Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences for the growth of cucumber under salt-stress conditions

Aims: After the determination of the toxic but nonlethal concentration of NaCl for cucumber, we examined the interaction between an ACC (1‐aminocyclopropane‐1‐carboxylate) deaminase producing bacterial strain and an arbuscular mycorrhizal fungus (AMF) and their effects on cucumber growth under salinity. Methods and Results: In the first experiment, cucumber seedlings were exposed to 0·1, 50, 100 or 200 mmol l^?1 NaCl, and plant biomass and leaf area were measured. While seeds exposed to 200 mmol l^?1 NaCl did not germinate, plant growth and leaf size were reduced by 50 or 100 mmol l^?1 salt. The latter salt cancentration caused plant death in 1 month. In the second experiment, seeds were inoculated with the ACC deaminase‐producing strain Pseudomonas putida UW4 (AcdS⁺), its mutant unable to produce the enzyme (AcdS^?), or the AMF Gigaspora rosea BEG9, individually or in combination and exposed to 75 mmol l^?1 salt. Plant morphometric and root architectural parameters, mycorrhizal and bacterial colonization and the influence of each micro‐organism on the photosynthetic efficiency were evaluated. The AcdS⁺ strain or the AMF, inoculated alone, increased plant growth, affected root architecture and improved photosynthetic activity. Mycorrhizal colonization was inhibited by each bacterial strain. Conclusions: Salinity negatively affects cucumber growth and health, but root colonization by ACC deaminase‐producing bacteria or arbuscular mycorrhizal fungi can improve plant tolerance to such stressful condition. Significance and Impact of the Study: Arbuscular mycorrhizal fungus and bacterial ACC deaminase may ameliorate plant growth under stressful conditions. It was previously shown that, under optimal growth conditions, Ps. putida UW4 AcdS⁺ increases root colonization by Gi. rosea resulting in synergistic effects on cucumber growth. These results suggest that while in optimal conditions ACC deaminase is mainly involved in the bacteria/fungus interactions, while under stressful conditions this enzyme plays a role in plant/bacterium interactions. This finding is relevant from an ecological and an applicative point of view.     

An ACC Deaminase Minus Mutant of Enterobacter cloacae UW4No Longer Promotes Root Elongation

The ACC deaminase gene (acdS) from Enterobacter cloacae UW4 was replaced by homologous recombination with the acdS gene with a tetracycline resistance gene inserted within the coding region. Upon characterization of this AcdS minus mutant, it was determined that both ACC deaminase activity and the ability to promote the elongation of canola roots under gnotobiotic conditions were greatly diminished. This result is consistent with a previously postulated model that suggests that a major mechanism utilized by plant growth-promoting bacteria involves the lowering of plant ethylene levels, and hence ethylene inhibition of root elongation, by bacterial ACC deaminase. Received: 20 January 2000 / Accepted: 22 February 2000     

ACC deaminase from plant growth-promoting bacteria affects crown gall development

In addition to the well-known roles of indoleacetic acid and cytokinin in crown gall formation, the plant hormone ethylene also plays an important role in this process. Many plant growth-promoting bacteria (PGPB) encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can degrade ACC, the immediate precursor of ethylene in plants, to alpha-ketobutyrate and ammonia and thereby lower plant ethylene levels. To study the effect of ACC deaminase on crown gall development, an ACC deaminase gene from the PGPB Pseudomonas putida UW4 was introduced into Agrobacterium tumefaciens C58, so that the effect of ACC deaminase activity on tumour formation in tomato and castor bean plants could be assessed. Plants were also coinoculated with A. tumefaciens C58 and P. putida UW4 or P. putida UW4-acdS- (an ACC deaminase minus mutant strain). In both types of experiments, it was observed that the presence of ACC deaminase generally inhibited tumour development on both tomato and castor bean plants.     

1-Aminocyclopropane-1-carboxylate deaminase from Pseudomonas putida UW4 facilitates the growth of canola in the presence of salt

The growth of canola plants treated with either wild-type Pseudomonas putida UW4 or a 1-aminocyclopropane-1-carboxylate (ACC) deaminase minus mutant of this strain was monitored in the presence of inhibitory levels of salt, i.e., 1.0 mol/L at 10 degrees C and 150 mmol/L at 20 degrees C. This strain is psychrotolerant with a maximal growth rate of approximately 30 degrees C and the ability to proliferate at 4 degrees C. Although plant growth was inhibited dramatically by the addition of 1.0 mol/L salt at 10 degrees C and only slightly by 150 mmol/L salt at 20 degrees C under both sets of conditions, the addition of the wild type but not the mutant strain of P. putida UW4 significantly improved plant growth. This result confirms the previous suggestion that bacterial strains that contain ACC deaminase confer salt tolerance to plants by lowering salt-induced ethylene synthesis.     

Effect of 1-aminocyclopropane-1-carboxylic acid deaminase producing bacteria on the hyphal growth and primordium initiation of Agaricus bisporus

The mechanism of casing soil stimulating the primordium formation of Agaricus bisporus is not well understood so far. Our results showed that 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (AcdS)-producing bacteria were abundant in the casing soil of A. bisporus and accounted for up to 20 % of total culturable bacteria. A. bisporus produced ACC and ethylene. The supplement of methionine increased the ACC concentrations within the hyphae, and aminooxyacetic acid displayed an opposite effect. Methionine and ACC promoted the ethylene production while CoCl₂ suppressed the production. The AcdS-producing bacterial strain Pseudomonas putida UW4 co-cultured with A. bisporus could attach to hyphae, stimulate the hyphal growth, and reduce the ethylene production of A. bisporus. Added in sterilized casing soil, it induced the primordium formation of A. bisporus. In comparison, its AcdS-deficient mutant UW4-AcdS^? displayed the opposite effects. These results indicated that the inhibitor to the primordium formation of A. bisporus was ethylene; the AcdS-producing bacteria within the casing layer cleaved ACC, lowered the ethylene level in mushroom hyphae, and relieved the inhibition of ethylene. This is a new model of the synergism between bacteria and fungi.     

The arbuscular mycorrhizal symbiosis attenuates symptom severity and reduces virus concentration in tomato infected by Tomato yellow leaf curl Sardinia virus (TYLCSV)

The arbuscular mycorrhizal (AM) symbiosis is considered a natural instrument to improve plant health and productivity since mycorrhizal plants often show higher tolerance to abiotic and biotic stresses. However, the impact of the AM symbiosis on infection by viral pathogens is still largely uncertain and little explored. In the present study, tomato plants were grown under controlled conditions and inoculated with the AM fungus Funneliformis mosseae. Once the mycorrhizal colonization had developed, plants were inoculated with the Tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus causing one of the most serious viral diseases of tomatoes in Mediterranean areas. Biological conditions consisted of control plants (C), TYLCSV-infected plants (V), mycorrhizal plants (M), and TYLCSV-infected mycorrhizal plants (MV). At the time of analysis, the level of mycorrhiza development and the expression profiles of mycorrhiza-responsive selected genes were not significantly modified by virus infection, thus indicating that the AM symbiosis was unaffected by the presence and spread of the virus. Viral symptoms were milder, and both shoot and root concentrations of viral DNA were lower in MV plants than in V plants. Overall F. mosseae colonization appears to exert a beneficial effect on tomato plants in attenuating the disease caused by TYLCSV.     

Influence of a Bacillus sp. on physiological activities of two arbuscular mycorrhizal fungi and on plant responses to PEG-induced drought stress

The effects of bacterial inoculation (Bacillus sp.) on the development and physiology of the symbiosis between lettuce and the arbuscular mycorrhizal (AM) fungi Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe and Glomus intraradices (Schenck and Smith) were investigated. Plant growth, mineral nutrition and gas-exchange values in response to bacterial inoculation after PEG-induced drought stress were also evaluated. In AM plants, inoculation with Bacillus sp. enhanced fungal development and metabolism, measured as succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities, more than plant growth. Under non-stressed conditions, G. intraradices colonization increased all plant physiological values to a higher extent when in dual inoculation with the bacterium. Under stress conditions, the bacterium had an important stimulatory effect on G. intraradices development. Under such conditions, the effects of the bacterium on photosynthetic rate, water use efficiency (WUE) and stomatal conductance of lettuce plants differed with the fungus species. Plant-gas exchange was enhanced in G. intraradices- and reduced in G. mosseae-colonized plants when co-inoculated with Bacillus sp. Thus, the effects of each fungus on plant physiology were modulated by the bacterium. Stress was detrimental, particularly in G. intraradices-colonized plants without the bacterium, reducing intra and extraradical mycelium growth and vitality (SDH), as well as plant-gas exchange. Nevertheless, Bacillus sp. inoculation improved all these plant and fungal parameters to the same level as in non-stressed plants. The highest amount of alive and active AM mycelium for both fungi was obtained after co-inoculation with Bacillus sp. These results suggest that selected free-living bacteria and AM fungi should be co-inoculated to optimize the formation and functioning of the AM symbiosis in both normal and adverse environments.     

Arbuscular mycorrhizal influence on growth,photosynthetic pigments,osmotic adjustment and oxidative stress in tomato plants subjected to low temperature stress

The influence of the arbuscular mycorrhizal (AM) fungus, Glomus mosseae, on characteristics of growth, photosynthetic pigments, osmotic adjustment, membrane lipid peroxidation and activity of antioxidant enzymes in leaves of tomato (Lycopersicon esculentum cv Zhongzha105) plants was studied in pot culture under low temperature stress. The tomato plants were placed in a sand and soil mixture at 25°C for 6 weeks, and then subjected to 8°C for 1 week. AM symbiosis decreased malondialdehyde (MDA) content in leaves. The contents of photosynthetic pigments, sugars and soluble protein in leaves were higher, but leaf proline content was lower in mycorrhizal than non-mycorrhizal plants. AM colonization increased the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) in leaves. The results indicate that the AM fungus is capable of alleviating the damage caused by low temperature stress on tomato plants by reducing membrane lipid peroxidation and increasing the photosynthetic pigments, accumulation of osmotic adjustment compounds, and antioxidant enzyme activity. Consequently, arbuscular mycorrhiza formation highly enhanced the cold tolerance of tomato plant, which increased host biomass and promoted plant growth.     

Effectiveness of mycorrhizal fungi on globe artichoke (Cynara cardunculus L. var. scolymus) micropropagation

The effectiveness of two arbuscular mycorrhizal (AM) fungal isolates (Glomus intraradices and Glomus viscosum) in sustaining plant growth and the physiological activities of the micropropagated globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori) were investigated during acclimatization and 90 days after plant establishment. All the mycorrhizal microplants survived transplant shock thus confirming the positive role of AM fungi colonization on ex vitro establishment. The growth increased in mycorrhizal plants, especially in plants inoculated with Glomus viscosum. Mycorrhizal plantlets showed higher stomatal conductance, which is probably necessary to supply the carbon needs of fungal symbionts. The SPAD (soil plant analysis development) data could be useful for plant management as a predictor for tissue nitrogen levels. The higher SPAD values in mycorrhizal plants are strictly related to a higher photosynthetic potential, and consequently to their better nitrogen nutrient status due to the symbiotic relationship. Regardless of the mycorrhizal performance in the host–fungus combination, the most efficient fungus for the artichoke microplants was Glomus viscosum.     

Arbuscular mycorrhizal symbiosis induces strigolactone biosynthesis under drought and improves drought tolerance in lettuce and tomato

Arbuscular mycorrhizal (AM) symbiosis alleviates drought stress in plants. However, the intimate mechanisms involved, as well as its effect on the production of signalling molecules associated with the host plant–AM fungus interaction remains largely unknown. In the present work, the effects of drought on lettuce and tomato plant performance and hormone levels were investigated in non‐AM and AM plants. Three different water regimes were applied, and their effects were analysed over time. AM plants showed an improved growth rate and efficiency of photosystem II than non‐AM plants under drought from very early stages of plant colonization. The levels of the phytohormone abscisic acid, as well as the expression of the corresponding marker genes, were influenced by drought stress in non‐AM and AM plants. The levels of strigolactones and the expression of corresponding marker genes were affected by both AM symbiosis and drought. The results suggest that AM symbiosis alleviates drought stress by altering the hormonal profiles and affecting plant physiology in the host plant. In addition, a correlation between AM root colonization, strigolactone levels and drought severity is shown, suggesting that under these unfavourable conditions, plants might increase strigolactone production in order to promote symbiosis establishment to cope with the stress.     

Engineered ACC deaminase-expressing free-living cells of Mesorhizobium loti show increased nodulation efficiency and competitiveness on Lotus spp

Ethylene inhibits the establishment of symbiosis between rhizobia and legumes. Several rhizobia species express the enzyme ACC deaminase, which degrades the ethylene precursor 1-cyclopropane-1-carboxilate (ACC), leading to reductions in the amount of ethylene evolved by the plant. M. loti has a gene encoding ACC deaminase, but this gene is under the activity of the NifA-RpoN-dependent promoter; thus, it is only expressed inside the nodule. The M. loti structural gene ACC deaminase (acdS) was integrated into the M. loti chromosome under a constitutive promoter activity. The resulting strain induced the formation of a higher number of nodules and was more competitive than the wild-type strain on Lotus japonicus and L. tenuis. These results suggest that the introduction of the ACC deaminase activity within M. loti in a constitutive way could be a novel strategy to increase nodulation competitiveness of the bacteria, which could be useful for the forage inoculants industry.     

Effect of arbuscular mycorrhizal (AM) colonization on terpene emission and content of Artemisia annua L

Plant roots interact with a wide variety of rhizospheric microorganisms, including bacteria and the symbiontic arbuscular mycorrhizal (AM) fungi. The mycorrhizal symbiosis represents a series of complex feedbacks between plant and fungus regulated by their physiology and nutrition. Despite the widespread distribution and ecological significance of AM symbiosis, little is known about the potential of AM fungi to affect plant VOC metabolism. The purpose of this study was to investigate whether colonization of plant roots by AM fungi and associated soil microorganisms affects VOC emission and content of Artemisia annua L. plants (Asteraceae). Two inoculum types were evaluated: one consisted of only an arbuscular mycorrhizal (AM) fungus species (Glomus spp.), and the other was a mixture of different Glomus species and associated soil bacteria. Inoculated plants were compared with non-inoculated plants and with plants supplemented with extra phosphorus (P) to obtain plants of the same size as mycorrhizal plants, thus excluding potentially-confounding mycorrhizal effects on shoot growth. VOC emissions of Artemisia annua plants were analyzed by leaf cuvette sampling followed by off-line measurements with pre-concentration and gas chromatography mass spectrometry (GC-MS). Measurements of CO(2) and H(2)O exchanges were conducted simultaneously. Several volatile monoterpenes were identified and characterized from leaf emissions of Artemisia annua L. by GC-MS analysis. The main components identified belong to different monoterpene structures: alpha-pinene, beta-pinene, camphor, 1,8-cineole, limonene, and artemisia ketone. A good correlation between monoterpene leaf concentration and leaf emission was found. Leaf extracts included also several sesquiterpenes. Total terpene content and emission was not affected by AM inoculation with or without bacteria, while emission of limonene and artemisia ketone was stimulated by this treatment. No differences were found among treatments for single monoterpene content, while accumulation of specific sesquiterpenes in leaves was altered in mycorrhizal plants compared to control plants. Growth conditions seemed to have mainly contributed to the outcome of the symbiosis and influenced the magnitude of the plant response. These results highlight the importance of considering the below-ground interaction between plant and soil for estimating VOC emission rates and their ecological role at multitrophic levels.     

Plant Responses to Drought Stress and Exogenous ABA Application are Modulated Differently by Mycorrhization in Tomato and an ABA-deficient Mutant (<Emphasis Type="Italic">Sitiens</Emphasis>)

The aims of the present study are to find out whether the effects of arbuscular mycorrhizal (AM) symbiosis on plant resistance to water deficit are mediated by the endogenous abscisic acid (ABA) content of the host plant and whether the exogenous ABA application modifies such effects. The ABA-deficient tomato mutant sitiens and its near-isogenic wild-type parental line were used. Plant development, physiology, and expression of plant genes expected to be modulated by AM symbiosis, drought, and ABA were studied. Results showed that only wild-type tomato plants responded positively to mycorrhizal inoculation, while AM symbiosis was not observed to have any effect on plant development in sitiens plants grown under well-watered conditions. The application of ABA to sitiens plants enhanced plant growth both under well-watered and drought stress conditions. In respect to sitiens plants subjected to drought stress, the addition of ABA had a cumulative effect in relation to that of inoculation with G. intraradices. Most of the genes analyzed in this study showed different regulation patterns in wild-type and sitiens plants, suggesting that their gene expression is modulated by the plant ABA phenotype. In the same way, the colonization of roots with the AM fungus G. intraradices differently regulated the expression of these genes in wild-type and in sitiens plants, which could explain the distinctive effect of the symbiosis on each plant ABA phenotype. This also suggests that the effects of the AM symbiosis on plant responses and resistance to water deficit are mediated by the plant ABA phenotype.     

Influence of arbuscular mycorrhiza on the growth and antioxidative activity in cyclamen under heat stress

The influence of the arbuscular mycorrhizal (AM) fungus, Glomus fasciculatum, on the growth, heat stress responses and the antioxidative activity in cyclamen (Cyclamen persicum Mill.) plants was studied. Cyclamen plants (inoculated or not with the AM fungus) were placed in a commercial potting media at 17–20 °C for 12 weeks in a greenhouse and subsequently subjected to two temperature conditions in a growth chamber. Initially, plants were grown at 20 °C for 4 weeks as a no heat stress (HS?) condition, followed by 30 °C for another 4 weeks as a heat stress (HS+) condition. Different morphological and physiological growth parameters were compared between G. fasciculatum-inoculated and noninoculated plants. The mycorrhizal symbiosis markedly enhanced biomass production and HS + responses in plants compared to that in the controls. A severe rate of leaf browning (80–100 %) was observed in control plants, whereas the mycorrhizal plants showed a minimum rate of leaf browning under HS + conditions. The mycorrhizal plants showed an increase activity of antioxidative enzymes such as superoxide dismutase and ascorbate peroxidase, as well as an increase in ascorbic acid and polyphenol contents. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity also showed a greater response in mycorrhizal plants than in the control plants under each temperature condition. The results indicate that in cyclamen plants, AM fungal colonisation alleviated heat stress damage through an increased antioxidative activity and that the mycorrhizal symbiosis strongly enhanced temperature stress tolerance which promoted plant growth and increased the host biomass under heat stress.     

Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles

In response to the colonization by arbuscular mycorrhizal (AM) fungi, plants reprioritize their phosphate (Pi)-uptake strategies to take advantage of nutrient transfer via the fungus. The mechanisms underlying Pi transport are beginning to be understood, and recently, details of the regulation of plant and fungal Pi transporters in the AM symbiosis have been revealed. This review summarizes recent advances in this area and explores current data and hypotheses of how the plant Pi status affects the symbiosis. Finally, suggestions of an interrelationship of Pi and nitrogen (N) in the AM symbiosis are discussed.     

丛枝菌根真菌通过上调根系及自身水孔蛋白基因表达提高玉米抗旱性

在模拟干旱条件下, 研究了接种丛枝菌根(AM)真菌Glomus intraradices对玉米(Zea mays)根部13种质膜水孔蛋白基因表达的影响, 同时观测了AM真菌自身水孔蛋白基因的表达情况。结果表明, 干旱条件下, 除Zm PIP1;3、Zm PIP1;4、Zm PIP1;5和Zm PIP2;2之外的接种处理能显著提高根部其他8种质膜水孔蛋白基因的表达(Zm PIP2;7表达量未检测出), 并且AM真菌菌丝中水孔蛋白基因GintAQP1表达也显著增强。与此同时, 接种处理明显改善了植物水分状况, 提高了叶片水势。AM真菌增强宿主植物根部及自身的水孔蛋白基因的表达对于提高植物抗旱性具有潜在的重要贡献。     

GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80% of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at subnanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination, and branching of germinating hyphae. In this study we show that treatment of Gigaspora rosea with a strigolactone analog (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity, and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching AM fungi toward full germination and a presymbiotic state.     

A petunia mutant affected in intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi

The regulation of the arbuscular mycorrhizal (AM) symbiosis is largely under the control of a genetic programme of the plant host. This programme includes a common symbiosis signalling pathway that is shared with the root nodule symbiosis. Whereas this common pathway has been investigated in detail, little is known about the mycorrhiza-specific regulatory steps upstream and downstream of the common pathway. To get further insight in the regulation of the AM symbiosis, a transposon-mutagenized population of Petunia hybrida was screened for mutants with defects in AM development. Here, we describe a petunia mutant, penetration and arbuscule morphogenesis1 (pam1), which is characterized by a strong decrease in colonization by three different AM fungi. Penetrating hyphae are frequently aborted in epidermal cells. Occasionally the fungus can progress to the cortex, but fails to develop arbuscules. The resulting hyphal colonization of the cortex in mutant plants does not support symbiotic acquisition of phosphate and copper by the plant. Expression analysis of three petunia orthologues of the common SYM genes LjPOLLUX, LjSYMRK and MtDMI3 indicates that pam1 is not mutated in these genes. We conclude that the PAM1 gene may play a specific role in intracellular accommodation and morphogenesis of the fungal endosymbiont.     

Impact of antifungals producing rhizobacteria on the performance of Vigna radiata in the presence of arbuscular mycorrhizal fungi

Plant growth-promoting rhizobacteria (PGPR) that produce antifungal metabolites are potential threats for the arbuscular mycorrhizal (AM) fungi known for their beneficial symbiosis with plants that is crucially important for low-input sustainable agriculture. To address this issue, we used a compartmented container system where test plants, Vigna radiata, could only reach a separate nutrient-rich compartment indirectly via the hyphae of AM fungi associated with their roots. In this system, where plants depended on nutrient uptake via AM symbiosis, we explored the impact of various PGPR. Plants were inoculated with or without a consortium of four species of AM fungi (Glomus coronatum, Glomus etunicatum, Glomus constrictum, and Glomus intraradices), and one or more of the following PGPR strains: phenazine producing (P⁺) and phenazine-less mutant (P⁻), diacetylphloroglucinol (DAPG) producing (G⁺) and DAPG-less mutant (G⁻) strains of Pseudomonas fluorescens, and an unknown antifungal metabolite-producing Alcaligenes faecalis strain, SLHRE425 (D). PGPR exerted only a small if any effect on the performance of AM symbiosis. G⁺ enhanced AM root colonization and had positive effects on shoot growth and nitrogen content when added alone, but not in combination with P⁺. D negatively influenced AM root colonization, but did not affect nutrient acquisition. Principal component analysis of all treatments indicated correlation between root weight, shoot weight, and nutrient uptake by AM fungus. The results indicate that antifungal metabolites producing PGPR do not necessarily interfere with AM symbiosis and may even promote it thus carefully chosen combinations of such bioinoculants could lead to better plant growth.