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Seed Treatment with Calcium Carbonate Containing Bacillus amyloliquefaciens PMB05 Powder Is an Efficient Way to Control Black Rot Disease of Cabbage
Seed Treatment with Calcium Carbonate Containing Bacillus amyloliquefaciens PMB05 Powder Is an...
Hsiao, Chia-Yu;Blanco, Sabrina Diana;Peng, An-Li;Fu, Ju-Yin;Chen, Bo-Wei;Luo, Min-Chia;Xie, Xing-Yu;Lin, Yi-Hsien
agriculture Article Seed Treatment with Calcium Carbonate Containing Bacillus amyloliquefaciens PMB05 Powder Is an Efﬁcient Way to Control Black Rot Disease of Cabbage 1 1 , 2 1 1 1 1 1 Chia-Yu Hsiao , Sabrina Diana Blanco , An-Li Peng , Ju-Yin Fu , Bo-Wei Chen , Min-Chia Luo , Xing-Yu Xie 1 , and Yi-Hsien Lin * Department of Plant Medicine, National Pingtung University of Science and Technology, Pingtung 912301, Taiwan Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung 912301, Taiwan * Correspondence: firstname.lastname@example.org Abstract: Black rot disease is a serious bacterial disease that harms vegetable crops of the Brassica genus (especially cabbage plants) worldwide. The causal agent, Xanthomonas campestris pv. campestris (Xcc), is a seed-borne pathogen that primarily infects seedlings. Previous studies suggest that the bacterial strain, Bacillus amyloliquefaciens PMB05, can intensify the plant immune responses of cabbage against black rot disease and reduce disease occurrence. In plant immunity, several reactions occur during a pathogen attack, but the elevation of calcium ion concentration in plant cells is essential in the induction of plant defense responses. Therefore, this study aims to investigate whether disease control of black rot disease in cabbage plants can be improved by integrating calcium carbonate in the formulation for preparing B. amyloliquefaciens PMB05. Firstly, we found the addition of calcium carbonate in the formulation revealed to have signiﬁcantly increased the cell and endospore populations of B. amyloliquefaciens PMB05 in the fermentation liquids. To increase the convenience of disease control in the ﬁeld, these fermentation liquids were converted to powder form for subsequent analysis. Results revealed that the grown seedlings from seeds, mixed with Citation: Hsiao, C.-Y.; Blanco, S.D.; PMB05 powders, signiﬁcantly intensiﬁed plant immune responses and improved black rot disease Peng, A.-L.; Fu, J.-Y.; Chen, B.-W.; control. We further compared distinct seed treatments using one PMB05 powder to evaluate its Luo, M.-C.; Xie, X.-Y.; Lin, Y.-H. Seed feasibility in ﬁeld application. The results demonstrated that the disease control efﬁcacy and yield of Treatment with Calcium Carbonate cabbage were signiﬁcantly improved in the seed treatment with the powder (SD-160C2) to 56.46% and Containing Bacillus amyloliquefaciens 5.91%, respectively, at 10 weeks post transplanting. Interestingly, the seed treatment combined with a PMB05 Powder Is an Efﬁcient Way to calcium-containing commercial fertilizer spraying treatment did not increase the control efﬁcacy of Control Black Rot Disease of black rot disease, but it signiﬁcantly increased the weight of cabbages after harvest. We concluded Cabbage. Agriculture 2023, 13, 926. that the seed treatment with calcium carbonate-containing Bacillus amyloliquefaciens PMB05 powder is https://doi.org/10.3390/ agriculture13050926 an efﬁcient way to control black rot disease in cabbage. Academic Editor: Alessandro Vitale Keywords: biological control; ﬁeld trial; plant immunity; powder; seed treatment Received: 17 March 2023 Revised: 18 April 2023 Accepted: 19 April 2023 Published: 23 April 2023 1. Introduction Cabbage (Brassica oleracea) is one of the vegetable crops with more than 8000 hectares planting area in Taiwan . In the growing stage of cabbage, black rot disease caused by Xanthomonas campestris pv. campestris (Xcc) often occurs in environments with high humidity Copyright: © 2023 by the authors. and temperature. This pathogen is mainly transmitted by seeds. It further invades the Licensee MDPI, Basel, Switzerland. plant through its water holes, stomata, roots and wounds on the leaf edge and causes the This article is an open access article typical symptoms of V-type gangrene on the leaf edge during plant growth. If the plant distributed under the terms and is left untreated, symptoms may lead to leaf drop. Black rot disease is considered one of conditions of the Creative Commons the most destructive diseases of cruciferous plants [2–4]. To control these diseases, copper- Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ containing fungicides and antibiotics are usually used in the field . However, under 4.0/). long-term fungicide and antibiotic treatment application, drug-resistant bacteria strains have Agriculture 2023, 13, 926. https://doi.org/10.3390/agriculture13050926 https://www.mdpi.com/journal/agriculture Agriculture 2023, 13, 926 2 of 15 gradually emerged [6–8]. Alternative control methods may include soaking seeds in hot water, but not only does this lead to a decrease in the germination rate of cabbage seeds, but it also fails to effectively eliminate pathogenic bacteria [9,10]. Therefore, the application of antagonistic microorganisms is considered to be an effective and environmentally friendly alternative in field application . Many microorganisms that can regulate plant defense responses have also been reported to control the occurrence of various diseases [12–15]. In recent years, Bacillus spp. strains with antagonistic activity have been proven to inhibit the growth of Xcc and effectively reduce the occurrence of black rot disease [16–19]. In addition, our previous research demonstrated that the application of Bacillus amyloliquefaciens PMB05 bacterial suspension or powder on Xcc-contaminated cabbage seeds was able to reduce the occurrence of black rot disease in greenhouse assay [1,20]. The ability of PMB05 to supplement plant disease resistance is in regard to its ability to intensify plant immune responses of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) [1,21–25]. PAMP-triggered immunity is the first layer of a plant’s defense system during a pathogen attack. This defense response is initiated by pattern recognition receptors (PRRs) on the plant cell membrane after recognizing the PAMP of the pathogen, which activates the cellular signal transduction to enhance plant disease resistance [22,26–29]. PAMPs are generally conserved and unique structures of microorganisms. For example, flagellin is a PAMP from bacterial pathogens or chitin from fungal pathogens [30–32]. When PAMPs induce PTI, extracellular calcium influx will occur to activate NADPH oxidase to generate reactive oxygen species (ROS) [33–37]. ROS is not only an important indicator in the PTI pathway, but it can also participate in the regulation of mitogen-activated protein kinase (MAPK) activity. It then further activates defense signals such as salicylic acid (SA) or jasmonate ethylene (JA)/ethylene (ET) to cause plants to exhibit disease resistance [28,38–41]. B. amyloliquefaciens PMB05 intensifies PTI response in this manner . Therefore, calcium plays a crucial role in the initial signal transduction of the plant defense response in the plant immune system. Integrating a calcium component in PMB05 formulations may increase the effect of PMB05 on PTI signal intensification in plants to attain improved disease control potential. In this study, we first used calcium channel blockers to confirm that the function of B. amyloliquefaciens PMB05 in intensifying plant immune signals in cabbage leaves does require the participation of calcium. Subsequently, calcium carbonate, which is a material commonly used in the field as a calcium fertilizer, was used as the calcium source of the most efficient formulation in plant immunity intensification and control of black rot disease. Our previous study showed that the use of PMB05 powder on Xcc-contaminated seeds still maintains excellent disease control efficacy , and this application method is convenient for field application. We used the fermented liquid of PMB05 to make powders for subsequent analysis. After confirming that the grown seedling obtained from seed treatment with PMB05 powder is efficient in intensifying plant immune response and controlling black rot disease in cabbage plants in the greenhouse, the actual feasibility in the field becomes very important. We thus used the treatment of powder-treated seeds as a basis to evaluate the difference in disease control and yield in the field. The feasibility of the above treatment with a commercial calcium-containing product, Meili Calcium, was also evaluated. In this study, we provided evidence to support that seed treatment with calcium carbonate-containing B. amyloliquefaciens PMB05 powder is efﬁcient to control black rot disease in cabbage. 2. Materials and Methods 2.1. Growth Conditions for Cabbage Plants The cabbage plant used in this study was Brassica oleracea L. var. capitata. Before assay, the seeds (Minghua Seed Co., New Taipei City, Taiwan) were sterilized with 1% sodium hypochlorite for 5 min and then washed three times with sterile water for 5 min each time. Afterwards, the seeds were regarded as clean seeds for use in further experiments. The seeds were sown individually in pots containing soil composed of vermiculite, pearlite and peat moss at a ratio of 10:1:1 (v/v/v). For plant immune response assays, the plants Agriculture 2023, 13, 926 3 of 15 were grown in a growth chamber (Model: F-1200, Hipoint, Kaohsiung, Taiwan) at 28 C under 16 h of light and 8 h of darkness. For disease evaluation, plants were grown in the greenhouse at around 30 C. 2.2. Growth Conditions of Bacterial Strains The bacterial strain, Xanthomonas campestris pv. campestris XCC15, was cultured on a nutrient agar (NA) plate and incubated at 28 C for 2 days. This was used as the source of inoculum. The bacterial suspension was prepared from colonies grown on the NA plates by washing them with sterilized distilled water containing 0.1% of CMC (Carboxymethylcellulose Sodium; Sigma, St. Louis, MO, USA). The OD value was adjusted to 0.3 (around 3 10 CFU/mL). Similarly, Bacillus amyloliquefaciens PMB05 was cultured on NA plates and incubated at 28 C for 2 days. This was the source of the beneﬁcial bacterium. To prepare the bacterial suspension of PMB05, one colony of PMB05 from the NA plate was inoculated in 5 mL of nutrient broth and incubated at 37 C under 200 rpm for 16 h. Then, the bacteria culture was centrifuged at 8000 rpm for 5 min and further resuspended with sterilized water containing 0.1 % of CMC to 0.3 of OD . 2.3. Effect of Calcium Influx on Bacillus amyloliquefaciens PMB05-Intensifying Plant Immune Signals To test if calcium is required for B. amyloliquefaciens PMB05 to intensify plant im- mune signals in cabbage leaves, the effect of Lanthanum chloride (LaCl , Sigma, Sigma, St. Louis, MO, USA) on ROS generation and callose deposition intensiﬁed by PMB05 were assayed upon treatment with the synthesized ﬂg22 . The ﬂg22 (QRLSSGLRIN- Xcc Xcc SAKDDAAGLAIS) derived from X. campestris pv. campestris B305 (GenBank accession number DQ356465) was purchased from Life Tein LCC (South Plainﬁeld, NJ, USA). In the assay, the ﬁnal concentration of ﬂg22 at 1 M was applied to the bacterial suspensions Xcc of PMB05. Then, the stock solution of LaCl was added immediately to reach a ﬁnal concentration of 40 M and 80 M. To evaluate the intensiﬁcation of plant immune re- sponses, rapid ROS generation and callose deposition were used as indicators and assayed according to the modiﬁed method . The leaves of 4-week-old cabbage seedlings were inﬁltrated with each test solution, and then rapid ROS generation and callose deposition on leaf strips were evaluated at 1 h and 8 h post inﬁltration, respectively. In addition, in the follow-up experiment of powder application, ﬂg22 was directly inﬁltrated into Xcc the leaves of seedlings grown from powder-treated seeds for analysis. The evaluation of rapid ROS generation was performed by staining leaf strips with 20 M of H DCFDA 0 0 (2 ,7 -dichlorodihydroﬂuorescein diacetate) (Molecular Probes, Eugene, OR, USA) in the dark for 20 min. Callose deposition was evaluated by staining leaf strips with 0.01% analine blue (Sigma, USA) in the dark for 2 h. The ﬂuorescent results of rapid ROS gen- eration and callose deposition were observed under a ﬂuorescent microscope with the Excitation/Emission ﬁlter set at 465–495 nm/515–555 nm and 340–380 nm/400–425 nm, respectively. All of the images were taken under consistent conditions, and the ImageJ software (https://imagej.nih.gov/ij/, 1 March 2023) [21,22] was used to calculate the ﬂuo- rescence intensities from each evaluation. In each experiment, 10 images were taken from each treatment as repeats, and three independent experiments were performed in this assay. 2.4. Effects of Calcium Inﬂux on Bacillus amyloliquefaciens PMB05 in the Control of Black Rot Disease in Cabbage To conﬁrm that calcium inﬂux is required for B. amyloliquefaciens PMB05 in the control of cabbage black rot, treatments with different ﬁnal concentrations of LaCl were used for a biocontrol assay. The bacterial suspensions of X. campestris pv. campestris XCC15 and B. amyloliquefaciens PMB05 were prepared by using 0.1% CMC in a ratio of 1:1 (v/v) mixed, and then LaCl was added to obtain ﬁnal concentrations of 0 M, 40 M and 80 M in the mixtures. The leaves of the 4-week-old seedlings, prepared from clean seeds, were cut and inoculated 0.5 cm from the leaf’s edge using scissors that were pre-sterilized with 75% alcohol and soaked in each mixture. The inoculated plants were bagged and placed in a Agriculture 2023, 13, 926 4 of 15 28 C growth chamber. The lesion length from the inoculated cutting site to the end of developed symptoms was measured after 7 days post-inoculation. Each treatment had ﬁve seedlings as repeats, and three independent experiments were performed in this assay. 2.5. Preparation of Xcc-Contaminated Seed and Disease Severity Assay To ensure the incidence and prevalence of black rot disease, cabbage seeds were contaminated with Xcc for all disease assays. A total of 1.6 g of clean seeds were soaked in a 10 mL bacterial suspension of X. campestris pv. campestris XCC15 for 1 h. The treated seeds were collected after drying in an air dryer (DHR-20TW, Cuisinart, Stamford, CT, USA). These seeds were regarded as “Xcc-contaminated seeds” for further experiments. The seeds were sown and grown in the greenhouse. The disease symptoms on the true leaves of 4-week-old plants were observed to calculate disease incidence and disease severity . Disease severity was calculated based on the disease index (0 is no disease symptoms; 1 is weak water-soaked or yellowing symptoms; 2 is obvious yellowing in a small area from leaf edge; 3 is a V-shaped gangrene with necrosis symptoms). Disease severity was calculated using the following formula: [(0 N + 1 N + 2 N + 3 N )/(3 N )] 0 1 2 3 Total 100%, whereby N -N represented the number of plants in each different disease index 0 3 and N represented the summation of plants from N to N (the total number of plants Total 0 3 tested in this experiment). Thirty plants were assayed in each treatment as repeats in a single experiment, and three independent experiments were performed in this assay. 2.6. Preparation of Bacillus amyloliquefaciens PMB05 Powder To evaluate whether the addition of calcium carbonate to the formulations affects the beneﬁcial effects of PMB05, the fermentation liquid was prepared based on the standard formulation, PMBFL-2A , with different concentrations of calcium carbonate: 0 mM, 2 mM, 10 mM, and 20 mM. The fermentation liquids were performed in a 30 L fermenter tank (BTF-B30L, Biotop Process & Equipment Inc., Nantou County, Taiwan) at 37 C under 120 rpm and 1.5 vvm. The fermentation process took 5 days, and after each distinct fermentation liquid was obtained. Additionally, 20% maltodextrin (w/v) was added into each fermentation liquid. The fermentation liquids were further prepared into powders (labeled SD-160, SD-160C2, SD-160C10, and SD-160C20) using a spray dryer (GB210-A, KOHSIEH, Taipei, Taiwan) under conditions of a ﬁxed ﬂow rate at an inlet temperature of 160 C . The bacteria populations in these powders were evaluated. 2.7. Biocontrol Efﬁcacy Assay of PMB05-Powder in Greenhouse and Field Trial Both greenhouse and ﬁeld trials were performed to test the efﬁcacy of the powder in controlling black rot disease. In the greenhouse trials, 1% of each powder was mixed with the Xcc-contaminated seeds before sowing. After 4 weeks, the disease severity of each treatment was calculated. In the ﬁeld trials, the experiment was conducted in Pingtung County (22.646191, 120.603503) from 28 October 2022 to 4 January 2023. Before starting the ﬁeld trials, 30-day-old seedlings obtained from powder (SD-160C2)-treated clean seeds were prepared. Similar sized seedlings were selected for ﬁeld planting. In order to ensure the consistent occurrence of black rot disease in the ﬁeld, the bacterial suspension of XCC15 was sprayed every two weeks after two weeks of ﬁeld planting. The disease control efﬁcacy of the powder treatment was compared with non-powder treatment (blank) in the ﬁeld. In order to conﬁrm whether the effect of powder treatment on disease control can be enhanced by the addition of a commercial calcium-containing fertilizer, 500-fold diluted Meili calcium (Diamond Nano Biochem, Taichung, Taiwan) was used for treatment once every two weeks from two weeks post-planting. Disease severity in the ﬁeld trial was calculated at 6, 8 and 10 weeks after ﬁeld planting based on the disease index (0, no disease symptoms; 1, 1–5% diseased area; 2, 5–25% diseased area; 3, 26–50% diseased area; 4, over 51% diseased area). Disease severity was calculated using the following formula: [(0 N + 1 N + 2 N + 0 1 2 3 N + 4 N )/(4 N )] 100%, where N –N represented the number of plants in 3 4 total 0 4 each different disease index and N represented the summation of plants from N to N . 0 4 Total Agriculture 2023, 13, 926 5 of 15 Control efﬁcacy = [1–(disease severity of treatment/disease severity of blank)] 100%. In addition, cabbages were harvested 10 weeks after planting. Cabbage yield was assessed by weighing the cabbage after cutting it from the base. In the above assays, data collected from six plants in one ﬁeld plot were regarded as one repeat, and ﬁve plots were repeated in each treatment. 2.8. Data Analysis Statistical analysis was performed using the SPSS Statistics software for Windows, version 25 (IBM Corp, Armonk, NY, USA). Analysis of variance (ANOVA) was used to assess differences between the treatments for all the assays to obtain F values. Then, the Post hoc tests (Tukey’s HSD) were performed to analyze signiﬁcant differences between treatments (p < 0.05). 3. Result 3.1. Effect of Calcium Influx on Bacillus amyloliquefaciens PMB05-Intensifying Plant Immune Signals To ascertain whether calcium inﬂux is required for B. amyloliquefaciens PMB05 in the intensiﬁcation of plant immune responses in cabbage, the calcium channel blocker, LaCl , was applied to assay its effects on rapid ROS generation and callose deposition. The results showed that the ﬂg22 -induced ROS generation was strongly intensiﬁed by the presence Xcc of PMB05 in cabbage leaves (Figure 1A). In the assay with the subsequent addition of LaCl , ROS generation was signiﬁcantly reduced. However, there were no signiﬁcant differences between the two concentrations tested (Figure 1B). For callose deposition, the ﬂg22 - Xcc induced callose signal was intensiﬁed by PMB05. More signiﬁcantly, in subsequent assays Agriculture 2023, 13, x FOR PEER REVIEW 6 of 16 with the addition of LaCl , both concentrations completely eliminated callose deposition in the presence of PMB05 (Figure 2). Figure 1. Eﬀect of LaCl3 on ROS generation intensiﬁed by Bacillus amyloliquefaciens PMB05 upon Figure 1. Effect of LaCl on ROS generation intensiﬁed by Bacillus amyloliquefaciens PMB05 upon activation with ﬂg22Xcc. Panel (A) indicates the ﬂg22Xcc-induced ROS generation was intensiﬁed by activation with ﬂg22 . Panel (A) indicates the ﬂg22 -induced ROS generation was intensiﬁed by Xcc Xcc B. amyloliquefaciens PMB05 on cabbage leaves. The symbols “+” and “−” indicate the application of B. amyloliquefaciens PMB05 on cabbage leaves. The symbols “+” and “