Structural and photosynthetic changes in‘Gannanzao’navel orange plants infected with Citrus tristeza virus

Abstract：【Objective】The objective of this study is to evaluate the effects of Citrus tristeza virus (CTV) infection on‘Gannanzao’navel orange plants from the perspective of its morphology and photosynthetic characteristics changes, which provides insights on how the pathogen causes damage to the host. The characteristics of CTV infection and the mechanism of the damage it caused in the host plant was analyzed, which could provide some useful information for the prevention and control of CTV. 【Methods】One-year-old healthy‘Gannanzao’navel orange seedlings were provided by the germplasm resource nursery of National Navel Orange Engineering Research Center. Seedlings were inoculated with CTV and cultured separately in an isolation net. The fresh leaves and roots from eighteen months old culture of‘Gannanzao’navel orange plants infected with CTV were collected with non- infected healthy leaves and roots served as a control. Preparing 40 samples per sample tissue and being embedded in paraffin, the leaves and the roots were cut into a thickness of 6 μm slices by using a Leica RM2245 slicing machine and the anatomical structure was observed under a Leica DMIL-LED fluorescence inverted microscope after being stained with saffron. The thickness of the phloem, xylem and medullary part, palisade tissue and spongy tissue of the leaves were measured by using the LAS V4.0and Image-Pro Plus 6.0 software. Taking 3rd to 4th leaves from top to bottom of healthy and CTV-infected plants, the blade tissues of about 1 mm ×1 mm on both sides of the central leaf veins of the leaves were quickly cut, fixed overnight in 2.5% glutaraldehyde at 4 ℃, phosphoric acid buffer (0.1 mol·L-1 , pH 7.0) was rinsed three times, 1% osmium tetroxide fixed at 4 ℃ for 2 hours, and penetrated in turn with acetone∶epoxy resin (2∶1), acetone∶epoxy resin (1∶1), epoxy resin overnight for 12 hours at 37 ℃. After being embeded by spur low viscosity resin, the sample was cut into slices by using a Leica EM UC7 slicing machine and the ultrastructure of chloroplast was observed under a FEI Tecnai G2 20 TWIN transmission electron microscope after double staining with uranium acetate and lead citrate. The absorbance values at 665 649 470 nm and 450 532 600 nm were determined by using an ultravioletvisible spectrophotometer, respectively, calculating the contents of photosynthetic pigments and malondialdehyde (MDA). The net photosynthetic rate (Pn) and intercellular CO2 concentration (Ci) of fully expanded leaves from the top to bottom of control and infected plants were measured by LI-6400 portable photosynthetic apparatus from 9:00 to 11:00 a.m. The experiment was repeated in triplicate manner, and results were graphically plotted using the SPSS 12.0 software.【Results】Fluorescence microscopic images revealed that the phloem, xylem, and pulp in the diseased leaves became significantly thinner than in the healthy ones, in which the thickness of the phloem in diseased leaves was 82.7% of that in healthy tissue and the thickness of xylem and pulp in diseased leaves was 87.4% of that in healthy tissue. There were no significant changes in the thickness of the palisade tissue and the sponge tissue, but the diseased leaves showed a loose arrangement of palisade tissue cells and a larger gap between the cells compared to the healthy leaves. The xylem of the plants infected with CTV showed the symptom of stem pitting. Electron microscopic observations revealed that the ultra-structure of xylem tissue was prominently damaged by the CTV infection. The root cells were found to be atrophied in diseased plants, with inclusions reduced and attached to the intima tissues. The content of photosynthetic pigments in the infected leaves was considerably reduced compared to that of the healthy leaves. The contents of chlorophyll a, chlorophyll b and total chlorophyll decreased significantly by 23.9%, 16.3%, and 21.4%, respectively. The content of MDA increased by 28.5%, and the net photosynthetic rate of CTV infected leaves decreased by 25.7%, and the intercellular CO2 concentration increased by 12.1%. The decrease in total chlorophyll content, net photosynthetic rate and the increase in intercellular CO2 concentration indicated that the photosynthetic perception of host leaves decreased, followed by the disease resistance ability of the infected host plant, compared to the normal healthy plant. The chloroplast structure in the CTV infected leaves changed significantly, characterized by swollen chloroplast, enlarged matrix area, and loosened lamellar structure. Enlargement in the chloroplast matrix area led to the osmophilic pressure changes inside the cells. The decrease in the photosynthetic rate of infected plant leaves was due to distortion in chlorophyll structure and chlorophyll pigment reduction.【Conclusion】Our results revealed that CTV infection in‘Gannanzao’navel orange plants drastically altered the morphology and anatomical structure of the leaves and roots. The total chlorophyll content and chloroplast structural changes considerably reduced the overall photosynthetic rate in the infected plants. Ultrastructure of xylem and phloem in infected plants exhibited the tissue atrophy and thereby it affected the inherent ability of nutrient and water conductivity of the plants, and caused the growth of branches and leaves to weaken. In general, CTV infection in‘Gannanzao’navel orange plants is dreadful damage to the citrus industry.