 ), and the fresh weight of S. oleracea was increased by 1.25 times and 1.55 times, respectively ( Table 4 ). These results indicate that Chlorella residue can function as an organic nitrogen fertilizer and/or bio-stimulant in soil.

The inhibitory effects of Chlorella residue on clubroot disease were investigated ( Figure 3 ). First, we compared the DI of clubroot disease between plants grown in soil with chemical fertilizer and those grown in soil containing organic fertilizer. In the plants growing in soil with chemical fertilizer, the DI was 100%, while in those growing in soil with organic fertilizer, the DI was 40%. This difference was related to differences in microbial biomass between the two soils. A similar

Different superscript letters within a column indicate significant differences (Tukey’s post-hoc test; p < 0.05) (n = 3).

Different superscript letters within a column indicate significant differences (Tukey’s post-hoc test; p < 0.05) (n = 3).

trend was observed in the organic soil, with a decrease in the DI from 40% to 20% upon addition of Chlorella residue. These results suggest that Chlorella residue positively affects the microbial community in the soil.

Next, we determined the effect of adding Chlorella residue on bacterial diversity in soils with chemical and organic fertilizers ( Figure 4 ). The bacterial diversity is reduced in both pathogenic chemical and organic soil. When Chlorella residue was added to both soils, the bacterial diversity returned to almost the same level as in non-infected soils. These results show that the addition of Chlorella residue, i.e., organic matter rich in nitrogen and mineral components, to the soil led to increased microbial biomass and bacterial diversity. The addition of Chlorella residue also contributed to increased plant yield and the suppression of clubroot disease.