Assessing microbial diversity in anthers of Eriobotrya japonica (Thunb.) Lindl. based on high-throughput sequencing
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摘要:
果树具有较高的经济价值，并大多依赖传粉者实现繁殖成功以保证产量。花药微生物能影响植物的花粉活力，其组成也可能受到传粉者访问的影响；理解花药微生物的多样性和群落构建模式对于提高果树的繁殖适合度具有潜在意义，但目前相关研究仍相对缺乏。本研究基于高通量测序技术分析了传粉者访问前（套袋组）与访问后（访问组）枇杷（Eriobotrya japonica (Thunb.) Lindl.）花药中真菌与细菌的组成及差异。结果显示，传粉者访问未显著改变花药微生物的多样性及组成，套袋组中优势真菌科为枝孢菌科与球腔菌科，访问组优势真菌科则为枝孢菌科与梅奇酵母科。此外，两组中优势细菌科均为产碱杆菌科与欧文菌科。研究结果表明，枇杷花药微生物具有相对稳定的群落结构，传粉者不是驱动花药微生物群落构建的主要因素。
Abstract:Fruit trees, vital to global agriculture, depend heavily on pollinators to facilitate successful reproduction and ensure optimal yield. Microorganisms associated with anthers can influence pollen viability, and their community composition may be affected by pollinator activity. While understanding the diversity and community assembly patterns of these microorganisms has potential implications for enhancing the reproductive fitness of fruit trees, research in this area remains relatively scarce. This study employed high-throughput sequencing to analyze the diversity and community structure of fungi and bacteria on the anthers of loquat (Eriobotrya japonica (Thunb.) Lindl.) before (bagged group) and after (nature group) pollinator visitation. Results showed that pollinator visitation had no significant effect on the diversity or composition of anther microbiomes. In the bagged group, dominant fungal taxa included Cladosporiaceae and Mycosphaerellaceae, whereas Cladosporiaceae and Metschnikowiaceae were dominant in the nature group. Bacterial communities in both groups were also dominated by Alcaligenaceae and Erwiniaceae. These findings indicate that the microbial community composition within loquat anthers is inherently stable and largely unaffected by pollinator activity.
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图 1 枇杷花药微生物群落稀释曲线
基于香农指数（Shannon）展示枇杷套袋组（红色）与访问组（蓝色）花药真菌（A）与细菌（B）群落的稀释曲线。
Figure 1. Rarefaction curves of microbial community in anthers of Eriobotrya japonica based on Shannon index
Rarefaction curves based on Shannon index, showing fungal (A) and bacterial (B) diversity in bagged (red) and nature (blue) groups.
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图 2 枇杷花药微生物群落结构（科水平）
A：以ITS2测序表征的平均丰度>1%的套袋组与访问组真菌科组成；B：以16S测序表征的平均丰度>1%的套袋组与访问组细菌科组成。图中仅展示各实验组中丰度占比前10的微生物，其余物种合并为“其他”。
Figure 2. Structure of microbial community in anthers of Eriobotrya japonica (family level)
A: Composition of fungal families with an average abundance greater than 1%, characterized by ITS2 sequencing, in nature and bagged groups; B: Composition of bacterial families with an average abundance greater than 1%, characterized by 16S sequencing, in nature and bagged groups. Only the top 10 most abundant microbes in each experimental group are shown in the figure, with all other species combined under “Others”.
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图 3 枇杷花药微生物Alpha多样性分析（Shannon指数）
基于香农指数展示枇杷访问组（蓝色）与套袋组（红色）花药真菌（A）与细菌（B）的Alpha多样性。利用Wilcoxon秩和检验比较两个实验组中真菌与细菌Alpha多样性的差异，箱线图显示了中值和四分位距，ns表示两个实验组间不存在显著差异。
Figure 3. Alpha diversity analysis (Shannon index) of microbial community in anthers of Eriobotrya japonica
Alpha diversity of fungal (A) and bacterial (B) communities in anthers of Eriobotrya japonica in nature (blue) and bagged (red) groups is presented based on Shannon index. Differences in fungal and bacterial alpha diversity between two experimental groups were assessed using Wilcoxon rank-sum test. Boxplot illustrates median and interquartile range, with “ns” signifying no significant difference.
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图 4 枇杷花药微生物群落的NMDS分析
基于Bray-Curtis距离对枇杷花药微生物群落进行非度量多维尺度（NMDS）排序，对真菌（A）和细菌（B）进行了单独排序。蓝色实心圆圈代表访问组样本，红色实心圆圈代表套袋组样本。绘制分组椭圆展示样本点之间的分布情况。基于Bray-Curtis距离算法，利用PERMANOVA比较套袋组与访问组真菌与细菌群落组成，P<0.05表示具有显著差异。
Figure 4. Non-metric multidimensional scaling (NMDS) of microbial community in anthers of Eriobotrya japonica
Non-metric multidimensional scaling (NMDS) ordination of microbial communities in anthers of Eriobotrya japonica based on Bray-Curtis distance was performed, with separate analyses for fungi (A) and bacteria (B). Blue solid circles represent samples from nature group, red solid circles represent samples from bagged group. Group ellipses are drawn to present distribution of sample points. Based on Bray-Curtis distance algorithm, PERMANOVA was used to compare fungal and bacterial community compositions between bagged and nature groups, with P<0.05 indicating significant differences.
表 1 使用不同距离算法的PERMANOVA分析汇总
Table 1 Summary of permutation multivariate analysis of variance (PERMANOVA) using different distance algorithms
Group df Bray-Curtis Unweighted-UniFrac Weighted-UniFrac F R2 P F R2 P F R2 P 真菌 1 1.423 0.262 0.098 1.387 0.257 0.100 1.233 0.236 0.200 细菌 1 2.167 0.351 0.100 0.991 0.198 0.500 0.473 0.106 0.900 
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