The relationship between microbial community structure, fungicide and plant health plays an important role in microbial ecology and tobacco disease control[11]. However, very few studies have been conducted on the interaction of fungicides, microbial community structure and tobacco health. This study investigated the responses of tobacco phyllospheric fungi and bacterial community structure and diversity to the application of Bordeaux Mixture at different application stages.
First, the relationship between fungicide and tobacco health should be determined by assessing the fungicide control efficacy on tobacco target spot. Based on field trial data, we can infer that the broad-spectrum fungicide Bordeaux Mixture was effective for tobacco target spot control at an early stage. However, it may not be suitable for long-term field tobacco target spot prevention and control. The pathogen T. cucumeris infects plants by producing extracellular hydrolytic enzymes to degrade plant cell walls and enter plant cells[12]. Bordeaux mixture controls the disease by disrupting the proteases of pathogen cells and blocking their metabolism, but once the pathogens enter the plant cells and produce propagules such as spores, Bordeaux Mixture may be powerless. Bordeaux Mixture is useful for surface pathogens, but it lacks curative or systemic activity and it is very leachable[13], so its protective effect on the plant is short-lived. Therefore, Bordeaux Mixture is not suitable for long-term field tobacco target spot prevention and control, which is consistent with the result of the study.
Second, we studied the change in tobacco phyllospheric microbial community composition and structure during the period of tobacco target spot damage. There are differences in phyllosphere dominant microbial communities when different tobacco leaf-spot diseases occur. Previous studies have reported that the dominant phyllospheric fungi of tobacco leaves infected by Didymella segeticola leaf spot disease were Boeremia, Meyerozyma, Phoma and Alternaria[9], the dominant phyllospheric fungi of leaves infected by tobacco brown spot included Alternaria and Phoma[14]. The dominant phyllospheric fungi of rotten tobacco leaves after curing included Aspergillus, Penicillium and Rhizopus[15]. Among the tobacco diseases mentioned above, Pseudomonas and Pantoea are both dominant bacteria. The dominant phyllospheric microorganisms of tobacco target spot in our study were not the same as the dominant phyllospheric fungi of the other diseases mentioned above. We could speculate that different tobacco diseases have different core fungal pathogenic microbiota.
Comparing the dominant fungal genus groups of the healthy and diseased groups, we found that there was a significantly higher relative abundance of Thanatephorus in the diseased group, the relative abundance of other genera accounted for less than 12%. The Alternaria and Sampaiozyma abundance of diseased leaves was significantly higher than the Alternaria and Sampaiozyma abundance of the healthy group. According to previous reports, Sampaiozyma was the common dominant fungus at all four tobacco processing stages (before flue-curing, after flue-curing, before redrying and after redrying), and the relative abundance at the four processing stages was 44.27%, 37.87%, 24.12% and 46.12%, respectively[16]. Some studies[17–20] have proven that Sampaiozyma is a wild yeast in the sediments of soil and lakes and can produce extracellular enzymes such as amylase, lipase, chitinase, and pectinase. Sampaiozyma may play a role in the process of disrupting plant defence barriers during pathogenic infestation by releasing extracellular enzymes, which can be further studied. Moreover, there was a study pointed that the yeast microbiota was not significantly affected by treatment of Bordeaux Mixture in field[21], which may link with the powerless effectiveness of Bordeaux Mixture. Alternaria is the typical pathogen of tobacco brown spot, and we could speculate that during the epidemic of tobacco target spot in the field, tobacco brown spot may also occur at the same time in some disease spots on tobacco leaves, so there might be some complex infection between Thanatephorus, Sampaiozyma and Alternaria. In the healthy group, the abundance of Cercospora was much higher than the abundance of Cercospora in the diseased group, and perhaps there is an antagonistic relationship between Cercospora and Thanatephorus. More studies should be conducted to determine the interactions among those pathogens in the next study.
For the bacterial community before application, Pseudomonas was the absolutely dominant genus in the diseased group, and Methylobacterium was the dominant bacterial genus in the healthy group. The relative abundance of Massilia in the diseased group was significantly higher than that in the healthy group. Pseudomonas and Methylobacterium were the common dominant bacteria of tobacco diseases, and they may have a synergistic effect on pathogen infection. Members of the genus Massilia are frequently isolated from soil such as forest soil, rhizosphere soil of rice, and soil of Pu-erh tea cellar and have meaningful biological roles, such as antibacterial activity against Ralstonia solanacearum and cellulose degradation[22–25]. Comparing the diseased group and healthy group, we can know that the diseased group has a higher richness and diversity of the bacterial community. The healthy group had a higher richness and diversity of the fungal community.
Then, we studied the responses of tobacco phyllospheric microbial community composition to Bordeaux Mixture application. After Bordeaux Mixture application, the indigenous microbial community on tobacco leaves changed, there was a difference between samples before application and after application. Although Thanatephorus predominated on both diseased and healthy tobacco leaves and Pseudomonas was the dominant genus of bacteria in the diseased group, the relative abundance of pathogen Thanatephorus was showing a significantly decreasing trend at different time-point after application. It has been documented that widespread copper-based fungicide application could affect the surrounding ecosystems[13], and research conducted by kandeler et al.[26] and kong et al.[27] found that the functional diversity of soil microbial communities decreased significantly with increasing concentrations of Cu and the microbial biomass and enzyme activities involved in cycling of C, N, P and S also decreased with increasing concentration of Cu. The negative effect of Bordeaux Mixture has been previously demonstrated in fungal species such as Beauveria bassiana[28] and Colletotrichum acutatum[29]. Results of those studies showed that Bordeaux Mixture inhibited the Spore formation and germination of fungi and the effect of Bordeaux mixture on fungi was probably due to the toxicity of the copper ion itself, and it also induced an oxidative state in fungal cells[28]. After application, the relative abundance of Thanatephorus in the diseased and healthy groups decreased by 79.35% and 19.95%, respectively, it could be speculated that there might be a negative influence of Bordeaux Mixture on pathogenic fungi Thanatephorus, too. Due to the pathogen are dominant fungus on diseased tobacco leaves, the impact of Bordeaux Mixture on microorganisms is mainly reflected in the relative abundance of pathogen, and significant inhibition of pathogen reduces the development of disease in diseased tobacco plants. In regard to the change in the dominant phyllosphere fungal and bacterial genera, this study found that with increasing application time, the diversity of the fungal and bacterial communities in diseased groups also increased. A higher diversity microbial community has a higher probability of containing antagonists to pathogens and a more stable microecology. Previous studies on soil communities have demonstrated that high diversity and species richness contribute to high functional diversity within the microbiome[30–31], due to antagonistic and exclusion relationship between microorganisms, higher community diversity is not conducive to the development of pathogen into dominant microorganism, thus facilitating resistance to disease development. This mechanism might also be applicable to the fungal and bacterial communities in the phyllosphere. Although the community diversity increased, the increase was not significant, which may be related to the unsatisfactory control effect of Bordeaux Mixture on tobacco target spot disease. According to the ecological function prediction results of the microbial community, the relative abundance of fungi belonged to pathotroph functions significantly decreased after Bordeaux Mixture application. Accordingly, the relative abundance of other unassigned fungi and pathotroph-symbiotroph had increased, and unassigned fungal communities ultimately occupied the vast majority. The increasing of relative abundance of pathotroph-symbiotroph indicated that there were more symbiotroph function group in phyllosphere. By comparing the functional predictions of the healthy and diseased groups, we can learn that when the tobacco plant is diseased, the dominant functional group on phyllosphere is pathotroph. With the application of fungicides, the pathogen was affected and their relative abundance decreases significantly, corresponding to the increase in the relative abundance of other functional group. It could be speculated that the significant reduction in the relative abundance of pathogenic allows for more ecological niches for other functional groups. The function of phyllosphere bacterial communities did not fluctuate significantly throughout the application period of Bordeaux Mixture. In summary, the present study showed the effects of Bordeaux Mixture on the phyllosphere microbial community and further revealed the potential relationships between the phyllosphere microbial community and fungicide’s application.