Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial interaction between fungi and land plants and promotes global phosphate cycling in terrestrial ecosystems. AM fungi are recognised as obligate symbionts that require root colonisation to complete a life cycle involving the production of propagules, asexual spores. Recently, it has been shown that Rhizophagus irregularis can produce infection-competent secondary spores asymbiotically by adding a fatty acid, palmitoleic acid. Furthermore, asymbiotic growth can be supported using myristate as a carbon and energy source for their asymbiotic growth to increase fungal biomass. However, the spore production and the ability of these spores to colonise host roots were still limited compared to the co-culture of the fungus with plant roots. Here we show that a combination of two plant hormones, strigolactone and jasmonate, induces the production of a large number of infection-competent spores in asymbiotic cultures of Rhizophagus clarus HR1 in the presence of myristate and organic nitrogen. Inoculation of asymbiotically-generated spores promoted the growth of host plants, as observed for spores produced by symbiotic culture system. Our findings provide a foundation for the elucidation of hormonal control of the fungal life cycle and the development of inoculum production schemes.

The Bacterial Wilt Reservoir Host Solanum dulcamara Shows Resistance to Ralstonia solanacearum Infection

Ralstonia solanacearum causes bacterial wilt, a devastating plant disease, responsible for serious losses on many crop plants. R. solanacearum phylotype II-B1 strains have caused important outbreaks in temperate regions, where the pathogen has been identified inside asymptomatic bittersweet (Solanum dulcamara) plants near rivers and in potato fields. S. dulcamara is a perennial species described as a reservoir host where R. solanacearum can overwinter, but their interaction remains uncharacterised. In this study, we have systematically analysed R. solanacearum infection in S. dulcamara, dissecting the behaviour of this plant compared with susceptible hosts such as tomato cv. Marmande, for which the interaction is well described. Compared with susceptible tomatoes, S. dulcamara plants (i) show delayed symptomatology and bacterial progression, (ii) restrict bacterial movement inside and between xylem vessels, (iii) limit bacterial root colonisation, and (iv) show constitutively higher lignification in the stem. Taken together, these results demonstrate that S. dulcamara behaves as partially resistant to bacterial wilt, a property that is enhanced at lower temperatures. This study proves that tolerance (i.e., the capacity to reduce the negative effects of infection) is not required for a wild plant to act as a reservoir host. We propose that inherent resistance (impediment to colonisation) and a perennial habit enable bittersweet plants to behave as reservoirs for R. solanacearum.

Environmental Stress Determines The Colonization and Impact of An Endophytic Fungus On Invasive Knotweed

There is increasing evidence that microbes play a key role in some plant invasions. A diverse and widespread but little understood group of plant-associated microbes are the fungal root endophytes of the order Sebacinales. They are associated with exotic populations of invasive knotweed (Reynoutria ssp.) in Europe, but their effects on the invaders are unknown. We used the recently isolated Sebacinales root endophyte Serendipita herbamans to experimentally inoculate invasive knotweed and study root colonisation and effects on knotweed growth under different environmental conditions. We verified the inoculation success and fungal colonisation through immunofluorescence microscopy and qPCR. We found that S. herbamans strongly colonized invasive knotweed in low-nutrient and shade environments, but much less under drought or benign conditions. At low nutrients, the endophyte had a positive effect on plant growth, whereas the opposite was true under shaded conditions. Our study demonstrates that the root endophyte S. herbamans has the potential to colonize invasive knotweed fine roots and impact its growth, and it could thus also play a role in natural populations. Our results also show that effects of fungal endophytes on plants can be strongly environment-dependent, and may only be visible under stressful environmental conditions.

Environmental stress determines the colonization and impact of an endophytic fungus on invasive knotweed

There is increasing evidence that microbes play a key role in some plant invasions. A diverse and widespread but little understood group of plant-associated microbes are the fungal root endophytes of the order Sebacinales. They are associated with exotic populations of invasive knotweed (Reynoutria ssp.) in Europe, but their effects on the invaders are unknown.We used the recently isolated Sebacinales root endophyte Serendipita herbamans to experimentally inoculate invasive knotweed and study root colonisation and effects on knotweed growth under different environmental conditions. We verified the inoculation success and fungal colonisation through immunofluorescence microscopy and qPCR.We found that S. herbamans strongly colonized invasive knotweed in low-nutrient and shade environments, but much less under drought or benign conditions. At low nutrients, the endophyte had a positive effect on plant growth, whereas the opposite was true under shaded conditions.Synthesis. Our study demonstrates that the root endophyte S. herbamans has the potential to colonize invasive knotweed fine roots and impact its growth, and it could thus also play a role in natural populations. Our results also show that effects of fungal endophytes on plants can be strongly environment-dependent, and may only be visible under stressful environmental conditions.

Linking plant growth promoting arbuscular mycorrhizal colonization with bacterial plant sulfur supply

Sulfur (S) exists in organically bound complexes (~95%), predominantly as sulfonates, and are not directly plant available. Specific soil bacteria can mobilise sulfonates but very little is known about these bacteria in the hyphosphere. Since mycorrhizal fungi support growth of the majority of land plants, hyphosphere desulfonating bacteria may be of substantial benefit to the plant host. This study analysed the effect of AM inoculation with Rhizophagus irregularis (former G. intraradices, Glomus) and a mix of six AM species (Mixed) on PGP, microbial communities and sulfonate mobilising bacteria with L. perenne, Agrostis stolonifera and Plantago lanceolata as plant hosts in bi-compartmental microcosms and A. stolonifera in PGP pot experiments. AM inoculation significantly increased plant growth, percentage root colonisation and the quantity of cultivable desulfonating bacteria in the hyphosphere over pre-inoculated soil for all plants. Community analysis via PCR-DGGE revealed significantly different bacterial and fungal communities post inoculation. Analysis of the sulfonate mobilising asfA gene revealed a significantly altered community and novel bacterial isolates with this important functional ability post-inoculation. The results demonstrate that AM inoculation increased plant biomass yield, AM root colonisation and altered bacterial and fungal community dynamics in the hyphosphere. AM inoculated microcosms had an increased abundance of desulfonating bacteria that may be beneficial for plant-S supply.

Rice Physiological Response with Bacillus subtilis and Saccharomyces cerevisiae Inoculation into Soil under Reclaimed Water–Fresh Water Combined Irrigation

The increasing soil salinity levels under reclaimed water irrigation have a negative effect on plant growth. Greenhouse experiments were conducted in 2018 and 2019 under reclaimed water–fresh water combined irrigation. After transplanting (Day 1), rice was irrigated with clean water (tap water) for 10 days to facilitate rice root colonisation. Subsequently, rice was irrigated with reclaimed water for 50 days (Day 11 to 60), and then irrigated with clean water. B. subtilis and S. cerevisiae were mixed with clean water (tap water) and irrigated into soil at Day 61. B. subtilis (20 billion colony-forming units/g) and S. cerevisiae (20 billion colony-forming units/g) were mixed at the following proportions: 5 g and 0 (J1), 3.75 g and 1.25 g (J2), 2.5 g and 2.5 g (J3), 1.25 g and 3.75 g (J4), and 0 and 5 g (J5), respectively; rice treated with reclaimed water (CK) and clean water (J0) with no microorganisms applied were also used. We measured NO3--N and NH4+-N concentrations and electrical conductivity (EC) in the soil at 0–5, 5–15, and 15–25 cm layers; root activity; and malondialdehyde (MDA), soluble sugar, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutamine synthetase (GS) activity in leaves at Day 71. B. subtilis and S. cerevisiae combination could promote rice physiological indices, and B. subtilis had a greater effect than S. cerevisiae. There are obvious differences in the physiological performance and soil N between 2018 and 2019 due to the EC of reclaimed water. Redundancy analysis revealed that soil NO3−-N and the mass of B. subtilis applied were major factors influencing leaf physiological indices. Five grams of B. subtilis is recommended to facilitate rice growth after irrigation with reclaimed water. Our research provides a new agronomic measure for the safe utilisation of reclaimed water.

Artificial intelligence enables the identification and quantification of arbuscular mycorrhizal fungi in plant roots

Soil fungi establish mutualistic interactions with the roots of most vascular land plants. Arbuscular mycorrhizal (AM) fungi are among the most extensively characterised mycobionts to date. Current approaches to quantifying the extent of root colonisation and the relative abundance of intraradical hyphal structures in mutant roots rely on staining and human scoring involving simple, yet repetitive tasks prone to variations between experimenters. We developed the software AMFinder which allows for automatic computer vision-based identification and quantification of AM fungal colonisation and intraradical hyphal structures on ink-stained root images using convolutional neural networks. AMFinder delivered high-confidence predictions on image datasets of colonised roots of Medicago truncatula, Lotus japonicus, Oryza sativa and Nicotiana benthamiana obtained via flatbed scanning or digital microscopy enabling reproducible and transparent data analysis. A streamlined protocol for sample preparation and imaging allowed us to quantify dynamic increases in colonisation in whole root systems over time. AMFinder adapts to a wide array of experimental conditions. It enables accurate, reproducible analyses of plant root systems and will support better documentation of AM fungal colonisation analyses. AMFinder can be accessed here: https://github.com/SchornacklabSLCU/amfinder.git

Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial interaction between fungi and land plants and promotes global phosphate cycling in terrestrial ecosystems. AM fungi are recognised as obligate symbionts that require root colonisation to complete a life cycle involving the production of propagules, asexual spores. Recently it has been shown that Rhizophagus irregularis can produce infection-competent secondary spores asymbiotically by adding a fatty acid, palmitoleic acid. Further, asymbiotic growth can be supported using myristate as a carbon and energy source for their asymbiotic growth to increase fungal biomass. However, spore production and the ability of these spores to colonise host roots were still limited compared to co-culture of the fungus with plant roots. Here we show that a combination of two plant hormones, strigolactone and methyl jasmonate, induces production of a large number of infection-competent spores in asymbiotic cultures of Rhizophagus clarus HR1 in the presence of myristate and organic nitrogen. Inoculation of asymbiotically-generated spores promoted the growth of Welsh onions, as observed for spores produced by symbiotic culture system. Our findings provide a foundation for elucidation of hormonal control of the fungal life cycle and development of new inoculum production schemes.

Mycorrhizal fungi enhance flooding tolerance of peach through inducing proline accumulation and improving root architecture

This study aimed to evaluate the effect of an arbuscular mycorrhizal fungus (AMF) Glomus mosseae on plant growth, root architecture, and proline metabolism in roots of peach (Prunes persica L.) under non-flooding and flooding conditions. The 12-day flooding dramatically inhibited root colonisation of G. mosseae, but induced a large number of extraradical mycelia. Although the flooding treatment also relatively inhibited growth and root architecture of peach, the mycorrhizal fungal inoculation dramatically increased shoot and root biomass, plant height, stem diameter, number of 1^st- and 2^nd-order lateral roots, root total length (mainly 0–1 cm and > 3 cm long), root surface area, and root volume under flooding. The study also revealed distinctly higher proline accumulation in the roots of mycorrhizal plants than non-mycorrhizal plants under both non-flooding and flooding conditions, accompanied by higher Δ¹-pyrroline-5-carboxylate synthase (P5CS) activity and lower δ-ornithine transaminase and proline dehydrogenase activities. In addition, the PpP5CS1 gene expression was up-regulated by flooding and mycorrhization. This study concluded that mycorrhizal fungi enhanced flooding tolerance of peach through inducing proline accumulation and improving root architecture.