Response of spring wheat (Triticum aestivum) to interactions between Pseudomonas species and Glomus clarum NT4

The effects of interactions between pseudomonads (Pseudomonas cepacia strains R55 and R85, P. aeruginosa strain R80, P. fluorescens strain R92, and P. putida strain R104) and the arbuscular mycorrhizal fungus Glomus clarum (Nicol. and Schenck) isolate NT4, on spring wheat (Triticum aestivum L. cv. Laura), grown under gnotobiotic and nonsterile conditions, were investigated. Although plant growth responses varied, positive responses to pseudomonad inoculants generally were obtained under gnotobiotic conditions. Shoot dry weight enhancement ranged from 16 to 48%, whereas root enhancement ranged from 82 to 137%. Shoot growth in nonsterile soil, however, was unaffected by pseudomonad inoculants, or reduced by as much as 24%. Shoot growth was unaffected or depressed by G. clarum NT4 whereas early root growth was enhanced by 38%. Significant interactions between the pseudomonad inoculants and G. clarum NT4 were detected. Typically, dual inoculation influenced the magnitude of response associated with any organism applied alone. The effect of these pseudomonads on G. clarum NT4 spore germination was investigated. Germination was inhibited when spores were incubated either on membranes placed directly on bacterial lawns of strains R85 and R104 (i.e., direct assay), or on agarose blocks separated from the bacteria by membranes (i.e., diffusion assay). When the agarose blocks were physically separated from the pseudomonad (i.e., volatile assay), there was no evidence of inhibition, suggesting that a nonvolatile, diffusible substance(s) produced by both strains R85 and R104 may inhibit G. clarum NT4 spore germination. Received: 11 December 1995     

Nature of antagonism of fungi by bacteria isolated from soils

The influence of 457 bacteria isolated from soil on spore germination by Aspergillus flavus was studied by light and scanning microscopy. Bacteria were found to be in physical association with the fungus. The bacteria were tested for antagonism against 11 fungal phytopathogens. A number of the bacterial antagonists displayed a wide spectrum in their activity against the fungi.     

影响丛枝菌根真菌孢子萌发的几种因素研究

对丛枝菌根真菌孢子萌发的几种影响因素进行了研究。结果表明,土壤是丛枝菌根真菌孢子萌发最适宜的培养基;寄主植物根的分泌物对孢子萌发有显著的促进作用。重金属Cd和Pb含量过高时(50mg kg)抑制真菌孢子的萌发。培养基中有效P含量较低时(KH2PO4添加量为0～80mg L),对孢子萌发影响较小,高浓度的有效P(KH2PO4添加量大于100mg L)对孢子萌发有一定的抑制作用。培养基的pH值过高(pH8.0以上)或过低(pH5.5以下)抑制孢子萌发。生长激素对孢子萌发率没有显著性影响。对于有休眠现象的丛枝菌根真菌,4℃低温处理4～6周,可打破休眠孢子的休眠,显著提高休眠孢子的萌发率。     

Factors that Influence the Transport of Bacillus cereus Spores through Sand

The goal of this study is to clarify the surface-chemical and microphysical variables that influence bacterial spore transport through soil, thereby defining the factors that may affect spore transport velocity. Bacillus cereus spores were continuously monitored in a soil column under saturated conditions with experimental variations in soil grain size (0.359 and 0.718 mm), pH (7.2 and 8.5), and water flow rate (1.3 and 3.0 mL/min). Increasing soil grain size, flow rate, and pH resulted in enhanced spore movement. Spore transport increased 82% when soil grain size was doubled. An increase in effluent flow rate from 1.3 to 3.0 mL/min increased spore movement by 71%. An increase in pH increased spore transport by 53%. The increase in hydrodynamic forces resulting from the larger grain size soil and higher flow rate functioned to overcome the hydrophobic nature of the spore’s coat, and the interparticle bonding forces between the spore and soil particles.     

Occurrence and infectivity of arbuscular mycorrhizal fungi in some norwegian soils influenced by heavy metals and soil properties

Mycorrhizae are ubiquitous symbiosis which can mediate uptake of some plant nutrients. In polluted soils they could be of great importance in heavy metal availability and toxicity to plants. Mycorrhizae have also been reported to protect plants against toxic metals. We investigated the occurrence and infectivity of arbuscular mycorrhizal (AM) spores as affected by heavy metal levels and other soil properties in Norwegian soils collected from heavy metal polluted, high natural background and non-polluted areas. Spore numbers, mycorrhizal infectivity and spore germination of indigenous mycorrhizal fungi and of a reference strain (Glomus mosseae) in soils showed lower values in two soils with high metal concentrations and in one soil with a low pH. Mycorrhizal infectivity was negatively correlated with extractable metals. Spore number and mycorrhizal infectivity in a soil with naturally high heavy metal content were not different to in non-polluted soils, and indigenous AM fungi appeared more tolerant to metals than those in non-polluted soils. Mycorrhizal infectivity, expressed as MSI₅₀ values, was significantly correlated (r′=0.89, P< 0.05) with the percentage of germinating G. mosseae spores in the soils. However, the number of spores per volume of soil was not significantly correlated with infectivity or spore germination of the reference strain. The spore germination method is discussed as a bioassay of heavy metal toxicity in soil.     

Association between Burkholderia species and arbuscular mycorrhizal fungus spores in soil

Burkholderia pseudomallei, the bacterial cause of the potentially fatal infection known as melioidosis, has a facultative intracellular lifestyle. The intracellular presence of B. pseudomallei in various eukaryotes including arbuscular mycorrhizal fungus (AMF) spores can be demonstrated in vitro. AMF spores were isolated from soils in a melioidosis-endemic area. B. pseudomallei and other Burkholderia spp. DNA was detected in these AMF spore samples, confirming an AMF spore-Burkholderia spp. association in soils which did not yield Burkholderia spp. by culture. This association may explain the environmental persistence, difficulty of recovery and dispersal of Burkholderia spp. in specific environments.     

Possible contributions of volatile-producing bacteria to soil fungistasis

Soil fungistasis can adversely affect the germination and growth of most fungal species in the field. Among the inhibitors, volatiles of microbial origins are potentially very important. In this study, we investigated the frequency and identity of bacteria producing fungistatic volatiles. Among the 1018 bacterial isolates tested, 328 were found to produce antifungal volatiles that could inhibit spore germination and mycelial growth of two nematicidal fungi Paecilomyces lilacinus and Pochonia chlamydosporia. A phylogenetic analysis based on restriction fragment length polymorphism (RFLP) and 16S rDNA sequence placed the 328 bacteria in five groups: Alcaligenaceae, Bacillales, Micrococcaceae, Rhizobiaceae and Xanthomonadaceae. Volatile compounds of 39 bacterial isolates were identified by gas chromatography/mass spectrum (GC/MS). Tests with commercially available antifungal compounds suggested that seven volatile compounds of bacterial origins (acetamide, benzaldehyde, benzothiazole, 1-butanamine, methanamine, phenylacetaldehyde and 1-decene) likely play important roles in soil fungistasis.     

Inoculation of seeds and soil with basidiospores of mycorrhizal fungi

It was demonstrated that basidiospores of the fungus Rhizopoyon luteolus, mycorrhizal for Pinus radiata, could be used successfully as seed inoculum after freeze-drying and storage for 3 months at 22°C, provided the inoculum level was increased 100-fold. Spore inoculum applied to seed could be held dry for at least 2 days before planting provided inoculum was increased 10-fold. On sowing freshly inoculated seed to sterile soil, 3 × 10³ basidiospores/seed were adequate for infection but maximum mycorrhizal infection occurred with 3 × 10⁴ spores/seed.A dose-response curve was obtained for mycorrhizal infection when basidiospores were applied to soil. As few as 100 spores/290 cm³ pot were sufficient for mycorrhizal infection although infection increased with greater spore dose to a maximum of 10⁵ spores/pot. Plant growth response was related to intensity of infection. It is suggested that the percentage germination of basidiospores in the rhizosphere may be considerably greater than those reported in studies with synthetic medium. A rhizosphere effect on germination of basidiospores was demonstrated and a method developed to facilitate studies of spore germination in the rhizosphere.     

内生环状芽孢杆菌Jcxy8对番茄灰霉病的防病机制研究

为明确从番茄植株体内筛选出的内生环状芽孢杆菌(Bacillus circulan)Jcxy8对番茄灰霉病菌的抑菌作用及防病的生理生化机制,采用平板打孔法测定了菌株Jcxy8对灰霉病菌(Botrytiscinerea)的拮抗力。结果表明:菌株Jcxy8对灰霉病菌的抑菌圈直径为35.6mm,抑菌圈边缘的产孢抑制率达到66.9%。当菌株培养滤液浓度为40%时,病菌孢子萌发完全被抑制。镜检发现抑菌圈周围的菌丝(或芽管)细胞消融,生长扭曲,中间或顶端膨大成泡囊状。Jcxy8菌株与灰霉病菌同时处理的番茄果体内可溶性蛋白含量比清水对照处理高12.7%,比单独接种灰霉病菌处理高39.1%;SOD、POD、CAT活性均较只经病菌处理低;O2-产生速率比清水对照和病菌处理低,而比菌株Jcxy8处理高。说明菌株Jcxy8对番茄果实有明显的诱导抗病作用。     

Arbuscular mycorrhizal fungal community structure and diversity in response to 3-year conservation tillage management in a sandy loam soil in North China

Purpose

Modern agricultural science has greatly reduced the use of tillage. Monitoring conservation versus conventional tillage effects on soil microbes could improve our understanding of soil biochemical processes and thus help us to develop sound management strategies. The objective of this study was to investigate the effects of conservation tillage on the spore community structure and the diversity of soil arbuscular mycorrhizal (AM) fungi and to find out the main factors that influence these parameters.

Materials and methods

A long-term field experiment established in a sandy loam soil in Northern China has received continuous tillage management treatments for 3?years, including conventional tillage (CT), no tillage (NT), and alternating tillage (AT). Topsoil samples (0?C15?cm) from four individual plots per treatment were collected for the analysis of chemical properties and fungal parameters. AM fungal spores were isolated using the wet-sieving method and identified to species level based on morphology by light microscopy. The community structure and the diversity of AM fungi were evaluated using the following parameters: spore density, relative abundance, species richness, Shannon?CWiener index (H??), evenness (E), and Simpson's index (D). Jaccard index (J) of similarity was calculated to compare AM fungal species composition under different treatments.

Results and discussion

Twenty-eight species of AM fungi within four genera, Glomus, Acaulospora, Scutellospora, and Entrophospora, were recovered from the 12 plots within the three tillage management treatments. Higher spore density, species richness, and species diversity (H??, E, and D) of AM fungi were observed in the two conservation tillage treatments, and the redundancy analysis showed that the species richness significantly correlated to soil organic carbon content (P?<?0.05). The positive effects of NT and AT on the species richness were very close, while the AT had relatively greater beneficial impacts on the spore density and the evenness of AM fungi compared to the NT. The lowest Jaccard index (J) of similarity in species composition was also observed between the AT and CT treatments.

Conclusions

Soil organic carbon, the spore density, and species richness and diversity of AM fungi increased in the two conservation tillage treatments. The species richness of AM fungi significantly correlated to soil organic carbon content (P?<?0.05). Compared with the CT treatment, the AT rather than the NT significantly increased the spore density and the evenness of AM fungi (P?<?0.05). Thus, alternating tillage practice may be more beneficial to agroecosystem in this region.     

Effect of Zn-tolerant bacterial strains on growth and Zn accumulation in Orychophragmus violaceus

Several Zn-tolerant bacterial strains were isolated from heavy-metal contaminated sludge, and their effects on root elongation, mobility, and accumulation of Zn in Orychophragmus violaceus were studied. The isolated strains included Bacillus subtilis, B. cereus, Flavobacterium sp. and Pseudomonas aeruginosa which were capable of stimulating root elongation in O. violaceus seedlings either in the presence or absence of Zn. The four bacterial strains significantly increased the concentration of water-extractable Zn compared with axenic soil. In addition, the four Zn-tolerant bacteria significantly increased the shoot biomass and Zn accumulation in O. violaceus compared to non-inoculated plants. The bacterial strains displayed different capacities to enhance plant Zn accumulation. Flavobacterium sp. was identified as the best candidate for enhancing Zn accumulation in plants, increasing Zn accumulation up to 1.21- and 1.19-fold in shoots and roots, respectively, compared to non-inoculated plants. It was indicated that Zn-tolerant bacteria played an important role in influencing the availability of water-soluble Zn in soil and Zn accumulation by plants. This study provides insight into the development of plant–microbe systems for phytoremediation.     

Monitoring biochemical changes in bacterial spore during thermal and pressure-assisted thermal processing using FT-IR spectroscopy

Pressure-assisted thermal processing (PATP) is being widely investigated for processing low acid foods. However, its microbial safety has not been well established and the mechanism of inactivation of pathogens and spores is not well understood. Fourier transform infrared (FT-IR) spectroscopy was used to study some of the biochemical changes in bacterial spores occurring during PATP and thermal processing (TP). Spore suspensions (approximately 10(9) CFU/mL of water) of Clostridium tyrobutyricum, Bacillus sphaericus, and three strains of Bacillus amyloliquefaciens were treated by PATP (121 degrees C and 700 MPa) for 0, 10, 20, and 30 s and TP (121 degrees C) for 0, 10, 20, and 30 s. Treated and untreated spore suspensions were analyzed using FT-IR in the mid-infrared region (4000-800 cm(-1)). Multivariate classification models based on soft independent modeling of class analogy (SIMCA) were developed using second derivative-transformed spectra. The spores could be differentiated up to the strain level due to differences in their biochemical composition, especially dipicolinic acid (DPA) and secondary structure of proteins. During PATP changes in alpha-helix and beta-sheets of secondary protein were evident in the spectral regions 1655 and 1626 cm(-1), respectively. Infrared absorption bands from DPA (1281, 1378, 1440, and 1568 cm(-1)) decreased significantly during the initial stages of PATP, indicating release of DPA. During TP changes were evident in the bands associated with secondary proteins. DPA bands showed little or no change during TP. A correlation was found between the spore's Ca-DPA content and its resistance to PATP. FT-IR spectroscopy could classify different strains of bacterial spores and determine some of the changes occurring during spore inactivation by PATP and TP. Furthermore, this technique shows great promise for rapid screening PATP-resistant bacterial spores.     

Early effects of tillage and crop rotation on arbuscular mycorrhizal fungal propagules in an Ultisol

The aim of this work was to study the early influence of conventional tillage (CT) and no-tillage (NT) on arbuscular mycorrhizal fungal (AMF) propagules. A short 2-year-course crop rotation, i.e. trial consisting of a succession of wheat and oat, was studied in a typic Chilean Ultisol from the second to fourth year after the beginning of the experiment. Measurements included mycorrhizal characteristics and some soil properties in order to explain their influence on AMF propagules. Soil samples were taken yearly in autumn (fallow period) and in early spring (flowering). Significant differences in AMF hyphal length were observed between NT and CT in the first year, but such differences disappeared thereafter. No differences in metabolically active hyphae were obtained with wheat or oat under the two tillage systems. Mycorrhizal root colonization was always higher under NT than under CT. The number of AMF spores was also higher under NT than under CT, ranging from 158 to 641 spores per 100 cm³. Twenty-two AMF species including eight Glomus spp., six Acaulospora spp., four Scutellospora spp., one Archaeospora sp., one Diversispora sp., one Entrophospora sp. and one Pacispora sp. were observed in both agro-ecosystems. Higher spore number of Acaulospora spp. was found under wheat than under oat and under CT than under NT, whilst more spores of Scutellospora spp. were observed under NT than under CT. From all mycorrhizal characteristics, spore number could be visualized as an early and useful indicator of the effect of tillage systems on mycorrhizal propagules in short-term experiments.     

Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere

The capability of native bacterial strains isolated from Lolium perenne rhizosphere to behave as plant growth promoting bacteria and /or biocontrol agents was investigated. One strain (BNM 0357) over 13 isolates from the root tips of L. perenne resulted proved to be nitrogenase positive (ARA test) and an IAA producer. Conventional tests and the API 20E diagnostic kit indicated that BNM 0357 behaves to the Enterobacteriaceae family and to the Enterobacter genus. Molecular identification by 16S rRNA sequence analysis indicated that BNM 0357 had the highest similarity to Enterobacter ludwigii (EN-119). Isolate BNM 0357 had the capability to solubilize calcium triphosphate and to antagonize Fusarium solani mycelial growth and spore germination. Strain BNM 0357 also showed the ability to improve the development of the root system of L. perenne. This study disclosed features of E. ludwigii BNM 0357 that deserve further studies aimed at confirming its putative importance as a PGPR.     

Quantitative evaluation of the microbial nutrient sink in soil in relation to a model system for soil fungistasis

A quantitative approach was devised to evaluate the influence of soil microbial activity as a sink for nutrients exuded by fungal spores as a factor in soil fungistasis. The approach was based on measuring the CO₂ evolved from microbial respiration of ¹⁴C-labelled exudates from conidia of Cochliobolus victoriae incubated on soil. The amount of exudate lost by spores on soil was greater than the amount lost by spores incubated on a bed of sand undergoing leaching at a flow rate of 110 ml h^?1. where restriction of germination was similar to that on soil. Increasing flow rates in the leaching system increased spore exudation and reduced germination. Germination of C. victoriae conidia on membrane filters floated on distilled water decreased as the volume of water increased. The results indicate that the microbial nutrient sink of soil is sufficient to impose soil fungistasis.     

Technique for visual demonstration of germinating arbuscular mycorrhizal spores and their multiplication in pots

We describe a simple technique for the germination of arbuscular mycorrhizal (AM)–fungal spores and their multiplication in pots. Glomus fasciculatum, G. mosseae, and Gigaspora margarita were used. A single wheat seedling was tied to a glass slide, previously covered with filter paper with the help of thread. One single surface‐sterilized AM‐fungal spore was placed on the middle portion of the root of the wheat seedling using a sterilized syringe. The slide was placed vertically in a 100 mL glass beaker filled with 25 mL of root exudates–water (1:4, v/v) solution, which was collected by growing twenty wheat seedlings in a 150 mL beaker filled with 100 mL sterilized distilled water for 7 d. The slide was observed daily using a compound microscope to follow the time course of germination. In this technique, the spore is directly in contact with the host root, and a visualization of spore germination, hyphal development, and appressorium formation is possible without disrupting fungal growth or the establishment of the symbiosis. The method allows to document the germination events and to assess hyphal‐elongation rates by photographing the same spore on consecutive days. The inoculated seedling was used to initiate single‐spore multiplication in a sterilized (autoclave on 3 alternate days at 120°C for 120 min at 1.05 kg cm^–2 pressure) potted sandy soil (150 mL volume) into which the slide with the inoculated seedling was inserted carefully through a previously made slit. The wheat seedlings in all pots (4 treatments and 15 replications) became colonized by mycorrhiza, confirming that the establishment of the AM‐fungal symbiosis is highly reproducible. Our technique permits the relatively undisturbed growth of the symbiotic partners, the visualization of germinating AM‐fungal spores, and their multiplication in pots. This simple and low‐cost method facilitates the production of pure lines of AM fungi from single spores, allowing for the study of intraspecific variation and potentiality for cytological, biochemical, physiological, and taxonomical studies.     

The Influence of Heavy Metals and Mineral Nutrient Supply on Bituminaria bituminosa

With a view to the selection of plants for the re-vegetation of contaminated, semi-arid land, two populations of the perennial species Bituminaria bituminosa (Fabaceae) from the south of Spain were studied: one (“LA”) from a non-contaminated soil and the other (“C2”) from a similar soil having elevated total levels of Pb and Zn (1,112 and 4,249 μg g^?1, respectively). For sand-cultured plants receiving nutrient solution, flow cytometry showed that heavy metals, at the concentrations measured in aqueous extracts from contaminated soils, had only slight genotoxic effects on root tip cell nuclei. Both populations were also grown in both soils, in two pot experiments. In the first, shoot biomass of LA and C2 in the contaminated soil was decreased to similar extents, with respect to the “clean” soil. Tissue heavy metal concentrations were unlikely to have been phytotoxic, except in the case of shoot Zn for population LA, but there were tissue deficiencies of P and K for populations LA and C2, respectively. In the second pot assay, the stimulation of growth by NPK fertiliser confirmed that even though this soil had high total heavy metal levels, nutrient availability was the principal factor limiting growth. The lesser transport of heavy metals (Cd, Mn and Zn) to the shoot by the population from the contaminated site is a factor that should be considered when selecting B. bituminosa lines for the phytostabilisation of such sites.     

The potential contribution of siderophore producing bacteria on growth and Fe ion concentration of sunflower (Helianthus annuus L.) under water stress

In the present study, 100 bacterial strains were isolated from the soil and tested for siderophore producing ability. Among siderophore producing bacteria, three superior isolates with the highest level of siderophore (F13, F58 and F66) selected for further study. Production of lecithinase, arginine dehydrolase, catalase, oxidase, hydrogen cyanide, indole acetic acid, solubilization of phosphate, gelatin hydrolysis, oxidative/fermentative, motility and gram staining by these strains was evaluated. In greenhouse experiment, the ability of selected isolates in solubilizing insoluble iron-containing compounds was examined at three moisture levels (0.45–0.6, 0.6–0.7 and 0.7–0.85 field capacity). Results showed that isolates F13 and F66 have greatest effects on chlorophyll amount and shoot dry weight compared to isolate F58. On average, iron concentration of sunflower in the presence of bacteria was 2.85-fold more than control, while it was 1.88-fold for roots. In addition, bacterial treatments enhanced resistance to water stress in sunflower. Based on biochemical tests and by comparison of 16S rRNA sequences, the F13, F58 and F66 isolates were closely related to Pseudomonas spp., Enterobacter spp. and Bacillus Sporothernodurans, respectively.     

Impact of electromagnetic microwaves on the germination of spores of <Emphasis Type="Italic">Streptomyces xanthochromogenes</Emphasis> in a peat soil and in a liquid nutrient medium

The impact of microwaves on the germination of spores of Streptomyces xanthochromogenes in a liquid nutrient medium and in a peat soil was studied. The treatment of inoculums with microwave radiation affected the development of the microorganisms from the stage of spore germination to the stage of the formation of microcolonies of actinomycetes upon the spore cultivation in the liquid medium. Typical hypnum-herbaceous peat was used to study the rate of germination of the actinomycetal spores in soil. The study of the dynamics of the Streptomyces xanthochromogenes population in the control soil (without treatment with microwaves) showed that the most active development of the culture took place in the soil moistened to 60% of the maximum water capacity. When the soil was moistened to the minimum adsorption capacity, the streptomyces did not complete their full cycle of development. The stimulation of the spore germination and mycelium growth with microwaves in the soil medium required a longer period in comparison with that for the liquid medium. The stimulation of the spore germination was observed in the liquid nutrient medium in the case of 30-s treatment and in the soil in the case of 60-s treatment.     

Influence of growth-promoting bacteria on the growth of wheat in different soils and temperatures

Plant-growth-promoting bacteria isolated from the rhizosphere, phyllosphere and soil of the root zone in different climatic regions of Germany and Uzbekistan were analysed for plant-growth-promoting effects and nutrient uptake on winter wheat on different soils and under different temperature regimes. The investigations were carried out in pot experiments using loamy sand and sandy loam soils from Müncheberg, Germany and Calcisol soil from Tashkent, Uzbekistan. The temperature and soil types were found to influence growth-promoting effects. Inoculation with bacterial strains Pseudomonas fluorescens PsIA12, Pantoea agglomerans 050309 and Mycobacterium sp. 44 isolated from Müncheberg (semi-continental climate) was found to significantly increase the root and shoot growth of winter wheat at 16 °C compared to 26 °C in loamy sand. Mycobacterium phlei MbP18 and Mycoplana bullata MpB46 isolated from Tashkent (semi-arid climate) were found to significantly increase the root and shoot growth of winter wheat in nutrient-poor Calcisol at 38 °C as well as in nutrient-rich loamy sand at 16 °C. Bacterial inoculation also resulted in significantly higher N, P, and K contents of plant components. The bacteria isolates were able to survive in the rhizosphere and in the soil of winter wheat after root and shoot inoculation.