Interactions between Pseudomonas fluorescens biocontrol agents and Glomus mosseae, an arbuscular mycorrhizal fungus, within the rhizosphere

Interactions between Pseudomonas fluorescens biocontrol agents and Glomus mosseae , an arbuscular mycorrhizal fungus, were studied. The biocontrol agents included the genetically modified strains CHA96 and CHA0 pME3424 which produced enhanced levels of antifungal compounds. Tomato ( Lycopersicum esculentum ) and leek ( Allium porrum ) host plants were grown in sterile Terra-Green (calcined attapulgite clay) with limited nutrients. Mycorrhizal activity was indicated by shoot dry weight and phosphorus content. In all experiments, plants grown in the presence of G. mosseae had a significantly higher shoot dry weight than those grown in the absence of G. mosseae . Colonisation and activity of G. mosseae was unaltered in the presence of P. fluorescens isolates and presence of G. mosseae increased the population of P. fluorescens in the rhizosphere.     

Interactions between Glomus mosseae and arbuscular mycorrhizal sporocarp-associated saprophytic fungi

The saprophytic fungi Wardomyces inflatus (Marchal) Hennebert, Paecilomyces farinosus (Holm & Gray) A. H. S. Brown & G. Sm., Gliocladium roseum Bain., sterile dark mycelium (SDM-54), Trichoderma pseudokoningii Rifai and Trichoderma harzianum Rifai were isolated from sporocarps of Glomus mosseae. The effect of saprophytic fungi on G. mosseae spore germination was tested on water agar. Wardomyces inflatus decreased the percent germination of G. mosseae spores; G. roseum, T. pseudokoningii and T. harzianum had no effect on germination; and P. farinosus and SDM-54 increased the percentage of spore germination of G. mosseae after 4 d. Wardomyces inflatus significantly decreased hyphal length of spores which germinated, but no other saprophytic fungi affected hyphal growth. Trichoderma pseudokoningii, T. harzianum, P. farinosus and SDM-54 increased the number of auxiliary cells formed by G. mosseae. The effect of saprophytic fungi on arbuscular mycorrhizal colonization of soybean was studied in a greenhouse trial. The percentage of soybean root length colonized was decreased by W. inflatus, unaffected by SDM-54 and T. harzianum, and increased by P. farinosus. Gliocladium roseum decreased root length colonized when plants were 12 wk old, and T. pseudokoningii increased colonization of roots when plants were 4 wk old. Antagonistic, synergistic and neutral actions of G. mosseae upon the saprophytic fungi were observed. The population of T. harzianum decreased and the populations of T. pseudokoningii and SDM-54 increased in the presence of G. mosseae. Our results indicate a complex interaction between G. mosseae and associated saprophytic fungi.     

Xyloglucanases in the interaction between saprobe fungi and the arbuscular mycorrhizal fungus Glomus mosseae

We studied the production of xyloglucanase enzymes of pea and lettuce roots in the presence of saprobe and arbuscular mycorrhizal (AM) fungi. The AM fungus Glomus mosseae and the saprobe fungi Fusarium graminearum, Fusarium oxysporum-126, Trichoderma harzianum, Penicillium chrysogenum, Pleurotus ostreatus and Aspergillus niger were used. G. mosseae increased the shoot and root dry weight of pea but not of lettuce. Most of the saprobe fungi increased the level of mycorrhization of pea and lettuce, but only P. chrysogenum and T. harzianum inoculated together with G. mosseae increased the dry weight of pea and lettuce respectively. The AM and saprobe fungi increased the production of xyloglucanases by plant roots. The level of xyloglucanase activities and the number of xyloglucanolytic isozymes in plants inoculated with G. mosseae and most of the saprobe fungi tested were higher than when both microorganisms were inoculated separately. The possible relationship between xylogucanase activities and the ability of AM and saprobe fungi to improve the dry weight and AM root colonization of plants was discussed.     

Bacterial associations with the mycorrhizosphere and hyphosphere of the arbuscular mycorrhizal fungus Glomus mosseae

Roots and mycorrhizal fungi may not associate with soil bacteria randomly, but rather in a hierarchical structure of mutual preferences. Elucidation of such structures would facilitate the management of the soil biota to enhance the stability of the plant-soil system. We conducted an experiment utilizing two isolates of soil bacteria to determine their persistence in distinct mycorrhizal regions of the root zone, and their effects on general rhizosphere populations of fluorescent pseudomonads (FP). Split-root sorghum (Sorghum bicolor L.) plants were grown in four-compartment containers, constructed so that the soils in individual compartments held either (1) roots colonized by the arbuscular-mycorrhizal (AM) fungus Glomus mosseae (M), (2) nonAM roots only (R), (3) hyphae of G. mosseae (H), or (4) no mycorrhizal structures (S). The soils were inoculated (10⁷ cells g^-1 dry soil) with antibiotic-resistant (rifampicin, rif; streptomycin, sm) strains of the soil bacteria, Alcaligenes eutrophus (rif^r50) or Arthrobacter globiformis (sm^r250), or were left uninoculated as control. A. eutrophus had been isolated from a specific source (hyphosphere soil of G. mosseae), and A. globiformis from mycorrhizosphere soils of two AM fungi. After 10 wk of growth, the presence of A. eutrophus was barely detectable (<10 cfu g^-1 dry soil) in nonAM (R and S) soils, but persisted well (10⁴ cfu g^-1 dry soil) in AM (H and M) soils. Numbers of A. globiformis were more evenly distributed between all soils, but were highest in the presence of AM roots (M soil). There were varied bacterial effects on root and AM-hyphal development: A. eutrophus decreased hyphal length in H soil, while A. globiformis stimulated root length in M soil. The two bacterial inoculants did not affect numbers of FP in H, R, and M soils, but the AM status of the soils did: the numbers of FP increased in the order M>R>H>S. There was a positive correlation of FP numbers with both bacterial inoculants in M and H soils. Numbers of FP changed with root or hyphal lengths, an effect that was related to changes in the numbers of the inoculated bacteria. The results indicate that the hyphosphere-specific A. eutrophus depended on the presence of G. mosseae, but that the nonspecific A. globiformis did not. The mycorrhizal status of soils may selectively influence persistence of bacterial inoculants as well as affecting the numbers of other native bacteria.     

Endoproteolytic activities in pea roots inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and/or Aphanomyces euteiches in relation to bioprotection

Arbuscular mycorrhizal (AM) symbioses are known to play a role in increased resistance of plants against soilborne pathogens. Mechanisms involved in this phenomenon are not yet well understood. This work investigates possible roles of endoproteolytic activities in bioprotection of Pisum sativum roots by Glomus mosseae against Aphanomyces euteiches . First, it is demonstrated that bioprotection occurs only in pre-mycorrhizal plants. Second, endoproteolytic activities were analysed qualitatively and quantitatively during AM symbiosis, in plants infected with either zoospores or mycelium of A. euteiches , and in mycorrhizal plants infected with the pathogen. In mycorrhizal symbiosis a progressive increase in endoproteolytic activities was observed following root colonization by G. mosseae . By contrast, in roots inoculated with A. euteiches , a drastic increase in endoproteolytic activities was observed which was correlated with the amount of pathogen occurring in roots. Qualitative differences were seen among the endoproteolytic activities detected in roots inoculated with zoospores or mycelium. The constitutive as well as mycorrhizal and pathogen-induced activities were further characterized as 'trypsin-like' serine endoproteases. Interestingly, in a situation of bioprotection, only low levels of the activities normally associated with the infection by A. euteiches were detected, suggesting that the synthesis of these proteins is directly linked to the growth or virulence of the pathogen.     

不同生态型摩西球囊霉菌株对棉花耐盐性的影响

在盆栽条件下研究了4个NaCl水平下(0、1、2和3g／kg)接种丛枝菌根真菌Glomus mosseae的2个菌株M1和M2对棉花耐盐性的影响。M1自非盐渍土壤分离,M2自盐渍土壤分离。结果表明,不同盐水平下2个菌株对棉花根系的侵染率为20％～40％,M2的侵染率高于M1;这两个菌株对棉花在盐胁迫环境下的生长状况都有一定的促进,其中Ml的促进作用明显大于M2的,并且在NaCl水平为2和3g／kg时,两菌株对棉花生长的效应之间的差异达到极显著水平。进一步的分析表明两菌株对植株吸收矿质元素的作用方面存在一定差异。接种M1的植株含磷量在4个盐水平下均显著高于对照,而接种M2处理的植株含磷量只是在0和1g／kg NaCl时显著高于对照。在2和3g／kg NaCl时略低于对照,并显著低于接种M1处理的。在4个NaCl水平下,接种M1的植株钠和氯含量与对照没有显著差异;接种M2的植株氯含量在1～3g／kg 3个NaCl水平下、钠含量在2和3g／kg 2个NaCl水平下不仅显著高于对照,同时也显著高于接种M1的植株氯和钠含量。这些差异是M1和M2两菌株对提高棉花耐盐性作用大小不同的主要原因。上述结果说明不同生态型AM真菌菌株对植物的耐盐性的影响力不同,这与真菌自身的生物学特性有关。     

Functional diversity of arbuscular mycorrhizal fungal isolates in relation to extraradical mycelial networks

We investigated the functional significance of extraradical mycorrhizal networks produced by geographically different isolates of the arbuscular mycorrhizal fungal (AMF) species Glomus mosseae and Glomus intraradices. A two-dimensional experimental system was used to visualize and quantify intact extraradical mycelium (ERM) spreading from Medicago sativa roots. Growth, phosphorus (P) and nitrogen (N) nutrition were assessed in M. sativa plants grown in microcosms. The AMF isolates were characterized by differences in extent and interconnectedness of ERM. Phenotypic fungal variables, such as total hyphal length, hyphal density, hyphal length per mm of total or colonized root length, were positively correlated with M. sativa growth response variables, such as total shoot biomass and plant P content. The utilization of an experimental system in which size, growth rate, viability and interconnectedness of ERM extending from mycorrhizal roots are easily quantified under realistic conditions allows the simultaneous evaluation of different isolates and provides data with a predictive value for selection of efficient AMF.     

Nitrogen assimilation in Lolium perenne colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum

To investigate nitrogen assimilation in Lolium perenne L. colonized by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.), nitrate uptake, key enzyme activities, and ¹⁵N incorporation into free amino acids were measured. After a 4-h labelling period with [¹⁵N]nitrate, ¹⁵N content was higher in roots and shoots of AM-plants than in those of control plants. Glutamine synthetase (GS) and nitrate reductase (NR) activities were increased in shoots of AM-plants, but not in roots. More label was incorporated into amino acids in shoots of AM plants. Glutamine, glutamate, alanine and γ-aminobutyric acid were the major sinks for ¹⁵N in roots and shoots of control and AM plants. Interactions between mycorrhizal colonization, phosphate and nitrate nutrition and NR activity were investigated in plants which received different amounts of phosphate or nitrate. In shoots of control plants, NR activity was not stimulated by high levels of phosphate nutrition but was stimulated by high levels of nitrate. At 4 m M nitrate in the nutrient solution, NR activity was similar in control and AM plants. We concluded that mycorrhizal effects on nitrate assimilation are not mediated via improved phosphate nutrition, but could be due to improved nitrogen uptake and translocation.     

Nested multiplex PCR—A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.