Plant volatiles play an important role in defending plants against insects and pathogens attack. Released volatiles from insect-damaged plants may result in direct or indirect defense against insect, and volatile accumulation in pathogen-infected plants may inhibit the movement of the pathogens within plant tissues.  However, available information about the response of citrus leaf volatiles to Asian citrus psyllids (ACP) feeding and ‘Candidatus Liberibacter asiaticus’ (Clas) infection is limited.  Here we investigate the effect of ACP feeding, Clas infection, and simultaneous attack by ACP and Clas on the volatile content of Valencia leaves.  Leaf volatiles were extracted using hexane and analyzed with gas chromatography-mass spectrometry (GC-MS).  Eighteen out of  twenty-seven detected volatiles were induced (2 to 10-folds) in ACP-infested plants.  On the other hand, only four volatiles were induced in Clas-infected plants (d-limonene, β-phelandrene, citronellal, and undecanl).  The abundance of latter compounds was induced 4-folds in Clas-infected plants, except for limonene, which was induced  more than 20-folds.  In addition, citrus plants that were subjected to  a simultaneous attack by ACP and Clas reduced their volatiles production compared to those that experience an attack only by ACP.  Our results suggested that insect and pathogen attack not only changes the profile of plant released volatiles, but also influences the volatile contents of the attacked plants. The result of this study might contribute to better understanding of citrus response to ACP and Clas attacks and provide more information about the relation between stored and released volatiles.

The honeydew composition of Asian citrus psyllids (ACP), the vector of citrus Huanglongbing (HLB), was studied using gas chromatography-mass spectrometry (GC-MS). Honeydew samples were collected from healthy ACP that were reared on one-year Valencia trees inside an insectary at a University of Florida’s Citrus Research and Education Center (CREC) grove in Lake Alfred, FL, USA. Dried samples (1 mg) were mixed with 30 μL of methoxyamine hydrochloride solution in pyridine (2%) and allowed to react for 17 h at room temperature. After methoximation, silylation reactions were induced by adding 80 μL of N-methyl-N-trimethylsilyl) trifluoracetamide (MSTFA) for 2 h at room temperature and 0.5 μL of derivatized sample was injected into the GC-MS running in the full scan mode. To check for amino acids, a 10 mg of alkaline honeydew sample was reacted with methylchloroformate (MCF) in a mixture of pyridine and methanol. The MCF derivatives were extracted with chloroform and analyzed with GC-MS. The moisure content was determined by drying the honeydew samples to a constant weight at a 100 °C. The major honeydew composition was as follows: 74.5 ± 2.8 sucrose, 12.4 ± 0.5 D-fructose, 6.4 ± 3.0 mannose,  1.8 ± 0.6 trehalose, myo-inositol 2.8 ± 0.8, ribitol 0.5 ± 1, galactose 0.4 ± 0.2, quinic acid 0.4± 0.3, and malic acid 0.3 ± 0.1. The moisture content of honeydew was 22.6 ± 2.6 No amino acids were detected as TMS or as MCF derivatives.

Huanglongbing (HLB), the most serious disease of citrus, is attributed in the United States to Candidatus Liberibacter asiaticus (CLas), a gram-negative, phloem-restricted α-proteobacterium transmitted by the Asian citrus psyllid (Diaphorina citri). Despite the fact that the psyllid is well recognized as the vector of CLas, to the best of our knowledge, little research has so far been conducted on molecular interactions between CLas and the psyllid. Many gram-negative bacterial pathogens have been shown to adhere to insect cell surface by interactions between receptors and ligands, establishing protein complexes that help them enter into insect cells. In the present study, Far-western (protein overlay assay) was used to seek receptors, two-dimensional blue native/SDS-PAGE to explore complexome (receptor-ligand), and MALDI TOF MS/MS to identify the receptors and ligands. We showed how Clas adhered to psyllid cells and which protein complexes were established on the cell membrane.  Understanding how CLas interacts with the insect cells should help in the development of new HLB control strategies.