Differential Physiological Response Of The Resistant And Susceptible Sunflower Varieties To Inoculation Of Black Stem Pathogen

Sunflower, a plant of Compositae, has a high economic and edible value, and is also one of the important oil crops in our country. In recent years, the risk that sunflower seeds imported from abroad may carry a variety of dangerous diseases that would spread into our country increases with the increase in the amount of the sunflower seeds imported from abroad. Sunflower black stem {Leptosphaeria lindquistii) is one of the important quarantined fungal diseases, which also occurred ocassionally in local regions of Xinjiang in our country. The research of sunflower black stem disease has been mainly focused on the areas of morphological identification and molecular detection of its pathogens; and although there have been statistical field surveys and analyses on the resistance of different sunflower genotypes to the disease reported, so far few reports has been seen concerning with the physiological stress responses of sunflowers upon the invasion of this fungal pathogen. There have been even fewer reports on the differential physiological stress responses between the resistant and susceptable sunflower varieties upon the inoculation of Leptosphaeria lindguistii under controlled conditions, such as the activity of antioxidant and related enzymes, the content of reactive oxygen species (ORS), the zymogram of antioxidant and related enzymes, and its relationship with its resistance.In this research, two previously reported to be susceptable and resistant sunflower varieties, Kangdi KWS303 and MT792G resepectively was used as plant materials. Under controlled laboratory conditions, seedlings of both were inoculated with the BXC1 isolate, which was previously isolated from sunflower field in Xingjiang and identified by ITS in our laboratory, to study the differential physiological responses of the susceptable and resistant sunflower varieties to this isolate. When the first 2 blades of true leaves (Ca. two weeks after seeding) was emmerged from the seedlings grown in growth chambers, the hypocotyls were artificially inoculated with one-week old mycelium of the BXC1 isolate on PDA medium. Two modes of inoculation, intact hypocotyl inoculation and needle-prick hypocotyl inoculation, together with two controlls, intact non-inoculation and needle-prick non-inoculation, were used in this research. Ten days of continuing growth in the growth chamber after inoculation, the infections (infection rates, dimensions of lesions) were observed and measured, to compare the infections of the the susceptable and resistant sunflower varieties; and then, a practical duplex PCR molecular detection method based on ITS was established in order to confirm at the molecular level its infection for later physiological response studies. After that, different plant parts (roots, stem with lesion, and leaves) were used to measure the activity of enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), phenylanaline ammonia-lyase (PAL) and glutathione peroxidase (GSH-Px), the contents of reactive oxygen species, such as superoxide radical (O2-), hydroxyl radical (·OH) and hydrogen peroxide (H2O2), and to analyze the zymograms of SOD, POD and esterase (EST). The main results are as follows.1. Lesion Difference between the Susceptable and Resistant Sunflower Varietie by Diffent Modes of Inoculation The annalyses on the lesion feature, infection rate of and lesion dimension (long and short diameters) revealed that needle prick inoculation was more prone to infection. In the same mode of inoculation, the susceptable variety resulted in deeper black colored lesions in the stem, higher rates of infection and larger dimensions of lesions than the the resistant one; and partial resistance of the sunflower to the black stem disease was observed. The effective mode of inoculation of the BXC1 isolate to the seedlings of both the susceptable and resistant sunflower varieties was found to be the needle prick inoculation, which laid the basis for later studies of physiological responses of the susceptable and resistant sunflower varieties to the disease and mechanisms for its resistance.2.ITS-based Practical Duplex PCR Detection of Black Stem Pathogen of Sunflowers was EstablishedSo far Leptosphaeria lindquistii has been mainly identified and characterized by its morphological traits at home and abroad. Conventional and multiplex PCR molecular detections of Leptosphaeria lindquistii based on its ITS and actin genes have also been reported. But most of them were methodological studies adopting purified fungal strains. The practical studies concerning with the detection sensitivity, actual detection of infected sunflower tissues etc have not yet been done. In this research, the strain BXC1 of Leptosphaeria lindquistii separated from the rural field in Xinjiang Province and preliminarily identified by ITS amplification and sequencing, was used for duplex PCR detection, sensitivity test and mock detection of mixed sunflower and the fungal DNAs. Then, the duplex PCR detections on the seedlings of two sunflower varieties challenged by BXC1 isolate with two different inoculation methods were conducted when its typical symptomic lesion appeared. Results showed that both the genomic DNA of BXC1 and its mixture with sunflower genomic DNA could produce the fungal ITS and the actin gene specific bands,580 bp and 255 bp respectively by the duplex PCR. In addition, the mixture DNA could also produce the sunflower ITS specific band (Ca.740 bp), indicating the excellent specificity of this established duplex PCR detection of Leptosphaeria lindquistii. Using the gradually diluted BXC1 DNA or sunflower DNA or the mixture of both as templates to perform the duplex PCR, a sensitivity of 0.05ng fungal DNA/μL in 20μL PCR reaction was detected; the stem portions of the inoculated seedlings of the two sunflower varieties all produced the fungal ITS and the actin gene specific bands by the established duplex PCR detection. These results indicate that the established duplex PCR detection of Leptosphaeria lindquistii is specific, reliable, conveniant and can be used directly for the quick detection of Leptosphaeria lindquistii pathogen in the infected sunflower tissues.3. The Differential Response in the Activity of Some Key Enzymes of the Susceptable and Resistant Sunflower Varieties upon Inoculation of BXC1 IsolateTests showed that the activity of SOD, POD, CAT, PAL and GSH-Px in roots, stem and leaves of the seedlings of both the susceptable and resistant sunflower varieties inoculated was all significantly higher than its corresponding control; and the activity of these enzymes in needle prick inoculation group was shown to be higher than that of the non-prick inoculation group. It was suggested that these were the responsive enzymes of the sunflowers to the black stem isolate. They might be closely related to the partial resistance in sunflowers to this disease.4. Difference in Contents of Reactive Oxygen Species (ROS) of the Susceptable and Resistant Sunflower Varieties upon the Inoculation of BXC1 IsolateIt was shown that the levels of reactive oxygen species (superoxide radical, hydroxyl radical and hydrogen peroxide) in roots, stem and leaves of the seedlings of both the susceptable and resistant sunflower varieties inoculated were all decreased and this was in accordance with the activity change of the related antioxidant enzymes. The amplitude of decrease in resistant sunflower was higher than in susceptable sunflower. It might be one of the reasons for its resistance.5. Difference in Zymograms of the Susceptable and Resistant Sunflower Varieties upon the Inoculation of BXC1 IsolatePreliminary analyses showed that the activity of band S3 of SOD might be related to the physiological response and partial restance of sunflowers to the black stem disease. In the lesioned stem, band P3 of POD in the susceptable variety appears gradually and the activity of bands P2 and P3 increased with respect to the controls. In the resistant variety, there were two specific POD bands (P4 and P5) in the stem; the activity of bands P2, P4 and P5 increased with the degree of lesion/inoculation. It was suggested that the activity of P4-5 might be related to the partial resitance in sunflowers to this disease; P2 and P3 might be related to pathogenic responses of sunflowers to the disease. The zymograms of EST showed that there were 3 bands (E3, E5 and E6) in the lesioned stem of the susceptable variety, whose activity increased with inoculation, while in the resistant variety, only bands E5 and E6 appeared, whose activity increased with inoculation and was significantly higher than those in the susceptable variety. This indicated that E3, E5 and E6 might be involved in the pathogenic response and partial resistance in sunflowers to the disease.6. Features of Enzyme Activity, ROS Levels and Zymograms in the Mycelium of Isolate BXC1 (Leptosphaeria lindguistii)Relatively high activity of SOD and very high activity of GSH-Px in the mycelium of BXC1 isolate were detected, however, the activities of POD, CAT and PAL were very low.In the BXC1 mycelium, the superoxide and hydroxyl radical levels were relatively high, and the level of hydrogen peroxide was very high. These might be due to its very low activity in POD and CAT. These special characteristics may have something to do with the infectablity of the mycelium and causing the black stem disease of sunflowers.In the zymograms, there were 2 SOD bands namely SLL1 and SLL2,6 EST bands (E1～6), among the later,3 bands (E3, E5 and E6) were relatively high in activity. However no POD bands were detected. These were in good accordance with its relatively high SOD and extremely low POD activity. Curiously, the two mycelial SOD bands did not appear in the lesioned stem. It is possible that the mycelium grown in the PDA medium is quite different from the mycelium growing in the host sunflower plant due to probably the physiological response/resistance of the host. Further studies are yet to be done to confirm this.