Studies On Mechanisms Of Salt Tolerance Improved By Arrbuscular Mycorrhizal Fungi (AMF) In Tomato

In this thesis, on the bases of studies on selecting AMF strains to suit to organic culture, a series of studies had been conducted on physiological and molecular mechanisms of salt tolerance improved by arbuscular mycorrhizal fungi in tomato (Lycopersicon esculentum L. Var. zhongza 9). The main results were as follows:1. when tomato seedlings inoculated with six AMF strains ( Glomus versiforme, Glomus mosseae-2, Glomus intraradices, Glomus diaphanum, Glomus mosseae, Glomus etunicatum1) under organic culture, positive effect on growth in AM plants was observed compared to CK and Glomus versiforme, and Glomus mosseae-2 were screened out as the best strains for tomato growth. Glomus versiforme, and Glomus mosseae-2 increased dry weight by 75% and 65% respectively which indicated that the two AMF strains may be potential in organic culture.2. The effects of Glomus mosseae-2 on salt tolerance of tomato were studied under 0.5% and 1% NaCl stress. The results showed that higher leaf area, root activity and stem sap flow were detected in AM tomato than corresponding non-AM plants under continuous salt stress which lead to higher shoot and root growth. Although formation of mycorrhizae was restrained by salt stress, the mycorrhizal benefit still increased with salt concentration increasing. Under the same NaCl concentration, coefficient of salt tolerance was improved , and so salt tolerance was enhanced.3. Nutrition absorbing in AM and non-AM tomato and mechanisms of ion damage to plant were studied under 0.5% and 1% NaCl stress. N, P, K +and Ca+ content improved and Na⁺ decreased significantly, but Cl^- content decreased indistinctively after AMF inoculation. Under the same salt stress, higher K⁺/ Na⁺, P/ Na⁺, Ca²⁺/ Na⁺ and P/Cl^- ratio were observed in AM plants compared to corresponding non-AM plants. These values of ratio had significantly positive relation with dry weight and K⁺/ Na⁺, P/ Na⁺, Ca²⁺/ Na⁺ were more closely related with dry weight. So enhanced salt tolerance in AM tomato were related with improved nutrition condition, especially higher K, P content, K⁺/ Na⁺,P/ Na⁺ and lower Na⁺ content which reduced Na⁺ damage to plant.4. To study mechanisms of the reduced Na⁺ damage to plant induced by AMF under salt stress, RT-PCR, Realtime-PCR were performed to assay the vacuolar Na⁺/H⁺ antiporter (LeNHX1) mRNA level in leaves and roots of AM and non-AM tomato. The results showed that expression of LeNHX1 gene in AM tomato was similar to that of non-AM plant under non- NaCl condition. Expression of LeNHX1 gene was induced by NaCl stress in AM and non-AM plants, but AMF had no significant effect on LeNHX1 mRNA level and lower expression was observed in AM tomato. So mechanisms of the reduced Na⁺ damage to plant induced by AMF was in little relation to LeNHX1 which can export Na⁺ from the cytosol to the vacuole across the tonoplast.5. The effects of AMF (Glomus mosseae-2) on osmotic adjustment matter content of tomato were studied under continuous NaCl stress (0.5% and 1%) for 40 days. The results showed that AMF-inoculation significantly promoted the accumulation of soluble sugar in leaves and roots, increased soluble protein in leaves and proline content in roots under salt stress, so salt tolerance of tomato was improved . The accumulation of soluble protein, soluble sugar and root proline induced by AMF played an important role in osmotic adjustment mechanism of enhanced salt tolerance in AM tomato.6. The effect of Glomus mosseae-2 on cell membrane damage and the antioxidants responses in leaves and roots of AM tomato and control were examined under different NaCl stress ( 0.5% and 1%) for 40 days. The results indicated that superoxide-dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), Glutathione peroxidase (GSH-PX) activity in leaves and roots of AM symbiosis were significantly higher than corresponding non-AM plants under NaCl stress or non- NaCl condition. However, CAT activity was transient significantly induced by AMF and then suppressed to a level similar with non-AM seedlings. AMF significantly reduced cell membrane osmosis and membrane peroxidation in leaves and roots in salinity. So, the salt tolerance of tomato was enhanced by AMF. This research suggested that the enhanced salt tolerance in AM symbiosis was mainly related with the elevated SOD, POD, APX and GSH-PX activity by AMF which degraded reactive oxygen species and so alleviated the cell membrane damages under salt stress.7. Under 0.3%, 0.6%, 1%NaCl stress, the net photosynthetic rate (Pn), light saturation point (LSP) was decreased in salinity, whereas AMF improved Pn, stomatal conductance (gs), transpiration rate (Tr), apparent quanturn yield (AQY), and carboxylation efficiency (CE) remarkly, although LSP in AM plants was not increased. At the same time, AMF improved photosphorylation activity in chloroplast which was favour to keeping higher ability to absorb light energy. So, AM tomato had higher capacity of CO₂ fixation and light energy exchange compared to corresponding non-AM tomato. Under 0.3-0.6% NaCl stress, the decline of Pn was caused mainly by stomatal factors, but under1% NaCl stress for 28 d, the decline of Pn was caused mainly by non-stomatal factors, whereas AMF improved photosynthesis in AM tomato and consequently improved plant growth and enhanced salt tolerance.8. Under 0.3%, 0.6%, 1%NaCl stress, the content of endogenous IAA, GA3, Zeatin in leaves and roots of AM and non-AM decreased and the content of ABA increased, whereas AMF inoculation improved the content of these endogenous hormones. AMF adjusted the balance of endogenous hormones and decreased the ratio of ABA/IAA,ABA/GA3,ABA/Zeatin and ABA/(IAA+GA3+Zeatin) compared to corresponding non-AM plants. The correlation analysis showed a significant reverse relation between gs and the ratio of ABA/Zeatin. AM tomato had higher gs and lower ratio of ABA/Zeatin than that of corresponding non-AM plants. So, higher ABA content did not led to lower gs compared to non-AM plants and ABA and Zeatin adjusted response of stoma to salt stress jointly and kept higher gs than non-AM ones which lead to higher photosynthesis. Studies on plant growth, dry weight, and other physiological item (soluble sugar, soluble protein and MDA content) when exdohormones (IAA, GA3) applied showed that IAA, GA3 improved salt tolerance of AM or non-AM tomato. Similarly, it also indicated that IAA, GA3 induced by AMF can also enhanced salt tolerance of AM tomato.9. Relative water content (RWC), water potential and water use efficient (WUE) in leaves of AM and non-AM tomato were examined under NaCl (0.5%, 1%) stress. The results showed that RWC,water potential,WUE declined with NaCl concentration increasing under continuous salt stress, however, AMF improved the value of these parameters. Water absence due to salt stress can be alleviated in AM tomato and the positive effect of AMF on water absorb was more obvious compared to non-AM plants especially under high NaCl (1%) stress. Consequently, AM tomato had higher capability to keep the water balance in plant under salt stress. At the same time, water condunce affected by AMF and salinity jointly. Salt stress decreased Lp and higher Lp in roots of AM plants also observed.To study molecular mechanism of water absorbing enhanced by AMF deeply, RT-PCR and Realtime-PCR was firstly used to study mRNA levels of five aquaporin genes (LePIP1, LePIP2, LeTRAMP, LeAQP2 and LeTIP)in leaves and roots of AM and non-AM tomato under 1% NaCl stress. The results indicated that the five aquaporin genes were all regulated at transcriptional level by AM fungi and salt stress. The expression of the five genes adjusted by AMF or salt stress was significantly different in leaves and roots. Higher level of the five genes mRNAs were observed in leaves compared to corresponding non-AM tomato under salt stress which indicated that overexpression of the five genes facilitated water transport across biomembranes, thus AM tomato can transport water faster and keep higher leaf water potential. However, LePIP1, LePIP2, LeTRAMP, LeTIP were down-regulated in roots of AM tomato in salinity compared to that of non-AM ones, but expression of LeAQP2 gene in AM tomato roots was higher than corresponding non-AM plants. Accordingly, overexpression of LeAQP2 gene in roots may be relation to enhanced root conductivity due to AMF. Under salt stress, other genes (LePIP1, LePIP2, LeTRAMP, LeTIP) in roots of AM or non-AM tomato may involve in osmosis adjustment in different ways by up-regulating or down-regulating the expression of these genes by salt stress and AMF jointly...