Table_2_Xylella fastidiosa Infection Reshapes Microbial Composition and Network Associations in the Xylem of Almond Trees.docx

Xylella fastidiosa represents a major threat to important crops worldwide including almond, citrus, grapevine, and olives. Nowadays, there are no efficient control measures for X. fastidiosa, and the use of preventive measures and host resistance represent the most practical disease management strategies. Research on vessel-associated microorganisms is gaining special interest as an innate natural defense of plants to cope against infection by xylem-inhabiting pathogens. The objective of this research has been to characterize, by next-generation sequencing (NGS) analysis, the microbial communities residing in the xylem sap of almond trees affected by almond leaf scorch disease (ALSD) in a recent X. fastidiosa outbreak occurring in Alicante province, Spain. We also determined community composition changes and network associations occurring between xylem-inhabiting microbial communities and X. fastidiosa. For that, a total of 91 trees with or without ALSD symptoms were selected from a total of eight representative orchards located in five municipalities within the X. fastidiosa-demarcated area. X. fastidiosa infection in each tree was verified by quantitative polymerase chain reaction (qPCR) analysis, with 54% of the trees being tested X. fastidiosa-positive. Globally, Xylella (27.4%), Sphingomonas (13.9%), and Hymenobacter (12.7%) were the most abundant bacterial genera, whereas Diplodia (30.18%), a member of the family Didymellaceae (10.7%), and Aureobasidium (9.9%) were the most predominant fungal taxa. Furthermore, principal coordinate analysis (PCoA) of Bray–Curtis and weighted UniFrac distances differentiated almond xylem bacterial communities mainly according to X. fastidiosa infection, in contrast to fungal community structure that was not closely related to the presence of the pathogen. Similar results were obtained when X. fastidiosa reads were removed from the bacterial data set although the effect was less pronounced. Co-occurrence network analysis revealed negative associations among four amplicon sequence variants (ASVs) assigned to X. fastidiosa with different bacterial ASVs belonging to 1174-901-12, Abditibacterium, Sphingomonas, Methylobacterium–Methylorubrum, Modestobacter, Xylophilus, and a non-identified member of the family Solirubrobacteraceae. Determination of the close-fitting associations between xylem-inhabiting microorganisms and X. fastidiosa may help to reveal specific microbial players associated with the suppression of ALSD under high X. fastidiosa inoculum pressure. These identified microorganisms would be good candidates to be tested in planta, to produce almond plants more resilient to X. fastidiosa infection when inoculated by endotherapy, contributing to suppress ALSD.

Xylella fastidiosa作为一种全球重要作物，如杏仁、柑橘、葡萄树和橄榄的主要威胁，其存在对农业生产构成了严重挑战。目前，针对X. fastidiosa尚无有效的控制措施，而采取预防措施和宿主抗性则成为最为实际且有效的疾病管理策略。随着对植物木质部相关微生物的研究日益受到重视，这些微生物被视为植物对抗木质部病原体感染的固有天然防御机制。本研究旨在利用下一代测序（NGS）技术，对西班牙阿利坎特省近期爆发X. fastidiosa所导致的杏仁叶片枯萎病（ALSD）受影响杏仁树的木质部汁液中居住的微生物群落进行表征。同时，本研究还确定了木质部居住微生物群落与X. fastidiosa之间的群落组成变化和网络关联。为此，从位于X. fastidiosa划定的区域内的五个市镇的八个代表性果园中，选取了91棵表现有或无ALSD症状的树木。通过定量聚合酶链反应（qPCR）分析验证了每棵树上的X. fastidiosa感染，其中54%的树木检测结果为X. fastidiosa阳性。在全球范围内，Xylella（27.4%）、Sphingomonas（13.9%）和Hymenobacter（12.7%）是最丰富的细菌属，而Diplodia（30.18%），属Didymellaceae家族（10.7%）和Aureobasidium（9.9%）则是最主要的真菌类群。此外，基于Bray-Curtis距离和加权UniFrac距离的主坐标分析（PCoA）显示，杏仁木质部细菌群落主要根据X. fastidiosa感染进行区分，而真菌群落结构则与病原体的存在关系不大。尽管影响较小，但当从细菌数据集中移除X. fastidiosa序列时，也获得了类似的结果。共现网络分析揭示了X. fastidiosa的四个扩增子序列变体（ASVs）与属于1174-901-12、Abditibacterium、Sphingomonas、Methylobacterium–Methylorubrum、Modestobacter、Xylophilus以及未鉴定属Solirubrobacteraceae的非识别成员之间的负关联。确定木质部居住微生物与X. fastidiosa之间的紧密关联，有助于揭示与抑制高X. fastidiosa接种量下ALSD的特定微生物。这些被确定的微生物将成为植病测试的良好候选者，通过内疗法接种，以提高杏仁树对X. fastidiosa感染的抗性，从而有助于抑制ALSD。