生物有机肥对菠萝根际真菌群落及心腐病发生率的影响

Effects of bio-organic fertilizers on rhizospheric fungal community and occurrence of heart rot disease in pineapple

【目的】以连续种植的、高发心腐病的菠萝园土壤为对象,研究根际土壤真菌群落与菠萝心腐病的关系,为利用生物有机肥防控菠萝心腐病提供理论依据。【方法】采用高通量测序技术手段,研究盆栽条件下三元复合肥(HF,3种载体菜籽饼、泥炭土、椰糠)和生防菌株(枯草芽孢杆菌HL2、链霉菌株HL3)与普通有机肥(YJ)、二次堆制而成的生物有机肥(KC、KN、KY、LC、LN、LY)对菠萝心腐病的防控效果,以及对根际土壤真菌群落和功能的影响。【结果】与三元复合肥(HF)处理相比,普通有机肥(YJ)处理和生物有机肥(KC、KN、KY、LC、LN、LY)处理的真菌群落丰富度和多样性更高,群落结构存在显著差异。生物有机肥(KC、KN、KY、LC、LN、LY)处理中,粪壳菌纲相对丰度显著低于HF和YJ,而座囊菌纲和散囊菌纲得到明显富集。网络分析表明,相比HF和YJ,生物有机肥处理形成了复杂连接的稳定真菌群落,但接种枯草芽孢杆菌的生物有机肥(KC、KN、KY)处理的真菌网络呈现出更强的相关性,接种链霉菌的生物有机肥(LC、LN、LY)次之。HF和YJ处理中,与发病率呈极显著正相关的真菌属青霉菌属、镰孢菌属、未定义毛壳科菌、木霉属和单胞瓶霉属的相对丰度显著高于生物有机肥(KC、KN、KY、LC、LN、LY)处理。相反,与发病率呈极显著负相关的真菌中,黑团球壳腔菌属在生物有机肥(KC、KN、KY;LC、LN、LY)处理中具有更高的相对丰度,真菌踝节菌属和未定义刺盾炱目菌属分别在KC处理、LY处理相对丰度达到最大。【结论】生物有机肥施用后,菠萝心腐病发病率下降,土壤真菌群落结构和组成发生变化。其中,KC处理有效减少了连作土壤菠萝心腐病发生,发病率为0,且土壤真菌群落丰富度和多样性达到最大,与发病率呈负相关的真菌属显著增加,土壤真菌群落的生态系统趋于稳定。

【Objective】Heart rot is a severe problem in pineapple orchards worldwide. In agricultural systems, the ecological imbalance of inter-root microorganisms becomes more serious with increasing planting years. However, the combination of suitable organic materials with beneficial microorganisms may become an effective countermeasure. To dig deeper into the elusive mechanisms of disease suppression, we conducted a study based on a monoculture, severely heart rot-infected pineapple cropping system as a test subject. The incidence of pineapple heart rot and the diversity and composition of the rhizospheric microbial community were followed up with the addition of bio-organic materials to reveal the effect of altered fungal abundance and community structure on disease incidence, and to explore the direct antagonistic effects of soil microbes and community changes on heart rot suppression under different biological control strategies.【Methods】A pot experiment in greenhouse was constructed with chemical, organic, and different bioorganic fertilizers. The chemical and organic fertilizers were directly purchased from local agricultural markets. The bioorganic fertilizers used in this experiment were obtained by composting three kinds of recycled materials(rapeseed cake, peat soil, coconut bran), two antagonistic microorganisms(Bacillus subtilis HL2, Streptomyces strain HL3), and the organic fertilizer.After 140 days of pineapple cultivation, the rhizosphere soil of each fertilizing treatment was collected from five random pots to form one mixture sample(about 100 g), and the roots and stones were removed. The soil samples were stored in an ultra-low temperature refrigerator at-80 ℃ for DNA extraction after being passed through a 2 mm sieve. Then the total DNA from 0.5g freeze soil samples was extracted by the Power Soil DNA Isolation Kit(MOBIO Co, USA). With 5’-TCCTCCGCTTATTGATATGC-3’and 5’-GCTGCGTTCTTCATCGATGC-3’as the primer pair, the fungal internal transcribed spacer(ITS) region of rDNA was amplified and then sequenced by means of Illumina MiSeq highthroughput sequencing technology. The per sample quality control and paired library preparation were completed by Biotech Company platform. After obtaining offline data, the raw sequencing sequences were refunctioned and primer sequences were removed using QIIME software. Forward and reverse sequences were then spliced using the USEARCH script and quality filtered to remove low quality base sequences, including de-redundancy and clustering, removing chimeras and non-bacterial sequences.The operational taxonomic units(otu) tables were converted to Biom format and annotated in QIIME software using the BLAST method against template sequences from the UNITE FUNGAL ITS database to obtain the taxonomic information of the fungus corresponding to each OTU and obtain the phylogenetic status of its representative sequence. The significance analysis and statistics visualization in fungal community were accomplished with R packages and Gephi.【Results】In our study, the richness and diversity of soil fungal community in organic fertilizer(YJ) and bio-fertilized treatments(KC, KN, KY;LC, LN, LY) were significantly higher, compared with that in HF(chemical fertilizer). The differences in fertilization methods reshaped the ecological structures of rhizospheric microbial community to some extent. According to the phylogenetic class level in LEfSe analysis, the relative abundance of Sordariomycetes was significantly lower in bio-fertilized treatments than those in HF and YJ, but the relative abundance of Dothideomycetes and Eurotiomycetes was significantly(p<0.05) higher. Among visualized network analysis, bio-fertilizing treatments showed complexly connected and stable networks of fungal communities in comparison with HF or YJ. Moreover, the bio-fertilized treatments inoculated with Bacillus subtilis HL2(KC, KN, KY) tended to have stronger correlations than those in other biofertilized treatments inoculated with Streptomyces strain HL3(LC, LN, LY). At genus level, fungal network centroids for HF were Penicillium, Fusarium, Chaetomiaceae_unidentified, Pulchromyces, Phialemonium;Central points for YJ were Dothideomycetes_unidentified, Bionectriaceae_unidentified, Penicillium, Clonostachys. After integration of the treatments, the network centroids of HL2 bioorganic fertilizer were Massariosphaeria, Trichocomaceae_unidentified, Talaromyces;HL3 bioorganic fertilizer network centroids were Massariosphaeria, Lasiodiplodia, Chaetothyriales_unidentified_1, Davidiella.And he relative abundance of Penicillium, Fusarium, Chaetomiaceae_unidentified, Trichoderma and Phialemonium in HF and YJ were significantly higher, which extremely positively correlated with the incidence. Conversely, the fungi genus Massariosphaeria highly correlated with incidence had greater abundance in bioorganic fertilizing treatments. Moreover, the relative abundance of the fungi Talaromyces and Chaetothyriales_unidentified_1 reached the maximum in the KC and LY treatments, respectively. However, the function guild of genus(Massariosphaeria & Talaromyces) was clarified to the undefined by FUNGuild.【Conclusion】The changes in fungal abundance and community structure after amendment with bio-fertilizers could contribute to reduce the density of hazardous microbiome group,such as Penicillium, Fusarium, Trichoderma, Chaetomiaceae_unidentified, Pulchromyces and Phialemonium highly related to the heart rot disease. In contrast, the relative abundance of Talaromyces and Massariosphaeria increased by bio-fertilizing. The bio-organic fertilizer inoculated with Bacillus subtilis using rapeseed cake and organic fertilizer as organic materials showed the best performance, effectively reducing the incidence of the heart rot of successive pineapple crops. Also, soil fungal community richness and diversity reached a maximum value, with a significant increase in fungal genera negatively correlated with the incidence and a stable community ecosystem. Consequently, the biofertilizers might reshape the soil fungal structure as increasing the proportion of beneficial microorganisms, which is of significance in reducing the incidence of heart rot disease in pineapple.