赣州橘园根系内生丛枝菌根真菌群落多样性鉴定及其受黄龙病菌侵染的影响

Identification of root endophytic arbuscular mycorrhizal fungi community diversity and its variations under the infection of Candidatus Liberibacter asiaticus in the citrus orchard of Ganzhou city

[目的]鉴定赣州橘园黄龙病菌侵染和健康柑橘根系内生丛枝菌根真菌(AMF)的多样性,并明确黄龙病菌的侵染对于AMF群落的影响。[方法]使用AMF18S小亚基核糖体特异引物AMV4.5NF/AMDGR对黄龙病菌侵染和健康柑橘根系DNA扩增建库,通过454高通量测序和生物信息学分析挖掘赣州橘园根系AMF多样性及其受黄龙病菌侵染的影响。[结果]从赣州橘园的柑橘根系中鉴定到80个AMF种,其中包括44个已知的AMF种和36个新种。进化分析发现,球囊霉属AMF占总AMF数的78.75%,是赣州柑橘根系内生AMF群落的优势菌属。鉴定到类球囊霉属的12个AMF,其中Paraglomus.N2和Paraglomus.N7的丰度位于总AMF的第二和第三,说明类球囊霉属AMF在赣州柑橘AMF群落中占有重要地位。PCoA分析表明黄龙病菌侵染显著改变了柑橘根系AMF的群落结构,而AMF群落的α多样性指数无显著性变化。通过丰度差异分析鉴定到6个在黄龙病菌侵染后丰度差异显著的AMF种,表明黄龙病菌侵染可能通过改变AMF菌种组成和相对丰度来影响其群落结构。[结论]全面揭示了赣州橘园根系内生AMF多样性,并且黄龙病菌侵染可通过改变菌种组成和相对丰度来影响AMF群落结构。

【Objective】Citrus trees have sparse root hairs and thus rely on Arbuscular Mycorrhizal Fungi (AMF) for mineral nutrient uptake. AMF play a significant role in plant growth, tolerance to biotic and abiotic stress and fruit quality. In this study, we employed a high- throughput sequencing of 18S rRNA gene colne library to determine the AMF community composition of Huanglongbing (HLB)-infected citrus roots and healthy citrus roots. The results would not only decipher the endophytic AMF diversity of citrus roots, but also reveal the effect of HLB infection on the citrus roots endophytic AMF community diversity.【Methods】The roots of the HLB-infected and healthy citrus trees (Mandarin(Citrus reticulata‘Unshiu’) grafted on Poncirus trifoliata) were sampled from a citrus orchard in Xunwu county of Ganzhou city. After removing the loose soil, lateral roots were collected and placed in sterile 50 mL tubes with 25 mL phosphate buffer (per liter: 6.33 g of NaH2PO4?H2O, 16.5 g of Na2HPO4?7H2O, 200 μL Silwet L-77). Subsequently, the tubes were vortexed at a maximum speed for 1 min to remove the attached soil, and the buffer was refreshed until the buffer was clear after vortex. Then the clean roots were washed in an ultrasonic cleaner for 6 min to remove the tiny attached soil. The total DNA of citrus roots and AMF were extracted from the clean citrus lateral roots using modified CTAB method, and the AMF 18S small subunit region of ribosomal RNA gene (18S SSU rRNA) specific primer pair AMV4.5NF/AMDGR was used to establish the sequencing libraries. After 454 GX FLX pyrosequencing, the raw sequencing data were trimmed by MOTHUR software to obtained the high-quality reads:(1) Reads which carried the correct barcode and forward primer sequences;(2) Reads were more than 200 bp in length. After denoising with PyroNoise and removing the chimeras sequences, the remaining sequences were considered as clean reads. QIIME (Quantitative Insights Into Microbial Ecology) software was utilized to generate OTUs with 97% identity threshold, and the most abundant sequence from each OTU was selected as the representative sequences. Then representative sequences were identified by searching in the Maarjam database. For phylogenetic analysis, the representative sequences of all AMF species were aligned using MAFFT (Multiple sequence alignment program), and a neighbor-joining phylogenetic analysis of all the AMF species was generated on the alignment result by TOPALi V2.5 (F84 model with gamma substitution rates and bootstrapping over 100 runs). For the α diversity analysis, Simpson Index, Shannon Index, Observed species (Sobs) and Chao1 Index were used. QIIME was utilized to calculate the AMF diversity (Shannon Index and Simpson Index) and richness (Observed species and Chao1 Index) indices of different samples. PCoA (Principal coordinates analysis) was used to compare the difference of endophytic AMF community structures between the HLB infected and healthy citrus roots. PCoA map was performed based on the unweighted Unifrac distance metrics by R software.【Results】Based on the high-throughput sequencing of AMF 18S SSU rRNA fragments, a total of 80 AMF molecular species were identified from the healthy and the HLB-infected citrus roots (Mandarin grafted on the Poncirus trifoliata) sampled from a citrus orchard in Xunwu county of Ganzhou city. Among which, 44 AMF molecular species were known deposited in Maarjam database and the remained 36 AMF molecular species were new species. From the phylogenetic analysis, the Glomus AMF were accounting for 78.75% of the total AMF species, indicating that Glomus was the dominant genus in the AMF community of citrus roots in Ganzhou city. In addition, 12 Paraglomus AMF species were also identified, and the Paraglomus.N2 and Paraglomus.N7 were the 2nd and 3rd abundant AMF species in this community, revealing the important position of Paraglomus in the AMF community of citrus roots in Ganzhou city. Based on the PCoA analysis, the HLB infection significantly altered the AMF community of citrus roots. However, no significant difference was observed in the total richness and diversity of AMF community between the HLB infected samples and the non-infected samples. Subsequently, the relative abundance of 6 AMF species were found to be changed significantly during the HLB infection. This phenomenon revealed that HLB infection might affect the citrus roots AMF community through altering the composition and relative abundance of AMF species.【Conclusion】Glomus AMF species were the dominant AMF species in the citrus roots of Ganzhou city, and Paraglomus AMF species also played an important role in the AMF community of citrus roots in Ganzhou city. In addition, the HLB infection significantly altered the endophytic AMF community structures of citrus roots (especially the composition and relative abundance of several AMF species), while the total α diversity was not changed. We hypothesized that the HLB infection might not affect the citrus roots AMF community through altering the composition of carbohydrates, but the actual pathway remains to be further explored.