## 4 Discussion  
Black spot disease caused by _A. alternata_ has devastating effects on broccoli cultivation. Black rot disease caused by _X. campestris_ also results in a similar phenotype that is manifested by a black spot in broccoli leaves. In the fields, it is a big challenge to distinguish the two pathogens, which to some extent restricts the disease control. Therefore, it is necessary to develop a simple identification method. With the development of molecular detection technology, researchers can study the relationships among various fungal groups at the nucleic acid level, such as via an Internally Transcribed Spacer (ITS) located among 18S, 5.8S, and 28S rRNA, which have highly contained abundant genetic variations. Given that the sequence of ITS can truly reflects the differences in different strains intra and extra species, it has been widely used for pathogen identification. Previous reports showed that RAPD (Randomly Amplified Polymorphic DNA) technology contributed to distinguishing genera and intra-genus species of _Alternaria_[35; 36]. Microsatellite primer pairs also have been used for pathogen identification, where five pairs of SSR markers can evaluate the genetic diversity of 64 isolates [37]. In this study, to compare the difference of _A. Alternaria_ and _X. campestris_, we screened five pairs of fungal and bacterial identification primers and found that two pairs of primers can distinguish the differences through agarose electrophoresis (Figure 1). Compared to previous research, the method is convenient for broccoli disease identification, and it will help to detect disease types in the early stage. Then, the experts can provide control measures to prevent a large-scale occurrence of black spot disease. Figure 5: Phenotypic comparison of susceptible materials before and after inoculation. The upper part belongs to susceptible; the lower part belongs to high susceptibility. Black spot disease can cause severe leaf yellowing and a decrease in yield, whether in crops or vegetables. Therefore, there are many researchers focused on the identification of resistance to black spot disease. The researchers identified black spot resistance in Chinese kale plants by spraying spore suspension (10\({}^{6}\) spores mL-1) on leaves. Then, the plants were grown under wet conditions in the greenhouse for 7 days. Compared to the control treatment, the plants with pathogenic induction showed serious diseases with large lesion areas [6]. [PERSON] et al. performed black spot disease identification on two months old Chinese kale and added 100 uL spore suspension (5 \(\times\) 10\({}^{5}\) spores mL-1) to the leaves to investigate the phenotype after 5 days [38]. In this study, we evaluated the black spot resistance of 173 broccoli accessions, but the identification method is different from that previously reported. We collected the leaves of mature plants and added 50 uL spore suspension (5 \(\times\) 10\({}^{5}\) spores mL-1) to the artificial wounds of leaves. After cultivating the leaves in a wet environment at 22 \({}^{\circ}\)C for 5 days, we then evaluated the infection situation of each accession. Through these means, we not only accurately identified the disease resistance of mature plants but also saved a lot of manpower and material resources. It is suitable for large-scale germplasm disease resistance identification. The current measures to prevent black spot disease in vegetables are seed disinfection treatment, biological control, and plant antibacterial active substances. The seeds of cauliflower treated with acetone and dichloromethane can completely inhibit the spore germination of _A. brassicicola_[39]. The mixture of iprodione and seeds also can effectively prevent black spot disease caused by _A. alternata_[40]. Although chemical reagents are effective in black spot disease control, they are harmful to humans and the environment and do not comply with green production standards.