Deciphering microbial diversity associated with Fusarium wilt-diseased and disease-free banana rhizosphere soil

Microorganisms play an important role in the formation and maintenance of healthy soil, improve soil conditions through many enzymatic activities that regulate the biogeochemical cycle, reorganization and mineralization of organic matter [1–6]. The microbiota present in soil are affected by climate, cultivation methods, soil nutrients, pathogens and soil management practices [7–10]. Rhizosphere microorganisms comprise a dynamic community with a complex interaction with plants [11]. Some intrinsic microbial communities or specific sub-populations have potential functions to suppress soilborne diseases caused by fungi, oomycetes, bacteria and nematodes [5, 6, 11]. Hence, understanding the microbial composition of the plant rhizosphere is key to controlling the spread of soilborne disease.

Banana (Musa spp.) is an important cash and food crop in the tropics and subtropics and is one of the world’s top 10 staple foods [12, 13]. Most of cultivated bananas multiplied by the vegetative propagation are prone to infection by various pests and diseases, particularly Fusarium wilt of banana. The disease is caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), which is one of the major constraints to banana production worldwide [14]. Foc infects the xylem through the roots, and causes extensive necrosis leading to of plant deaths [14]. Currently, management of the Fusarium wilt disease mainly involves rotation, selection of resistant varieties, and chemical or biological methods. Nonetheless, there is no an effective control for Fusarium wilt of banana, as evidenced by the continuous spread among continents, countries and regions [15, 16]. In general, biocontrol is considered as a safe, environmentally friendly and cost-effective method for disease control [5, 8, 16].

Multiple pathogen-plant systems have been widely used to suppress different innate diseases in the agricultural soil [6, 17–19]. Once attacked by root pathogens, plants can exploit microbial consortia from soil for protection against pathogen infection. Comparative microbiome analysis indicated that the relative abundance of bacterial and fungal diversity was significantly higher in the suppressive soil than that in the conducive soil [6]. High richness and diversity indices of bacterial communities were also detected in the soil naturally suppressive to Fusarium wilt of banana [18]. In addition, known plant-beneficial rhizobacteria such as Azospirillum, Gluconacetobacter, Burkholderia, Comamonas and Sphingomonadaceae were more prevalent in soil suppressive to tobacco black root rot disease [19]. Furthermore, application of Paenibacillus polymyxa NSY50 reduced the abundance of Fusarium and increased the population of beneficial microbes, including Bacillus, Actinobacteria, Streptomyces, Actinospica, Catenulispora and Pseudomonas genera [20]. These beneficial microorganisms inhibit pathogen infection through metabolites such as pyoverdins and iron-chelating siderophores [21]. Hence, novel bacterial and fungal taxa can serve as indicators of disease suppression in soil-quality assessments. A previous study revealed that microbial species composition and abundance, which are also significantly influenced by the chemical properties of soil, are vital for favorable microecology [22]. However, how soil microbial species are affected by mutual constraints among disease occurrence, pathogenic abundance, soil chemical properties and beneficial microorganisms are still an open question.

In the present study, we performed a comparative microbiome analysis of the Fusarium wilt-diseased and Fusarium wilt-suppressive (disease-free) rhizosphere soils. Based on 454-pyrosequencing of the fungal internal transcribed spacer (ITS) region and the bacterial 16S rRNA gene, changes in dominant bacterial and fungal species were investigated in both types of soils. Additionally, correlations between soil chemical properties and microbial distributions were analyzed. Our results offer new insight into identifying fungal and bacterial genera associated with Fusarium wilt disease-free soils and suggest the microbial genera and mechanisms involved in Fusarium wilt suppressiveness.

Understanding of microbial species and distributions is essential for controlling plant diseases [23]. In our study, lower colonization of Foc was detected in the disease-free soils, suggesting that the severity of banana Fusarium wilt accompanied by the increase of Foc colonization in the field. Previous reports have indicated that 1000 CFU/g can be considered a critical index of Foc diagnosis in the pot experiments [23, 24]. However, we revealed that lower than 1300 CFU/g of Foc colonization will not cause Fusarium wilt of banana in the field. It was supported by that over 1700 CFU/g of Foc colonization were detected in the diseased soils. This difference may be due to the complex field environment.

Although the ACE index was much higher in the diseased soils than in the disease-free soils, the species diversity based on the analysis of Shannon and Simpson had no significant differences, indicating that the species structure was similar in both types of soil samples. Higher fungal OTUs in the diseased soils may be due to an increase of Foc colonization, which might provoke the changes of microbial species and distributions. Thus far, a few reports have demonstrated the relationship between the incidence of fungal disease and the type of dominant genera. In our study, the dominant fungal phyla were also similar in the diseased and disease-free soils. Ascomycota and Zygomycota were the most abundant fungal phyla, accounting for 93.46 and 88.59% of total fungal ITS sequences. The abundance of Ascomycota in the diseased soils was much higher (p = 0.002) than that in the disease-free soils. It was consistent with previous study that the decrease of Proteobacteria and Ascomycota were associated with some fungal diseases suppression [25–27].

By contrast, Mortierella, Fusarium, Pseudallescheria, Nectriaceae, Chloridium, Chaetomium, Humicola, Trichoderma and Ascomycota were the dominant genera in both types of soil samples. Although the soil conditions or plant varieties could cause different distributions of the dominant fungal genera, Fusarium is frequently considered as one of the most dominant genera in some studies [6, 18, 28]. Nonetheless, the dominant genera Trichoderma and Mortierella were enriched in the disease-free soils, whereas Fusarium, Pseudallescheria, Nectriacea, Chaetomium, Humicola, Sordarionmyetes and Gibellulopsis demonstrated a decreasing tendency. Trichoderma has been widely used as a biological control agent against various pathogens [29], and although Mortierella has not been as a biological control agent, some antifungal and antibacterial metabolites proved to be produced by isolates of Mortierella [30–33]. Considering the only 8.30% of Fusarium abundance in the disease-free soils, we speculated that Trichoderma and Mortierella may be associated with suppression of banana Fusarium wilt disease. However, whether the regulation mechanism is through direct antagonism or resource competition, especially in the case of Mortierella is currently unknown.

Our results indicated that the higher abundance and diversity of bacteria were detected in the diseased soils. However, opposite results showed that a large number of OTUs were observed in the Fusarium wilt disease-free soils [25]. No significant difference of bacterial community distribution and diversity was exhibited in the rhizosphere soil samples of diseased and disease-free apple tree [34]. It may be caused by the field sampling time or soil microorganism changes in the presence of pathogens. Although many studies has shown that increased microbial diversity played an important role in the control of diseases [35–37], the relative abundance of several bacterial taxa is a more important indicator of disease suppression than the exclusive presence of specific bacterial taxa [17, 38]. It was supported by that most of abundant phyla such as Proteobacteria, Acidobacteria, Chloroflexi, Firmicutes, Actinobacteria, Gemmatimonadetes, Bacteroidetes, Nitrospirae, Verrucomicrobia and Planctomycete were abundant in both types of soil samples. Proteobacteria and Firmicutes were the most dominant phyla, which were similar to the previous studies on diseases infected by Rhizoctonia solani [38, 39]. It might be related to the different growth rate of bacteria [40, 41]. In addition, the higher abundance of Firmicutes in the disease-free soils supported that the disease incidence was negatively correlated with the richness of Firmicutes [18]. Similarly, the abundances of Acidobacteria and Firmicutes in a wheat rhizosphere were positively correlated with disease suppression [42]. Moreover, the abundances of Bacillus, Lactococcus and Pseudomonas in the disease-free soils were two folds than those in the diseased soils (Table 3). Bacillus and Pseudomonas were proved to be responsible for natural suppression of Fusarium wilt disease [25, 43–47]. Streptococcus was also enriched in disease-free soils probably because of the antagonist relationship between Streptococcus and Fusarium oxysporum [48], suggesting that Streptococcus may participate resistance to Fusarium wilt disease. Actually, the complex phenomenon of disease suppression in soils cannot be simply ascribed to a single bacterial taxon or group, but was governed by microbial consortia [38]. Plants and microbiota established a microenvironment on the roots with a complex interaction. A model of seven-species community evidenced that beneficial microbes inhibited the infection of phytopathogenic fungus Fusarium verticillioides in plant roots [11]. This model system research provides a useful method for future research on the banana-microbe interaction. However, we should fully recognize the effect of beneficial species on Fusarium suppression before assembling model community on banana. Comparisons of bacterial communities from banana Fusarium wilt diseased and diseased-free soils will prove to be essential for constructing disease suppressive soil in the future.

Based on the results of RDA analysis, disease status may accelerate soil environmental variables that affect the distribution profiles of bacterial and fungal communities [25, 49–51]. Higher AP and pH were detected in the disease-free soil samples. In addition, AP or pH exhibited a negative correlation with abundance of Fusarium, but was a positive correlation with abundance of Bacillus. A previous study reported that a high pH enhanced the suppression of Fusarium wilt disease [52]. Similarly, high soil AP was also associated with the inhibition incidence of stem rot disease caused by Rhizoctonia solani [53]. Therefore, the soil management strategies were related to the inhibition of Fusarium wilt disease of banana [54]. Our findings provided an evidence that higher pH and AP may enhance the inhibition of banana Fusarium wilt disease. It may be related to directly increased populations of beneficial genera or indirectly to altered soil nutrient availability to host plants. Additionally, TOC and TON showed significant differences between disease and diseased-free soils, with a positive correlation with the fungal taxon Fusarium and a negative correlation with the bacterial taxon Bacillus. Previous study demonstrated that bacterial and fungal communities were mainly driven by soil organic matter [55]. Indeed, due to the resource use preference of different microbes, contents of TOC and TON may alter the microbial community. For example, the soil microorganisms responded differently to the inputs of inorganic and organic fertilizers in paddy and banana soils [56–58]. Based on these findings, we can effectively suppress Fusarium wilt disease in practice by optimizing the structure of fertilization.

We compared the distribution of the microbial communities in Fusarium wilt-diseased and disease-free soils. The bacterial and fungal distributions in the diseased soils were different from those in the disease-free soils. Regardles, a higher abundance of Fusarium was observed in the diseased soils, whereas the Firmicutes phylum and the Bacillus, Lactococcus and Pseudomonas genera were enriched in the disease-free soils. Regarding the fungal distribution, the abundance of Ascomycota in the diseased soils was much greater than that in the diseased-free soils. Mortierella was most dominant genera in the disease-free soils. We also found that Bacillus abundance, AP concentration and pH value were positively correlated with suppression of banana Fusarium wilt disease. These results will provide a theoretical basis for evaluating soil disease-free conditions and controlling plant diseases.