Ahmed, Ayesha

Abstract Background Phloem limited non-culturable bacteria Candidatus Liberibacter asiaticus (CLas) affects the worldwide citrus production through causing citrus Huanglongbing (HLB). Despite the efficient colonization of citrus endophyte in the phloem as same niche as CLas pathogen, citrus microbiome manipulation and recruitment as well as citrus defense mechanisms in the presence of indigenous citrus endophyte against this pathogen are still unknown.Results Endophyte-mediated microbiome manipulation may potentially play a significant role in restoration of disease suppressive endophytic microbiome in vascular pathogen affected diseased plants and positively influence the citrus defense. For this, citrus endophyte Bacillus subtilis L1-21 was introduced in CLas-infected citrus groves for one year and pathogen reduction from 105 to 10 copies/gram/leaves was recorded. Resident bacterial community composition in diseased host dramatically changed after introduction of B. subtilis L1-21 and positive enrichment of certain bacteria was recorded in diseased citrus host. These enrichments were predominantly driven by high and low relative abundance of Bacillus and CLas pathogen respectively, after one year of endophyte application. Moreover, endophyte application resulted in citrus defence gene induction against CLas pathogen and demonstrated key resistance genes (PR-1, PR-4, RPS5, RBOHD) in endophyte-pathogen interaction pathway in infected citrus. Upon introduction of B. subtilis L1-21 in the diseased citrus plants, we identified high level of up-regulated genes (> 2-fold) involved in defense pathway (padj < 0.05) underpinning the fundamental defense mechanisms.Conclusion Thorough evaluation of disease suppressive mechanism of endophyte against pathogen requires further exploration. However, introduction of B. subtilis L1-21 restructured citrus microbiome by regulating key bacterial communities which might help plant to control this pathogen. In addition, we highlight advanced insights regarding activation of multiple disease resistance and secondary metabolites encoding genes in endophyte treated HLB-infected citrus plants showing potential resistance against CLas pathogen. Conclusively, endophyte-mediated manipulation could play decisive role in restoration of microbiome to positively influence the citrus defense.

Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas) brings a great concern about the phloem nutrient transport in diseased plants. There is an urgent need to find the best management strategies to reduce the losses in the citrus industry worldwide. Endophytic bacteria are negatively affected by CLas pathogen, and these endophytes are associated with improved availability of nutrients and pathogen resistance. This study underpins the relationship between CLas pathogen, endophyte population and nutrients availability in citrus plants. The citrus plants were treated with Bacillus subtilis L1-21 and Hoagland solution to find out synergism efficacy to mitigate citrus HLB. We showed that citrus shoots in the presence of 50% Hoagland solution displayed maximum number of endophytes with 6.28 × 103 to 3.04 × 105 CFU/g. Among 50 candidate strains, B. subtilis L1-21 emerged as potential antagonist against surrogate strain Xanthomonas citri subsp. citri. The citrus half-leaf method identified that application of endophyte L1-21 with 50% Hoagland solution successfully reduces the CLas abundance. We point out that this combination results in a higher number of endophytes population with 2.52 × 104 to 9.11 × 106 CFU/g after 60 days, and reduces CLas pathogen abundance in asymptomatic HLB plants. In HLB symptomatic citrus plants, B. subtilis L1-21 potentially increases the endophyte population from 1.11 × 104 to 5.26 × 107 CFU/g in the presence of Hoagland solution, and pathogen abundance was reduced from 9.51 × 105 to 1.06 × 104 copies/g. Altogether, we suggested that the presence of endophyte L1-21 with Hoagland solution is more effective in HLB asymptomatic citrus plants, but a slight reduction of pathogen was observed in symptomatic plants. The findings revealed the role of indigenous citrus endophyte B. subtilis L1-21 along with other nutrients in the reduction of CLas pathogen abundance inside symptomatic and asymptomatic plants in citrus endophyte–nutrient–pathogen interplay.