Arbuscular mycorrhizal (AM) fungi are one of the most common fungal organisms to exist in symbiosis with terrestrial plants, facilitating the growth and maintenance of arable crops. Wheat has been studied extensively for AM fungal symbiosis using the carcinogen trypan blue as the identifying stain for fungal components, namely arbuscles, vesicles and hyphal structures. The present study uses Sheaffer blue ink with a lower risk as an alternative to this carcinogenic stain. Justification for this is determined by stained wheat root sections ( n =120), with statistically significant increases in the observed abundance of intracellular root cortical fungal structures stained with Sheaffer blue ink compared to trypan blue for both Zulu ( P =0.003) and Siskin ( P =0.0003) varieties of winter wheat. This new alternative combines an improved quantification of intracellular fungal components with a lower hazard risk at a lower cost.

Zero till cropping systems typically apply broad-spectrum herbicides such as glyphosate as an alternative weed control strategy to the physical inversion of the soil provided by cultivation. Glyphosate targets 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in plants. There is growing evidence that this may have a detrimental impact on non-target organisms such as those present in the soil microbiome. Species of commercial importance, such as arbuscular mycorrhizal (AM) fungi that form a symbiotic relationship with plant roots are an important example. This study investigates the impact of soil cultivation and glyphosate application associated with conventional tillage (CT) and zero tillage (ZT) respectively on AM fungi populations under field and glasshouse conditions. Topsoil (<10 cm) was extracted from CT and ZT fields cropped with winter wheat, plus non-cropped control plots within the same field boundary, throughout the cropping year. Glyphosate was applied in glasshouse experiments at rates between 0 and 350 g L−1. Ergosterol, an indicator of fungal biomass, was measured using high performance liquid chromatography before and after glyphosate application. Fungal root arbuscules, an indicator of AM fungi–root symbiosis, were quantified from the roots of wheat plants. Under glasshouse conditions root arbuscules were consistently higher in wheat grown in ZT field extracted soils (P = 0.01) compared to CT. Glyphosate application however inhibited fungal biomass in both the ZT (P < 0.00001) and CT (P < 0.001) treatments. In the absence of glyphosate, the number of stained root arbuscules increased significantly. Ergosterol levels, used as a proxy for fungal biomass, remained lower in the soil post glyphosate application. The results suggest that CT has a greater negative impact on AM fungal growth than ZT and glyphosate, but that glyphosate is also detrimental to AM fungal growth and hinders subsequent population recovery.

Arbuscular Mycorrhizal (AM) fungi form mutualistic symbiotic relationships with approximately 80% of terrestrial plant species, while producing the glycoprotein glomalin as a structural support molecule along their mycelial network. Glomalin confers two benefits for soils: (1) acting as a carbon and nitrogen storage molecule; (2) the binding of soil microaggregates (<250 µm) to form larger, more stable structures. The present study aimed to test the hypothesis that a correlation between glomalin and soil aggregation exists and that this is influenced by the method of seedbed preparation. The soils from two crops of winter wheat in Hertfordshire, UK, practising either conventional (20 cm soil inversion) or zero tillage exclusively, were sampled in a 50 m grid arrangement over a 12 month period. Glomalin and water stable aggregates (WSA) were quantified for each soil sample and found to be significantly greater in zero tillage soils compared to those of conventional tillage. A stronger correlation between WSA and glomalin was observed in zero tillage (Pearson’s coeffect 0.85) throughout the cropping year compared to conventional tillage (Pearson’s coeffect 0.07). The present study was able to conclude that zero tillage systems are beneficial for AM fungi, the enhancement of soil glomalin and soil erosion mitigation.

Soils and plant root rhizospheres have diverse microorganism profiles. Components of this naturally occurring microbiome, arbuscular mycorrhizal (AM) fungi and plant growth promoting rhizobacteria (PGPR), may be beneficial to plant growth. Supplementary application to host plants of AM fungi and PGPR either as single species or multiple species inoculants has the potential to enhance this symbiotic relationship further. Single species interactions have been described; the nature of multi-species tripartite relationships between AM fungi, PGPR and the host plant require further scrutiny. The impact of select Bacilli spp. rhizobacteria and the AM fungus Rhizophagus intraradices as both single and combined inoculations (PGPR[i] and AMF[i]) within field extracted arable soils of two tillage treatments, conventional soil inversion (CT) and zero tillage (ZT) at winter wheat growth stages GS30 and GS39 have been conducted. The naturally occurring soil borne species (PGPR[s] and AMF[s]) have been determined by qPCR analysis. Significant differences (p < 0.05) were evident between inocula treatments and the method of seedbed preparation. A positive impact on wheat plant growth was noted for B. amyloliquefaciens applied as both a single inoculant (PGPR[i]) and in combination with R. intraradices (PGPR[i] + AMF[i]); however, the two treatments did not differ significantly from each other. The findings are discussed in the context of the inocula applied and the naturally occurring soil borne PGPR[s] present in the field extracted soil under each method of tillage.

Arbuscular mycorrhizal (AM) fungi establish close interactions with host plants, an estimated 80% of vascular plant species. The host plant receives additional soil bound nutrients that would otherwise not be available. Other components of the microbiome, such as rhizobacteria, may influence interactions between AM fungi and the host plant. Within a commercial arable crop selected rhizobacteria in combination with AM fungi may benefit crop yields. The precise nature of interactions between rhizobacteria and AM fungi in a symbiotic relationship overall requires greater understanding. The present study aims to assess this relationship by quantifying: (1) AM fungal intracellular root structures (arbuscules) and soil glomalin as an indicator of AM fungal growth; and (2) root length and tiller number as a measure of crop growth, in response to inoculation with one of three species of Bacillus: B. amyloliquefaciences, B. pumilis, or B. subtilis. The influence of soil management, conventional (CT) or zero tillage (ZT) was a further variable evaluated. A significant (p < 0.0001) species-specific impact on the number of quantifiable AM fungal arbuscules was observed. The inoculation of winter wheat (Triticum aestivum) with B. amyloliquefaciences had a positive impact on AM fungal symbiosis, as indicated by an average of 3226 arbuscules per centimetre of root tissue. Bacillus subtilis increased root length significantly (p < 0.01) but decreased fungal symbiosis (p < 0.01). The inoculation of field soils altered the concentration of glomalin, an indicator of AM fungal growth, significantly (p < 0.00001) for each tillage treatment. The greatest increase was associated with B. amyloliquefaciences for both CT (p < 0.0001) and ZT (p < 0.00001). Bacillus subtilis reduced measured glomalin significantly in both tillage treatments (p < 0.0001 and p < 0.00001 for CT and ZT respectively). The interaction between rhizobacteria and AM fungi is variable, being beneficial or detrimental depending on species. This relationship was evident in both tillage treatments and has important implications for maximizing symbiosis in the crop plant-microbiome present in agricultural systems.

Arbuscular mycorrhizal (AM) fungi are biotrophic symbionts forming close relationships with an estimated 80% of terrestrial plants suitable as their host. Via an established AM fungal–host relationship, soil-bound nutrients are made available to the host plant through root cortical arbuscules as the site of exchange. At these sites, photosynthetic carbohydrates are provided to the AM fungus—carbohydrates that cannot be produced by the fungus. AM fungal–host symbiosis is very sensitive to soil disturbance, for example, agricultural tillage practices can damage and reduce AM fungal abilities to interact with a host and provide plant growth-promoting properties.

Sheaffer blue ink is an effective method to stain arbuscular mycorrhizal (AM) fungi in a variety of plant species. It has, however, received criticism for its potential rapid degradation and short-term viability. The long and medium term storage and viability of stained samples has not, to date, been described for this particular staining method. This short communication reports on the viability of 730 samples stained with Sheaffer blue ink stored for the duration of 4 years in microscope slide boxes out of direct sunlight. There was no significant difference in micrograph image quality and presence of stain between years as indicated by the number of AM fungal structures quantified. In conclusion Sheaffer blue ink stain does not deteriorate in the medium term.

Ergosterol is a component of the cell membrane of mycorrhizal fungi and is frequently used to quantify their biomass. Arbuscular mycorrhizal (AM) fungi and ectomycorrhizal (ECM) fungi establish a symbiotic relationship with a respective host plant. Several methods are currently employed for quantification of ergosterol; however, these utilise a series of potentially hazardous chemicals with a varying degree of exposure times to the user. The present method comparative study aims to ascertain the most reliable method to extract ergosterol whilst limiting hazard exposure to the user. Chloroform, cyclohexane, methanol and methanol hydroxide extraction protocols were applied to a total of 300 samples of root samples and a further 300 growth substrate samples across all protocols. Extracts were analysed via HPLC methodologies. Chromagraphic analysis showed chloroform based extraction procedures produced a consistently higher concentration of ergosterol in both root and growth substrate samples. Methanol hydroxide, without the addition of cyclohexane, produced very low concentration of ergosterol, with a reduction of quantified ergosterol of between 80 and 92% compared to chloroform extractions. Hazard exposure was greatly reduced following chloroform extraction protocol when compared with other extraction procedures.