Enhancement Of Paxillus Involutus And Hydrophilic Polymers On Salt Tolerance Of Tree Species

Soil salinization and drought are two serious factors restricting the development of agriculture and forestry in China. Biotechnological protocols have a great potential to improve salt and drought tolerance of crop and woody species. Recent studies show that mycorrhiza formed by fungi and roots and hydrophilic polymers are able to improve salt and drought resistance of plants. The enhancement mechanisms of mycorrhiza and polymers on salt tolerance have been investigated, however, the contribution of ectomycorrhiza (ECM) to ionic homeostasis of host plants, and the effects of different types of polymers on salt and drought tolerance are less known. In this thesis, we investigated salt-tolerance of ECM fungi, ion distribution and dynamic transport in mycorrhizal roots. Moreover, different polymers were used to improve the drought and salt-tolerance of a woody species. Main results are listed below:1.The effects of different concentration of NaCl on growth, biomass, ion content (Na+, Cl-, K+, Mg2+ and Ca2+) and anti-oxidative enzymes activity (SOD, CAT, APX, POD and GPX) in Paxillus involutus (strains MAJ and NAU) were investigated. (1) The growth and biomass of the two strains MAJ and NAU were both improved under 100 mM NaCl treatment. SOD, CAT and POD were increased in strain MAJ after a short-term 100 and 200 mM NaCl stress (1d). However, activity of SOD, CAT and POD declined with the duration of salt treatment. Under a long-term salinity (9d), strain NAU remained activities of anti-oxidative enzymes, especially APX, GPX and POD. (2) Strain MAJ included Na+ and Cl- under salt stress, which was defined as a salt-includer, and NAU exhibited a greater capacity to exclude Na+ and Cl-, which was a salt-excluder. For ion homeostasis, K+/Na+, Ca2+/Na+ and Mg2+/Na+ were not reduced in two strains under 100 and 200mM NaCl, but markedly declined at 500 mM NaCl. This reveals that the two strains, especially MAJ, could increase nutrient uptake to balance the increased Na+ under a moderate salinity. Due to strategies to maintain ionic homeostasis in Paxillus under salt stress, we supposed that the two tested strains (MAJ and NAU) may enhance salt tolerance of host relying on different pathways. a. Strain MAJ is an includer, but keeps a high ability of absorbing nutrients under salinity, accordingly, the hosts may enhance nutrient uptake via the fungi MAJ. b. Strain NAU, a salt-excluder, may increase salt tolerance of hosts by salt exclusion. Therefore, the ectomycorrhiza would protect host plants from uptake of excessive NaCl.2.By scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDX), ion distribution in ectomycorrhizal symbiosis formed by Paxillus involutus (strains MAJ and NAU) and Populus×canescens roots were investigated under salt stress conditions. Results show that ECM decreased Na+ concentrations in roots under 50 mM NaCl treatment (7d). Noteworthy, salt exclusion in leaves and roots was more pronounced in NAU-mycorrhizal plants, suggesting that ectomycorrhiza-NAU (ECM-NAU) had higher ability to exclude Na+. Moreover, ECM could improve ionic homeostasis in leaves and roots of host plants. K+/Na+, Ca2+/Na+ and Mg2+/Na+ in leaves and roots of ectomycorrhizal plants were less affected under salt stress, compared with non-mycorrhizal plants. In addition, higher Ca2+ level retained in leaves and roots could maintain integrity and selectivity of biomembranes. Therefore, in addition to salt exclusion, salt-tolerance of P.×canescens was also enhanced by nutrient selectivity, due to high ability of nutrient absorption of fungi (especially MAJ).3. The scanning ion-elective electrode technique (SIET) was used to measure net fluxes of Na+, H+, K+, Ca2+ and Cl- in roots of mycorrhizal and non-mycorrhizal (NM) plants. According to the established relationship between ion flux and salt tolerance of woody plants, we found the acclimation strategies of mycorrhizal roots upon NaCl stress. (1) salt shock:a. Enhancement of H+ influx was observed in mycorrhizal roots. This is presumably due to the rapid activation of plasma membrane H+-ATPase. In host cells, the H+-ATPase activity was stimulated by the growth of hypha (MAJ and NAU) even under non-saline conditions. This enabled H+-coupling transporters (such as Na+/H+ antiporter and 2H+/Cl- symporter) to transport salt ions (i.e., Na+, Cl-) across the PM, resulting a net H+ influx.b. The salt shock-induced efflux of K+ was lesser in mycorrhizal than NM roots. It is possibly due to a higher activity of H+-ATPase in mycorrhizal roots, which inhibits the salt-induced PM depolarization and the opening of KORC and NSCC. Futhermore, a more drastic Ca2+ efflux in mycorrhizal roots, which possibly comes from more acute cell wall Na+/Ca2+ exchange, may contribute to the increase of extracellular Ca2+ concentration and simultaneously help to the decrease of NaCl-induced K+ loss via KORC and NSCC. (2) long-term salt-stress:a. Under LT salt treatment, a more drastic H+ influx was found in NAU-mycorrhizal roots, which was possibly caused by the stimulation of PM Na+/H+ antiport. The antiport was promoted by a higher H+-ATPase activity, which was stimulated via a speculated H2O2-Ca2+ signaling pathway. Compared to NAU-mycorrhizal roots, MAJ-mycorrhizal roots exhibit a less H+ influx under LT salt stress and seem to posses another transport mechanism responsible to improved plant tolerance. We speculated that it was due to an improved vacuole membrane Na+/H+ antiport system and thus accumulated more Na+ in the vacuole. This speculation is consistent with the salt-includer characteristic in strain MAJ. b. K+ efflux was restricted in mycorrhizal roots under LT salt treatment. We speculated that salt stress induced a drastic H+ influx, thus caused apoplastic alkalization and led to a H2O2 burst, which stimulates Ca2+ entry via Ca2+-permeable channels. The elevated [Ca2+]cyt stimulates the PM Na+/H+ antiporters through SOS signalling pathway; on the other hand, H2O2 induced a Ca2+-dependent increase of PM H+-ATPase activity. The upward-regulated H+ pumps can:(1) sustain an H+ gradient to drive the Na+/H+ antiport across the PM; and (2) preserve a less depolarized MP, thus restricting the K+ efflux through DA-KORCs and DA-NSCCs. As a result, the K+ efflux was obviously limited in mycorrhizal roots, and K+/Na+ homeostasis was retained in salinized plants.c. Under a 24 h or LT salt stresses, Ca2+ efflux was more pronounced in mycorrhizal roots compared to non-mycorrhizal roots. It is possibly due to the Na+/Ca2+ exchange in the cell wall and activation of PM Ca2+-ATPase. PM Ca2+-ATPase was able to reduced cytosolic Ca2+ because the long-term sustained Ca2+ in the cytosol would induce a programmed cell death. Therefore, ECM had a protective strategy after subjected to a long term of salt stress.4. Stockosorb 500XL (a granular type, cross-linked poly potassium-co-(acrylic resin polymer)-co-polyacrylamide hydrogel) manufactured by Stockhausen GmbH Krefeld in Germany, Luquasorb(?) product (a powder type of potassium polyacrylate) produced by BASF Corporation in Germany and Konjac flour (a powder type of Konjac Glucomannan, raw material of Konjac superabsorbent polymer) were used to improve salt-and drought-tolerance of Metasequoia glyptostroboides Hu et Cheng. (1) Hydrophilic polymers held Na+ and Cl- in the soil solution due to their high water-holding capacity under saline conditions, thus limiting an excessive accumulation of toxic ions in the plant organs; furthermore, the exchangeable K+ that contained in Stockosorb and Luquasorb resulted in an improved K+/Na+ homeostasis in salinized plants; and Konjac flour held abundant nutrients supplied for plants. (2) Roots took up the retained water from Stockosorb, Luquasorb and Konjac flour when water was deficient in the soil. (3) Hydrophilic polymers aided the plants to tolerate an interactive impacts of drought and salt stresses, which was mainly accounted for their water-and salt-holding capacities. In comparison, the growth and survival enhancement effects of the polymers on drought+NaCl-treated plants was more evident by Luquasorb and Konjac flour application, because it supplied water to plants at a lower rate during soil drying, thus prolonging the duration of water supply and allowed roots to grow in an environment of lower salinity for a long period of salt and drought stresses.