The Mechanism Of Resistance To Dieback Of Eucalyptus Cultivars

Eucalyptus was an important fast-growing tree species in the world. Cylindrocladium quinqueseptatum with the characteristics of wide distribution, strong spreading ability, damage degree and great output reduction was a kind of disease in the world which harm to the seedlings and young trees of eucalyptus. It was a ultimate way to select resistant cultivars of eucalyptus to prevent this disease because the disease was difficult to prevent and cure by chemical measures. The current study on the C. quinqueseptatum only focus on characteristics of C.quinqueseptatum and its occurrence regularity, there were not systemically study on the mechanism of resistance to dieback of eucalyptus cultivars in recent years. Therefore, 11 cultivars planted mainly in Fujian Province were selected to identify their resistance, and then the resistant mechanism to the dieback were analyzed by the view of morphological structure, physiology, biochemistry and resistant gene in order to enrich the base theory of early identifying and finding resistant clones of eucalyptus. The results were as follows:(1) There was significant difference between the resistant abilities of the 11 eucalyptus cultivars. According their disease incidence and infection indexes at natural condition, the 11 cultivars were classified as high resistant cultivars(Eucalyptus grandis×urophylla 9224, E .urophylla×tereticornis M1), middle resistant cultivars(E. grandis×camaldulensis 201-2, E. saligna, E. dunnii, E. grandis×urophylla Guang 9, E. urophylla×grandis Ug9), middle susceptible cultivars(E. benthamii, E.urophylla xgrandis 3229), high susceptible cultivars(E. grandis 3, E. grandis 5).(2) The stoma densities of upper leaf epidermis of high resistant cultivars were clearly greater than that of lower epidermis. The stoma densities of upper leaf epidermis of all the 11 cultivars were greater than that of lower epidermis. The sizes and stoma densities of lower epidermis were not related to their resistance.(3) The wax contents and corneal layer thickness of upper and lower epidermis were positively related to their resistance to Clindrocladium quinqueseptatum. The wax and corneal layer were the outermost layers for resisting and delaying the infection of pathogens. The thicker leaf, orderly and close palisade tissue and close sponge tissue of the high resistant cultivars were another barrier to resist the pathogens. The high resistant cultivars had more chloroplast, accumulating more starch making the resistance stronger. Therefore, the content of leaf wax layer could be used as a quick measure for identifying the resistance of different cultivars. The thickness of leaf, palisade tissue and palisade tissue density also could be used as one of the assistant indexes for the resistance to eucalyptus dieback.(4) No matter in healthy leaves or diseased leaves, the contents of polyphenols and flavonoids, and the level of polyphenoloxidase (PPO) activities, as well as PPO isozyme were at the rank orderly as: high resistant cultivars > middle resistant cultivars >middle susceptible cultivars >high susceptible cultivars. Before infected, polyphenoloxidase isozyme activities of high resistant cultivars and middle resistant cultivars were evidently stronger than that of middle susceptible cultivars and high susceptible cultivars; after infected, the eucalyptus cultivars except the high susceptible ones appears 1-2 new bands of polyphenoloxidase isozyme. The contents of polyphenols and flavonoids, the activities level of PPO and PPO isozyme may be used as important biochemistry indexes to estimate the resistance of eucalyptus to the dieback.(5) The activities of two kinds of major defense-enzymes to clear away oxyradical: peroxidase(POD) and superoxide dismutase(SOD) showed that no matter in healthy leaves or diseased ones after inoculation, the activities of POD and SOD of different eucalyptus cultivars was in sequence of resistant cultivars > middle resistant cultivars > middle susceptible cultivars>susceptible cultivars, which changed orderly. The bands of POD isozyme showed no regularity with resistance to eucalyptus dieback before or after inoculation while that of SOD isozyme were relative to resistance to eucalyptus dieback positively. The activities of POD and SOD before and after inoculation and the bands of SOD isozyme after inoculation could be selected as early identifying biochemistry indexes from the view of breeding for improving resistance to eucalyptus dieback.(6) The chlorophyll contents and chlorophyll a/b ratio didn't vary directly with the resistance of eucalyptus to C. quinqueseptatum before inoculation. After inoculation, the chlorophyll contents and chlorophyll a/b ratio of the resistant cultivars showed small decreasing degrees, and increased quickly with larger values than that before inoculation. The chlorophyll contents of the susceptive cultivars decreased markedly with the inoculation time, and their chlorophyll a/b ratio continued to decrease after increased with small degree at the middle and latter half of inoculation period. The chlorophyll contents and changing percentage of chlorophyll a/b ratio after inoculation in eucalyptus could reflect the resistance. The chlorophyll contents and changing percentage of chlorophyll a/b ratio after inoculation in eucalyptus might be selected as supplementary indexes to appraise the resistance of eucalyptus to C. quinqueseptatum .(7) To understand the molecular mechanism of Eucalyptus grandis×urophylla 9224 defending against Clindrocladium quinqueseptatum, suppression subtractive hybridization (SSH) method was utilized to analyze the differential expression genes in Eucalyptus grandis×urophylla 9224 after inoculated with C. quinqueseptatum. Nine hundred and twenty-eight clones were obtained in the forward SSH-cDNA library of Eucalyptus grandis×urophylla 9224, some of these clones were confirmed by reverse northern hybridization. A total of 25 ESTs from SSH-cDNA library were sequenced, and all of the sequences of inserted fragments were analyzed with the aid of bioinformatics. Twenty-three genes were sorted into five groups such as photosynthesis, phytoalexin synthesis, energy-delivering, plant growth regulation, resistance and signal transduction, protein metabolism, which were speculated in the pathways of enhancing the resistance. The result showed us a way for the eucalyptus breeding with high resistance.In a word, the wax and corneal layer of eucalyptus leaf were the outermost layers to resist and delay the infection of pathogens. The thicker leaf, orderly and close palisade tissue and close sponge tissue of the high resistant cultivars of the wax and corneal layer were another barrier to resist the pathogens. When the C. quinqueseptatum penetrated the body of eucalyptus, membrane of eucalyptus could identify this signals, and this signals were magnified by the IP₃,DG and tyrosine kinase, Up-regulation of photosynthetic and respiratory enzyme were promoted, and the photosynthetic ability of Eucalyptus were tone up, organics and energy were accumulated, the material which used to compound defense enzymes, anti-fungus substance and cytoderm were safeguarded, so as to promote the resistant ability of eucalyptus. These may be the main mechanism of resistance to dieback of eucalyptus.