Identification of a Vesicular-Arbuscular Mycorrhizal Fungus by Using Monoclonal Antibodies in an Enzyme-Linked Immunosorbent Assay

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains.     

Synergistic effect of colonization with arbuscular mycorrhizal fungi improves growth and drought tolerance of Plukenetia volubilis seedlings

The objective of this study was to investigate the effects of arbuscular mycorrhizal fungus (AMF) inoculation on plant growth and drought tolerance in seedlings of a promising oilseed crop, Sacha Inchi (Plukenetia volubilis L.), under well-watered or drought conditions. AMF inoculation was applied in four treatments: without AMF inoculation, Glomus versiforme, Paraglomus occultum, or combination of both microorganism inoculations. The results showed that AMF colonization significantly enhanced the growth of Sacha Inchi seedlings regardless of soil water conditions, and the greatest development was reached in plants dually inoculated under well-watered conditions. G. versiforme was more efficient than P. occultum. Plants inoculated with both symbionts had significantly greater specific leaf area, leaf area ratio and root volume when compared with the uninoculated control, G. versiforme, and P. occultum treatments alone, indicating a synergistic effect in the two AMF inoculation. Photosynthetic rate and water-use efficiency were stimulated by AMF, but not stomatal conductance. Inoculation with AM fungus increased antioxidant enzymes activities including guaiacol peroxidase and catalase, thus lowering hydrogen peroxide accumulation and oxidative damage, especially under drought stress conditions. However, proline content showed little change during drought stress and AMF colonization conditions, which suggested that proline accumulation might not serve as the main compound for osmotic adjustment of the studied species. These results indicate that AMF inoculation stimulated growth and enhanced drought tolerance of Sacha Inchi seedlings, through alterations in morphological, physiological and biochemical traits. This microbial symbiosis might be an effective cultivation practice in improving the performance and development for Sacha Inchi plants.     

Soil Fungicides for Controlling Chytridiaceous Mycoparasites of Gigaspora margarita and Glomus fasciculatum

The percentage of azygospores of Gigaspora margarita with zoosporangia of chytridiaceous fungi (CF) was reduced significantly after agitating them in fenaminosulf before incubation in soil. Fenaminosulf did not affect zoosporangia development on chlamydospores of Glomus fasciculatum. Metalaxyl and ethazol were not effective against CF on spores of either mycorrhizal fungus. Azygospores of G. margarita were treated with fenaminosulf and used as the inoculum for pot cultures. After 19 weeks, the percentage of azygospores containing CF was reduced significantly by this treatment, whereas root colonization and sporulation by the mycorrhizal fungus were unaffected. Pot cultures of G. margarita, either drenched with fenaminosulf or not, did not differ in the percentage of azygospores containing CF. However, root colonization and sporulation by the mycorrhizal fungus was temporarily delayed when pots were drenched with fenaminosulf.     

Fungal Growth Stimulation by CO2 and Root Exudates in Vesicular-Arbuscular Mycorrhizal Symbiosis

Transformed roots of carrot were used to determine the effects of root metabolites on hyphal development from spores of the vesicular-arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal growth of this obligately biotrophic symbiont was greatly stimulated by a synergistic interaction between volatile and exudated factors produced by roots. Root volatiles alone provided little stimulation, and root exudates alone had no effect. For the first time, carbon dioxide was demonstrated to be a critical root volatile involved in the enhancement of hyphal growth. ¹⁴C-labeled root volatiles were fixed by the fungus and thus strongly suggested that CO₂ served as an essential carbon source.     

Proof for the Production of Cutinase by Fusarium solani f. pisi during Penetration into Its Host, Pisum sativum

Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this ferritin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.     

Towards Growth of Arbuscular Mycorrhizal Fungi Independent of a Plant Host

When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Sy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G. Bécard and J. A. Fortin (New Phytol. 108:211-218, 1988), slime-forming bacteria, identified as Paenibacillus validus, frequently grew up. These bacteria were able to support growth of the fungus on the agar plates. In the presence of P. validus, hyphae branched profusely and formed coiled structures. These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA from Agrobacterium rhizogenes. The presence of P. validus alone also enabled G. intraradices to form new spores, mainly at the densely packed hyphal coils. The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei. In these experiments P. validus could not be replaced by bacteria such as Escherichia coli K-12 or Azospirillum brasilense Sp7. Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) from P. validus which stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.     

ROLE OF MYCORRHIZAL FUNGUS ORIGIN IN GROWTH AND NUTRIENT UPTAKE BY GERANIUM ROBERTIANUM

Prior field studies have shown that populations of forest herbs on relatively nutrient poor soils have higher vesicular-arbuscular mycorrhizal (VAM) infection intensity than plants on rich soils. However, the growth responses and ability to take up P against the soil nutrient gradient are often not linearly related to infection intensity. To determine if intraspecific differences among populations of the common VAM fungus Glomus occultum could differentially affect growth and nutrient uptake, Geranium robertianum seedlings were inoculated with Glomus occultum isolated from four forest types along a gradient of soil fertility, and grown in a greenhouse at P levels typical of the extremes of that gradient. Plants given inoculum from relatively infertile forest sites generally produced greater root, shoot, and total mass than plants given inoculum from fertile sites or uninoculated plants, especially at the low P supply rate. Total P uptake and both P and N uptake efficiency were also highest in plants given inocula from low fertility sites. These results indicate that local adaptation and intraspecific variations in the ability of VAM fungi to induce growth and nutrient uptake effects on host plants may be as important as interspecific differences among VAM fungus species.     

Contribution of different arbuscular mycorrhizal fungal inoculum to <Emphasis Type="Italic">Elymus nutans</Emphasis> under nitrogen addition

Arbuscular mycorrhizal (AM) fungi are known to promote plant growth and nutrient uptake, but their role in nitrogen (N) uptake still remains unclear. Therefore, a pot experiment was set up to evaluate the impacts of N addition and AM inoculation (Diversispora eburnea, Claroideoglomus etunicatum, Paraglomus occultum, and their mixture) on AM root colonization, plant biomass, N and P nutrition in Elymus nutans. Our results showed that AM root colonization was unaffected by N addition but was significantly affected by different AM fungal species. D. eburnea and C. etunicatum showed significant higher root colonization than P. occultum. The E. nutans exhibited the highest biomass when inoculated with D. eburnea and significantly higher than non-mycorrhizal (the control) regardless of N addition. Under N addition treatment, D. eburnea significantly enhanced P content of roots, N content of shoots and roots, while AM mixture significantly enhanced shoot P content compared with non-mycorrhizal. However, N and P content in shoots and roots did not significantly vary among treatments when no N was added. In addition, inoculation with C. etunicatum and P. occultum showed no significant effect on plant biomass, N and P content regardless of N addition. In conclusion, this study revealed that the plant response to N addition depends on AM fungal species and also confirmed that significant functional diversity exists among AM fungal species.     

Identification and Isolation of Two Ascomycete Fungi from Spores of the Arbuscular Mycorrhizal Fungus Scutellospora castanea

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.     

Effect of activated charcoal and pruning of the taproot on the in vitro mycorrhization of Hevea brasiliensis Müll. Arg.

In vitro mycorrhization of Hevea brasiliensis under autotrophic culture conditions is a promising methodology to produce plantlets adapted to overcome stresses during acclimatization. However, to succeed in the in vitro production of mycorrhizal plantlets, root production and subsequent colonization by the mycorrhizal fungus need to be optimized. Plantlets of H. brasiliensis clone PB 260 were grown in contact with the extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833. Addition of activated charcoal to the medium and pruning of the taproot were evaluated for their effects on root growth and colonization. None of the treatments stimulated the early formation of new roots. However, total root length, total root colonization, and production of arbuscules and intraradical spores/vesicles were significantly higher in plantlets grown in the presence of activated charcoal (especially after 13 wk of culture). In contrast, total root colonization was significantly lower in the pruned plantlets, while total root length and arbuscule formation were not affected. None of the treatments affected activities of succinate dehydrogenase and alkaline phosphatase measured in the extraradical mycelium of the fungus. It appeared that the addition of activated charcoal to the culture medium favored root growth and mycorrhization of rubber plantlets under in vitro culture conditions, while taproot pruning did not favor these parameters.     

Indigenous Arbuscular Mycorrhizal Fungi Associated with Acacia albida Del. in Different Areas of Senegal

The influences of seasons, plant age, and physicochemical properties of the soil on surface and deep biological arbuscular mycorrhizal fungus parameters associated with Acacia albida were assessed in different areas of Senegal. More indigenous arbuscular mycorrhizal propagules were found in the localities of the Sudano-Guinean zone (Djinaki and Kabrousse) than in those of the Sahelian zone (Louga and Diokoul), and species belonging to the genera Glomus, Gigaspora, Acaulospora, and Sclerocystis prevailed. The numbers of total and viable spores increased more during the rainy season than during the dry season (about 108% more total spores and 262% more viable spores). Similarly, both total and viable spores were more prevalent around young Acacia trees than old trees. However, the intensities of root colonization did not differ in each ecoclimatic zone.     

Influence of arbuscular mycorrhizal fungi on microbes and enzymes of soils from different cultivated densities of red clover

Effects of arbuscular mycorrhizal fungus (AMF)Glomus mosseae on plant growth, soil microbial populations and enzymes activities of soils were studied in red clover (Trifolium pratense L.) grown in pots at different cultivated densities. Seeds of red clover were sown with 50 g inoculums ofG. mosseae per pot. After a week, the plants were thinned to 20, 30, 40, 50 and 60 seedlings per pot. Three months after treatment, AMF inoculation significantly stimulated plant growth. Quantities of vesicles and spores, arbuscules and hyphae were the highest when 30 and 50 seedlings were grown per pot, respectively. However, no root was infected in control plants. In all the soil sites, the numbers of fungi and bacteria were followed in the order: root > root surface > rhizospheric. It was indicated that arbuscular mycorrhizal fungus decreased the numbers of fungi and bacteria but improved growth of actinomycetes. Compared to control plants, AMF stimulated activities of phosphatase and urease but decreased invertase.     

In vitro mycorrhization of the rubber tree Hevea brasiliensis Müll Arg

In vitro cultivation systems of arbuscular mycorrhizal fungi are useful tools to study the interaction between plants and their fungal symbiont, and also to develop new biotechnologies. Plantlets of the latex-producing species Hevea brasiliensis clone PB 260 were grown in a dense extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 developed from a mycelium donor plant (Medicago truncatula A17). The factors indole-3-butyric acid (IBA), 2-morpholineoethanesulfonic acid monohydrate (MES) buffer, and carbon dioxide (CO₂) were tested on root development and colonization by the fungus. No colonization was observed in the presence of plantlets pre-treated with IBA. The highest levels of root colonization were obtained when plantlets were mycorrhized under a high CO₂ concentration (1,000 μmol?mol^?1) with MES (10 mM) added to the growth medium. Widespread root colonization (with presence of arbuscules, intraradical mycelium, and spores/vesicles) was predominantly observed in newly produced roots. Therefore, it appears essential to improve root initiation and growth for improving in vitro mycorrhization of H. brasiliensis. We demonstrated the potential of the “mycelium donor plant” in vitro culture system to produce colonized H. brasiliensis plantlets before their transfer to ex vitro conditions.     

Identification of arbuscular mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland

 Analysis of the community of arbuscular mycorrhizal (AM) fungi in roots of Fragaria vesca growing in a heavy metal contaminated site was carried out on a Zn waste site near Chrzanow (southern Poland). The waste substratum was characterized by high contents of Pb, Zn, Cd, Cu and As, and by low levels of N, P and organic matter. Spores of Glomales were isolated by wet sieving and DNA was isolated from individual spores. Nested polymerase chain reaction (PCR) with taxon-specific primers was used to identify the species Glomus mosseae, Glomus intraradices and Glomus claroideum. Spores of other fungi were morphologically characterized and new taxon-discriminating molecular probes were developed for two of them (Glomus sp. HM-CL4 and HM-CL5) based on variations in the large ribosomal subunit (25S rDNA). High sequence similarities were found between Glomus sp. HM-CL4 and Glomus gerdemanii, and between Glomus sp. HM-CL5 and Glomus occultum. The designed primers were used to characterize the population of AM fungi colonizing the roots of F. vesca collected from the Zn waste site. The analysis, carried out on roots stained with trypan blue, showed that the most effective colonizer was closely related to G. gerdemannii. G. claroideum and the G. occultum-like fungus were slightly less common whilst frequencies of G. intraradices and G. mosseae in roots were much lower. The analysis of mycorrhiza stained with rhodizoniate to localize heavy metal accumulation showed that the stain does not influence the PCR reaction. Seventy percent of the root samples containing positively stained fungal hyphae were found to be colonized by G. mosseae. The data obtained demonstrate the usefulness of nested PCR for studies carried out in polluted areas. It will enable selection of AM fungi which are able to colonize plant roots under heavy metal stress conditions, as well as the identification of fungi showing high in situ accumulation of potentially toxic elements. Accepted: 7 July 2000     

丛枝菌根真菌和根瘤菌对白三叶氮同化的影响

为揭示丛枝菌根真菌(AMF)和根瘤菌在白三叶氮(N)同化中的作用,该研究对白三叶进行单一或联合接种隐类球囊霉(Paraglomus occultum)和三叶草根瘤菌(Rhizobium trifolii),分析其对白三叶的生长、光合作用、叶片N和氨基酸含量以及N同化相关酶活性的影响。结果表明:(1)单一接种AMF或根瘤菌以及联合接种AMF和根瘤菌均显著增加了白三叶的株高、匍匐茎长度、叶片数、地上部生物量、总生物量、叶绿素b和总叶绿素含量、稳态光量子效率和叶片N含量,这种增强效应是联合接种>单一AMF>单一根瘤菌>未接种处理。(2)联合接种AMF和根瘤菌显著增加了白三叶叶片中丙氨酸、精氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸和组氨酸的含量,显著提升了叶片N同化相关酶如硝酸还原酶、亚硝酸还原酶、谷氨酰胺合成酶、谷氨酸合成酶、谷氨酸脱氢酶、天冬酰胺合成酶和天冬氨酸转氨酶的活性,显著促进AMF对白三叶根系的侵染。综上认为,联合接种AMF和根瘤菌通过激活N同化相关酶活性有效促进N同化,产生更多氨基酸,进一步促进白三叶植株生长; 联合接种AMF和根瘤菌具有协同作用,有效促进了白三叶的N同化。     

Contributions of arbuscular mycorrhizal fungi to growth,photosynthesis, root morphology and ionic balance of citrus seedlings under salt stress

A pot study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi (Glomus mosseae and Paraglomus occultum) and salt (NaCl) stress on growth, photosynthesis, root morphology and ionic balance of citrus (Citrus tangerine Hort. ex Tanaka) seedlings. Eighty-five-day-old seedlings were exposed to 100 mM NaCl for 60 days to induce salt stress. Mycorrhizal colonization of citrus seedlings was not affected by salinity when associated with P. occultum, but significantly decreased when with G. mosseae. Compared with the non-mycorrhizal controls, mycorrhizal seedlings generally had greater plant height, stem diameter, shoot, root and total plant biomass, photosynthetic rate, transpiration rate and stomatal conductance under the 0 and 100 mM NaCl stresses. Root length, root projected area and root surface area were also higher in the mycorrhizal than in the non-mycorrhizal seedlings, but higher root volume in seedlings with G. mosseae. Leaf Na⁺ concentrations were significantly decreased, but leaf K⁺ and Mg²⁺ concentrations and the K⁺/Na⁺ ratio were increased when seedlings with both G. mosseae and P. occultum. Under the salt stress, Na⁺ concentrations were increased but K⁺ concentrations decreased in the mycorrhizal seedlings. Under the salt stress, Ca²⁺ concentrations were increased in the seedlings with P. occultum or without AM fungi (AMF), but decreased with G. mosseae. Ratios of both Ca²⁺/Na⁺ and Mg²⁺/Na⁺ were also increased in seedlings with G. mosseae under the non-salinity stress, while only the Mg²⁺/Na⁺ ratio was increased in seedlings with P. occultum under the salt stress. Our results suggested that salt tolerance of citrus seedlings could be enhanced by associated AMF with better plant growth, root morphology, photosynthesis and ionic balance.     

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10⁴ B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.     

Arbuscular mycorrhizal fungi can alter some root characters and physiological status in trifoliate orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.) seedlings

Citrus plants strongly depend on mycorrhizal symbiosis because of less or no root hairs, but few reports have studied if their root traits and physiological status could be altered by different arbuscular mycorrhizal fungi (AMF). In a pot experiment we evaluated the effects of three AMF species, Glomus mosseae, G. versiforme and Paraglomus occultum on the root traits and physiological variables of the trifoliate orange (Poncirus trifoliata L. Raf.) seedlings. Root mycorrhizal colonization was 58–76% after 180 days of inoculation. AMF association significantly increased plant height, stem diameter, leaf number per plant, shoot and root biomass. Mycorrhizal seedlings also had higher total root length, total root projected area, total root surface area and total root volume but thinner root diameter. Among the three AMFs, greater positive effects on aboveground growth generally ranked as G. mosseae > P. occultum > G. versiforme, whilst on root traits as G. mosseae ≈ P. occultum > G. versiforme. Compared to the non-mycorrhizal seedlings, contents of chlorophyll, leaf glucose and sucrose, root soluble protein were significantly increased in the mycorrhizal seedlings. In contrast, root glucose and sucrose, leaf soluble protein, and activity of peroxidase (POD) in both leaves and roots were significantly decreased in the mycorrhizal seedlings. It suggested that the improvement of root traits could be dependent on AMF species and be related to the AMF-induced alteration of carbohydrates and POD.     

"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A

A simple and efficient procedure was employed for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to sheets of pure, unmodified nitrocellulose. Immobilized proteins could then be radiographically visualized in situ by reaction with specific antibody and the subsequent binding of radioiodinated Staphylococcus protein A to the immune complexes. The detection of murine leukemia virus antigens in complex cellular lysates was used to demonstrate the efficacy of this technique.     

Development,Distribution, and Host Studies of the Fungus Macrobiotophthoira vermicola (Entomophthorales)

The life cycle and host range of Macrobiotophthora vermicola were studied. Secondary spores produced from forcibly ejected primary spores adhered to the cuticle of Cruznema tripartitum, germinated, and penetrated the cuticle within 30 minutes. New primary spores were produced within 24 hours of initial spore adhesion. In a host range study, species of Rhabditidae, Diplogasteridae, and Aphelenchoidea were hosts, but not species of Bunonematidae, Tripylidae, Cephalobida, or Tylenchina. Numbers of second-stage Meloidogyne incognita juveniles were not decreased when added to soil seeded with infected C. tripartitum. In six Tennessee soybean fields, Macrobiotophthora vermicola was the most commonly encountered nematode-destroying fungus, followed by a sterile, nonseptate fungus and Arthrobotrys conoides. Nematophagous fungi were isolated more frequently from silt loam soils than from clay soils. Addition of C. tripartitum to soil extract plates as a bait nematode did not increase isolations of nematophagous fungi.