A comparative study on resistance between‘Gannanzao'and‘Newhall'navel orange varieties to citrus canker

Abstract: 【Objective】To explore the resistance differences of‘Newhall'navel orange and‘Gannanzao'selected by bud mutation of‘Newhall'to citrus canker, leaf micro- morphological characteristics and physio-biochemical differences were comparatively analyzed. The current research will be helpful in guiding the layout of early and late matured navel oranges and promoting the extension of the‘Gannanzao'variety.【Methods】The resistance differences to citrus canker between‘Newhall'and‘Gannanzao'varieties were evaluated by in vitro leaf inoculation test. Xanthomonas citri subsp. citri(Xcc), the pathogen of citrus canker, was inoculated with 1.0×108 CFU·mL^-1 on the back of the blade by using syringe injection.The disease incidence and lesion size were collected after 7 days post-inoculation(dpi) on inoculated leaves. To find the susceptible Xcc concentration of two varieties, the lesion expansion and formation data were collected from inoculated leave with a series concentration of Xcc from 1.0×1011CFU·mL^-1 to 1.0×106CFU·mL^-1. The leaves of‘Newhall'and‘Gannanzao'were cut into 1 μm thick slices by a Leica RM2245 slicing machine, and were performed on paraffin embedded sections. The leaf anatomical structure was observed by Leica DMIL-LED fluorescence inverted microscope after staining with saffron. The leaf thickness and epithelial layer, palisade tissue, sponge tissue and hypodermal layer thickness were measured by the software of LAS V4.0 and Image-Pro Plus 6.0. The leaf tissue(excluding vein) from leaf center was cut to 5 mm × 5 mm, and observed by the scanning electron microscope Quanta 450. The leaf lower epidermis and stomatal morphology were photographed. The stomatal density of randomly 30 different fields of vision was counted, and the stomatal length and width of randomly 40 stomata using were measured with Pro Qcapture 6.0 software. The fully expanded leaves of healthy‘Gannanzao'and‘Newhall'varieties from 2 years old plants were needling inoculated with 1.0×108CFU·m L-1of Xcc. The leaves were collected before inoculation and 1, 2 and 3 days after inoculation, and then frozen with liquid nitrogen and conserved in ultra-low temperature refrigerator for enzyme activity determination. The change of the phenylalanine ammonia lyase(PAL), peroxidase(POD), catalase(CAT), polyphenol oxidase(PPO) and superoxide dismutase(SOD) activities of the leaves of‘Newhall'and‘Gannanzao'varieties were measured.【Results】Water soaked phenotype was observed on the inoculation points of‘Gannanzao'and‘Newhall'leaves after 2 dpi, and lesion formation appeared after 3dpi and 5dpi, respectively. The disease incidence of the inoculation points in‘Gannanzao'leaves was lower than that of‘Newhall'leaves. The average lesion size of‘Gannanzao'and‘Newhall'reached to 7.2 mm² and 9.5 mm² after 7 dpi, respectively. The susceptible Xcc concentration of‘Gannanzao'and‘Newhall'was 1.0×107 CFU·mL^-1 and 1.0×106 CFU·mL^-1 in syringe injection test. The leaf stomatal density between ‘Gannanzao' and ‘Newhall' was significantly different. Scanning electron microscopy results revealed that ‘Newhall' has rough leaf lower epider, sunken stomata, uneven stomata peripheral and larger pore diameter and chamber space of stomata compared with‘Gannanzao'variety. The structure of leaf palisade tissue cell between‘Gannanzao'and‘Newhall'was also different. The measurement of thickness of‘Gannanzao'and‘Newhall'showed that the thickness of leaf spongy tissue of‘Gannanzao'was significantly higher than that of‘Newhall'. In contrast, the thickness of palisade tissue of‘Newhall'was higher than that of‘Gannanzao'. The change of the phenylalanine ammonia lyase(PAL), peroxidase(POD), catalase(CAT), polyphenol oxidase(PPO)and superoxide dismutase(SOD) activities of the leaves of‘Newhall'and‘Gannanzao'varieties were measured after the plants was inoculated with Xcc. The five enzyme activity of the leaves of‘Newhall'and‘Gannanzao'were increased with different degrees, suggesting the enhancement of the resistance to citrus canker. The SOD and CAT enzyme activity reached the maximum at 2 days after inoculation, and then decrease gradually. The PAL, PPO and POD activity increased gradually from one to three days. The enzyme activity changes revealed that induced resistance to Xcc was not significantly different between‘Gannanzao'and‘Newhall'varieties.【Conclusion】The susceptible Xcc concentration of‘Newhall'was less than that of‘Gannanzao'. Lesion expansion and formation on‘Newhall'was much easier to be observed compared with that of‘Gannanzao'. The differences in tissue structure between two Citrus species were mainly on the stomatal density, leaf thickness, palisade tissue and spongy tissue tightness. Slight activity change on five enzymes was found in the two varieties.‘Gannanzao'had higher resistance to citrus canker than‘Newhall'.