Abstract [Objectives] This study was conducted to investigate the spoilage microorganisms in the storage process of Chinese flowering cabbage. [Methods] Pathogenic bacteria were separated and purified from rotted Chinese flowering cabbage during storage. The gradient dilution culture method and streaking purification method were applied to selectively cultivate spoilage microorganisms for separation and observation. The isolated strains were identified through the ITS and sequence analysis of 16S rDNA combined with the morphological characteristics and physiological and biochemical properties of the microbes. On the basis of morphology， combined with gene sequence analysis， the isolated pathogenic bacteria A1， A2， A3， and A4 were identified by PCR using the bacterial universal primer 16S rDNA sequences， and B1 was amplified using the fungal universal primer ITS sequence. The gene sequences obtained by sequencing were subjected to homologous sequence alignment in the NCBI gene library to determine the biological classification of the spoilage bacteria. [Results] The results showed that the four bacteria numbered A1， A2， A3， and A4 were Klebsiella， Acinetobacter baylyi， Staphylococcus epidermidis， and Pseudomonas， respectively. The saprophytic fungus labeled B1 was Streptomyces albus. Re-contacting it to Chinese flowering cabbage caused the cabbage to rot， so it was the main saprophytic fungus that caused the cabbage to rot after picking. Therefore， the main spoilage microorganisms during storage of Chinese flowering cabbage were Klebsiella， A. baylyi， S. epidermidis， Pseudomonas， and S. albus. [Conclusions] This study provides a certain scientific basis and theoretical basis for the storage and preservation of Chinese flowering cabbage.
　　Key words Decay; Bacteria; Microbial detection; PCR
　　Chinese flowering cabbage （Brassica campestris L.） is a vegetable of Brassica in Cruciferae. It has a shallow root system， and its stem becomes shorter before bolting and is green in color. The leaves are wide and oval， green or yellow-green， and have wavy leaf margins， and the petioles are light green. The flower stems are round in cross section， and have thereon small leaves， which have a short leaf stalk at the lower part and no leaf stalk at the upper part. It is a popular vegetable with a very wide eating habit in southern China. With the rapid development of social economy and frequent personnel exchanges， especially the rapid economic development in the south， people spread all over the country. As a result， the living habits and food requirements of southerners have gradually spread throughout the country. Their favorite cabbage has gradually expanded from the south to the national market. The demand for Chinese flowering cabbage has increased， which naturally began to stimulate economic development. 　　Damage， senescence， dehydration， wilting and decay are all problems that easily occur after picking Chinese flowering cabbage， which seriously affects the storage and transportation quality of Chinese flowering cabbage. In the main production areas， because of the low level of commercial storage and transportation of vegetables after processing and the lack of storage and transportation equipment， most of the products are put on the vegetable market without packaging[1-4]. Moreover， due to the characteristic of the Chinese flowering cabbage product that integrates green leaves， tender stems and flower stalks， it is more difficult to store， transport and keep fresh than simple vegetables， causing very high loss rate of Chinese flowering cabbage after picking and thus high economic loss.
　　Food is easily contaminated by microorganisms， so microbiological testing is an important part of food inspection. With the rapid development of biotechnology and the combination of various new technologies and PCR technology， and the in-depth research on how to use PCR technology to identify whether food has produced pathogenic microorganisms in the future， it will certainly provide new ideas for the wider application of PCR technology in the field of food safety. With the development of domestic science and technology， using PCR technology to detect microorganisms in food has become a new stage of microbial detection technology. Whether for a testing center or a scientific research unit， when a rapid detection method is needed， the accuracy of the selected method， the length of time， the economy and the ease of operation will be considered. Applying PCR amplification technology to identify the microorganisms extracted from Chinese flowering cabbage improves the shortcomings of the traditional microorganism detection method， such as tedious operation， long operation time， false negatives and low sensitivity， so it is of great significance for preventing and controlling fruit and vegetable diseases and improving the quality of fruit and vegetable output[5-8]. Therefore， the use of PCR technology to research and expand the detection methods of key pathogenic microorganisms related to fruit and vegetable spoilage is simple to operate and has great convenience， which is far better than traditional detection methods[9-13]. It has a good guiding role in the storage of fruits and vegetables. Combining with gene sequence analysis， strains can be identified to species and genera on a molecular level， providing a certain scientific basis and theoretical basis for the storage and preservation of Chinese flowering cabbage in the later stage. 　　Materials and Methods
　　Experimental materials
　　Experimental sample
　　Chinese flowering cabbage.
　　Main instruments and equipment
　　SPX-250B-Z constant temperature biochemical incubator： the medical equipment factory of Shanghai Boxun Industrial Co.， Ltd.; KDC-2046 centrifuge： Anhui USTC Zongke Scientific instruments Co.， Ltd.; BC-power300 electrophoresis apparatus： Beijing Liuyi Instrument Factory; DSX-24L high-pressure steam sterilizer： Shanghai ShenAn Medical Instrument Factory; SW-CJ-2FD ultra-clean workbench： Suzhou Huayu Purification Equipment Co.， Ltd.; ALC-1100.2 electronic balance： Beijing Sartorius Instrument System Co.， Ltd.; BIO-RAD gel imaging analyzer： Lenovo Biotechnology Co.， Ltd.
　　Main reagents and media
　　Nutrient agar medium： peptone 10 g， beef powder 3.0 g， sodium chloride 5.0 g， agar 15.0 g; potato dextrose agar medium： potato powder 6.0 g， dextrose 20.0 g， agar 20.0 g; rose bengal medium： peptone 5.0 g， glucose 10.0 g， monopotassium phosphate 1.0 g， magnesium sulfate 0.5 g， agar 20.0 g， red bengal 0.033 g， chloramphenicol 0.1 g.
　　Experimental methods
　　The experiment used cabbage leaves as the material， which was homogenized to extract the leaf bacteria. The sampling amount of the leaves was generally 4-5 g. The sample was added with 30 ml of sterile saline and homogenized with a homogenizer， and then centrifuged in a centrifuge tube at a speed of 9 000 r/min and a temperature of 30 ℃ for 15 min. The supernatant was diluted in gradients in sterile test tubes， and then inoculated to a culture medium by the streak plate method.
　　Separation and purification of strains
　　After culturing the diluted microbes on plates for 48 h， characteristic colonies were selected and streaked with sterile inoculation loops to the newly prepared nutrient agar medium， potato dextrose agar medium and red bengal medium， respectively. They were cultured in a constant temperature incubator at 37 ℃ for 48 h. After culturing the microbes for 48 h， obvious single colonies were streak-inoculated with sterile inoculation loops or inoculating needles separately to new plates for purification. Single colonies with obvious culture characteristics and the same colony characteristics as the diluted plate culture were picked and observed under a microscope. If the shape is consistent， it could be identified as a single strain. If it is not possible to pick a single colony， isolation by plate streaking should be performed again until a single strain is selected[14-17]. Finally， four sets of bacterial genomes and one set of fungal genomes were extracted. 　　The four groups of bacterial genomic DNA extracted above were used as templates， and the primers of 16S rRNA gene were used for PCR amplification.
　　The sequences of the two primers used were： （27F）： 5′AGA GTT TGA TCC TGG CTC AG 3′ and （1492R）：5′TAC GGY TAC CTT GTT ACG ACT T 3′.
　　The reaction system included appropriate template， primer l27F 0.5 μl， primer 1492R 0.5 μl， TaqPolymerase dNTP： 15 μl， sterile ddH2O： 14 μl. The PCR program started with pre-denaturation for 10 min and denaturation for 30 s at 94 ℃， followed by 30 cycles of annealing at 52 ℃ for 30 s and extension at 72 ℃ for 1.5 min， and ended with extension at 72℃ for 10 min. After cooling at 40 ℃ for 10 min， the product was taken out[18-19].
　　Electrophoresis
　　In the part of mounting the gel-making plate part and the electrophoresis system， for gels of different specifications， water was used instead of the gel solution to find a suitable volume adjustment device to adjust the volume to the best. Then， 30 μl of PCR sample containing 20% loading buffer was added into the sample well of each gel， and the voltage of the electrophoresis instrument was adjusted to 120 V for electrophoresis， to allow full staining， and the electrophoresis was completed after 30 min. The stained gel was analyzed with a gel imaging analyzer， and the electrophoresis bands of the sample were observed and photographed.
　　The obtained samples with complete band imaging were sent to Wuhan Taisheng Biotechnology Co.， Ltd. for detection， and the products after electrophoresis detection were sent to the company for sequencing to obtain the 16S rDNA gene sequence. The sequencing results were subjected to BLAST analysis and comparison in NCBI gene bank， and homologous sequences were searched. According to the homologous sequence search results， the 16S rDNA gene sequences of related model strains with more than 99% homology were derived to construct phylogenetic trees[20-22].
　　Analysis Results of Gene Sequence
　　16S rDNA and ITS universal primers were used for PCR amplification of the target product. The DNA fragments extracted from strains A1， A2， A3， A4， and B1 generated clear bands with a length of about 750 bp， respectively. The electrophoresis results are shown in Fig. 1. The sequences of 850， 890， 865， 830 and 810 bp were obtained.
　　Gene Alignment and Identification
　　Identification of bacteria
　　Bacteria colony morphology and microscopic examination results 　　Through the isolation of the bacteria in decayed Chinese flowering cabbage and simple morphological analysis， 4 strains of different morphology were obtained， which were numbered A1， A2， A3 and A4. Strains A1， A2， A3 and A4 were streak-inoculated on nutrient agar medium and observed after culturing at 37 ℃ for 48 h. The colonies of strain A1 were about 3 mm in diameter， off-white， creamy， oval， and showed smooth surface， neat edges， with spores. The colonies of strain A2 were 1-2 mm in diameter， white， oval， and had irregular edges， without spores. Strain A3 showed colonies 1-2 mm in diameter， round or approximately round， yellow， without spores， and the edges were irregular. Strain A4 showed colonies 2-4 mm in diameter， round or approximately round， white， with spores.
　　Construction of phylogenetic trees
　　The PCR sample was submitted and subjected to homologous sequence comparison by BLAST in NCBI gene database， and the MEGA7.0 software was used to download similar reference sequences to construct a phylogenetic tree by the NJ method. The relationship between the extracted spoilage bacteria and other strains of the same genus was analyzed to determine their molecular taxonomic status.
　　Homology alignment of bacterial 16S rDNA gene sequence
　　The amplified gene sequences of strains A1， A2， A3， and A4 were aligned with the corresponding sequences of known strains in the gene bank， and phylogenetic trees were constructed based on the homology comparison results. The phylogenetic trees of the 4 bacterial strains with 16S rDNA gene sequence as the molecular marker is shown in Fig. 2. Strain A1 and Klebsiella were on the same branch， and Bootstrap verification showed that they had a high degree of confidence， with a support degree of 66% （Fig. 2）， so the isolated strain A1 can be identified as Klebsiella. Strain A2 and Acinetobacter were on the same branch， and Bootstrap verification showed that they had a high degree of confidence， with a support degree of 71% （Fig. 3）， so the isolated strain A2 could be identified as Acinetobacter baylyi. Strain A3 and Staphylococcus were on the same branch， with a support degree of 78% （Fig. 4）， so the isolated strain A3 could be identified as Staphylococcus epidermidis. Strain A4 and Pseudomonas in the gene bank showed a support rate of 76% on the same branch （Fig. 5）， so the isolated strain A4 could be identified as Pseudomonas.
　　Agricultural Biotechnology2020
　　Identification of the fungus 　　Colony morphology and microscopic examination results of the fungus
　　A fungus was isolated and purified from spoiled Chinese flowering cabbage， numbered B1. As shown in Fig. 5， after the fungus was aerobically cultured on a red bengal medium at 28 ℃ for 3 d， the colonies were white， moist， dense， uniform in texture and smooth in surface.
　　The genomic DNA extracted from the above fungus was used as a template， and the primer used to amplify the ITS gene was used for PCR amplification reaction. The sequence of the ITS universal primer was： TCC GTA GGT GAA CCT GCG G.
　　The PCR system included appropriate amount of template， primer ITS 1 μl， TaqPolymerase， dNTP 15 μl， sterile ddH2O 14 μl. The program started with pre-denaturation for 3 min and denaturation for 30 s at 94 ℃， followed by 30 cycles of annealing at 55 ℃ for 30 s and extension at 72 ℃ for 2 min， and ended with extension at 72 ℃ for 10 min. The product was cooled at 40 ℃ for 10 min and taken out[23-25].
　　ITS homology comparison and phylogenetic analysis of the fungus
　　The amplified gene sequence of strain B1 was aligned with the corresponding sequences of known strains in the gene bank， and a phylogenetic tree was constructed through the results of homology comparison. Strain B1 shared more than 80% homology with multiple strains of Streptomyces albus. Strain B1 was on the same branch as S. albus strain MG5147 and S. albus strain JQ6573. Multi-level verification showed that they had a high degree of confidence， with a support rate of 85% （Fig. 6）. Therefore， the isolated strain B1 was determined to be S. albus.
　　Conclusions and Discussion
　　In this study， the microorganisms in the decay process of Chinese flowering cabbage were separated and purified， and 4 strains bacteria and one fungus with different morphology were obtained. Further morphological observation， 16S rDNA molecular sequence sequencing， phylogenetic tree construction of the isolated microorganisms were performed， and combining with physiological and biochemical identification， bacteria A1， A2， A3 and A4 were Klebsiella， A. baylyi， S. epidermidis and Pseudomonas， respectively. The identification results showed that Bacillus was the main spoilage microorganisms in Chinese flowering cabbage. A single-celled fungus B1 was isolated and purified from spoiled Chinese flowering cabbage， and it was morphologically identified. A phylogenetic tree was further constructed. It was determined that the fungus was S. albus. Therefore， attention should be paid to prevention of the harm of such microorganisms during the processing and storage of Chinese flowering cabbage. The identification of the types of pathogenic bacteria harming Chinese flowering cabbage after harvest lays a theoretical foundation for further strengthening the transportation of fresh Chinese flowering cabbage， the prevention of microbial diseases during storage and the suppression of the rot of Chinese flowering cabbage in the future. 　　References
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