Arbuscular mycorrhizal fungi stimulate organic phosphate mobilization associated with changing bacterial community structure under field conditions

The extraradical hyphae of arbuscular mycorrhizal fungi (AMF) harbour and interact with a microbial community performing multiple functions. However, how the AMF‐microbiome interaction influences the phosphorus (P) acquisition efficiency of the mycorrhizal pathway is unclear. Here we investigated whether AMF and their hyphal microbiome play a role in promoting organic phosphorus (P) mineralizing under field conditions. We developed an AMF hyphae in‐growth core system for the field using PVC tubes sealed with membrane with different size of pores (30 or 0.45 μm) to allow or deny AMF hyphae access to a patch of organic P in root‐free soil. AMF and their hyphae associated microbiome played a role in enhancing soil organic P mineralization in situ in the field, which was shown to be a function of the change in bacteria community on the hyphae surface. The bacterial communities attached to the AMF hyphae surface were significantly different from those in the bulk soil. Importantly, AMF hyphae recruited bacteria that produced alkaline phosphatase and provided a function that was absent from the hyphae. These results demonstrate the importance of understanding trophic interactions to be able to gain insight into the functional controls of nutrient cycles in the rhizosphere.     

丛枝菌根真菌伴生细菌的研究进展

在丛枝菌根真菌（Arbuscular mycorrhizal fungi,AMF）的孢子、菌丝的表面或内部栖息着细菌,称之为AMF伴生细菌。AMF伴生细菌种类多样、分布广泛,生态位点包括孢子壁的表面或内部、细胞质、菌丝、孢子果等。其可能的生物学意义包括影响AMF孢子萌发、菌丝生长、菌根形成等过程。由于伴生细菌与AMF联系紧密,其对AMF和土壤微生物生态学具有重要的意义。国际上在该领域的研究已有30多年的历史,就其研究进展进行综述。     

丛枝菌根真菌状巨孢囊霉孢子伴生细菌的绿色荧光蛋白标记及定殖分析

摘要：【目的】分析丛枝菌根（Arbuscualr Mycorrhizal, AM）真菌珠状巨孢囊霉(Gigaspora margarita) MAFF 520054孢子伴生细菌的定殖情况,明确其生态位点,以及为进一步分析其种群生态或功能提供信息。【方法】以载体pNF8（gfp-mut1）对6株珠状巨孢囊霉MAFF 520054 孢子伴生细菌进行绿色荧光蛋白（GFP）标记,并通过荧光显微镜和平板计数的方法研究标记菌株对真菌宿主的定殖位点和不同条件下的定殖数量动态。【结果】对粘状芽孢杆菌（Peanibacillus spp.）M060106-1和M061122-6、芽孢杆菌（Bacillus sp.）M061122-10和短小芽孢杆菌（Brevibacillus sp.）M061122-12成功进行了GFP标记,其均具有较好的质粒稳定性,且与出发株的基本性状一致,适合短期内进行环境定殖研究。所有菌株均能定殖珠状巨孢囊霉MAFF 520054孢子壁,而M061122-6和M061122-12还能够定殖其菌丝;不同pH值条件下,各菌株定殖珠状巨孢囊霉MAFF 520054孢子的数量动态均为先上升后下降,pH值对各菌株的定殖数量有不同的影响;各GFP菌株对低活力的珠状巨孢囊霉孢子定殖数量高于高活力的孢子,且对高活力孢子的定殖数量动态不同。【结论】分离的珠状巨孢囊霉孢子伴生细菌能够重新定殖其孢子,菌株的定殖能力受其特性及外界因子的影响,为进一步分析AM真菌伴生细菌的种群生态及功能提供了信息。     

Bacteria associated with spores of the arbuscular mycorrhizal fungi Glomus geosporum and Glomus constrictum

Spores of the arbuscular mycorrhizal fungi (AMF) Glomus geosporum and Glomus constrictum were harvested from single-spore-derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated with taxonomic groups known to degrade biopolymers (Cellvibrio, Chondromyces, Flexibacter, Lysobacter, and Pseudomonas). Scanning electron microscopy of G. geosporum spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.     

Isolation and analysis of bacteria associated with spores of Gigaspora margarita

Aims:  The aim of this work was to observe bacteria associated with the spores of Gigaspora margarita , an arbuscular mycorrhizal fungus (AMF).
Methods and Results:  First, a direct analysis of DNA from sterilized spores indicated the bacteria belonging to the genus Janthinobacterium . In the second assay, two bacterial strains were isolated by osmosis from protoplasts, which were derived from spores by using two particular enzymes: lysing enzymes and yatalase. After isolation, cultivation and identification by their DNA as performed in the first experiment, the species with the closest relation were Janthinobacterium lividum (KCIGM01) and Paenibacillus polymyxa (KCIGM04) isolated with lysing enzymes and yatalase respectively. Morphologically, J. lividum was Gram negative and oval, while P. polymyxa was also oval, but Gram positive. Both strains had antagonistic effects to the pathogenic fungi Rosellimia necatrix, Pythium ultimum , Fusarium oxysporum and Rhizoctonia solani . In particular, J. lividum was much stronger in this role. However, in phosphorus (P) solubilization P. polymyxa functioned better than J. lividum.
Conclusions:  This experiment had revealed two new bacteria species ( P. polymyxa and J. lividum ), associated with AMF spores, which functioned to suppress diseases and to solubilize P.
Significance and Impact of the Study:  AMF spores could be a useful source for bacterial antagonists to soil-borne diseases and P solubilization.     

Bacteria Associated with Spores of the Arbuscular Mycorrhizal Fungi Glomus geosporum and Glomus constrictum

Spores of the arbuscular mycorrhizal fungi (AMF) Glomus geosporum and Glomus constrictum were harvested from single-spore-derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated with taxonomic groups known to degrade biopolymers (Cellvibrio, Chondromyces, Flexibacter, Lysobacter, and Pseudomonas). Scanning electron microscopy of G. geosporum spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.     

Elemental composition of arbuscular mycorrhizal fungi at high salinity

We investigated the elemental composition of spores and hyphae of arbuscular mycorrhizal fungi (AMF) collected from two saline sites at the desert border in Tunisia, and of Glomus intraradices grown in vitro with or without addition of NaCl to the medium, by proton-induced X-ray emission. We compared the elemental composition of the field AMF to those of the soil and the associated plants. The spores and hyphae from the saline soils showed strongly elevated levels of Ca, Cl, Mg, Fe, Si, and K compared to their growth environment. In contrast, the spores of both the field-derived AMF and the in vitro grown G. intraradices contained lower or not elevated Na levels compared to their growth environment. This resulted in higher K:Na and Ca:Na ratios in spores than in soil, but lower than in the associated plants for the field AMF. The K:Na and Ca:Na ratios of G. intraradices grown in monoxenic cultures were also in the same range as those of the field AMF and did not change even when those ratios in the growth medium were lowered several orders of magnitude by adding NaCl. These results indicate that AMF can selectively take up elements such as K and Ca, which act as osmotic equivalents while they avoid uptake of toxic Na. This could make them important in the alleviation of salinity stress in their plant hosts.     

钦州湾红树林丛枝菌根初步研究

海漆 (Excoecaria agallocha)、桐花树 (Aegiceras corniculatum)、秋茄 (Kandlia candel)、白骨壤 (Avi-cennia marina)是钦州湾分布广、生长量大的 4种红树植物 ,该项研究初步调查了丛枝菌根真菌在这四种植物根系的定居情况。在这四种植物的根际土壤均分离到丛枝菌根真菌孢子 ,其孢子密度以海漆根际的最高 ,其次为桐花树、秋茄、白骨壤。同时 ,海漆根系的侵染率和物种丰富度也最高 ,但在所取的白骨壤根样中没有观察到丛枝菌根的侵染。菌根侵染主要是以根内菌丝、胞内菌丝膨大扭曲、泡囊等形态出现。海水和土壤质地是影响菌根侵染率的主要因素。在所采土样中仅发现球囊霉属 (Glomus)、无梗囊霉属 (Acaulospora)丛枝菌根真菌 ,计有 1 7种和多个未确定种。     

Allelic Differences within and among Sister Spores of the Arbuscular Mycorrhizal Fungus Glomus etunicatum Suggest Segregation at Sporulation

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF are made up of coenocytic networks of hyphae through which nuclei and organelles can freely migrate. In this study, we investigated the possibility of a genetic bottleneck and segregation of allelic variation at sporulation for a low-copy Polymerase1-like gene, PLS. Specifically, our objectives were (1) to estimate what allelic diversity is passed on to a single spore (2) to determine whether this diversity is less than the total amount of variation found in all spores (3) to investigate whether there is any differential segregation of allelic variation. We inoculated three tomato plants with a single spore of Glomus etunicatum each and after six months sampled between two and three daughter spores per tomato plant. Pyrosequencing PLS amplicons in eight spores revealed high levels of allelic diversity; between 43 and 152 alleles per spore. We corroborated the spore pyrosequencing results with Sanger- and pyrosequenced allele distributions from the original parent isolate. Both sequencing methods retrieved the most abundant alleles from the offspring spore allele distributions. Our results indicate that individual spores contain only a subset of the total allelic variation from the pooled spores and parent isolate. Patterns of allele diversity between spores suggest the possibility for segregation of PLS alleles among spores. We conclude that a genetic bottleneck could potentially occur during sporulation in AMF, with resulting differences in genetic variation among sister spores. We suggest that the effects of this bottleneck may be countered by anastomosis (hyphal fusion) between related hyphae.     

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.     

Analysis of bacterial communities associated with spores of Gigaspora margarita and Gigaspora rosea

The spores of arbuscular mycorrhizal fungi (AMF) form a unique microhabitat that is suitable for the colonization by many species of bacteria. The aim of the current study was to analyze the bacterial communities associated with the surface of spores of the AMF species Gigaspora margarita MAFF 520054 and Gigaspora rosea JP1. The two AMF species were propagated with tobacco (Nicotiana tabacum) grown in a mixture of sand and soil. In another experiment, G. margarita was propagated with tobacco or alfalfa (Medicago sativa) grown in vermiculite or a mixture of sand and soil. The bacterial community composition of the new-formed spores and sand/soil substrate was analyzed using PCR of 16S rDNA fragments and denaturing gradient gel electrophoresis (DGGE). Clustering analysis revealed that the bacterial communities on the surface of G. margarita spores was different form that in the substrate or on the surface of the G. rosea spores, and both the host plant and the substrate could influence the composition of spore-associated bacterial populations of the G. margarita. Sequence analysis of the major DGGE bands of G. margarita spore samples revealed that most of the bacterial sequences were affiliated with the phyla Proteobacteria (Azospirillum, Azovibrio, Polyangium, Ramlibacter, Rubrivivax, Sphingomonas, and Rhizobium) and Actinobacteria (Streptomyces, Amycolatopsis, and Pseudonocardia).     

SeSaMe:Metagenome Sequence Classification of Arbuscular Mycorrhizal Fungi-associated Microorganisms

Arbuscular mycorrhizal fungi (AMF) are plant root symbionts that play key roles in plant growth and soil fertility. They are obligate biotrophic fungi that form coenocytic multinucleated hyphae and spores. Numerous studies have shown that diverse microorganisms live on the surface of and inside their mycelia, resulting in a metagenome when whole-genome sequencing (WGS) data are obtained from sequencing AMF cultivated in vivo. The metagenome contains not only the AMF sequences, but also those from associated microorganisms. In this study, we introduce a novel bioinformatics program, Spore-associated Symbiotic Microbes (SeSaMe), designed for taxonomic classification of short sequences obtained by next-generation DNA sequencing. A genus-specific usage bias database was created based on amino acid usage and codon usage of a three consecutive codon DNA 9-mer encoding an amino acid trimer in a protein secondary structure. The program distinguishes between coding sequence (CDS) and non-CDS, and classifies a query sequence into a genus group out of 54 genera used as reference. The mean percentages of correct predictions of the CDS and the non-CDS test sets at the genus level were 71% and 50% for bacteria, 68% and 73% for fungi (excluding AMF), and 49% and 72% for AMF (Rhizophagus irregularis), respectively. SeSaMe provides not only a means for estimating taxonomic diversity and abundance but also the gene reservoir of the reference taxonomic groups associated with AMF. Therefore, it enables users to study the symbiotic roles of associated microorganisms. It can also be applicable to other microorganisms as well as soil metagenomes. SeSaMe is freely available at www.fungalsesame.org.     

The importance of being regular: Caenorhabditis elegans and Pristionchus pacificus defecation mutants are hypersusceptible to bacterial pathogens

Bacterial pathogens have shaped the evolution and survival of organisms throughout history, but little is known about the evolution of virulence mechanisms and the counteracting defence strategies of host species. The nematode model organisms, Caenorhabditis elegans and Pristionchus pacificus, feed on a wealth of bacteria in their natural soil environment, some of which can cause mortality. Previously, we have shown that these nematodes differ in their susceptibility to a range of human and insect pathogenic bacteria, with P. pacificus showing extreme resistance compared with C. elegans. Here, we isolated 400 strains of Bacillus from soil samples and fed their spores to both nematodes. Spores of six Bacillus strains were found to kill C. elegans but not P. pacificus. While the majority of Bacillus strains are benign to nematodes, observed pathogenicity is restricted to either the spore or the vegetative stage. We used the rapid C. elegans killer strain (Bacillus sp. 142) to conduct a screen for hypersusceptible P. pacificus mutants. Two P. pacificus mutants with severe muscle defects and an extended defecation cycle that die rapidly on Bacillus spores were isolated. These genes were identified to be homologous to C. elegans, unc-22 and unc-13. To test whether a similar relationship between defecation and bacterial pathogenesis exists in C. elegans, we used five known defecation mutants. Quantification of the defecation cycle in mutants also revealed a severe effect on survival in C. elegans. Thus, intestinal peristalsis is critical to nematode health and contributes significantly to survival when fed Gram-positive bacteria.     

益生菌地衣芽孢杆菌治疗鸡腹泻机理初探

目的：探讨益生菌地衣芽孢杆菌饲喂鸡腹泻的治疗效果以及分子机理。方法：从养鸡场土壤中选择性分离芽孢杆菌,通过优 化条件提高产生芽孢的比例;利用芽孢和大米混合后饲喂腹泻鸡,通过克隆测序分析腹泻鸡肠道微生物群落在饲喂前后的差异。 结果：我们分离到一株地衣芽孢杆菌,通过优化条件使产芽孢的比例提高到60 %。利用芽孢饲喂一周后能够有效地治疗鸡腹泻。 分析鸡肠道微生物群落发现：鸡腹泻后其肠道细菌群落的多样性和均匀度降低。造成腹泻鸡肠道主要菌群为动胶杆菌（Zoogloea sp.）;经芽孢饲喂后,主要菌群转变为拟杆菌（Bacteroides sp.）,与健康鸡的肠道微生物主要菌群相似。结论：我们筛选到一株可能 治疗鸡腹泻的芽孢杆菌,通过优化培养条件能够提高其产芽孢比例;腹泻鸡经芽孢饲喂后,其肠道中致病菌被益生菌所代替,形 成正常的肠微生物菌群而恢复健康。     

Artificial wind-gust liberation of microbial bioaerosols previously deposited on plants

Summary A bioaerosol research chamber was constructed and used to evaluate wind-gust release of previously depositedPseudomonas syringae, spores ofBacillus subtilis var.niger, and fluorescent microspheres (FM) on oat plants, and the airborne survival of the releasedP. syringae. Observations of wind gusts on the releasedBacillus spores and FM showed they had similar particle size distributions and therefore FM could act as bacterial sized surrogates in particle dispersion. Microscopic examination of the released FM containing particles revealed that 84% were associated with either fungal spores and hyphae were epiphytic on the plant, or with plant and soil debris. The release rate ofBacillus spores decreased as the number of gusts experienced by the plants increased, with a greater proportion of larger particles removed. The larger the particle associated with the releasedP. syringae, the longer the bacteria survived.     

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.     

Interactions between Streptomyces coelicolor and Bacillus subtilis: Role of surfactants in raising aerial structures

Using mixed-species cultures, we have undertaken a study of interactions between two common spore-forming soil bacteria, Bacillus subtilis and Streptomyces coelicolor. Our experiments demonstrate that the development of aerial hyphae and spores by S. coelicolor is inhibited by surfactin, a lipopeptide surfactant produced by B. subtilis. Current models of aerial development by sporulating bacteria and fungi postulate a role for surfactants in reducing surface tension at air-liquid interfaces, thereby removing the major barrier to aerial growth. S. coelicolor produces SapB, an amphipathic peptide that is surface active and required for aerial growth on certain media. Loss of aerial hyphae in developmental mutants can be rescued by addition of purified SapB. While a surfactant from a fungus can substitute for SapB in a mutant that lacks aerial hyphae, not all surfactants have this effect. We show that surfactin is required for formation of aerial structures on the surface of B. subtilis colonies. However, in contrast to this positive role, our experiments reveal that surfactin acts antagonistically by arresting S. coelicolor aerial development and causing altered expression of developmental genes. Our observations support the idea that surfactants function specifically for a given organism regardless of their shared ability to reduce surface tension. Production of surfactants with antagonistic activity could provide a powerful competitive advantage during surface colonization and in competition for resources.     

Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface

The filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growing aerial hyphae but not in spores. Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer. Disruption of both rdlA and rdlB in S. coelicolor and S. lividans (DeltardlAB strains) did not affect the formation and differentiation of aerial hyphae. However, the characteristic rodlet layer was absent. Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid. Attachment to this substratum was greatly reduced in the DeltardlAB strains. Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.     

Isolation and properties of pili from spores of Bacillus cereus.

Structures whose morphology is identical to that of bacterial pili have been isolated from spores of Bacillus cereus. The structures are absent from log-phase and sporulating cells. The pili are 6.8 nm in diameter, are of variable length, and appear to emanate randomly from the exosporium. Examination of spores from 12 Bacillus species showed that only those from B. cereus and B. thuringiensis have pili. Although isolated spore pili were shown to be composed of protein, their subunit nature was not discernible due to the extreme insolubility of the structure. An antiserum to spore pili was labeled with ferritin and used to examine the distribution of pilus antigen on the outer spore surface.     

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.