Abstract
Citrus Decline Disease was recently reported to affect several citrus species in Iran when grafted on a local rootstock variety, Bakraee. Preliminary studies found “Candidatus Phytoplasma aurantifoliae” and “Candidatus Liberibacter asiaticus” as putative etiological agents, but were not ultimately able to determine which one, or if an association of both, were causing the disease. The current study has the aim of characterizing the microbiota of citrus plants that are either asymptomatic, showing early symptoms, or showing late symptoms through amplification of the V1–V3 region of 16S rRNA gene using an Illumina sequencer in order to (i) clarify the etiology of the disease, and (ii) describe the microbiota associated to different symptom stages. Our results suggest that liberibacter may be the main pathogen causing Citrus Decline Disease, but cannot rule out the possibility of phytoplasma being involved as well. The characterization of microbiota shows that the leaves show only two kinds of communities, either symptomatic or asymptomatic, while roots show clear distinction between early and late symptoms. These results could lead to the identification of bacteria that are related to successful plant defense response and, therefore, to immunity to the Citrus Decline Disease.
Highlights
Since 2010, a declining condition on trees belonging to different citrus species has widely appeared in various groves of the Southern Kerman region of Iran and has subsequently killed around 10% of cultivated citrus trees
Liberibacter were detected in all five symptomatic roots samples, regardless of symptom stage (Table 1). These amplified fragments were sequenced and the comparison with the database showed that the phytoplasma sequences belonged to the “Ca. P. aurantifolia” species while the liberibacter sequences belonged to the “Ca. L. asiaticus” species
For each plant sanitary status and organ, rarefaction curves were described (Figure S2). Of these operational taxonomic units (OTUs), most were exclusive to the root compartment (1486), followed by those shared among leaf and root (687), and the fewest were identified in leaf only (526) (Figure 1a). These results indicate that relatively few bacterial OTUs are shared among the leaf and root compartments, identifying very diverse microbiota in the different organs
Summary
Since 2010, a declining condition on trees belonging to different citrus species has widely appeared in various groves of the Southern Kerman region of Iran and has subsequently killed around 10% of cultivated citrus trees This condition, called Citrus Decline Disease (CDD), causes symptoms that have been classified as (i) early CDD symptoms, which include leaves developing a pale green color, limited production of fresh sprouts, and in general a retardation of growth (Figure S1a), and (ii) late CDD symptoms which manifest as an evident tree decline along with reduction and decay of the root system (Figure S1b) [1]. Affected plants die approximately 5 years after the development of the first symptoms These symptoms, similar to those associated with obligate bacterial parasites, brought to the identification of putative etiological agents of the disease in both phytoplasma and liberibacter, as these microorganisms were detected by PCR in infected plants and not in healthy plants [1]. The bacterial microbiota of asymptomatic plants, early symptomatic plants, and late symptomatic plants, determined both from leaf and root, was described and compared with the aims of (i) clearing the etiology of the disease and (ii) identifying the shifts in the microbial community linked to the development of the disease, in order to identify possible taxonomical units associated to a diseased or healthy state
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