Early detection of Huanglongbing with EESI-MS indicates a role of phenylpropanoid pathway in citrus.

Huanglongbing (HLB), a devastating disease for citrus worldwide, is caused by Candidatus Liberibacter asiaticus (CLas). In this study, we employed a novel extractive electrospray ionization-mass spectrometry (EESI-MS) method to analyze the metabolites in leaves of uninfected and HLB-infected Newhall navel orange. The results showed that uninfected and HLB-infected leaves could be readily distinguished based on EESI-MS combined by multivariable analysis. Nine phenolic compounds involved in phenylpropanoid pathway, such as p-coumaric acid, naringin, and apigenin, were principal components to distinguish the leaves of uninfected and HLB-infected Newhall navel orange. Gene expression was also conducted to further explore the molecular mechanism of phenylpropanoid branch pathway in HLB. The expression of genes (4CL, HCT, CHI, CHS, CYP, and C12R) involved in phenylpropanoid branch pathway was increased in asymptomatic and early period of HLB-infected leaves, while decreased in later period of HLB-infected leaves. This study provides a novel method for early detection of citrus HLB and suggests the regulation mechanism of phenylpropanoid pathway in the interaction between citrus and CLas.

Metabolic response of Citrus limon to Asian citrus psyllid infestation revealed by EESI-MS and HPLC.

Asian citrus psyllid (ACP) causes direct and indirect damage to the citrus industry. Extractive electrospray ionization mass spectrometry (EESI-MS) and high performance liquid chromatography (HPLC) were used to detect the metabolites of C. limon leaves at 0, 12, 24, and 72 h after ACP treatment. The EESI-MS results showed that ACP infestation significantly affected metabolites within a short feeding duration with 8 metabolites identified. The metabolites in leaves of these four groups could be distinguished, with 55 peaks showing significant differences including methyl N-methylanthranilate, caffeic acid, and syringic acid. The quantification of 15 phenolic compounds with HPLC-UV method in C. limon leaves after ACP infestation showed that the total content of them reached a peak of 3504.69 µg g-1 at 12 h, with 9 phenolic compounds changing significantly (P < 0.05). A total of 21 metabolites identified in this study were involved in the biosynthesis pathways of flavonoid, flavone and flavonol, isoflavonoid and phenylpropanoid, and the degradation of aminobenzoate. Contents of epicatechin and caffeic acid increased with the feeding time of ACP as detected by both EESI-MS and HPLC. This may be related to plant defense. This study provides novel insights into the biochemical relationship of ACP and its host plants.

Direct identification and metabolomic analysis of Huanglongbing associated with Candidatus Liberibacter spp. in navel orange by MALDI-TOF-MS.

Citrus Huanglongbing (HLB) is one of the most destructive citrus diseases worldwide, and is associated with the phloem-limited plant pathogenic bacteria Candidatus Liberibacter species. However, there is a latency period during which newly infected trees do not show symptoms, creating challenges for the early detection of HLB. In order to establish a method for rapid detection and to assess the metabolite differences between healthy and HLB-affected Newhall navel oranges, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study asymptomatic and symptomatic leaf extracts compared with healthy leaves. The results showed that healthy, asymptomatic, and symptomatic leaves were distinguishable using MALDI-TOF-MS combined with multivariable analysis. Discriminant analysis (DA) results showed that the accuracy of discrimination of the training set and the success rate of cross-validation analysis were both 100%. Blind testing revealed that nine validation samples were correctly classified to their corresponding group. In addition, 32 MS peaks of metabolites had significant differences between healthy and different HLB-affected navel orange leaves. Among 32 MS peaks, the relative content of nine substances showed an upward trend, and 19 metabolites showed a downward trend, including a significant decrease in malate and citrate, with an increase in HLB bacterial populations. Malate and citrate were found to be specifically reduced in infected leaves regardless of whether symptoms appeared. Thus, they could be used as potential biomarkers for early detection of HLB, reducing false-negative results caused by uneven distribution of the HLB bacterial pathogen. This novel method can efficiently differentiate HLB-affected samples from the host population and provide new information for early detection of HLB in citrus. The entire process from metabolite extraction to the collection of mass spectra of citrus leaves was a simple operation, with no need for sterile conditions. We believe this strategy has significant implications for rapid and effective HLB screening. Graphical abstract.

In Situ Study of Metabolic Response of Arabidopsis thaliana Leaves to Salt Stress by Neutral Desorption-Extractive Electrospray Ionization Mass Spectrometry.

Salt stress is one of the most common factors limiting plant cultivation. In this study, metabolic responses to salt stress in Arabidopsis thaliana (A. thaliana) leaves were analyzed in situ by neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) without any sample pretreatment. Metabolic changes of A. thaliana leaves were observed in response to salt stress conditions, including the levels of serine, glutamic acid, arginine, cinnamic acid, ferulic acid, caffeic acid, protocatechuic acid, epicatechin, morin, myricetin, apigravin, and ß-cotonefuran. The content of serine increased under 50, 100, and 200 mM NaCl salt stress, reaching the highest level at 200 mM NaCl, but decreased under the maximum concentration of 300 mM NaCl. A similar phenomenon was observed for arginine, glutamic acid, cinnamic acid, caffeic acid, ferulic acid, and epicatechin, respectively, involved in the metabolic pathway of shikimate-phenylpropanoid. Both principal component analysis (PCA) and partial least-squares discrimination analysis (PLS-DA) showed that the salt stress treatment groups of the higher concentrations (200 and 300 mM) could be well distinguished from those of the lower concentrations (50 and 100 mM) and the control. Marker metabolites, like m/z 261 (apigravin) and m/z 305 (ß-cotonefuran), were assistantly selected from the fingerprints by variable importance for the projection (VIP). Our results indicated the potential of the ND-EESI-MS method for the rapid recognition of metabolic conditions in plant leaves under salt stress.

Effect of Dibutyl Phthalate on the Tolerance and Lipid Accumulation in the Green Microalgae Chlorella vulgaris.

In the present study, Chlorella vulgaris were cultured in the presence of the common plasticizer dibutyl phthalate (DBP) with different concentrations for 10 days. The cell density, DBP concentrations, neutral lipid concentrations, and lipid morphology in C. vulgaris were studied using optical microscopy, gas chromatography (GC), fluorescence spectrophotometry, and laser scanning confocal microscopy (LSCM). We observed that the neutral lipid contents and cell density of C. vulgaris were negatively influenced by DBP of high concentrations (50 and 100 mg/L), but significantly stimulated by DBP of low concentrations (5, 10, and 20 mg/L). Lipid bodies were destroyed into pieces by DBP of high concentrations (50 and 100 mg/L), but were slightly suppressed by DBP at low concentrations (5, 10, and 20 mg/L). Chlorella vulgaris treated with DBP (50 mg/L) for 2 days showed the highest removal efficiency (31.69%). The results suggested that C. vulgaris could be used in practice to remove DBP and has the potential of being oleaginous microalgae in DBP contaminated water.