Rapid screening for citrus canker resistance employing pathogen-associated molecular pattern-triggered immunity responses

Citrus canker,caused by the bacterial pathogen Xanthomonas citri ssp.citri(Xcc),has been attributed to millions of dollars in loss or damage to commercial citrus crops in subtropical production areas of the world.Since identification of resistant plants is one of the most effective methods of disease management,the ability to screen for resistant seedlings plays a key role in the production of a long-term solution to canker.Here,an inverse correlation between reactive oxygen species(ROS)production by the plant and the ability of Xcc to grow and form lesions on infected plants is reported.Based on this information,a novel screening method that can rapidly identify citrus seedlings that are less susceptible to early infection by Xcc was devised by measuring ROS accumulation triggered by a 22-amino acid sequence of the conserved N-terminal part of flagellin(flg22)from X.citri ssp.citri(Xcc-flg22).In addition to limiting disease symptoms,ROS production was also correlated with the expression of basal defense-related genes such as the pattern recognition receptors LRR8 and FLS2,the leucine-rich repeat receptor-like protein RLP12,and the defense-related gene PR1,indicating an important role for pathogen-associated molecular pattern-triggered immunity(PTI)in determining resistance to citrus canker.Moreover,the differential expression patterns observed amongst the citrus seedlings demonstrated the existence of genetic variations in the PTI response among citrus species/varieties.

Molecular mechanisms underlying heat or tetracycline treatments for citrus HLB control

Huanglongbing(HLB),a destructive plant bacterial disease,severely impedes worldwide citrus production.In our previous reports,we revealed the molecular mechanisms of host plant responses that underlie thermotherapy against HLB.In this study,we investigated the molecular mechanism underlying heat or tetracycline treatments on the HLB bacterium,‘Candidatus Liberibacter asiaticus’(Las)by focusing on Las prophage/phage conversion under stress conditions.By comparing the prophage FP1 and FP2 copy number to the copy number of 16S rDNA in HLB-affected plants,we found that the relative copy number of both FP1 and FP2 increased significantly,ranging from 3.4-to 6.7-fold change when Las-infected samples underwent a temperature shift from 23 to 37,42 or 45°C.When treated with tetracycline at 50–150 and 200–250µg/ml,respectively,the relative copy number of both FP1 and FP2 increased by 3.4-to 6.0-fold.In addition,analyses of Las prophage structural gene and antirepressor gene copy numbers showed similar trends for all treatments.Furthermore,transmission electron microscopy provided direct evidence of lysogenic to lytic conversion upon temperature increase.These results not only provide new insight into the molecular mechanisms underlying heat or tetracycline treatment but also suggest a novel HLB control strategy by enhancing the endogenous conversion from Las prophages to phages.

Molecular mechanisms behind the accumulation of ATP and H_(2)O_(2) in citrus plants in response to‘Candidatus Liberibacter asiaticus’infection

Candidatus Liberibacter asiaticus(Las)is a fastidious,phloem-restricted pathogen with a significantly reduced genome,and attacks all citrus species with no immune cultivars documented to date.Like other plant bacterial pathogens,Las deploys effector proteins into the organelles of plant cells,such as mitochondria and chloroplasts to manipulate host immunity and physiology.These organelles are responsible for the synthesis of adenosine triphosphate(ATP)and have a critical role in plant immune signaling during hydrogen peroxide(H2O2)production.In this study,we investigated H2O2 and ATP accumulation in relation to citrus huanglongbing(HLB)in addition to revealing the expression profiles of genes critical for the production and detoxification of H2O2 and ATP synthesis.We also found that as ATP and H2O2 concentrations increased in the leaf,so did the severity of the HLB symptoms,a trend that remained consistent among the four different citrus varieties tested.Furthermore,the upregulation of ATP synthase,a key enzyme for energy conversion,may contribute to the accumulation of ATP in infected tissues,whereas downregulation of the H2O2 detoxification system may cause oxidative damage to plant macromolecules and cell structures.This may explain the cause of some of the HLB symptoms such as chlorosis or leaf discoloration.The findings in this study highlight important molecular and physiological mechanisms involved in the host plants’response to Las infection and provide new targets for interrupting the disease cycle.

Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus and enhances canopy growth by altering gene expression profiles in HLB-affected citrus plants

Huanglongbing(HLB),a systemic and destructive disease of citrus,is associated with‘Candidatus Liberibacter asiaticus’(Las)in the United States.Our earlier work has shown that Las bacteria were significantly reduced or eliminated when potted HLB-affected citrus were continuously exposed to high temperatures of 40 to 42°C for a minimum of 48 h.To determine the feasibility and effectiveness of solar thermotherapy in the field,portable plastic enclosures were placed over commercial and residential citrus,exposing trees to high temperatures through solarization.Within 3–6 weeks after treatment,most trees responded with vigorous new growth.Las titer in new growth was greatly reduced for 18–36 months after treatment.Unlike with potted trees,exposure to high heat did not eradicate the Las population under field conditions.This may be attributed to reduced temperatures at night in the field compared to continuous high temperature exposure that can be maintained in growth chambers,and the failure to achieve therapeutic temperatures in the root zone.Despite the presence of Las in heat-treated commercial citrus,many trees produced abundant flush and grew vigorously for 2 to 3 years after treatment.Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls for most differentially expressed genes and that genes involved with plant-bacterium interactions are upregulated after heat treatment.Overall,these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB.

Beneficial horticultural responses from the application of solar thermotherapy to mature Huanglongbing-affected citrus trees

The detrimental effects of Huanglongbing(HLB)on citrus are well known and so is the need for effective methods to combat this disease.Solar thermotherapy(ST),one of the management methods to help alleviate some of the negative effects of HLB,is the process of heating trees to therapeutic temperatures by encompassing the tree within a plastic structure(Visqueen)to harness the sun’s natural energy.ST was applied to mature‘Valencia’citrus trees in three locations.Tree vigor,yield,and Candidatus Liberibacter asiaticus(Las)titer in leaves were monitored for two consecutive seasons post ST treatment,while fruit and juice quality were evaluated at the end of the second season.ST promoted an increase in canopy density in most groves tested and did not have a significant effect on fruit quantity,despite the prolonged exposure of the trees to increased temperatures.Moreover,Las titer was reduced in both leaves and juice processed from affected trees post ST.With respect to fruit quality,the ratio of total soluble solids to titratable acidity was higher post ST compared to controls as was the sucrose content of the juice in most treated groves,whereas limonin was higher in juice from control trees.Principal components analysis of aroma volatiles showed significant differences between juice from treated versus control trees with volatiles imparting top-note and freshness to orange juice,such as acetaldehyde,hexanal,Z-3-hexenol and linalool,being higher in juice fromtreated trees.Taste panels confirmed that flavor differences existed,ultimately showing that ST resulted in improved juice flavor in well-managed groves.Although the effects of ST on the trees appear temporary,such outcomes emphasize its utility when used as part of an integrated management strategy for HLB-affected citrus,imparting beneficial horticultural responses with minimal to positive effects on subsequent juice flavor.