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‘Candidatus Liberibacter asiaticus’ (Las) is the prevalent species of three different Liberibacter associated with citrus huanglongbing (HLB). Two psyllid species, Diaphorina citri and Trioza erytreae, are currently known to transmit Liberibacter bacteria. In this study, we tested the acquisition and transmission efficiency of Las by striped mealybugs (Ferrisia virgata) (Pseudococcidae; Hemiptera), another phloem-sap feeding insect with a broad host range of 264 species in 68 plant families. In our previous report, 63.0 % of striped mealybugs collected from the Las-infected periwinkle plants in USHRL greenhouse tested positive for Las using the HLBasp primers and probe, and the Las populations were estimated at 3.11 x 103 to 2.32 x 105 cells per mealybug. This was confirmed using conventional PCR with six primer sets targeting different Las loci and by the 100% identity of all seven PCR amplicons to the known Las sequences. However, attempts to transmit the disease in periwinkle and citrus using Las-infected mealybugs were not successful. To reveal the reason why Las-infected mealybugs were not able to transmit the disease, we used a leaf-disc bioassay in conjunction with typing of Las populations. Positive Las results were found in 100% of the mealybugs after feeding for 1-2 weeks on infected leaf discs that were obtained from infected periwinkle and citrus leaves. In addition, Las bacteria were detected in mealybug gut, salivary glands and body cavity, with the titer in the gut and body being higher than that in the salivary glands. It is of interest to note that mealybugs grown on infected leaf-discs for 1 week and then transferred to non-infected leaf-discs did not test positive for Las. However, mealybugs grown on infected leaf-discs for 2 weeks and then transferred to non-infected leaf-discs for 2 more weeks remained positive for Las. These results indicate that striped mealybugs share similarities and differences in comparison with the Asian citrus psyllids in terms of acquisition and/or transmission of the Las bacterium.

Background Candidatus Liberibacter solanacearum (Lso) is a phloem-limited alphaproteobacterium associated with the devastating zebra chip disease of potato (Solanum tuberosum). Like other members of Liberibacter, Lso-ZC1 encodes a flagellin domain-containing protein (FlaLso,) with a conserved 22 amino-acid peptide (flg22Lso). To understand the innate immune responses triggered by this unculturable intracellular bacterium, we studied the pathogenassociated molecular patterns (PAMPs) that triggered immunity in Nicotiana benthamiana, using the flg22Lso peptide and the full length flaLso gene. Results Our results showed that the expression of flaLso via Agrobacterium-mediated transient expression induced a slow necrotic cell death in the inoculated leaves of N. benthamiana, which was coupled with a burst of reactive oxygen species (ROS) production. Moreover, the expression of several representative genes involved in innate immunity was transiently upregulated by the flg22Lso in N. benthamiana. The FlaLso, however, induced stronger upregulation of these representative genes compared to the flg22Lso, especially that of flagellin receptor FLAGELLIN SENSING2 (FLS2) and respiratory burst oxidase (RbohB) in N. benthamiana. Although neither cell death nor ROS were induced by the synthetic flg22Lso, a weak callose deposition was observed in infiltrated leaves of tobacco, tomato, and potato plants. Conclusion The flagellin of Lso and its functional domain, flg22Lso share characteristics of pathogenassociated molecular patterns, and trigger unique innate immune responses in N. benthamiana. Slow and weak activation of the innate immune response in host plants by the flagellin of Lso may reflect the nature of its intracellular life cycle. Our findings provide new insights into the role of the Lso flagellin in the development of potato zebra chip disease and potential application in breeding for resistance. [ABSTRACT FROM AUTHOR]

‘Candidatus Liberibacter asiaticus’ (Las) is the primary causal agent of huanglongbing (HLB), the most devastating disease of citrus worldwide. There are three known insect vectors of the HLB-associated bacteria, and all are members of the Hemiptera: Diaphorina citri (Psyllidae), Trioza erytreae (Triozidae), and Cacopsylla (Psylla) citrisuga (Psyllidae). In this study, we found that another hemipteran, the striped mealybug Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae), was able to acquire and retain Las bacteria. The bacterial titers were positively correlated with the feeding acquisition time on Las-infected leaf discs, with a two-weeks feeding period resulting in Ct values ranging from 23.1 to 36.1 (8.24×107 to 1.07×104 Las cells per mealybug). We further discovered that the prophage/phage populations of Las in the mealybugs were different from those of Las in psyllids based on Las prophage-specific molecular markers: infected psyllids harbored the Las populations with prophage/phage FP1 and FP2, while infected mealybugs carried the Las populations with the iFP3 being the dominant prophage/phage. As in the psyllids, Las bacteria were shown to move through the insect gut wall to the salivary glands after being ingested by the mealybug based on a time-course quantitative polymerase chain reaction (qPCR) assay of the dissected digestive systems. However, Las populations transmitted by the mealybugs did not cause disease in host plants. This is the first evidence of genetic difference among Las populations harbored by different insect vectors and difference among Las populations with respect to whether or not they cause disease in host plants. [ABSTRACT FROM AUTHOR]

Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease. [ABSTRACT FROM AUTHOR]

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed. To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant's trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed. Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for testing and facilitate research and screening purposes.

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 (H 2 O 2 ) production. In this study, we investigated H 2 O 2 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 H 2 O 2 and ATP synthesis. We also found that as ATP and H 2 O 2 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 H 2 O 2 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., Competing Interests: Competing interestsThe authors declare no conflict of interest., (© The Author(s) 2017.)

Background: 'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited alphaproteobacterium associated with the devastating zebra chip disease of potato (Solanum tuberosum). Like other members of Liberibacter, Lso-ZC1 encodes a flagellin domain-containing protein (Fla Lso ) with a conserved 22 amino-acid peptide (flg22 Lso ). To understand the innate immune responses triggered by this unculturable intracellular bacterium, we studied the pathogen-associated molecular patterns (PAMPs) that triggered immunity in Nicotiana benthamiana, using the flg22 Lso peptide and the full length fla Lso gene., Results: Our results showed that the expression of fla Lso via Agrobacterium-mediated transient expression induced a slow necrotic cell death in the inoculated leaves of N. benthamiana, which was coupled with a burst of reactive oxygen species (ROS) production. Moreover, the expression of several representative genes involved in innate immunity was transiently up-regulated by the flg22 Lso in N. benthamiana. The Fla Lso , however, induced stronger up-regulation of these representative genes compared to the flg22 Lso , especially that of flagellin receptor FLAGELLIN SENSING2 (FLS2) and respiratory burst oxidase (RbohB) in N. benthamiana. Although neither cell death nor ROS were induced by the synthetic flg22 Lso , a weak callose deposition was observed in infiltrated leaves of tobacco, tomato, and potato plants., Conclusion: The flagellin of Lso and its functional domain, flg22 Lso share characteristics of pathogen-associated molecular patterns, and trigger unique innate immune responses in N. benthamiana. Slow and weak activation of the innate immune response in host plants by the flagellin of Lso may reflect the nature of its intracellular life cycle. Our findings provide new insights into the role of the Lso flagellin in the development of potato zebra chip disease and potential application in breeding for resistance.

Aphids are economically important pests that predominantly feed from the plant phloem. Genome, transcriptome, and proteome data are being generated for these insects, and predicted secreted proteins in aphid saliva have been identified. These secreted proteins are candidate effectors that may modulate plant processes and aid aphid colonization of plants. The next step is to develop post-genomics strategies to study the functions of identified aphid genes. One such strategy is to express aphid effector genes in planta to assess whether aphid effectors alter plant development and aphid survival and fecundity. A second strategy is to knock down the expression of aphid target genes by RNA interference (RNAi). In this chapter, we describe how to knock down aphid gene expression using plant-mediated RNAi. This strategy is useful for assessing the contribution of aphid effectors to aphid colonization of plants.