Micro-ecological Mechanism Of Soil-borne Tomato Fusarium Wilt In The Nitrogen-rich Facilities Cultivation Soil

Facilities cultivation technique has been becoming the important way to obtain high-yield, and high-quality vegetables currently. However, to pursue the higher economic interests, excessively high levels of fertilizers, especially the nitrogen fertilizer, are applied into the soil. With the planting years increasing, the N P K nutrients in the soil were accumulated, leding to severe nutrient imbalance in the soil, and the nitrogen-rich phenomenon is very obvious. This situation leads to a series of serious problems, such as the soil acidification, soil salinization, and the high levels of soil-borne diseases (e.g. the wilt disease of tomato). Current studies mainly focus on the plant protection, prevention and control of soil-borne diseases, while the research about mechanism of soil-borne diseases in nutrient-rich soil is relatively rare. In our work, pathogen of the wilt disease of tomato was selected as the research object, and different nitrogen treatments were designed. We tried to explore the the mechanism of the soil-borne tomato Fusarium wilt in the nitrogen-rich soil from the macro (soil microbial ecology) perspective and micro (microbial physiological and biochemical perspective) perspective. Through the soil culture experiment and the pure culture (liquid fermentation), effects of nitrogen contents and its forms on the numbers of soil microbes, microbial community dynamics and changes in the microflora were explored. Meanwhile, we also studied the incidence of a disease and disease index under the different nitrogen conditions after inoculation of pathogen. In our study, we proposed a hypothesis towards the mechanism of the soil-borne tomato Fusarium wilt. With the soil microflora balance (mainly proportion of soil microorganisms) is broken, the tomato Fusarium wilt will take place when the imbalance exceeds a "threshold"; And with the imbalance is farther off the "threshold", incidence of the disease is higher accordingly. The survey found that the soil with long-term cultivation or the soil with long-term application of nitrogen fertilizers were more susceptible to soil borne diseases. The accumulation of nitrate nitrogen in the soil was, advantageous to the growth of pathogenic bacteria, but bad for the growth of their antagonistic microrganisms. Our work quantitatively studied the effects of nitrogen accumulation on the soil microbes and amount of tomato Fusarium wilt from the perspectives of soil microbes and microbial biochemistry. We found that different microorganisms responded to the forms and concentrations of soil nitrogen. Then we found that the proportions of the bacteria and actinomycetes declined seriously with the accumulation of nitrogen, while the ratio of fungi and the pathogen increased. Meanwhile, the original balance of microbial flora was broken and the microbial diversity was reduced, resulting the increase of incidence of tomato Fusarium wilt. We included that the accumulation of soil nitrogen is one of the important reasons affecting increased incidence of tomato Fusarium wilt. Therefore, our conclusion is consistent with the practical production. Our research will provide the theoretical support for the rational application of fertilizer and control soil-borne disease in the facility cultivation. The main results of our work were as follows:1. In soil experiments, different nitrogen-rich test (Nitrogen content was setted as 95.96, 203.56,257.62,279.36,319.59,348.94 mg·kg-1, effectively.) were carried out. With the extension of incubation time, the numbers of culturable bacteria, fungi and actinomycetes in the soil showed the increase trend in the early period, then reduced slowly. Numbers of the tomato Fusarium wilt pathogen showed rapid increase in the early period, while it showed the slow increasing trend in the late period. When the nitrogen content was 279.36 mg·kg-1-～ 319.59 mg·kg-1, culturable bacteria numbers reached the maximum. Numbers of soil culturable fungi, actinomyces, and pahtogen could reached highest when the nitrogen content of soil were 279.36 mg·kg-1,251.62 mg·kg-1,319.59 mg·kg-1, respectively. Therefore, the riched nitrogen in the soil can significantly influence the numbers of soil culturable microorganisms.2. The nitrogen-rich phenomenon not only significantly affected the amounts of soil microbes, but also caused change of microbial proportions. With the nitrogen content increasing, B/F (proportion of bacteria to fungi) and A/F (proportion of actinomyces to fungi) in the soil all decreased firstly and increased subsequently. When the soil nitrogen content was 203.56 mg·kg-1, the B/F and A/F in the soil all reached highest, showing proportion of bacteria and actinomycetes highest. As the amount of nitrogen in the soil increased, both numbers and proportion of bacteria and actinomycetes all decreased.16S rRNA gene amplicon sequencing indicated that the diversity and abundance of bacteria decreased with the increase of nitrogen content.3. The nitrogen-rich phenomenon could cause the significant decrease of soil pH and increase of soil nitrate nitrogen, ammonium nitrogen, alkali-hydrolyzable nitrogen, and available nitrogen. We also found that soil pH negatively correlated with numbers of soil culturable microorganisms (bacteria、fungi) and pathogen, positively correlated with numbers of culturable actinomyces. But other soil chacteristics positively correlated with numbers of soil culturable microorganisms (bacteria、fungi) and pathogen, negatively correlated with numbers of culturable actinomyces. Thus, changes of soil chacteristics caused by the nitrogen-rich factor soil could dramatically affect the number of soil microflora and pathogen.4. In soil experiment of inoculated with pathogen, compared with the treatment without pathogen, the varied trend of numbers of culturable bacteria, fungi and actinomycetes and pathogen showed the similar trend in the nitrogen-rich conditions. When the nitrogen content was 279.36 mg·kg-1, culturable bacteria numbers reached the maximum. Numbers of soil culturable fungi, actinomyces, and pahtogen could reached highest when the nitrogen content of soil were 319.59 mg·kg-1,257.62mg·kg-1,319.59 mg·kg-1, respectively. Extra addition of pathogen could result in the significant number changes of three mian microbial populations in soil, it could increase the numbers of fungi significantly, and inhibit the increase of numbers of bacteria and actinomycete, thus causing the severe changes of microflora balance, which caused the occurrence of tomato Fusarium wilt eventually.5. In soil experiment of inoculated with pathogen, disease incidence of tomato Fusarium wilt all took place under the condition of different nitrogen-rich treatment. When the nitrogen content in soil was 203.56 mg·kg-1, the disease incidence and disease index all researched the lowest, with the value being 41.82% and 16.22%, respectively. At this situation, the proportion of bacteria and actinomycetes in the soil were highest, while the proportion of fungi was lowest. As the proportion of soil bacteria and actinomyces became lower, and that of fungi rised, the disease incidence and disease index of tomato Fusarium wilt all showed rising trend. Therefore, we can include that the broken microbial balance in the nitrogen soil (reduced proportion of bacteria and actinomyces, increased proportion of fungi) is the important causing higher incidence of tomato Fusarium wilt.6. In the pure culture experiment, the abilities of breeding, producing acid, secreting toxins and secreting enzyme of plant cell wall degradation were all the appropriate when the nitrate nitrogen was selectedd as the only nitrogen source for the pathogen. When the ammonium nitrogen as the only nitrogen source for the pathogen, some representatives (Bacillus amyloliquefaciens, Bacillus subtilis and Penicillium oxalicum) showing antagonistic activity against the pathogen had the highest abilities of breeding, producing acid and secreting hydrolytic enzyme. However, microbial growth and metabolism were all suppressed when the nitrogen concentration were the high levels. Fusarium oxysporum can produce acidoid by itself, and they are more likely to grow in the acidic environment compared with other microorganisms. The accumulation of nirtrate nitrogen in the soil can lead to the decrease of soil pH, which is advantageous to the growth of pathogenic bacteria.