Citrus Huanglongbing is a pathogen-triggered immune disease that can be mitigated with antioxidants and gibberellin

Abstract

Huanglongbing (HLB) is a devastating disease of citrus, caused by the phloem-colonizing bacterium Candidatus Liberibacter asiaticus (CLas). Here, we present evidence that HLB is an immune-mediated disease. We show that CLas infection of Citrus sinensis stimulates systemic and chronic immune responses in phloem tissue, including callose deposition, production of reactive oxygen species (ROS) such as H2O2, and induction of immunity-related genes. The infection also upregulates genes encoding ROS-producing NADPH oxidases, and downregulates antioxidant enzyme genes, supporting that CLas causes oxidative stress. CLas-triggered ROS production localizes in phloem-enriched bark tissue and is followed by systemic cell death of companion and sieve element cells. Inhibition of ROS levels in CLas-positive stems by NADPH oxidase inhibitor diphenyleneiodonium (DPI) indicates that NADPH oxidases contribute to CLas-triggered ROS production. To investigate potential treatments, we show that addition of the growth hormone gibberellin (known to have immunoregulatory activities) upregulates genes encoding H2O2-scavenging enzymes and downregulates NADPH oxidases. Furthermore, foliar spray of HLB-affected citrus with gibberellin or antioxidants (uric acid, rutin) reduces H2O2 concentrations and cell death in phloem tissues and reduces HLB symptoms. Thus, our results indicate that HLB is an immune-mediated disease that can be mitigated with antioxidants and gibberellin.

Candidatus Liberibacter asiaticus reduces callose and reactive oxygen species production in the phloem

“…Previous research demonstrated that ROS was elevated in citrus leaves following C Las infection (Ma et al, 2022; Pitino et al, 2017). However, ROS has not been quantified in infected seed vasculatures, where our results indicated that bacteria are present in large numbers.…”

“…This corresponded with a lower expression of the CsCalS and CsRBOH genes. Previously it was shown that callose and ROS metabolism increase in infected plants (Ma et al, 2022; Pitino et al, 2017; Granato et al, 2019), but these observations were made in the leaf tissue, where C Las levels are low. Our results suggest that these increases include both the activation by the host and inhibition by the pathogen, but because the pathogen is absent from most SE in the leaf, the net effect is an increase.…”

A chromosome-level phased Citrus sinensis genome facilitates understanding Huanglongbing tolerance mechanisms at the allelic level in an irradiation-induced mutant

“…Downregulation of a few HSPs has been reported in HLB susceptible citrus after CLas infection 46,[61][62][63] , while upregulation of them was putatively related to enhanced HLB tolerance 46 . Ma et al (2022) inferred the accumulation of reactive oxygen species (ROS) to be caused by chronic immune responses in the phloem tissue 64 . However, there is no proof that CLas could be recognized by citrus within the phloem, which is a prerequisite for inducing immune responses.…”

“…Enhanced NPR1 dependent defense 41,42 or high-level antimicrobial peptide expression 43–45 ; B. Promoted protein and cellular homeostasis due to upregulation of molecular chaperones (mainly heat shock proteins); C. Inhibition of programmed cell death by high-level of antioxidant metabolites or enhanced detoxication pathways 46,48,64 ; D. Increased functional phloem ratio from novel high phloem regeneration rate 15,49 .…”

An Overview of the Mechanisms Against “Candidatus Liberibacter asiaticus”: Virulence Targets, Citrus Defenses, and Microbiome

“…Recent research indicates that elevated levels of manganese (Mn) promote better tree response to the effects of HLB increasing citrus tree lifespan ( Morgan et al, 2016 ; Zambon et al, 2019 ). Sufficient Mn in the rhizosphere is critical for scavenging ROS ( Alscher et al, 2002 ), which is known to be produced extensively in C Las-damaged cells ( Ma et al, 2022 ). Although many nutrients can mitigate symptoms of HLB-affected trees, the mechanisms of how these nutrients trigger citrus defenses are still unclear and warrant investigation.…”