Ameliorative symbiosis of endophyte (Penicillium funiculosum LHL06) under salt stress elevated plant growth of Glycine max L.

Experiments were conducted to investigate the role of a newly isolated endophytic fungus GMC-2A on physiology of host plant (Glycine max. L cv. Hwangkeum-kong) growing under salinity stress. GMC-2A was identified as a new strain of Penicillium funiculosum on the basis of sequence homology and phylogenetic analysis of D1/D2 regions of 28S rDNA. Preliminary screening experiment showed that the culture filtrate (CF) of GMC-2A promoted the growth of Waito-C, a dwarf gibberellin (GA) biosynthesis mutant rice cultivar. Analysis of fungal CF revealed the presence of GAs (GA₁ 1.53 ng/ml; GA₄ 9.34 ng/ml; GA₈ 1.21 ng/ml; GA₉ 37.87 ng/ml) and indole acetic acid (14.85 μg/ml). GMC-2A also showed high phosphate solubilization of tricalcium phosphate. Besides that, GMC-2A application enhanced soybean seed germination as compared to control. Under salinity stress (70 and 140 mM), GMC-2A significantly promoted the soybean growth attributes (shoot length, shoot fresh/dry biomass, chlorophyll content, photosynthesis rate and leaf area) in comparison to control treatments. We also observed low endogenous abscisic acid and elevated jasmonic acid contents in GMC-2A treated plants under salt stress. GMC-2A treatment significantly enhanced levels of isoflavones (34.22% and 75.37%) under salinity stress as compared to control. In conclusion, P. funiculosum LHL06 has significantly ameliorated the adverse effects of salinity induced abiotic stress, and re-programmed soybean to higher growth and isoflavone biosynthesis.     

Gibberellin-producing endophytic fungi isolated from Monochoria vaginalis

The role of endophytic fungi in plant growth and development is well documented. However, endophytic fungi with growth promotion capacity have never been isolated from weeds previously. In the current study, we isolated 8 fungal endophytes from the roots of Monochoria vaginalis, a serious weed of rice paddy in Korea. These isolates were screened on Waito-C, in order to identify plant growth promoting metabolites. Two fungal isolates (M5.A & M1.5) significantly promoted the plant height and shoot length of Waito-C during preliminary screening experiments. The culture filtrates (CFs) of M5.A and M1.5 also promoted the shoot length of Echinocloa crusgalli. Gibberellins (GAs) analysis of the CFs of M5.A and M1.5 showed that these endophytic fungi secrete higher quantities of GAs as compared with wild-type G. fujikuroi KCCM12329. The CF of M5.A contained bioactive GAs (GA3, 2.8 ng/ml; GA4, 2.6 ng/ml, and GA7, 6.68 ng/ml) in conjunction with physiologically inactive GA9 (1.61 ng/ml) and GA24 (0.18 ng/ml). The CF of M1.5 contained physiologically active GAs (GA3, 1.64 ng/ml; GA4, 1.37 ng/ml and GA7, 6.29 ng/ml) in conjunction with physiologically inactive GA9 (3.44 ng/ml), GA12 (0.3 ng/ml), and GA24 (0.59 ng/ml). M5.A and M1.5 were identified as new strains of Penicillium sp. and Aspergillus sp., respectively, based on their 18S rDNA sequence homology and phylogenetic analysis.     

Fungal endophyte Penicillium janthinellum LK5 improves growth of ABA-deficient tomato under salinity

An endophytic fungus was isolated from the roots of tomato (Solanum lycopersicum Mill) and identified as Penicillium janthinellum LK5. The culture filtrate (CF) of P. janthinellum significantly increased the shoot length of gibberellins (GAs) deficient mutant waito-c and normal Dongjin-beyo rice seedlings as compared to control. The CF of P. janthinellum contained GAs (GA₃, GA₄, GA₇ and GA₁₂). To assess endophyte-growth promoting and stress-tolerance potential, the CF along with the propagules of endophyte was applied to tomato-host and abscisic acid (ABA)-deficient mutant Sitiens plants under sodium chloride (NaCl) induced salinity stress. Sitiens plants had retarded growth under normal and salinity stress however its growth was much improved during P. janthinellum-association. The endophyte inoculation reduced the membrane injury by decreasing lipid peroxidation as compared to non-inoculated control under salinity. Endophyte-associated Sitiens plants have significantly higher catalase, peroxidase and glutathione activities as compared to control. Endophyte-infected host and Sitiens plants had low level of sodium ion toxicity and high calcium contents in its root as compared to control. P. janthinellum LK5 helped the Sitiens plants to synthesis significantly higher ABA and reduced the level of jasmonic acid to modulate stress responses. The results suggest that endophytes-association can resist salinity stress by producing gibberellins and activating defensive mechanisms of host and Sitiens plants to achieve improved growth.     

Exophiala sp. LHL08 reprograms Cucumis sativus to higher growth under abiotic stresses

Endophytic fungi are potential sources of secondary metabolites; however, they are little known for phytohormones secretion and amelioration of plant growth under abiotic stresses. We isolated a novel endophyte from the roots of Cucumis sativus and identified it as a strain of Exophiala sp. by sequencing internal transcribed spacer/large subunit rDNA and phylogenetic analysis. Prior to identification, culture filtrate (CF) of Exophiala sp. has shown significant growth promotion of Waito‐C [a gibberellins (GAs)‐deficient mutant cultivar] and Dongjin‐byeo (normal GAs biosynthesis cultivar) rice seedlings. CF analysis of Exophiala sp. showed the presence of physiologically active GAs (GA₁, GA₃, GA₄ and GA₇) and inactive GAs (GA₅, GA₈, GA₉, GA₁₂ and GA₂₀). Exophiala sp. had higher GAs in its CF than wild‐type strain of Gibberella fujikuroi except GA₃. Influence of Exophiala sp. was assessed on cucumber plant's growth and endogenous abscisic acid (ABA), salicylic acid (SA) and bioactive GAs under salinity and drought stresses. Exophiala sp.‐treated plants have shown significantly higher growth and rescued the host plants from stress promulgated water deficit, osmotic and cellular damage. The altered levels of stress‐responsive ABA showed low level of stress confined to endophyte‐applied plants than control. Elevated levels of SA and bioactive GAs (GA₃ and GA₄) in endophyte‐associated plants suggest stress‐modulating response toward salinity and drought. In conclusion, symbiotic relations between Exophiala and cucumber have reprogrammed the host plant growth under abiotic stresses, thus indicating a possible threshold role of endophytic fungi in stress alleviation. This study could be extended for improving agricultural productivity under extreme environmental conditions.     

Gibberellin production and plant growth promotion from pure cultures of Cladosporium sp. MH-6 isolated from cucumber (Cucumis sativus L.)

Gibberellin (GA) production by soil fungi has received little attention, although substantial work has been carried out on other aspects of plant growth promoting fungi (PGPF). In our studies we investigated GA production and growth-promoting capacity of a novel fungal strain isolated from the roots of soil-grown cucumber. Pure cultures of 19 endophytic fungi were tested for shoot length promotion of Waito-C rice to identify the GA production capacity of these fungal isolates. Isolate MH-6 significantly increased shoot length (12.9 cm) of Waito-C, in comparison to control treatments. Bioassay with culture filtrate (CF) of MH-6 also significantly promoted growth attributes of cucumber plants. Analysis of MH-6 CF showed the presence of physiologically active (GA1, 1.97 ng/mL; GA3, 5.18 ng/mL; GA4, 13.35 ng/mL and GA7, 2.4 ng/ mL) in conjunction with physiologically inactive (GA9 [0.69 ng/mL], GA12 [0.24 ng/mL], GA15 [0.68 ng/ mL, GA19 [1.94 ng/mL and GA20 [0.78 ng/mL]) gibberellins. The CF of MH-6 produced greater amounts of GA3, GA4, GA7 and GA19 than wild type Fusarium fujikuroi, a fungus known for high production of GA. The fungal isolate MH-6 was identified as a new strain of Cladosporium sp. on the basis of sequence homology (99%) and phylogenetic analysis of 18S rDNA sequence.     

Endophytic Cephalotheca sulfurea AGH07 reprograms soybean to higher growth

Abstract

Gibberellins (GAs) are well known for plant growth promotion. GAs production by fungi has received little attention, although substantial work has been carried out on other aspects of plant growth-promoting fungi (PGPF). We investigated GAs production and plant growth-promoting capacity of an endophytic fungus isolated from the roots of soil grown soybean plants. The endophytic fungus is reported as GAs producer and as PGPF for the first time in this study. Nine endophytic isolates were collected from the roots of soybean, and culture filtrates (CFs) obtained from their pure cultures were screened on Waito-C, a dwarf rice cultivar, for the presence of GAs. Of these, seven fungal isolates promoted shoot length as compared to control (distilled water), while one inhibited it. Three fungal isolates were selected on the basis of higher shoot elongation as compared to wild type Gibberella fujikuroi, which was used as positive control. The growth-prompting capacity of selected fungal isolates SB5-1, SB3-2, and SB3-3 was bio-assayed on soybean cv. Hwangkeumkong. Fungal isolate SB5-1 provided maximum plant height (31.6 cm), shoot length (21.1 cm), whole plant fresh biomass (2.41 g), shoot fresh biomass (1.99 g), and leaf area (24.37 cm²). The CF of isolate SB5-1 was analyzed for the presence of GAs, and it was found that all physiologically active GAs were present (GA₁, 0.15 ng/ml, GA₃, 1.2 ng/ml, GA₄, 7.37 ng/ml, and GA₇, 3.18 ng/ml) in conjunction with physiologically inactive GA₅, GA₉, GA₁₅, GA₁₉, GA₂₀, and GA₂₄. The fungal isolate SB5-1 was identified as a new strain of Cephalotheca sulfurea through molecular and phylogenetic approaches.     

Pure culture of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> LHL07 reprograms soybean to higher growth and mitigates salt stress

Little is known about the role of endophytic fungi against abiotic stresses and isoflavonoids (IF) contents of soybean. In current study, we investigated the role of fungal endophytes on the growth of soybean under salt stress conditions. Pure cultures of nine endophytic fungi were isolated from the roots of field-grown soybean plants, and their culture filtrates were screened on Waito-C and Dongjin-byeo rice cultivars; for identification of plant growth promoting fungal strains. It was observed that fungal isolate GMC-2B significantly promoted the growth of both Waito-C and Dongjin-byeo. GMC-2B was later identified as a new strain of Metarhizium anisopliae LHL07 on the basis of 18S rDNA sequences and phylogenetic analysis. Metarhizium anisopliae LHL07 inoculated soybean plants recorded significantly higher shoot length, shoot fresh and dry biomass, chlorophyll contents, transpiration rate, photosynthetic rate and leaf area; under sodium chloride induced salt stress as compared to non-inoculated control plants. An elevated proline and reduced superoxide dismutase and malondialdehyde contents in M. anisopliae LHL07 inoculated soybean plants demonstrated mitigation of salt induced oxidative stress. Furthermore, reduced abscisic acid and elevated jasmonic acid contents in soybean plants confirmed that lesser stress was convened to M. anisopliae inoculated-plants under salinity stress. We also assessed the role of M. anisopliae interaction on IF biosynthesis of soybean, and found significantly higher IF contents in M. anisopliae inoculated soybean plants. In conclusion, endophytic fungal interactions with soybean can be beneficial to improve soybean quality and quantity under salt affected agricultural systems.     

赤霉素调节植物对非生物逆境的耐性

赤霉素（GAs）是一类重要的植物激素,调控植物生长发育的诸多方面．最近的研究表明,GA也参与对生物与非生物胁迫的响应,然而GA参与非生物胁迫响应的遗传学证据及其机制有待于进一步研究．本实验室前期研究证明,水稻EullfELONGATEDUPPERMOSTINTERNODE）通过一个新的生化途径降解体内的活性赤霉素分子,并参与调控水稻对病原菌的基础抗病性．本研究发现,euil突变体对盐胁迫能力降低,而超表达EUll基因的水稻和拟南芥耐盐性显著提高．进一步研究发现,积累高含量赤霉素的水稻euil突变体对脱落酸（ABA）的敏感性下降,而赤霉素缺失的EUll超表达转基因水稻和拟南芥均改变了对于ABA的敏感性．EUll基因的转录受逆境诱导,其功能缺失与超表达调控了逆境标志基因的表达．综上推测,GA可能是通过影响ABA的信号途径从而改变了植物对非生物胁迫的响应．     

Tissue-specific localization of gibberellins and expression of gibberellin-biosynthetic and signaling genes in wood-forming tissues in aspen

Bioactive gibberellins (GAs) are known regulators of shoot growth and development in plants. In an attempt to identify where GAs are formed, we have analyzed the expression patterns of six GA biosynthesis genes and two genes with predicted roles in GA signaling and responses in relation to measured levels of GAs. The analysis was based on tangential sections, giving tissue-specific resolution across the cambial region of aspen trees (Populus tremula). Gibberellin quantification by GC/MS-SRM showed that the bioactive GA1 and GA4 were predominantly located in the zone of expansion of xylem cells. Based on co-localization of the expression of the late GA biosynthesis gene GA 20-oxidase 1 and bioactive GAs, we suggest that de novo GA biosynthesis occurs in the expanding xylem. However, expression levels of the first committed GA biosynthesis enzyme, ent-copalyl diphosphate synthase, were high in the phloem, suggesting that a GA precursor(s) may be transported to the xylem. The expression of the GA signaling and response genes DELLA-like1 and GIP-like1 coincided well with sites of high bioactive GA levels. We therefore suggest that the main role of GA during wood formation is to regulate early stages of xylem differentiation, including cell elongation.     

Endophytic fungal pre-treatments of seeds alleviates salinity stress effects in soybean plants

In the present study, four endophytic fungi (GM-1, GM-2, GM-3, and GM-4) were tested for their ability to improve soybean plant growth under salinity stress conditions. The seed germination and plant growth were higher in seeds pretreated with endophytic fungal cultures than their controls. The positive influence of fungi on plant growth was supported by gibberellins analysis of culture filtrate (CF), which showed wide diversity and various concentrations of GAs. Specifically, GA₄, GA₇, GA₈, GA₉, GA₁₂, and GA₂₀ were found in fungal CFs. Under salinity stress conditions, GM-1 significantly enhanced the length and fresh weight of soybean plants relative to other fungal treatments. GM-1 effectively mitigated the adverse effects of salinity by limiting lipid peroxidation and accumulating protein content. GM-2, GM-3, and GM-4 also counteracted the salinity induced oxidative stress in soybean plants through reduction of lipid peroxidation and enhancement of protein content, maintaining the length and fresh weight of shoots. The activities of the antioxidant enzymes catalase, superoxide dismutase and peroxidase were inhibited in salinity exposed plants, while GM-1 significantly enhanced these antioxidant enzyme activities in plants under salt stress. GM-1 treatment also showed lower levels of abscisic acid and elevated levels of salicylic acid in plants under salinity stress. Hence, GM-1 was identified as Fusarium verticillioides (teleomorph Gibberella moniliformis) isolate RK01 based on its DNA sequence homology. These results suggest that endophytic fungal (F. verticillioides) pre-treatment of soybean seeds would be an effective method to promote soybean plant growth under salinity stress conditions.     

Inhibition of growth of microcultured <Emphasis Type="Italic">Hancornia speciosa</Emphasis> shoots by 3β-hydroxylated gibberellins and one of their C-3 deoxy precursors

Gibberellins (GAs) A(1), A(3), A(4) and A(7), all 3beta-hydroxylated, growth-active GAs, significantly inhibited shoot elongation and the formation of nodes in in vitro-grown Hancornia speciosa, as did GA(20), a 3-deoxy precursor of GA(1). Ancymidol, an early-stage inhibitor of GA biosynthesis, significantly retarded shoot elongation without affecting the formation of nodes. Co-application of ancymidol and GA(1 )did not overcome the ancymidol-induced growth retardation. Trinexapac-ethyl, which can inhibit 3beta-hydroxylation (GA activation) and 2beta-hydroxylation (GA inactivation), gave no significant response on either shoot elongation or node formation, while two isomers of 16,17-dihydro GA(5), also inhibitors of GA 3beta-hydroxylation, significantly inhibited both shoot growth and the formation of nodes. These unusual results may indicate a unique metabolism for GAs in microcultured shoots of H. speciosa.     

Vernalization and Gibberellin Physiology of Winter Canola (Endogenous Gibberellin (GA) Content and Metabolism of [3H]GA1 and [3H]GA20

Winter canola (Brassica napus cv Crystal) is an oilseed crop that requires vernalization (chilling treatment) for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization-induced events, endogenous GA content and the metabolism of [3H]GAs were examined in 10-week vernalized and nonvernalized plants. Shoot tips were harvested 0, 8, and 18 d postvernalization (DPV), and GAs were purified and quantified using 2H2-internal standards and gas chromatography-selected ion monitoring. Concentrations of GA1, GA3, GA8, GA19, and GA20 were 3.1-, 2.3-, 7.8-, 12.0-, and 24.5-fold higher, respectively, in the vernalized plants at the end of the vernalization treatment (0 DPV) relative to the nonvernalized plants. Thermoregulation apparently occurs prior to GA19 biosynthesis, since vernalization elevated the concentration of all of the monitored GAs. [3H]GA20 or [3H]GA1 was applied to the shoot tips of vernalized and nonvernalized plants, and after 24 h, plants were harvested at 6, 12, and 15 DPV. Following high-performance liquid chromatography analyses, vernalized plants showed increased conversion of [3H]GA20 to a [3H]GA1-like metabolite and reduced conversion of [3H]GA1 or [3H]GA20 to polar 3H-metabolites, putative glucosyl conjugates. These results demonstrate that vernalization influences GA content and GA metabolism, with GAs serving as probable regulatory intermediaries between chilling treatment and subsequent stem growth.     

Improved growth of salinity-stressed soybean after inoculation with salt pre-treated mycorrhizal fungi

Two sets of experiments to determine the effect of mycorrhiza on soybean (Glycine max) growth under saline conditions and to investigate the salt acclimation of mycorrhizal fungi were conducted. In the first experiment, the effect of an arbuscular mycorrhizal (AM) fungus Glomus etunicatum on mineral nutrient, proline and carbohydrate concentrations and growth of soybean. Under different NaCl concentrations (0, 50, 100, 150 and 200mM) was evaluated. Salinity decreased AM colonization. In both the M and nonAM plants shoot and root proline and shoot Na and Zn concentrations were increased under salinity. Soybean plants inoculated with the AM fungus had significantly higher fresh and dry weight, root proline, P, K and Zn but lower shoot proline and Na concentrations compared to the non inoculated plants. In the second experiment, the AM fungus was pre-treated with NaCl (salt acclimation) then was used as inoculum for soybean plants subjected to 100mM NaCl. Root colonization, fresh and dry weight, root proline, P, K and Zn concentrations were greater in soybean plants inoculated with the salt pre-treated fungus, compared to those inoculated with the nonsalt pre-treated fungus. However, for Na, the situation was the opposite. Based on these results, the AM inoculation helps the growth of soybean plants grown in saline conditions. When the AM fungus was pre-treated with NaCl with a gradual increase of concentration, and then exposed to a sudden salt stress, their efficiency was increased. This may be due to the acclimation of the AM fungus to salinity.     

Growth-active gibberellins overcome the very slow shoot growth of Hancornia speciosa, an important fruit tree from the Brazilian “Cerrado”

Shoot elongation of Hancornia speciosa, an endangered tree from the Brazilian savannah “Cerrado”, is very slow, thus limiting nursery production of plants. Gibberellins (GAs) A₁, A₃, and A₅, and two inhibitors of GA biosynthesis, trinexapac-ethyl and ancymidol were applied to shoots of Hancornia seedlings. GA₁ and GA₃ significantly stimulated shoot elongation, while GA₅ had no significant effect. Trinexapac-ethyl and ancymidol, both at 100 μg per seedling, inhibited shoot elongation up to 45 days after treatment, though the effect was statistically significant only for ancymidol. Somewhat surprisingly, exogenous GA₃ more effectively stimulated shoot elongation in SD-grown plants, than in LD-grown plants. The results from exogenous application of GAs and inhibitors of GA biosynthesis imply that Hancornia shoot growth is controlled by GAs, and that level of endogenous growth-active GAs is likely to be the limiting factor for shoot elongation in Hancornia. Application of GAs thus offer a practical method for nursery production of Hancornia seedlings for outplanting into the field.     

Neotyphodium endophyte strain and superoxide dismutase activity in perennial ryegrass plants under water deficit

The presence of Neotyphodium endophyte in forage grass tillers has been associated with increased tolerance of abiotic stresses. The effect of four endophyte treatments (plants with three different strains of Neotyphodium lolii compared with plants without endophyte) on superoxide dismutase (SOD) (EC1.15.1.1) activity in Lolium perenne cv ‘Grasslands Samson’ was measured under high and low dehydration regimes in a glasshouse experiment. SOD activity was assayed by a microplate method utilising the inhibition of reduction of a tetrazolium dye by superoxide radicals. A progressive increase in dehydration over 2 weeks reduced shoot fresh weight, dry weight and shoot water content for high, compared with low, moisture-stressed plants. Mean shoot fresh weight was significantly lower for plants with strain AR37 endophyte than for plants with strain AR1 endophyte, wild-type or endophyte-free plants, but there was no interaction between endophyte treatment and dehydration treatment. There were no differences in mean SOD activity between the dehydration treatments, and the four endophyte treatments at any of the harvests. All harvest mean SOD levels for plants in both stress groups, however, were significantly different from the preceding harvest value. Between the first and second week of moisture stress there was a significant endophyte by harvest interaction for mean percentage change in SOD activity when activity in plants with wild-type strain endophyte increased more rapidly than in AR1, AR37 or endophyte-free plants. The results are in agreement with earlier reports suggesting that Neotyphodium endophytes do not have major effects on the water stress physiology of perennial ryegrass, although water deficits applied were not extreme.     

Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis.

The gibberellin (GA) 20-oxidase encoded by the GA5 gene of Arabidopsis directs GA biosynthesis to active GAs, whereas that encoded by the P16 gene of pumpkin endosperm leads to biosynthesis of inactive GAs. Negative feedback regulation of GA5 expression was demonstrated in stems of Arabidopsis by bioactive GAs but not by inactive GA. In transgenic Arabidopsis plants overexpressing P16, there was a severe reduction in the amounts of C20-GA intermediates, accumulation of large amounts of inactive GA25 and GA17, a reduction in GA4 content, and a small increase in GA1. However, due to feedback regulation, expression of GA5 and GA4, the gene coding for the subsequent 3beta-hydroxylase, was greatly increased to compensate for the effects of the P16 transgene. Consequently, stem height was only slightly reduced in the transgenic plants.