Samples of lotus root
Lotus root and rhizosphere soil materials were sampled from the main lotus root planting area of Hongzhong Village, Huishi Town, Xiangtan, Hunan, China in 2020 and 2021. The lotus seed used in field cultivation was the Xiangtan Cunsanlian variety.
Pathogens
F. oxysporum f. sp. nelumbicola, a pathogen of lotus root spoilage disease, was provided by the Plant Protection Institute of Hunan Academy of Agricultural Sciences.
Test medium
PDA medium was used for isolation, preservation, activation, and antagonistic confrontation cultures of pathogenic fungi. LB medium was used to isolate, preserve, and activate biocontrol bacteria. NB medium was used for the expanded culture of the seed solution in a shaking flask. The PDA medium contained peeled potato (200 g), glucose (20 g), agar (15 g), and deionized water to a volume of 1000 mL. The pH is not adjusted. The LB medium contained peptone (10 g), yeast powder (5 g), sodium chloride (10 g), agar (15 g), and deionized water to a volume of 1000 mL; the pH ranged between 7.2 and 7.4. The NB medium contained peptone (10 g), sodium chloride (5 g), beef powder (3 g), and deionized water to a volume of 1000 mL, with a pH of 7.0.
Isolation of rhizosphere biocontrol bacteria
A total of 1 g of rhizosphere soil taken from Xiangtan was weighed into a 50-mL sterile centrifuge tube, and 9 mL of sterile water was added. Next, a gradient dilution to 105 was established using a 10-fold dilution method (100 µL of the bacterial solution was added to 900 µL of sterile water). Each step was mixed using shaking, and four concentration coating plates of 102, 103, 104, and 105 were obtained. Later, 100 µL of the bacterial solution was uniformly coated on LB solid medium plates and cultured at 25°C for 72 h. Morphological identification was conducted without a stereomicroscope (naked eye). After purification, the Bacillus was isolated and inoculated in the middle of the PDA solid medium plate inoculated with an F. oxysporum cake with a diameter of 4 mm. The culture was maintained at 25°C for 48 h when the experimental observations were made. The inhibition zone indicated that the bacterium had an inhibitory effect on F. oxysporum, and the single colony was purified and frozen with 50% glycerol at − 20°C for use.
Bacterial isolation from lotus root tissues
Fresh lotus roots were cleaned and disinfected. Next, 1 g of lotus root tissue was sterilized with 1% mercury solution for 5 min. The sample was then rinsed with sterile water three times, sterilized with 70% ethanol for 30 s, and rinsed a further three times with sterile water. Next, 9 mL of sterile water and an appropriate amount of quartz sand were added, and the mixture was ground into pulp with mortar. Finally, the sample was diluted to 10− 3 using a 10-fold dilution gradient. Three concentration gradient coatings of 10− 1, 10− 2, and 10− 3 were selected. The other operations were the same as described above.
Screening of biocontrol bacteria
Biocontrol bacteria with an inhibitory effect on F. oxysporum were screened using the confrontation culture method. First, the activated F. oxysporum colonies were drilled with a hole punch with an inner diameter of 4 mm, and the mycelium mass was picked up with an inoculum hook and inoculated in the center of the PDA medium. Next, the biocontrol group was inoculated with sterile water around the center of the mycelium mass at a distance of 2.5 cm. Finally, the plate was cultured in a constant-temperature incubator at 25°C until the F. oxysporum mycelia of the control group grew to the inoculation site, and the colony diameters of the control and experimental groups were measured. The bacteriostatic rate of the sample was calculated as follows:
Bacteriostatic rate = (colony diameter of control group − colony diameter of experimental group) / (colony diameter of the control group − diameter of pathogenic bacteria hypha) × 100%.
Morphological identification
Several strains with a high inhibition rate of F. oxysporum on the plate were inoculated on an LB plate and cultured at a constant temperature in an incubator at 30°C. The color and morphology of the colonies were observed, and Gram staining was performed. Additionally, the morphological characteristics of the bacteria were observed using an optical microscope, and the biocontrol bacteria were observed using a scanning electron microscope.
Physiological and biochemical identification
Physiological and biochemical characteristics of biocontrol bacteria were identified according to the Manual of Systematic Identification of Common Bacteria (Dong Xiuzhu 2001)
Molecular biological identification using 16S rDNA
The DNA of the strain was prepared using the bacterial liquid PCR method. The specific operation steps were as follows: 25 µL PCR reaction system, which consisted of 12.5 µL of 2× Master Mix, 1 µL upstream primer 27F (5′-AGA GTTTGA TCCTGGCTCAG-3′), 1 µL downstream primer 1492R (5′-GGTTACCTTGTT ACGACTT-3′), 9 µL of ddH2O, and 1.5 µL of template DNA.
The PCR conditions were pre-denaturation at 94°C for 5 min; 30 cycles of 94°C denaturation for 30 s, annealing at 53°C for 30 s, and 1 min extension at 72°C; a final extension at 72°C for 5 min, and storage at 4°C. The PCR products were sent to Hunan Musculus Biotechnology Co., Ltd. for sequencing. The obtained nucleotide sequences were analyzed using NCBI-BLAST. Then, homologous sequences with high similarity were obtained, and MEGA-X software was used to reconstruct a phylogenetic tree.
Determination of the antimicrobial spectrum of the biocontrol bacterium LS8
Small pieces of paper stained with raw control LS8 bacteria liquid were inoculated 2.5 cm from the center of plates containing the causative agents of lotus root rot disease, lotus root black spot disease, yam black skin disease, flax fusarium wilt disease, lily fusarium wilt disease, lily root rot disease, camphor wood disease, and honeysuckle disease, respectively, which were used at the target pathogens. Each treatment was repeated three times in the control group without LS8 bacteria inoculation. The plates were placed upside down in an incubator at 25°C. When pathogenic fungi in the control group overgrew the plate, the diameter of the biocontrol candidate bacteria and the diameter of the bacteriostatic zone against each pathogenic fungus were measured, and the antagonistic index was calculated according to the diameter of the bacteriostatic zone. The calculation of the bacteriostatic rate was the same as described above.
In vitro control test of biocontrol bacteria
The lotus root used in the in vitro control experiment was purchased from the local market. The surface of the lotus root was washed with tap water, and the lotus root was cut into small round slices with a diameter of about 4 cm parallel to the lotus root hole. The lotus root slices were put in the bacterial solution of the raw control bacteria for 0.5 h. The control group was placed in ultra-pure water for 0.5 h. The sample was dried and included in a 9-cm plate containing a filter paper. The activated F. oxysporum mycelium blocks were inoculated into the center of the lotus root block with a hole punch. One F. oxysporum mycelium block was inoculated into each lotus root block. The lotus root block was placed in a 25°C constant-temperature incubator and cultured until the mycelium of the control group was about to fill the lotus root block after inoculation. The colony diameter of the control group and the experimental group was measured by cross method (Wei et al. 2009). The calculation of the bacteriostatic rate was the same as described above.
Plant early treatment
Lotus seeds were stored in a 4°C refrigerator. The lotus seeds at both ends of the drilling diameter of 2 mm holes were cultivated with 20 L of water in the pot to promote budding, with a culture temperature range of 20–25°C. The samples were watered once every morning and evening to prevent bacterial water pollution. After three days of cultivating most lotus seeds, water absorption expansion, and lotus shell splitculture after 5d most lotus seeds germination, germinated and were shipped to the field to prepare for cultivation.
Preparation of pathogenic bacteria and LS8 biocontrol bacteria in the lotus root
The pathogen F. oxysporum was activated in PDA plates, several plates were scraped, the mycelia were picked into sterile water, and the concentration of F. oxysporum was 106 CFU. Next, LS8 biocontrol bacteria were cultured in liquid fermentation at 150 rpm at 25°C for two days. After that, the bacterial solution was diluted to 108 CFU for later use.
Inoculation of biocontrol bacteria and pathogens
A total of 200 lotus seeds with similar growth were selected and divided into four groups: blank group, biocontrol bacteria group, pathogen group, and biocontrol bacteria + pathogen group. The blank group was soaked in clean water without treatment. The biocontrol group was soaked in biocontrol bacteria at a concentration of 108 CFU of LS8 for 12 h. The pathogen group was soaked in Fusarium oxysporum solution with a concentration of 106 CFU for 3 h. The biocontrol bacteria + pathogen group was first soaked with a concentration of 108 CFU. The LS8 biocontrol bacteria were soaked for 12 h and then washed with clean water. Finally, the bacteria were soaked for 3 h with F. acarium at a concentration of 106 CFU.
Field cultivation
The lotus field was divided into four 2 m × 4 m squares with a depth of approximately 20 cm. The soil between the squares was covered with plastic films. The irrigation depth of each grid was water about 10 cm. The cultivated grid was divided into four groups: blank group, biocontrol bacteria group, pathogen group, and biocontrol bacteria + pathogen group. Each grid was divided into three areas for parallel and uniform cultivation of 20 treated lotus seeds. The whole lotus seeds were buried in the soil at a depth of about 3 cm. The culture temperature ranged from 15 to 25°C. The disease was recorded and classified by sampling after 14 days of planting.
The disease grading standard was as follows: 0, no symptoms on the leaves; 1, 1–5 light red lesions on the leaves; 3, 6–10 light red spots on the leaves; 5, 11–20 red spots on the leaves; 7, more than 20 red lesions on the leaves, or a small amount of missing mesophyll; 9, the whole leaf was infected with plaque, or the entire leaf has a large portion of the missing mesophyll.
The disease index was calculated as follows:
Disease index = ∑ (number of disease grade × representative value of disease grade) / (total number of plants × representative value of highest disease grade )× 100%.
Confirmation that the biocontrol bacteria colonized lotus plants
After the lotus seeds were soaked using the biocontrol bacterial strain LS8, the whole lotus seedlings should be infected by the biocontrol bacteria in this test. Endophytic bacteria in the biocontrol treatment group were isolated using tissue separation. Morphological recognition was used to obtain and purify Bacillus and transfer them to an LB liquid medium for culture at 28°C for 48 h. Strains of different groups and colony morphology were selected and marked for preservation. The DNA of the strains was prepared using liquid PCR and sequenced. A phylogenetic tree was reconstructed by sequencing the primary research strain Bacillus velesiensis LS8 and other isolated bacteria.