In this study, high-throughput sequencing identified a high level of fungal diversity in the phyllosphere of maize leaves. The dominant fungi at the class level were Dothideomycetes and Sordariomycetes, both members of the phylum Ascomycota. This finding is similar to previous reports that Ascomycota was the most common phyllosphere fungi in many crops (Angelini et al. 2012; Ding et al. 2022; Janakiev et al. 2019). At the genus level, the main fungal group was Alternaria with different abundance among the four maize varieties. The Alternaria fungi, as saprophytes or pathogens (Lee et al. 2015), can colonize the phyllosphere of multiple plants (Chen et al. 2020b; Liu et al. 2022). Moreover, these fungi are commonly regarded as a highly efficient lignocellose decomposer (Song et al. 2010). In our study, Alternaria in the phyllosphere of maize is assumed to be a saprophyte as no associated disease symptoms were observed on maize leaves. Nigrospora was the second dominant fungal group in the phyllosphere of all four maize varieties. It is well known that Nigrospora are widely distributed in various environments and are endophytes or pathogens of plants (Liu et al. 2021; Thanabalasingam et al. 2015). In our study, Nigrospora was not a maize-specific pathogen, but rather a saprophyte similar to Alternaria in the phyllosphere of maize. Of note, Exserohilum was found in the four maize varieties, which was not surprising as significant northern leaf blight was observed in our experimental field. Moreover, the Exserohilum was the biomarker taxa in the HS varieties, suggesting that Exserohilum survived easily on the leaves of the HS maize variety.
In terms of Beta diversity, the fungal communities were not only different among the four maize varieties, but also grouped according to the four resistance levels, reflecting that maize resistance to Exserohilum may shape the fungal communities. Our four maize varieties were planted in a one field to reduce any environmental influence on the fungi in the phyllosphere. As previously mentioned, some plants such as poplar (Bálint et al. 2013), trees in rainforest (Kembel and Mueller 2014) and cereal (Sapkota et al. 2015) can structure the fungal community in the phyllosphere at the plant species level. At the cultivar level, grape plants are important in shaping the phyllosphere fungal community (Bokulich et al, 2014). Moreover, cereal cultivars had significant influence on fungal communities in their phyllospheres. On the other hand, disease resistances patterns at the cultivar level had no clear correlation with the fungal communities (Sapkota et al. 2015), which is inconsistent with our results. This difference could be attributed to the phyllosphere fungi samples from the previous studies containing endophytic and epiphytic fungi, which impacted community assembly (Yao et al. 2019). The combination of epiphytic and endophytic fungi probably prevented researchers from uncovering the true effect of disease resistance on the phyllosphere fungal community. Previous reports demonstrated that maize cultivar with different resistance to pathogenic fungi can structure the microbial community (Balint-Kurti et al. 2010). In our study, the resistance phenotype of a maize variety may directly affect the fungal community by suppressing or promoting some pathogenic maize fungi of maize to a certain extent. For example, Exserohilum, Bipolaris, Cercospora and Curvularia were only observed from the highly susceptible variety.
It is well known that plant genotypes determine leaf structure (such as cutin and cuticular wax properties) and leaf physiology (including leaf exudates and volatiles), which together significantly shape the microbial communities in a phyllosphere and microbe–microbe interactions (Bodenhausen et al. 2014; Chen et al. 2020a; Farré-Armengol et al. 2016; Horton et al. 2014; Xin et al. 2016). Principally, the leaf cell wall controls the quantity and quality of exudates and strongly influences on the microbe community of a phyllosphere. The absence of a cuticular membrane in single gene mutants of Arabidopsis thaliana dramatically affected the phyllosphere microbiota (Bodenhausen et al. 2014). The maize resistant gene Htn1 against E. turcicum infection, encodes a cell wall-associated receptor-like kinase that regulates the cell wall structure (Hurni et al. 2015; Kohorn and Kohorn 2012), and changes the chemical substances secreted by the leaves. Therefore, we measured the main chemical constituents in leaves, which were nitrogen, phosphorus and soluble sugar as well as the two secondary metabolites tannin and flavonoid. The HS and S varieties leaves had higher nitrogen content, which is related to the nitrogen-induced susceptibility to plant pathogen (Huang et al. 2017). RDA revealed that nitrogen was closely associated with the fungal communities of the S and HS varieties, also demonstrating that high nitrogen levels in S and HS varieties shape their fungal communities. This aligns well with the fact that phyllosphere bacteria are also influenced by nitrogen in susceptible varieties (Tian et al. 2020). Sugars determine the total microbial population in the phyllosphere (van der Wal and Leveau 2011). We found that soluble sugars played minor roles in structuring the phyllosphere fungal communities of all four maize varieties, probably due to the absence of marked differences in soluble sugar contents in the leaves of the four maize varieties.
The HR variety had high phosphorus concentrations in the leaves when compared to the other varieties and this closely correlated with the fungal community in the HR leaf phyllosphere, suggesting that phosphorus affects the fungal communities. It is well known that phosphorus can promote plant tolerance to biotic and abiotic stresses (Pan 2004), and our findings suggest that to some extent, high phosphorus levels are linked to high infection resistance. Phosphorus is also a key factor in shaping the microbes on a leaf surface (Yadav et al. 2005). An epiphytic microbe growth is limited by the availability of phosphorus (Scho¨nherr and Baur 1996), as it is difficult for ATP and polar P-containing compounds to penetrate the cuticles. Therefore, a high phosphorus concentration in the HR variety leaves provides a suitable environment for fungi and promote fungal growth.
Tannins and flavonoid contents also positively correlated with the fungal communities in the phyllosphere of HR and R varieties. Tannins are the most abundant secondary metabolites produced by plants and assist leaves in defending against insect herbivores by deterrence and/or toxicity (Barbehenn and Constabel 2011). Flavonoids are also a vital secondary metabolite synthesized by plants and have important biological activity (Lewis and Ausubel 2006; Song et al. 2021). Thus, the high content of tannins and flavonoids in the HR and R varieties also enhances their resistance to pathogens, and also regulates the fungal community in the phyllosphere. Two-factor correlation network analysis in this study revealed that flavonoids and tannins could alter the composition of the fungal community by specifically promoting or inhibiting certain types of fungi. As different plant varieties exhibit specific host attributes such as resistance, and higher nitrogen, potassium or phosphorus concentrations (Bodenhausen et al. 2014; Kembel and Mueller 2014; Kembel et al. 2014), we speculated that the resistance of these maize varieties and their leaf chemical substances jointly shape the fungal community in the phyllosphere, which is in concordance with the view that the microbial community in a phyllosphere is controlled by multiple factors (Bokulich et al. 2014 ).
Microbial co-occurrence networks display the interaction between different species in a community (Deng et al. 2012). In this study, the composition of the fungal communities among the four maize varieties was similar, while the network structure was different, which indicated differences in the organization of the fungal community. Similar results were also found for microbes in the maize phyllosphere (Kong et al. 2020), which demonstrated that different maize varieties had different networks of microbial communities. We found that the complexity of the fungal community networks for the HR variety was relatively higher than the other three varieties, which indicated higher stability of the fungal community. This is supported by the notion that a resistant plant genotype frequently possessed a more complex microbial network in the rhizophsere and phyllosphere (Xu et al. 2020; Zhong et al. 2019). More positive associations in the HR fungal community included more cross feeding, co-aggregation, co-colonization and niche overlap in the community (Faust and Raes 2012), suggesting a relatively healthy community. More negative relationships in the network of the S and HS fungal communities reflected amensalism, competition and antagonism in the community (Faust and Raes 2012), which probably resulted from the colonization of fungal pathogen Exserohilum that disturbed the balance of the community.
The present study revealed that maize varieties with different resistance to northern corn leaf blight possessed various leaf chemical traits and distinct fungal communities in their phyllosphere. The maize variety and leaf chemical substances, such as phosphorus, nitrogen, tannins and flavonoids jointly shape the fungal community in a phyllosphere. The HR variety had a more complex network compared to the others varieties, suggesting that it was more stable. Future research is necessary to reveal the functional roles of key genes that control the leaf chemical traits that shape the fungal community in a phyllosphere.