Response of Candidatus Liberibacter Asiaticus-Infected Citrus Plants to Bacillus amyloliquefaciens GJ1

Research Article

Austin J Plant Biol. 2018; 4(1): 1018.

Response of Candidatus Liberibacter Asiaticus-Infected Citrus Plants to Bacillus amyloliquefaciens GJ1

Tang JZ¹, Ding YX¹, Deng L¹, Nan J¹, Yang XY¹, Zhao XY2 and JIANG L¹*

¹Key Laboratory of Horticultural Plant Biology of Ministry of Education, Huazhong Agricultural University, China

²State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, China

*Corresponding author: JIANG L, Key Laboratory of Horticultural Plant Biology of Ministry of Education, Huazhong Agricultural University, National Indoor Conservation Center of Virus-free Germplasm of Fruit Crops, Wuhan 430070, China

Received: December 26, 2017; Accepted: March 16, 2017; Published: March 23, 2018


Huanglongbing (HLB) is a major disease limiting citrus production worldwide. In this study, a new potential biocontrol agent Bacillus amyloliquefaciens GJ1 against HLB was employed to resistance disease experiment. We utilized isobaric tags for relative and absolute quantification-based quantitative proteomics approach for comparative analysis of protein abundances with Candidatus Liberibacter asiaticus-infected leaves treated and untreated by solution of B. amyloliquefaciens GJ1 (OD600 nm ≈ 1). The result revealed Differential Abundance Protein (DAP) of 21 expression-increased (FC>1.2, P-value<0.05) and 43 expression-decreased (FC=0.83, P-value<0.05) and COG classification annotation proteins were elucidated. Among them, the proteins involved in carbohydrate transport and metabolism were analyzed. Bacillus amyloliquefaciens GJ1 treatment brought about decreasing both of the content of the starch and the soluble sugar in leaves was proved, the function of starch degradation caused by B. amyloliquefaciens GJ1 treatment was proven via I2-KI test in roots. This study highlight one of the reasons in HLB biocontrol in citrus.

Keywords: Huanglongbing (HLB); Bacillus amyloliquefaciens GJ1; Candidatus Liberibacter Asiaticus; isobaric Tags for Relative and Absolute Quantification (iTRAQ); Degradation starch


COG: Orthologous Groups of proteins; DAP: Differential Abundance Protein; GC: Gas Chromatography analysis; HLB: Huanglongbing; KEGG: Kyoto Encyclopedia of Genes and Genomes; qPCR: Real time quantitative PCR detecting system; iTRAQ: isobaric Tags for Relative and Absolute Quantification


Citrus Huanglongbing (Huanglongbing, HLB) is considered to be one of the most devastating diseases threatening citrus production worldwide, and all cultivated citrus are suspceptible to the disease in different degree. The pathogen mainly are vectored by the Asian psyllid [1], it is caused by three species of Gram-negative, a-proteobacteria, Candidatus Liberibacter spp. “Ca. L.asiaticus”, “Ca. L.africanus”, “Ca. L.americanus” [2, 3 & 4]. The disease can also be transmitted to healthy trees by grafting of diseased bud wood [5]. Typical symptoms of Huanglongbing disease in leaves include shoot yellowing and blotchy mottled leaves, and in fruits express small, asymmetric, acidic, with many aboorted seeds. The callose deposition occurs specifically in midribs of leaves [6]. Disordered cambial tissue and massive accumulation of starch in phloem have also been reported [6, 7 & 8]. HLB has led to the devastating harm of citrus in more than 40 countries [1,9]. HLB has not only decreased citrus production but also greatly increased production costs [10]. HLB has reached epidemic proportions in Florida and has caused more than $4 billion in economic losses between 2005 to 2011.

A variety of ways have been adopted to control HLB over the past few decades, for instance, heavy pruning [11], inputting antibiotic agents [12], inhibiting the LdtR gene by an inhibitor [13] and screening the combination of stock and scion [14]. However, the above mentioned techniques used for solving the problem of HLB present some limitations. Management of HLB to enable the continued economic production of citrus is the largest challenge ever faced by the citrus industry, and new control strategies are urgently needed.

Recent advances on host–pathogen interactions, genetics of different varieties, and resistance mechanism are discussed in HLB [15]. Reported that Pathogen-Associated Molecular Patterns (PAMP) activity of flg22 in Las is weaker than those in other wellstudied plant pathogenic bacteria [16]. Predecessors thought that host citrus plants have not evolved sufficient immune responses to effectively prevent infection [17]. Given the availability of highthroughput sequencing, transcriptomes and proteomes have been applied to Las-infected plants and treated plants; as a result, numerous characteristic innate immunity elicitors, transcription factors, defense responsive components, and signaling molecules have been discovered [18 - 22]. However, the resistance metabolism of HLB remains unknown. Bacillus sp. Is a biological control agent that can control diseases to a host animal or plant, thereby preventing the development of disease by a pathogen [23]. Bacillus sp. is widely distributed in nature and harmless to humans and animals. Bacillus belongs mostly to endogenous spores, reproduces rapidly, and can be easily colonized on the surface of plant roots. It can produce a series of metabolites during growth. The antibacterial substances of Bacillus include antimicrobial protein, lipid peptide and polyketone, secretory proteins, enzymes etc. [24] The different genes involved in the metabolite synthesis in B. amyloliquefaciens FZB42 were determined. Bacillus bacillus is the dominant microbial population in soil and nature. Biological control agents have developed numerous success stories in the field of plant disease resistance. Previous studies have shown that B. amyloliquefaciens FZB42 is a highly successful example for plant disease resistance [25, 26]. The above mentioned reports have proven that B. amyloliquefaciens is a powerful biocontrol agent against plant diseases.

Carbohydrate transport and metabolism are closely related to the infection and control of HLB disease. Sucrose phosphate synthase is an important control point in the synthesis of sucrose [27]. The enzymes of sucrose degradation are sucrose invertase and sucrose synthase. The direct precursor of starch synthesis is adenosine diphosphate glucose. Starch is formed via ADP-glucose pyrophosphosrylase, which catalyzes the reaction between glucose- 1-phosphate and ATP. Amylose synthase is directly related to starch content and the ratio of amylose and amylopectin [28]. The degradation of starch in leaves involves three pathways: maltose pathway, glucose pathway, and G-1-P pathway. The phosphorylation of starch granules in leaves influences the hydrophilicity of starch granules, thereby affecting starch degradation [29]. The enzymes related mainly to phosphorylation are glucan water dikinase and phosphate glucan hydration kinase. During starch degradation, a-amylase and β-amylase play key roles. The expression levels of many proteins involved in carbohydrate metabolism are affected by Las infection. Proteins that participate in starch synthesis and starch granules synthesis are unregulated by Las infection [6, 7 & 22].

In this study, we attempted to investigate the responses of Lasinfected host citrus plants to a newly screened B. amyloliquefaciens GJ1 with the aid of iTRAQ-based proteomics analysis, In particular, the annotation proteins related to “carbohydrate transport and metabolism” were concerned. We also detected several physiological indices of soluble sugar, starch, fructose, sucrose and glucose, and the I2-KI reaction, in order to clarify the affect on the carbohydrates, while the infected plants treated by B. amyloliquefaciens GJ1. Understanding of the mechanisms is crucial for the isolation of effective biocontrol agents and the further development of biocontrol strategies for HLB.

Materials and Methods

Preparation of plant material

The infected plants by Ca. L. asiaticus-infected (Las) and health plants of Citrus madurensis Lour were used for measurement of the physiological index, all the plants were growed under the same soil condition and management. Las-infected plants treated and untreated (CK) by B. amyloliquefaciens GJ1 were carried out in Citrus sinensis (L). Osbeck grown in a greenhouse. HLB was detected with QPCR and PCR in advance, and the Las positive plants were used for subsequent tests. Both treated and control plants were grown in the same soil condition in a plastic pot, the normal soil irrigated the supplementary 1/8 MS mineral element 0.5 L/plant and 200 mg/L polypeptide (patent number: CN1827560A) once a month. Above mentioned the supplementary solution can be called “A solution”. Bacillus was applied via root irrigation once every 7 days. Each plant was irrigated with 1.5 L of treatment bacteria solution.

Protein preparation and iTRAQ labeling

The leaves were milled to powder in mortar with liquid nitrogen. Subsequently, 150 mg of powder from each sample was mixed with 1mL of lyses buffer containing Tris-base pH=8, 7M Urea, 2 M Thiourea, 0.1% SDS, 2 mM EDTA, Protease inhibitor cocktail (Roche), 1 mM phenyl methyl sulfonyl fluoride in a glass homogenizer. Homogenates were incubated on the ice for 20 min and then centrifuged at 12000 g for 15 min at 4°C; the supernatant was transferred to a new tube. Protein concentrations were determined by Bradford assay.

The supernatant containing precisely 100 μg protein of each sample was digested with Trypsin (Promega, Madison) at 37°C for 16 h. After trypsin digestion, peptides were dried by vacuum centrifugation. Desalted peptides were labeled with iTRAQ reagents (iTRAQ® Reagent-8PLEX Multiplex Kit, 4381663) according to the manufacturer’s instructions (AB Scitex, Foster City, CA). For 100 μg peptide 1 units of labeling reagent were used. Peptides were dissolved in 20 μl of 0.5 M TEAB (Triethylammonium Bicarbonate) pH 8.5 solution and labeling reagent was added in 70 μl of isopropanol. After 1h incubation the reaction was stopped with 50 mM Tris-HCl pH 7.5. The labeled peptides were incubated for 2h at room temperature. Differentially labeled peptides were mixed.

HPLC fractionation and LC-MS/MS analysis

RP separation in the first dimension by micro LC was performed on an L-3000 HPLC System (Rigol) using a Dura shell RP column. Mobile phases A (2% acetonitrile, 20 mM NH4FA, pH was adjusted to 10.0 using NH3•H2O) and B (98% acetonitrile, 20 mM NH4FA, pH was adjusted to 10.0 using NH3•H2O) were used to develop a gradient elution. Fractions from the first RPLC dimension were dissolved with loading buffer and then separated by a C18 column (75 μm inner diameters, 360 μm outer diameter × 10 cm, 3 μm C18). Mobile phase A consisted of 0.1% formic acid in water solution, where as mobile phase B consisted of 0.1% formic acid in acetonitrile solution. A series of adjusted linear gradients was applied based on the hydrophobicity of fractions eluted in 1D LC with a flow rate of 300 nL/min. For Orbitrap Q Exactive, the source was operated at 1.8 kV. For full MS survey scan, the AGC target was 3e6 and the scan range was from 350 to 1800 with a resolution of 70,000. The 20 most intense peaks with charge state 2 and above were selected for fragmentation by HCD with normalized collision energy of 32% for iTRAQ-labeled peptides. The MS2 spectra were acquired with 17, 500 resolution.

Data analysis of proteins

The MS raw data files from Q-Exactive were first searched by Mascot (Matrix Science, London, UK; version 2.6.0). Mascot was set up to search the Swiss rot database assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine and iTRAQ8plex of lysine and the n-terminus were specified in Mascot as fixed modifications. Oxidation of methionine, acetyl of the n-terminus and iTRAQ8plex of tyrosine were specified in Mascot as variable modifications. Protein identifications were accepted if they could be established at greater than 90.0% probability to achieve an FDR less than 10.0% and contained at least 2 identified peptides, We only used ratios with p-values = 0.05, and only ratio of >1.2 were considered as significant. We used databases, namely, Clusters of Orthologous Groups ( COG/), and ( fa.tar.gz) to predict gene functions.

Determination of soluble sugar by anthrone colorimetry

Anthrone colorimetry was conducted following previously reported instructions [30]. Anthrone reagent and stock standard were prepared, and the working standard was obtained. A blank solution was simultaneously prepared, and a calibration curve was constructed. The sugar concentration in the samples was computed from the calibration curve. Samples were collected for five biological repeats. Statistical analyses of differences among groups were determined using the t-test. The correlation index R² was 0.9959.

Determination of total starch content via iodization method

The total starch content was determined via iodization method [30]. The standard curve and iodine were prepared. Soluble starch was extracted, and color measurement was performed. In brief, 2 mL of sample extract was drawn in a calibration tube and added with 0.1 mL of I2-KI solution. The following steps were the same as those in the determination of the standard curve. Finally, absorbance values of samples were measured, and the total starch content was calculated. Samples were collected for three biological repeats. Statistical analyses were performed using the t-test. The correlation index R²=0.9988 of the standard curve.

Determination of amylose and amylopectin via the dual wavelength method

T?he dual wavelength method was conducted to analyze the amylose and amylopectin contents severally [31]. Standard liquid and starch scanning liquid were prepared. Amylose standard and amylopectin standard curves were constructed, and samples were then prepared. Approximately 0.1000 g of samples was mixed with 10 mL of 0.5 M KOH, stirred in a 60°C water bath, heated for 10 min, transferred to a 50 mL flask, and added with distilled water to reach a constant volume. Subsequently, 2.5 mL of the sample mixture, to a 50 mL flask, added 30 distilled water and adjusted pH with hydrochloric acid solution to 3.5, added 0.5 mL of iodine solution and filled to scale with distilled water. Af?ter 30 min, the solution was shaken well. And the starch scanning fluid was scanned by a spectrophotometer. The measurement wavelength of amylose was 603 nm, and the reference wavelength of amylose was 450 nm. By contrast, the measurement wavelength of amylopectin was 526 nm, and the reference wavelength of amylopectin was 730 nm. Samples were collected for three biological repeats. Statistical analyses were conducted using t-test. In the study, the correlation exponent of the standard curve of amylose was R²=0.9938, where as the correlation index of the standard curve of amylopectin was R²=0.9968.

Determination of fructose, sucrose, and glucose contents by gas chromatography analysis (GC)

We prepared a single sample and mixed samples of fructose, sucrose, and glucose severally. Derivatization and preparation of samples was performed, dry material was crushed, blended, weighed the sample of 0.05 gand placed in a test tube, added with7 mL of distilled water, and sealed with plastic film. After boiling water bath extraction for 30 min, the filtrate was extracted and transferred to a 10 mL volumetric flask. Take 200 u l internal standard and add the distilled water to a calibration tube. Shake well, place for 1 hour, and take 1 ml solution into 1.5 ml centrifuge tube, and perform centrifugation 20 mins (10000 g) (repeat one time). GC analysis was conducted using a chromatographic column under the following conditions: HP-5 capillary column (5%-phenyl-methyl polysiloxane, 30 m×25 μm i.d.×0.1 μm); injection port temperature, 270°C; test temperature, 300°C; high purity nitrogen as carrier gas, flow rate 25 mL/min; hydrogen, flow rate 30 mL/min; air, flow rate 400 mL/ min; cylinder head pressure, 12.00 psi; sample, 1 μL; and shunt ratio, 60:1 [32]. Gas chromatography analysis with Agilent 6890N gas chromatograph (USA). The samples were collected for three biological repeats. Statistical analyses were conducted using the t-test.

Characteristic observation of root and leaves with I2-KI and measure of photosynthetic rate

Collect the fresh roots and leaves from plants of treated and untreated by GJ1, at least 4 biology repetitions. The roots were washed clear and staining with I2-KI for 30 mins. The veins via a hand slice and staining with I2-KI for 30 mins, and then observed with Olympus SZX16 Microscope (Japan). The photosynthetic rate was detected with Li-6400XT Portable Photosynthesis system. The technical route is shown below (Figure 1).