Subcellular effects of drought stress in Rosmarinus officinalis

The use of Rosmarinus officinalis, and other wild plant species, in the Mediterranean area is an interesting solution in order to avoid the desertification and rapid soil erosion, because of their good resistance to environmental conditions. Previous articles have described experiments designed to determine the impact of water stress at the plant level in this species, but more knowledge is required at the subcellular and ultrastructural levels. An anatomic and ultrastructural study of the leaves was conducted on Rosmarinus officinalis plants growing under different water treatments. In the leaves of water-stressed plants, the leaf water potential and turgor decreased, and leaf osmotic potential became more negative with respect to control plants. The anatomic investigations showed that both the mesophyll intercellular spaces and the epidermal cell size were reduced significantly under the more intense drought stress conditions. At the subcellular level, chloroplasts accumulated plastoglobuli and lipid bodies, and cuticle thickness was increased under water stress. In our experiment, the anatomic and ultrastructural modifications of Rosmarinus officinalis could be considered an additional adaptation to drought stress together with physiological and biochemical modifications as antioxidant accumulation.     

Effects of relative humidity and temperature conditions on pollen fluorochromatic reaction of Rosmarinus officinalis L. (Lamiaceae)

Summary. Mediterranean ecosystems are characterized by seasonal and annual fluctuations in humidity and temperature which are considered limiting factors for plant growth and might have played a key role in the selection of species that compose the present vegetation. After anther release, pollen is generally exposed to various changes of temperature and humidity conditions, therefore its viability and consequently successful fruit set are strongly affected by these environmental parameters. The aim of this research was to study the effect of different combinations of humidity and temperature on pollen membrane integrity of Rosmarinus officinalis L. in order to investigate possible relations between pollen features and climatic conditions during flowering. This species is an evergreen perennial shrub, occurring among the sclerophyllous vegetation of Mediterranean maquis. In many areas of Southern Italy, it shows a flowering period starting from the beginning of winter and spreading throughout spring months. The analysis of data showed that duration of pollen viability of R. officinalis is enhanced by the co-occurrence of low temperature and high humidity. Moreover, these conditions are able to newly raise the viability of pollen kept at higher temperatures and lower humidity. These observations indicate that reactivation of the pollen membrane depends on low temperature apart from high humidity. Therefore “vernalization” of rosemary pollen enhances its viability, supporting that pollen behavior is adapted to winter condition and allows flowering in winter and early spring. Correspondence and reprints: Laboratorio di Botanica ed Ecologia Riproduttiva, Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici, Naples, Italy.     

Reduction of ambient UV-B radiation does not affect growth but may change the flowering pattern of Rosmarinus officinalis L.

The effects of sub-ambient levels of UV-B radiation on the shrub Rosmarinus officinalis L. were investigated in a field filtration experiment in which the ambient UV-B was manipulated by a combination of UV-B transmitting and UV-B absorbing filters. As a result, the plants were receiving near-ambient or drastically reduced UV-B radiation doses. Drastic reduction of UV-B radiation had no effect on mean, total and maximum stem length, number of stems per plant, dry mass of leaves, stems and roots and leaf nitrogen and phenolic contents. However, flowering was more pronounced under reduced UV-B radiation during the winter period which coincides with ascending ambient UV-B radiation. In contrast, during autumn and early winter, a period which coincides with descending ambient UV-B radiation, flowering was unaffected by reduced UV-B radiation. We can conclude that natural UV-B radiation does not affect growth of Rosmarinus officinalis, but its reduction could influence the flowering pattern of the species.     

Aquaporin genes GintAQPF1 and GintAQPF2 from Glomus intraradices contribute to plant drought tolerance

Arbuscular mycorrhizal (AM) symbiosis, established between AM fungi (AMF) and roots of higher plants, occurs in most terrestrial ecosystems. It has been well demonstrated that AM symbiosis can improve plant performance under various environmental stresses, including drought stress. However, the molecular basis for the direct involvement of AMF in plant drought tolerance has not yet been established. Most recently, we cloned two functional aquaporin genes, GintAQPF1 and GintAQPF2, from AM fungus Glomus intraradices. By heterologous gene expression in yeast, aquaporin localization, activities and water permeability were examined. Gene expressions during symbiosis in expose to drought stress were also analyzed. Our data strongly supported potential water transport via AMF to host plants. As a complement, here we adopted the monoxenic culture system for AMF, in which carrot roots transformed by Ri-T DNA were cultured with Glomus intraradices in two-compartment Petri dishes, to verify the aquaporin gene functions in assisting AMF survival under polyethylene glycol (PEG) treatment. Our results showed that 25% PEG significantly upregulated the expression of two aquaporin genes, which was in line with the gene functions examined in yeast. We therefore concluded that the aquaporins function similarly in AMF as in yeast subjected to osmotic stress. The study provided further evidence to the direct involvement of AMF in improving plant water relations under drought stresses.     

Quantification of water uptake by arbuscular mycorrhizal hyphae and its significance for leaf growth, water relations, and gas exchange of barley subjected to drought stress

Arbuscular mycorrhizal fungi alleviate drought stress in their host plants via the direct uptake and transfer of water and nutrients through the fungal hyphae to the host plants. To quantify the contribution of the hyphae to plant water uptake, a new split-root hyphae system was designed and employed on barley grown in loamy soil inoculated with Glomus intraradices under well-watered and drought conditions in a growth chamber with a 14-h light period and a constant temperature (15 degrees C; day/night). Drought conditions were initiated 21 days after sowing, with a total of eight 7-day drying cycles applied. Leaf water relations, net photosynthesis rates, and stomatal conductance were measured at the end of each drying cycle. Plants were harvested 90 days after sowing. Compared to the control treatment, the leaf elongation rate and the dry weight of the shoots and roots were reduced in all plants under drought conditions. However, drought resistance was comparatively increased in the mycorrhizal host plants, which suffered smaller decreases in leaf elongation, net photosynthetic rate, stomatal conductance, and turgor pressure compared to the non-mycorrhizal plants. Quantification of the contribution of the arbuscular mycorrhizal hyphae to root water uptake showed that, compared to the non-mycorrhizal treatment, 4 % of water in the hyphal compartment was transferred to the root compartment through the arbuscular mycorrhizal hyphae under drought conditions. This indicates that there is indeed transport of water by the arbuscular mycorrhizal hyphae under drought conditions. Although only a small amount of water transport from the hyphal compartment was detected, the much higher hyphal density found in the root compartment than in the hyphal compartment suggests that a larger amount of water uptake by the arbuscular mycorrhizal hyphae may occur in the root compartment.     

Variations in abscisic acid, indole-3-acetic acid and zeatin riboside concentrations in two Mediterranean shrubs subjected to water stress

Water stress induced an increase in endogenous concentrations of ABA in Lavandula stoechas L. plants to 13100 pmol ABA g^–1 FW, which may contribute to the maintenance of water relations between the second and the third day of water stress treatment. After the third day, a sharp decrease in ABA levels was observed to 2630 pmol ABA g^–1 FW, together with a decrease in water content and water potential and a loss of plant response to water stress. Water deficit did not induce an increase in endogenous ABA concentration, which remained at 514 pmol ABA g^–1 FW in Rosmarinus officinalis L., which is more sclerophyllous than L. stoechas. Nevertheless, the relative water content of Rosmarinus officinalis L. after seven days of water stress decreased more than 40% and reached values of –3.2 MPa. R. officinalis showed lower levels of ABA, but significantly higher levels of IAA and ZR than L. stoechas (4 times and 6 times respectively in well watered-plants). The increase in ABA levels is not a common mechanism in these two Mediterranean shrubs which survive under water stress conditions.Abbreviations ABA abscisic acid - d days of water stress treatment - DW dry weight - FW fresh weight - IAA indole-3-acetic acid - RP Reversed Phase - RWC relative water content - TW turgid weight - WC water content - ZR zeatin riboside - water potential     

Mycorrhizal association of Glomus aggregatum with palmarosa enhances growth and biomass

Palmarosa (Cymbopogon martinii var. motia) was found to be associated with a vesicular-arbuscular mycorrhizal (VAM) fungus, Glomus aggregatum. Glasshouse experiments showed that inoculation of palmarosa with G. aggregatum caused a two-fold growth and three-fold biomass production as compared to non-mycorrhizal plants. These findings indicate the potential use of VAM-fungi for improving the production of this essential oil bearing plant.CIMAP Publication No. 879.CIMAP Publication No. 879.     

Early developmentally regulated genes in the arbuscular mycorrhizal fungus Glomus mosseae: identification of GmGIN1, a novel gene with homology to the C-terminus of metazoan hedgehog proteins

The life cycle of the obligate biotrophic arbuscular mycorrhizal fungi comprises several well-defined developmental stages whose genetic determinants are still unknown. With the aim of understanding the molecular processes governing the early developmental phase of the AM fungal life cycle, a subtractive cDNA library was constructed using a suppressive subtractive hybridization technique. The library contains more than 600 clones with an average size of 500 bp. The isolated cDNAs correspond to genes up-regulated during the early development of the AM fungus Glomus mosseaeversus genes expressed in extraradical hyphae. The expression of several of the isolated genes was further confirmed by RT-PCR analysis. Among the isolated clones, a novel gene named GmGIN1 only expressed during early development in G. mosseae was found. The full-length GmGIN1 cDNA codes for a protein of 429 amino acids. The most interesting feature of the deduced protein is its two-domain structure with a putative self-splicing activity. The N-terminal domain shares sequence similarity with a novel family of GTP binding proteins while the C-terminus has a striking homology to the C-terminal part of the hedgehog protein family from metazoa. The C-terminal part of hedgehog proteins is known to participate in the covalent modification of the N-terminus by cholesterol, and in the self-splicing activity which renders the active form of the protein with signalling function. We speculate that the N-terminal part of GmGIN1, activated through a similar mechanism to the hedgehog proteins, has GTP-binding activity and participates in the signalling events prior to symbiosis formation.     

干旱胁迫对两种油桐幼苗生长、气体交换及叶绿素荧光参数的影响

为了探究干旱胁迫对两种油桐(三年桐和千年桐)幼苗光合生理特性的变化及响应,采用盆栽试验,研究不同水分处理(正常供水、轻度干旱、中度干旱、重度干旱)对油桐幼苗生长、叶片气体交换及叶绿素荧光参数的影响。结果表明:与对照相比,轻度干旱胁迫对两种油桐生长、气体交换及叶绿素荧光参数无明显影响(P0.05);中度干旱及重度干旱使两种油桐的叶绿素SPAD值、生长量、净光合速率(P_n)、气孔导度(G_s)、蒸腾速率(T_r)、气孔限制值(L_s)、最大净光合速率(P_(nmax))、光饱和点(LSP)、表观量子效率(AQY)、暗呼吸速率(R_d)、最大光化学效率(F_v/F_m)、实际光化学量子效率(Φ_(PSⅡ))、电子传递速率(ETR)及光化学猝灭系数(q_P)显著下降(P0.05),且在重度干旱胁迫下迅速下降,胞间CO_2浓度(C_i)、水分利用效率(WUE)、光补偿点(LCP)、初始荧光(F_o)、非光化学猝灭系数(NPQ)显著升高(P0.05);中度干旱胁迫下油桐幼苗P_n的降低是由气孔因素及光合机构活性降低的非气孔因素共同引起的,而重度干旱胁迫下光合作用的下降主要是由光合机构活性降低的非气孔因素引起的。三年桐的光合机构活性及光合效率高于千年桐,对干旱胁迫的适应性较千年桐强。     

Mycorrhizal impact on drought stress tolerance of rose plants probed by chlorophyll a fluorescence, proline content and visual scoring

Micropropagated rose plants (Rosa hybrida L., cv. New Dawn) were inoculated with the arbuscular mycorrhizal (AM) fungus Glomus intraradices (Schenk and Smith) and subjected to different drought regimens. The dual objectives of these experiments were to investigate the mechanism and the extent to which AM can prevent drought damages and whether physiological analyses reveal enhanced drought tolerance of an economically important plant such as the rose. In a long-term drought experiment with four different water regimens, visual scoring of wilt symptoms affirmed that AM in a selected host–symbiont combination increased plant performance. This effect was mostly expressed if moderate drought stress was constantly applied over a long period. In a short-term experiment in which severe drought stress was implemented and plants were allowed to recover after 4 or 9 days, no visual differences between mycorrhizal and non-mycorrhizal roses were observed. Therefore, the early physiological steps conferring drought tolerance were prone to investigation. Proline content in leaves proved to be an unsuitable marker for AM-induced drought tolerance, whereas analysis of chlorophyll a fluorescence using the JIP test (collecting stress-induced changes of the polyphasic O-J-I-P fluorescence kinetics in a non-destructive tissue screening) was more explanatory. Parameters derived from this test could describe the extent of foliar stress response and help to differentiate physiological mechanisms of stress tolerance. AM led to a more intense electron flow and a higher productive photosynthetic activity at several sites of the photosynthetic electron transport chain. A K step, known as a stress indicator of general character, appeared in the fluorescence transient only in drought-stressed non-mycorrhizal plants; conversely, the data elucidate a stabilising effect of AM on the oxygen-evolving complex at the donor site of photosystem (PS) II and at the electron-transport chain between PS II and PS I. If drought stress intensity was reduced by a prolonged and milder drying phase, these significant tolerance features were less pronounced or missing, indicating a possible threshold level for mycorrhizal tolerance induction.     

Leaf water and carbohydrate status of VA mycorrhizal rose exposed to drought stress

Summary Shoot water relations and carbohydrate levels were compared for droughted nonmycorrhizal and vesicular-arbuscular (VA) mycorrhizalRosa hybrida L. cv ‘Samantha’ plants grown with high and low phosphorus fertilization. Leaf diffusive conductance (g _i ) of plants colonized byGlomus intraradices Schenk and Smith andGlomus deserticola Trappe, Bloss and Menge were 2 × and 1.5× greater, respectively, than in nonmycorrhizal plants. Regardless of P fertilization, leaf osmotic and bulk water potentials were 0.5 to 1.1 MPa higher in mycorrhizal than in nonmycorrhizal plants. Leaf starch, chlorophyll and water contents while fructose, glucose and total soluble carbohydrates were lower. Level of P fertilization had no effect on water relations or soluble carbohydrate content of nonmycorrhizal roses. The water status of droughted rose was impoved more byG. intraradices than byG. deserticola. Washington State University College of Agriculture and Home Economics Research Center Scientific Paper No. 7375.     

Influence of leaf water status on stomatal response to humidity,hydraulic conductance,and soil drought in Betula occidentalis

Whole-canopy measurements of water flux were used to calculate stomatal conductance (g _s ) and transpiration (E) for seedlings of western water birch (Betula occidentalis Hook.) under various soil-plant hydraulic conductances (k), evaporative driving forces (ΔN; difference in leaf-to-air molar fraction of water vapor), and soil water potentials (Ψ_s). As expected, g _s dropped in response to decreased k or Ψ_S, or increased ΔN(> 0.025). Field data showed a decrease in mid-day g _s with decreasing k from soil-to-petiole, with sapling and adult plants having lower values of both parameters than juveniles. Stomatal closure prevented E and Ψ from inducing xylem cavitation except during extreme soil drought when cavitation occurred in the main stem and probably roots as well. Although all decreases in g _s were associated with approximately constant bulk leaf water potential (ψ_l), this does not logically exclude a feedback response between Ψ_L and g _s . To test the influence of leaf versus root water status on g _s , we manipulated water status of the leaf independently of the root by using a pressure chamber enclosing the seedling root system; pressurizing the chamber alters cell turgor and volume only in the shoot cells outside the chamber. Stomatal closure in response to increased ΔN, decreased k, and decreased Ψ_S was fully or partially reversed within 5 min of pressurizing the soil. Bulk Ψ_L remained constant before and after soil pressurizing because of the increase in E associated with stomatal opening. When ΔN was low (i.e., < 0.025), pressurizing the soil either had no effect on g _s , or caused it to decline; and bulk Ψ_L increased. Increased Ψ_l may have caused stomatal closure via increased backpressure on the stomatal apparatus from elevated epidermal turgor. The stomatal response to soil pressurizing indicated a central role of leaf cells in sensing water stress caused by high ΔN, low k, and low Ψ_S. Invoking a prominent role for feedforward signalling in short-term stomatal control may be premature.     

Influences of Arbuscular Mycorrhizal Fungus Glomus mosseae on Growth and Nutrition of Lentil Irrigated with Arsenic Contaminated Water

Arsenic (As) contamination of irrigation water represents a major constraint to Bangladesh agriculture. While arbuscular mycorrhizal (AM) fungi have their most significant effect on P uptake, they have also been shown to alleviate metal toxicity to the host plant. This study examined the effects of As and inoculation with an AM fungus, Glomus mosseae, on lentil (Lens culinaris L. cv. Titore). Plants were grown with and without AM inoculum for 9 weeks in a sand and terra green mixture 50:50 v/v and watered with five levels of As (0, 1, 2, 5, 10 mg As L⁻¹ arsenate). Inoculum of Rhizobium leguminosarum b.v. Viceae strain 3841 was applied to all plants. Plants were fed with modified Hoagland solution (1/10 N of a full-strength solution and without P). Plant height, leaf number, pod number, plant biomass and shoot and root P concentration/offtake increased significantly due to mycorrhizal infection. Plant height, leaf/ pod number, plant biomass, root length, shoot P concentration/offtake, root P offtake and mycorrhizal infection decreased significantly with increasing As concentration. However, mycorrhizal inoculation reduced As concentration in roots and shoots. This study shows that growing lentil with compatible AM inoculum can minimise As toxicity and increase growth and P uptake.     

Effects of drought stress on photosynthetic gas exchange, chlorophyll fluorescence and stem diameter of soybean plants

Changes in plant growth, photosynthetic gas exchange, chlorophyll fluorescence and stem diameter of soybean [Glycine max (L.) Merr.] plants under drought stress were studied. Total plant dry mass was reduced by 30 % compared to well-watered control plants. Leaf water potential was slightly decreased by water stress. Water stress induced daytime shrinkage and reduced night-time expansion of stem. Photosynthetic rate, stomatal conductance and transpiration rate were significantly declined by water stress, while the intercellular CO₂ concentration was changed only slightly at the initiation of stress treatment. The maximum photochemical efficiency of photosystem 2 and apparent photosynthetic electron transport rate were not changed by water stress.     

Effects of the mycorrhizal fungus Glomus intraradices on uranium uptake and accumulation by Medicago truncatula L. from uranium-contaminated soil

Phytostabilization strategies may be suitable to reduce the dispersion of uranium (U) and the overall environmental risks of U-contaminated soils. The role of Glomus intraradices, an arbuscular mycorrhizal (AM) fungus, in such phytostabilization of U was investigated with a compartmented plant cultivation system facilitating the specific measurement of U uptake by roots, AM roots and extraradical hyphae of AM fungi and the measurement of U partitioning between root and shoot. A soil-filled plastic pot constituted the main root compartment (C_A) which contained a plastic vial filled with U-contaminated soil amended with 0, 50 or 200 mg KH₂PO₄−P kg^–1soil (C_B). The vial was sealed by coarse or fine nylon mesh, permitting the penetration of both roots and hyphae or of just hyphae. Medicago truncatula plants grown in C_A were inoculated with G. intraradices or remained uninoculated. Dry weight of shoots and roots in C_A was significantly increased by G. intraradices, but was unaffected by mesh size or by P application in C_B. The P amendments decreased root colonization in C_B, and increased P content and dry weight of those roots. Glomus intraradices increased root U concentration and content in C_A, but decreased shoot U concentrations. Root U concentrations and contents were significantly higher when only hyphae could access U inside C_B than when roots could also directly access this U pool. The proportion of plant U content partitioned to shoots was decreased by root exclusion from C_B and by mycorrhizas (M) in the order: no M, roots in C_B > no M, no roots in C_B > M, roots in C_B > M, no roots in C_B. Such mycorrhiza-induced retention of U in plant roots may contribute to the phytostabilization of U contaminated environments.     

Genetic differentiation in Plantago major L. in growth and P uptake under conditions of P limitation

To study possible adaptive mechanisms inbred lines from three populations of Plantago major from sites that were found to differ in P availability were compared. In a pot experiment the growth and P uptake either in the presence or absence of Glomus fasciculatum was determined. Under these P-limited conditions it was shown by partitioning the relative growth rate (RGR, in mg g^-1 day^-1) in the components root weight ratio (RWR, in g_roots g_plant ^-1), specific P uptake rate (SPUR, in mol P g_roots ^-1 day^-1), and P-efficiency (PEFF, in mg mol P^-1), that the increase in RGR of mycorrhizal infected plants was related to an increase in SPUR, and a decrease in RWR and PEFF. P. major ssp. major had a lower RGR (related to a lower PEFF and SPUR) and a higher RWR than P. major ssp. pleiosperma. In a second experiment three inbred lines were compared upon P depletion in a nutrient solution. The P. major ssp. major line had a lower RGR and higher RWR, and a higher accumulation of P in the roots than the P. major ssp. pleiosperma lines under optimal growing conditions. There were no differences among the inbred lines in the relative contribution of inorganic P to the total P concentration in the shoot. The results are discussed in relation to the characteristics of the habitats of the investigated P. major populations.     

Leaf water characteristics and drought acclimation in sunflower genotypes

The responses of leaf water parameters to drought were examined using three sunflower (Helianthus annuus L.) genotypes. Osmotic potential at full water saturation (π¹⁰⁰), apoplastic water fraction (AWF) and bulk elastic modulus (BEM) were determined by pressure-volume curve analysis on well watered or on water-stressed plants (−1.0 MPa Ψ₁ < −1.5 MPa) previously drought-pretreated or not. The drought-pretreated plants were subjected to a 7-day drought period (predawn leaf water potential reached −0.9 MPa) followed by 8 days of rewatering. In well watered plants, all genotypes in response to drought acclimation displayed a significantly decreased π¹⁰⁰ associated with a decrease in the leaf water potential at the turgor-loss point (decrease in Ψ_tlp was between 0.15 and 0.21 MPa, depending on the genotype). In two genotypes, drought acclimation affected the partitioning of water between the apoplastic and symplastic fractions without any effect on the total amount of water in the leaves. As a third genotype displayed no modification of AWF and BEM after drought acclimation, the decreased π¹⁰⁰ was only due to the net accumulation of solutes and was consistent with the adjustment of the photochemical efficiency observed previously in this genotype in response to drought acclimation. In water-stressed plants, the osmotic adjustment (OA) can increase further beyond that observed in response to the drought pretreatment. However, the maintenance of photosynthetic rate and stomatal conductance at low leaf water potentials not only depends on the extent of osmotic adjustment, but also on the interaction between OA and AWF or BEM. Adaptative responses of leaf water parameters to drought are thus quite contrasted in sunflower genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in carotenoids, tocopherols and diterpenes during drought and recovery, and the biological significance of chlorophyll loss in Rosmarinus officinalis plants

Two-year-old rosemary (Rosmarinus officinalis L.) plants were subjected to severe stress by exposure to prolonged drought during a Mediterranean summer. Severely stressed plants recovered completely after the autumn rainfalls although the relative water content remained below 35% for 3 months and the chlorophyll content of leaves was reduced by up to 85% during the drought. In severe stress: (i) α-tocopherol increased 9-fold per g dry weight and 20-fold per unit of chlorophyll; (ii) lutein and β-carotene contents decreased on a dry-weight basis, but an 80% increase in lutein and constant levels of β-carotene were observed on a chlorophyll basis; (iii) there were transient and sustained increases in the de-epoxidation state of the xanthophyll cycle; and (iv) the highly oxidised abietane diterpene isorosmanol increased 8-fold as a result of the oxidation of carnosic acid. With the autumn rainfalls, water status, α-tocopherol and violaxanthin recovered first and the levels of photosynthetic pigments and abietane diterpenes increased later. The photoprotection conferred by the xanthophyll cycle and the antioxidant function of tocopherols, lutein and diterpenes may help to avoid irreversible damage in severe drought, making possible the recovery of functional membranes after the autumn rainfalls. Besides, chlorophyll loss reduces the amount of photons absorbed by leaves, which enhances the photoprotective and antioxidant capacity of leaves per amount of photons absorbed, since the ratios of xanthophylls, α-tocopherol and abietane diterpenes to chlorophyll increase. Received: 12 July 1999 / Accepted: 25 November 1999     

Cold-storage of mixed inoculum of Glomus intraradices enhances root colonization,phosphorus status and growth of hot pepper

Growth response of hot pepper (Capsicum annuum L.) inoculated with the arbuscular mycorrhizal (AM) fungus, Glomus intraradices Schenck and Smith was evaluated in a greenhouse study. Three treatments in a soil-based medium amended with rock phosphate were: (1) control (CON), (2) inoculation of G. intraradices as a freshly prepared soil mixture of spores, hyphae and colonized roots of Sorghum vulgare (FM), and (3) inoculation of the fungus as cold-stored mixed inoculum (CM). Colonization at 14 weeks after inoculation with CM was 42.5%, but was significantly lower with FM (14.5%). Inoculation with G. intraradices as FM and CM increased growth of pepper, and total phosphorus and nitrogen uptake in shoots and roots compared with the CON treatment. Inoculation with CM resulted in significant increases in plant dry weight and chlorophyll concentration compared to the FM and CON treatments. Acid phosphatase activity in the rhizosphere was generally increased by AM fungal treatments. Highest acid phosphatase activity occurred at 14 weeks after inoculation with CM. Alkaline phosphatase activity in the CM treatment was significantly higher compared to that in CON and FM treatments throughout the growth period. Thus, cold storage of mixed inoculum enhanced colonization and growth-promoting activity of G. intraradices compared to freshly prepared inoculum.