Study on Screening and Degradation Effect of Autotoxin-Degrading Bacteria in Muskmelon

: The autotoxins of muskmelon are one of the most important reasons for the continuous cropping obstacle of muskmelon, of which the main components are phenolic acids. Phenolic acids can inhibit the growth and development of muskmelon plants. The purpose of this study was to screen the strains that can degrade phenolic acids in soil. Using phenolic acids as the sole carbon source, the strains were isolated and screened by the dilution plate method, which could efﬁciently degrade various phenolic acids. The abilities of the strains to degrade phenolic acids were measured by HPLC, and the effects of degrading phenolic acids in soil were veriﬁed by a pot experiment. After identiﬁcation, strain T58 was identiﬁed as Burkholderia sp., strain T79 was identiﬁed as Burkholderia sp., strain H16 was identiﬁed as Pseudomonas sp., and strain T15 was identiﬁed as Burkholderia sp. The results showed that, after 21 days of culture, the degradation rates of ferulic acid, p-coumaric acid, vanillin and sinapic acid by strain H16, strain T79, strain T15 and strain T58 were 100%, respectively. Additionally, the degradation rates of gallic acid by the four strains were also 100%. In this study, it was found that the four strains of autotoxin-degrading bacteria had good degradation effects on various phenolic acids, which could not only alleviate the toxic effects of phenolic acids on muskmelon, but also promote the growth of muskmelon seedlings.


Introduction
Muskmelon (Cucumis melon L.), as an economically important vegetable crop, is widely cultivated worldwide [1]. As the demand for muskmelon continues to grow, its planting area in China is also increasing. The continuous cropping obstacle in the process of intensive muskmelon planting is becoming increasingly serious [2]. Continuous cropping obstacles can lead to weakened growth, reduced yields, lower quality and increased soil-borne pests and diseases in successive crops of the same or closely related crops, even if they are under normal cultivation management [3,4]. The main reasons for continuous cropping obstacles include the deterioration of the soil's physical and chemical properties, the changes in the soil microbial community, and the autotoxicity of autotoxins [5,6]. Autotoxicity is when crops release substances to inhibit the growth of the same plant or family plants through root exudates, aboveground leaching and plant stubble decomposition [7,8]. Phenolic acids are the main components of autotoxins [9][10][11][12][13]. Phenolic acids can inhibit plant growth by affecting the plant membrane system, photosynthesis, enzyme activity, soil microbial activity and the soil's physical and chemical properties [14][15][16][17]. The accumulation of phenolic acids can change the structure of the soil microbial community, transform soil microorganisms from bacterial to fungal, and increase pathogenic microorganisms [18,19]. It is difficult to eliminate phenolic acids through physical or chemical methods. Reducing the accumulation of phenolic acids has become an urgent problem to be solved [20].
Studies have shown that the autotoxins of phenolic acids secreted by muskmelon accumulate with the increase in planting years, and the accumulation of these phenolic acids is an important reason for the continuous cropping obstacles of muskmelon [21][22][23][24].

Isolation and Preliminary Screening of Strains
Muskmelon rhizosphere soil bacteria: The muskmelon rhizosphere soil (10 g) was added to a triangular flask containing 90 mL of sterile water, and a soil solution with a concentration of 10 −1 was prepared after fully shaking and mixing. After the soil solution had stood and precipitated, 1 mL of the supernatant was taken, and soil solutions with concentrations of 10 −2 , 10 −3 , 10 −4 , 10 −5 , and 10 −6 were prepared by the gradient dilution method. A total of 100 µL of soil solution at each concentration were added to the solid screening medium plate containing ten phenolic acids, which were evenly coated. Each concentration was replicated three times, and sterile water was used as the control group. The culture dishes were placed in a 37 • C incubator, and the results were observed after 48 h.
Muskmelon endophytic bacteria: The stems and leaf parts of muskmelon plants at four true leaf stages were selected and then rinsed with sterile water and dried. The plants were cut to about 1 cm, disinfected with 75% alcohol and 0.5% sodium hypochlorite for 1 min and 5 min, respectively, and rinsed with sterile water 3 times. The plant fragments were placed in a mortar, ground into a slurry, and diluted with sterile water into a solution. A total of 100 µL of the solution were added to the solid screening medium plate containing ten phenolic acids, and evenly coated. The sterile water was used as the control group. All Agronomy 2023, 13, 1334 3 of 14 treatments were replicated three times. The culture dishes were placed in a 37 • C incubator, and the results were observed after 48 h.

Rescreening of Strains
After the colonies grew on the primary screening plate, single colonies were picked and streaked on a solid screening medium plate containing ten phenolic acids. The single colonies were streaked and separated 3-4 times, then purified and cultured.
After the purified strains were activated by NB medium, the bacteria fermentation broth was placed in a centrifuge tube, centrifuged at 5000 r/min for 10 min, and the supernatant was discarded. The bacteria were washed and diluted with the basic salt medium solution, and the NB medium was removed to prepare a bacteria solution with a concentration of 10 8 cfu/mL. The bacteria solution was inoculated into the screening medium solution containing ten phenolic acids at 5% inoculation amount and was cultured in a constant-temperature oscillation incubator at 37 • C in the dark. The control group was inoculated with the same amount of sterile water. After 7 days of culture, 1 mL of the degradation solution was taken and filtered with a 0.22 µm microporous membrane. The degradation rates of phenolic acids were determined by HPLC. The strains were screened for use, which could degrade phenolic acids.

Identification of Strains
After the screened strains were activated, the DNA of the strains was extracted by the Shanghai Sangon Ezup column bacterial genomic DNA extraction kit. After the DNA sample extraction was complete, DNA concentration was detected by an ultra-micro spectrophotometer. PCR amplification was performed using bacterial universal primers, and the amplified products were detected by gel electrophoresis. The primers were 27F: 5 -AGAGTTTGATCCTGGCTCAG-3 ; 1492R: 5 -GGTTACCTTGTTACGACTT-3 . The PCR reaction conditions were as follows: pre-denaturation at 94 • C for 4 min; 30 cycles (94 • C denaturation 45 s, 55 • C annealing 45 s, 72 • C extension 1 min); an extended culture at 72 • C for 10 min; 4 • C heat preservation [35,36]. A total of 10 µL of PCR amplification products were detected by 1% agarose gel electrophoresis and viewed in the gel imaging system.
The PCR products were sent to Shanghai Sangon Bioengineering Co., Ltd. (Shanghai, China) for sequencing. The 16S rDNA sequences of the determined strains were submitted to GenBank for Blast homology comparison, and the 16S rDNA sequences of the model strains with higher homology were found for comparison. The sequences of the model strains that had high similarity with the measured strains were downloaded and imported into Mega11 software (version 11.0.13). The phylogenetic tree was constructed by the neighbor-joining method. The number of self-expansions was set to 1000, and the genetic relationship with the model strains was analyzed. The 16S rDNA sequences of the strains were submitted to GenBank to obtain the sequence accession numbers.

Preparation of Phenolic Acid Solution
Each standard of phenolic acid in Section 2.1 was dissolved in methanol to prepare standard solutions with mass concentrations of 1000 mg/L. The standard solutions of 1000 mg/L were diluted with methanol to 10 mg/L, 20 mg/L, 40 mg/L, 80 mg/L and 160 mg/L, respectively, filtered with a 0.22 µm microporous membrane, and stored at 4 • C in the dark.

Standard Curve Drawing of Phenolic Acids
Using the Agilent off-line working platform (C.01.07SR2), according to the data of the standard solution detection in Section 2.3.1, the standard curves of phenolic acids were drawn. The concentration of the standard solution was taken as the abscissa, and the signal area detected by the standard solution was taken as the ordinate. The standard curves were drawn by the five-point drawing method, and the standard curves and regression equations of each standard solution were obtained.

Determination of the Degradation Effect of Phenolic Acids by Autotoxin-Degrading Bacteria
After the strain was activated by the NB medium, the bacteria fermentation broth was placed in a centrifuge tube, centrifuged at 5000 r/min for 10 min, and the supernatant was discarded. The bacteria were washed and diluted with the basic salt medium solution, and the NB medium was removed to prepare a bacteria solution with a concentration of 10 8 cfu/mL.
Phenolic acid was added to the basic salt medium to a concentration of 25 mg/L, and then 50 mL of solution were placed in the 150 mL triangular flask. Each triangular flask was inoculated with 10 8 cfu/mL bacteria solution at a 5% inoculation amount and cultured in a constant-temperature oscillation incubator at 37 • C in the dark. The control group was inoculated with the same amount of sterile water. After 0 day, 7 days, 14 days and 21 days, the degradation solution was taken and filtered with a 0.22 µm microporous membrane. The content of phenolic acid in the degradation solution was detected by HPLC, and the degradation rate of each phenolic acid was calculated, as shown below: Degradation rate (%) = C ck −C t C ck × 100, where C ck is the phenolic acid concentration of the control group, and C t is the phenolic acid concentration of the treatment group. The soil of the unplanted cucurbitaceae plants was air-dried and fully mixed. Each pot was placed in 5 kg of soil, and ten phenolic acids were added to the soil at concentrations of 25 mg/kg. The treatment of adding the same amount of sterile water without phenolic acids was used as the control group CK. Each pot except the CK group was inoculated with 10 8 cfu/mL bacteria solution at a 5% inoculation amount. The treatment of adding the same amount of sterile water without inoculating the bacteria solution was used as the control group CKP. There were 6 treatments in this experiment, with 10 pots in each treatment. The seedlings were raised in nutrient pots. When the muskmelon seedlings grew to two true leaves, they were selected for transplanting with basically the same growth and plant height. Three seedlings were transplanted into each pot. After 21 days of transplanting, the plant height, root length, fresh weight, dry weight and phenolic acid content in the soil were measured.

Determination of Degradation Effect of Phenolic Acids by Autotoxin-Degrading Bacteria in the Soil
The soil samples were naturally dried in the shade after removing impurities, and then ground and sieved. The phenolic acids in the soil were extracted according to the method of Dalton [37]. A total of 25 g of each soil sample were placed in a 50 mL centrifuge tube, and 25 mL of 1 mol/L sodium hydroxide solution were added. The samples were shaken for 1 h, ultrasonically oscillated for 30 min, and fully mixed. After standing for 24 h, the samples were shaken at 200 r/min for 30 min and centrifuged at 8000 r/min for Agronomy 2023, 13, 1334 5 of 14 10 min. The supernatant was separated, and the pH value of the samples was adjusted to 2.5 with 12 mol/L hydrochloric acid to precipitate humic acid. After standing for 2 h, the supernatant was centrifuged at 8000 r/min for 10 min to remove the precipitate. The supernatant was stored at 4 • C for analysis. The chromatographic conditions and methods for the determination of phenolic acids by HPLC were the same as in Section 2.3.4.

Stability Detection of Degradation Ability of Autotoxin-Degrading Bacteria
The strains were subcultured once every 3 days for 20 generations in the laboratory. In order to detect the stability of the degradation ability of the strains, the degradation rates of phenolic acids by the strains that were subcultured for 0-20 generations were determined.

Isolation and Screening of Autotoxin-Degrading Bacteria
According to the growth status of the initial screening, 234 strains of bacteria were obtained. Among them, 75 strains were isolated from the 1-year continuous cropping soil of muskmelon, 80 strains were isolated from the 2-year continuous cropping soil, 62 strains were isolated from the 3-year continuous cropping soil, and 17 strains of endophytic bacteria were isolated from muskmelon plants. A total of nine strains of bacteria were obtained by re-screening, all of which had obvious degradation effects on phenolic acids.

Identification of Autotoxin-Degrading Bacteria
The PCR amplification product was detected by gel electrophoresis, and a product with the length of about 1500 bp was obtained. Under the gel imaging system, the electrophoresis bands were clear, and there was no degradation trace ( Figure 1). method of Dalton [37]. A total of 25 g of each soil sample were placed in a 50 mL centrifuge tube, and 25 mL of 1 mol/L sodium hydroxide solution were added. The samples were shaken for 1 h, ultrasonically oscillated for 30 min, and fully mixed. After standing for 24 h, the samples were shaken at 200 r/min for 30 min and centrifuged at 8000 r/min for 10 min. The supernatant was separated, and the pH value of the samples was adjusted to 2.5 with 12 mol/L hydrochloric acid to precipitate humic acid. After standing for 2 h, the supernatant was centrifuged at 8000 r/min for 10 min to remove the precipitate. The supernatant was stored at 4 °C for analysis. The chromatographic conditions and methods for the determination of phenolic acids by HPLC were the same as in Section 2.3.4.

Stability Detection of Degradation Ability of Autotoxin-Degrading Bacteria
The strains were subcultured once every 3 days for 20 generations in the laboratory. In order to detect the stability of the degradation ability of the strains, the degradation rates of phenolic acids by the strains that were subcultured for 0-20 generations were determined.

Isolation and Screening of Autotoxin-Degrading Bacteria
According to the growth status of the initial screening, 234 strains of bacteria were obtained. Among them, 75 strains were isolated from the 1-year continuous cropping soil of muskmelon, 80 strains were isolated from the 2-year continuous cropping soil, 62 strains were isolated from the 3-year continuous cropping soil, and 17 strains of endophytic bacteria were isolated from muskmelon plants. A total of nine strains of bacteria were obtained by re-screening, all of which had obvious degradation effects on phenolic acids.

Identification of Autotoxin-Degrading Bacteria
The PCR amplification product was detected by gel electrophoresis, and a product with the length of about 1500 bp was obtained. Under the gel imaging system, the electrophoresis bands were clear, and there was no degradation trace ( Figure 1). According to the results of the 16S rDNA sequence homology and phylogenetic tree, all strains except for H16 belonged to Burkholderia sp. and had a high similarity with strain H16. Strain H16 belonged to Pseudomonas sp.; T58(OQ733247) and T194(OQ733254) were preliminarily identified as Burkholderia sp.; T79(OQ733251) and T147(OQ733253) were According to the results of the 16S rDNA sequence homology and phylogenetic tree, all strains except for H16 belonged to Burkholderia sp. and had a high similarity with strain H16. Strain H16 belonged to Pseudomonas sp.; T58(OQ733247) and T194(OQ733254) were preliminarily identified as Burkholderia sp.; T79(OQ733251) and T147(OQ733253) were Burkholderia sp.; T15(OQ733249), T31(OQ733248), T113(OQ733252) and T182(OQ733255) were Burkholderia sp.; H16(OQ733257) was Pseudomonas sp. (Figure 2).

Standard Curve of Phenolic Acid Solution
According to the results of the HPLC, the regression equation of each phenolic acid (Table 1) could be obtained. The results showed that when the concentration of each phenolic acid ranged from 10 mg/L to 160 mg/L, the peak area had a good linear relationship with its concentration. This regression equation can be used in future detection work. Note: x represents the concentration of phenolic acids (mg/L), y represents the peak area.

Degradation of Phenolic Acids by Autotoxin-Degrading Bacteria
With the extension of culture time, the degradation rates gradually increased. The four strains had significant degradation effects on the ten phenolic acids. After 21 days of culture, the degradation rates of gallic acid by the four strains were 100%. The degradation rates of ferulic acid and p-coumaric acid by strain H16 and strain T79 were 100%. The degradation rate of vanillin by strain T15 was 100%. The degradation rate of sinapic acid by strain T58 and strain T79 was 100%. Table 2 shows that strain T58 had different degradation effects on the ten phenolic acids after seven days of culture, but the degradation effects were not obvious. After 14

Standard Curve of Phenolic Acid Solution
According to the results of the HPLC, the regression equation of each phenolic acid (Table 1) could be obtained. The results showed that when the concentration of each phenolic acid ranged from 10 mg/L to 160 mg/L, the peak area had a good linear relationship with its concentration. This regression equation can be used in future detection work. Note: x represents the concentration of phenolic acids (mg/L), y represents the peak area.

Degradation of Phenolic Acids by Autotoxin-Degrading Bacteria
With the extension of culture time, the degradation rates gradually increased. The four strains had significant degradation effects on the ten phenolic acids. After 21 days of culture, the degradation rates of gallic acid by the four strains were 100%. The degradation rates of ferulic acid and p-coumaric acid by strain H16 and strain T79 were 100%. The degradation rate of vanillin by strain T15 was 100%. The degradation rate of sinapic acid by strain T58 and strain T79 was 100%. Table 2 shows that strain T58 had different degradation effects on the ten phenolic acids after seven days of culture, but the degradation effects were not obvious. After 14 days of culture, the concentration of phenolic acids changed significantly, and the degradation rate of sinapic acid was the highest, reaching 63.77%. After 21 days of culture, the degradation rates of the different phenolic acids were significantly different. The degradation rates of sinapic acid and gallic acid were the highest (100%), and there were significant differences between the degradation rates of other phenolic acids. The degradation rate of salicylic acid was 98.09%. The degradation rates of cinnamic acid and p-coumaric acid were 89.04% and 75.35%, respectively. The degradation rates of ferulic acid, caffeic acid, vanillin, protocatechuic acid and vanillic acid were between 30.44% and 41.77%, and all were lower than 50%. The results showed that strain T58 could not only efficiently degrade sinapic acid, gallic acid, salicylic acid, cinnamic acid and p-coumaric acid, but also have different degrees of degradation effects on the other five phenolic acids.  Table 3 shows that strain T79 had different degrees of degradation effects on the ten phenolic acids after seven days of culture, and the degradation rate of ferulic acid was significantly higher than those of the other phenolic acids. The concentration of ferulic acid in the degradation solution decreased rapidly, and the degradation rate was the highest. After 14 days of culture, the concentration of phenolic acids changed significantly, and the degradation rates of ferulic acid, sinapic acid, p-coumaric acid, cinnamic acid, gallic acid and salicylic acid were higher than those of the the other phenolic acids. After 21 days of culture, the degradation rates of ferulic acid, sinapic acid and p-coumaric acid were the highest, which were 100%, and there were significant differences between the degradation rates of other phenolic acids. The degradation rates of gallic acid, salicylic acid and cinnamic acid were 98.47%, 98.27% and 92.38%, respectively. The degradation rates of vanillin, protocatechuic acid, caffeic acid and vanillic acid were between 34.29% and 46.23%, and all were lower than 50%. The results showed that strain T79 could not only efficiently degrade ferulic acid, sinapic acid, p-coumaric acid, gallic acid, salicylic acid and cinnamic acid, but also have different degrees of degradation effects on the other four phenolic acids.  Table 4 shows that strain H16 had different degrees of degradation effects on the ten phenolic acids after seven days of culture, and the degradation rates were significantly different. The concentration of ferulic acid in the degradation solution decreased rapidly, and the degradation rate was the highest. After 14 days of culture, the concentration of each phenolic acid changed significantly, and the degradation rates of ferulic acid, p-coumaric acid, salicylic acid, gallic acid and sinapic acid were higher than those of the other phenolic acids. After 21 days of culture, the degradation rates of the different phenolic acids were significantly different. The degradation rates of ferulic acid and p-coumaric acid were the highest, which were 100%, and there were significant differences between the degradation rates of other phenolic acids. The degradation rates of gallic acid and salicylic acid were 99.20% and 95.94%, respectively. The degradation rates of sinapic acid, cinnamic acid, vanillin and protocatechuic acid were 69.97%, 69.63%, 52.85% and 51.61%, respectively. The degradation rates of caffeic acid and vanillic acid were 43.80% and 42.26%, which were lower than 50%. The results showed that strain H16 could not only efficiently degrade ferulic acid, p-coumaric acid, gallic acid, salicylic acid, sinapic acid and cinnamic acid, but also have different degrees of degradation effects on the other four phenolic acids.  Table 5 shows that strain T15 had different degrees of degradation effects on the ten phenolic acids after seven days of culture, and the degradation rates were significantly different. The concentration of vanillin in the degradation solution decreased rapidly, and the degradation rate was the highest. After 14 days of culture, the concentration of phenolic acids changed significantly, and the degradation rates of vanillin, gallic acid, sinapic acid and salicylic acid were higher than those of the other phenolic acids. After 21 days of culture, the degradation rates of different phenolic acids were significantly different. The degradation rate of vanillin was 100%, and there were significant differences between the degradation rates of other phenolic acids. The degradation rates of gallic acid, salicylic acid and cinnamic acid were 99.00%, 96.38% and 91.55%, respectively. The degradation rates of coumaric acid, sinapic acid, ferulic acid and protocatechuic acid were 80.65%, 69.99%, 66.48% and 54.70%, respectively. The degradation rates of vanillic acid and caffeic acid were 48.37% and 38.90%, which were all lower than 50%. The results showed that strain T15 could not only efficiently degrade vanillin, gallic acid, salicylic acid, cinnamic acid, p-coumaric acid, sinapic acid and ferulic acid, but also have different degrees of degradation effects on the other three phenolic acids.  Figure 3 shows that the plant height, root length, fresh weight and dry weight of muskmelon seedlings in the treatment group were significantly better than those in the control group CKP and CK. The physiological indexes of CKP in the control group were the lowest, and there were significant differences between other groups. The physiological Agronomy 2023, 13, 1334 9 of 14 indexes of strain T79 in the treatment group were the highest, and there were significant differences with other groups. The results showed that the four strains of autotoxindegrading bacteria could not only alleviate the toxic effect of phenolic acids on muskmelon, but also promote the growth of muskmelon seedlings. Among the four strains of autotoxindegrading bacteria, strain T79 had the most significant growth-promoting effects.

Autotoxin-Degrading Bacteria Alleviate the Toxic Effects of Phenolic Acids on Muskmelon
Different lowercase letters in rows indicate significant differences at p < 0.05. Figure 3 shows that the plant height, root length, fresh weight and dry weight of muskmelon seedlings in the treatment group were significantly better than those in the control group CKP and CK. The physiological indexes of CKP in the control group were the lowest, and there were significant differences between other groups. The physiological indexes of strain T79 in the treatment group were the highest, and there were significant differences with other groups. The results showed that the four strains of autotoxin-degrading bacteria could not only alleviate the toxic effect of phenolic acids on muskmelon, but also promote the growth of muskmelon seedlings. Among the four strains of autotoxin-degrading bacteria, strain T79 had the most significant growth-promoting effects.

Degradation of Phenolic Acids by Autotoxin-Degrading Bacteria in the Soil
The degradation effects of four strains of autotoxin-degrading bacteria on phenolic acids in soil were obtained by HPLC. The degradation rate of CKP in the control group was 16.29%, which was significantly lower than those in the four treatment groups. The degradation rate of strain T79 was not significantly different from that of strain T15 in the treatment group. The degradation rate of strain T79 was significantly higher than those of strain H16 and strain T58. The results showed that the content of phenolic acids in the

Degradation of Phenolic Acids by Autotoxin-Degrading Bacteria in the Soil
The degradation effects of four strains of autotoxin-degrading bacteria on phenolic acids in soil were obtained by HPLC. The degradation rate of CKP in the control group was 16.29%, which was significantly lower than those in the four treatment groups. The degradation rate of strain T79 was not significantly different from that of strain T15 in the treatment group. The degradation rate of strain T79 was significantly higher than those of strain H16 and strain T58. The results showed that the content of phenolic acids in the control group decreased slowly after 21 days, while the four treatment groups had significant degradation effects on the phenolic acids, and the degradation rates were between 77.18% and 86.76%. Figure 4 shows that strain T79 was the best, which was consistent with the degradation effect under shaking flask culture in the laboratory. control group decreased slowly after 21 days, while the four treatment groups had significant degradation effects on the phenolic acids, and the degradation rates were between 77.18% and 86.76%. Figure 4 shows that strain T79 was the best, which was consistent with the degradation effect under shaking flask culture in the laboratory.

Stability Detection of Degradation Ability of Autotoxin-Degrading Bacteria
The degradation rates of phenolic acids by the four strains of subculture for 0-20 generations were measured, which began to decrease slowly with the increase in subculture generations. However, the degradation rates of generation 20 were higher than 79.85% (the lowest degradation rate of generation 0 was 82.66%). For strains T58, H16 and T15, the degradation rates did not change significantly from generation 0 to generation 15 and began to change significantly from generation 20. For strain T79, the degradation rates did not change significantly from generation 0 to generation 10 but changed significantly

Stability Detection of Degradation Ability of Autotoxin-Degrading Bacteria
The degradation rates of phenolic acids by the four strains of subculture for 0-20 generations were measured, which began to decrease slowly with the increase in subculture generations. However, the degradation rates of generation 20 were higher than 79.85% (the lowest degradation rate of generation 0 was 82.66%). For strains T58, H16 and T15, the degradation rates did not change significantly from generation 0 to generation 15 and began to change significantly from generation 20. For strain T79, the degradation rates did not change significantly from generation 0 to generation 10 but changed significantly from generation 15. However, the degradation ability of four strains after subculture was still relatively high ( Figure 5), which indicates that four strains had a relatively stable ability to degrade phenolic acids.

Stability Detection of Degradation Ability of Autotoxin-Degrading Bacteria
The degradation rates of phenolic acids by the four strains of subculture for 0-20 generations were measured, which began to decrease slowly with the increase in subculture generations. However, the degradation rates of generation 20 were higher than 79.85% (the lowest degradation rate of generation 0 was 82.66%). For strains T58, H16 and T15, the degradation rates did not change significantly from generation 0 to generation 15 and began to change significantly from generation 20. For strain T79, the degradation rates did not change significantly from generation 0 to generation 10 but changed significantly from generation 15. However, the degradation ability of four strains after subculture was still relatively high ( Figure 5), which indicates that four strains had a relatively stable ability to degrade phenolic acids.

Discussion
In recent years, domestic and foreign scholars have studied the biological control of continuous cropping obstacles. These studies have shown that autotoxin-degrading strains can effectively degrade phenolic acids in soil and alleviate the toxic effects of phenolic acids on plants [31]. Three strains of bacteria were screened from the continuous cropping soil of tomato, which could efficiently degrade benzoic acid, cinnamic acid and vanillic acid [38]. Cinnamic acid could be degraded up to 99% by a bacterial strain R30, which was isolated from watermelon continuous cropping soil, and was identified as Exiguobacterium sp. [39]. The degradation rates of phthalic acid, p-hydroxybenzoic acid and pyrogallic acid by the autotoxin-degrading bacteria, which were screened from the continuous cropping soil of apple, were higher than 50% [40]. The degradation rate of salicylic acid by the strain FD-21, which was screened from vanilla rhizosphere soil, was 93.62% [41]. Five strains of autotoxin-degrading bacteria were screened from the continuous cropping soil of ginseng, which could grow with palmitic acid as the sole carbon source, and the degradation rate of palmitic acid was close to 80% [42]. A strain of fungus was isolated from the continuous cropping soil of Chinese goldthread, which could degrade ferulic acid [43]. Phanerochaete chrysosporium could effectively degrade p-hydroxybenzoic acid, vanillic acid and ferulic acid under laboratory conditions, and the degradation rates were 58.20%, 98.38% and 97.88%, respectively [44]. The above studies showed that the strains that could degrade autotoxins were screened from the continuous cropping soil of plants, and most of these strains were fungi or bacteria. In this study, four strains of autotoxin-degrading bacteria that could efficiently degrade various phenolic acids were screened from the continuous cropping soil of muskmelon and muskmelon plants. This was consistent with the results of previous studies, that many strains could efficiently degrade a variety of phenolic acids with a broad substrate spectrum. Compared with the previous studies, it was found that many autotoxins of plants were the same, such as salicylic acid, cinnamic acid, ferulic acid, gallic acid, etc. Therefore, the four strains of autotoxin-degrading bacteria in this study may be able to alleviate the continuous cropping obstacles of other plants, which needs to be further verified.
Studies have shown that phenolic acids usually undergo biochemical reactions such as decarboxylation, oxidation and hydroxylation under the degradation of microorganisms. The substituents on the benzene ring in the molecular structure are easily degraded and converted. These reactions usually lead to the degradation of macromolecular phenolic acids into small molecular phenolic acids or other small molecular organic compounds containing benzene rings [3].
Studies showed that bacteria with the ability to degrade p-hydroxybenzoic acid could significantly improve growth indexes such as the root length, leaf area, plant height and dry weight of strawberry seedlings [45]. An autotoxin-degrading strain was isolated from sludge, which could alleviate the toxic effects of phenylacrylic acid on cucumber growth by hydroponic and soil culture experiments [46]. A strain of actinomycetes was screened, which could effectively alleviate the toxic effects of ferulic acid on the growth of watermelon seedlings [47]. In the pot experiment of our study, the plant height, root length, fresh weight and dry weight of muskmelon seedlings inoculated with four autotoxin-degrading bacteria were significantly better than the control group without inoculation. In the present study, four strains of autotoxin-degrading bacteria could not only alleviate the toxic effects of phenolic acids on plants, but also promote the growth of muskmelon seedlings, which was similar to the previously reported growth-promoting effects of autotoxin-degrading bacteria on other crops.
Pseudomonas sp. is one of the preferred strains for plant rhizosphere control and growth promotion [48]. The strains of this genus have the effects of promoting plant growth, inhibiting pathogenic microorganisms and degrading autotoxins. The strains of Pseudomonas sp. are prominent in the degradation of autotoxins. The degradation rates of p-hydroxybenzoic acid, cinnamic acid, syringic acid, benzoic acid and ferulic acid by Pseudomonas putida, which was screened from peanut rhizosphere soil, were 99.85%, 17.44%, 90.04%, 98.69% and 38.89%, respectively [49]. P. putida, which was screened from cucumber rhizosphere soil, could degrade p-hydroxybenzoic acid [50]. In this study, Pseudomonas sp. strain H16 could promote the growth of muskmelon seedlings, which was consistent with the growth-promoting effects of some strains in Pseudomonas oryzihabitans [51].
Burkholderia sp. is a kind of beneficial microorganism that has been found in recent years to have growth-promoting effects on plant rhizosphere soil [52][53][54]. Burkholderia sp. could promote sugarcane growth through biological nitrogen fixation [55]. In this study, Burkholderia sp. strain T58, Burkholderia sp. strain T79 and Burkholderia sp. strain T15 could promote the growth of muskmelon seedlings, which was similar to the results of previous studies on Burkholderia sp. YZU-S230 [56], Burkholderia tropica MTo-293 [57], and Burkholderia sp. [58], which could promote the growth of different crops.
Previous studies showed that phenolic acids could inhibit the growth of muskmelon plants [27]. Therefore, the growth of muskmelon was promoted in this study, which might be due to the growth-promoting effects of autotoxin-degrading bacteria and the reduction in phenolic acids in the soil.

Conclusions
In this study, four strains of autotoxin-degrading bacteria that could efficiently degrade various phenolic acids were screened from the continuous cropping soil of muskmelon and muskmelon plants. After 21 days of culture under laboratory conditions, four strains of autotoxin-degrading bacteria had significant degradation effects on various phenolic acids. The results of the pot experiment showed that four strains of autotoxin-degrading bacteria could alleviate the toxic effects of phenolic acids on muskmelon and promote the growth of muskmelon seedlings. The four strains demonstrated good degradation ability for various phenolic acids in the soil environment. The four strains of autotoxin-degrading bacteria had a relatively stable ability to degrade phenolic acids after subculture. The results of this study can provide a relevant theoretical basis for using autotoxin-degrading bacteria to alleviate continuous cropping obstacles of muskmelon.