Exudate

The collection of phloem and xylem saps from wheat seedlings was performed according to the method of Hazama et al. (2015) (link). Treated and untreated seedlings of 27 days cut on surfaces of the stems near the petioles of mature leaves with a razor blade and exuded drops (excluding the first drop) collected as phloem sap using micropipettes. Samples of phloem sap were stored in eppendorf previously washed with 0.1 M HNO₃ for 2 days and then with double distilled water three times to eliminate metals. The samples were stored at -20°C until analysis.
After the collection of phloem sap, stems were cut at 2 cm above the interface of the shoot and root, and xylem sap exudates were collected for 30 min using micropipettes. The measured pH of the xylem sap was 6.0–6.4. Samples of xylem sap were also stored at -20°C until analysis.
For the estimation of Zn in phloem and xylem saps above, the described procedure (for Zn) was followed.

The isolates included in this project were carefully selected to be representative of the S. cerevisiae whole species. All the isolate details, including ecological and geographical origins, providers and references, are listed in Supplementary Table 1. We maximized the isolate ecological origins by including both human-associated environments such as wine and sake fermentation, brewing and dairy products, as well as natural environments such as soil, insects, tree exudate and fruit. Geographical origins are also highly diverse and have a worldwide distribution (Supplementary Table 1). In addition to the 918 isolates provided by research laboratories and yeast collections, we included 93 strains sequenced in previous studies^{6 (link)–8 (link)}, to give a total of 1,011 samples analysed in this study. We sought to keep the isolates in their natural state before sequencing to provide a global picture of the ploidy and level of heterozogosity. However, among the 918 selected isolates, 124 were non-natural haploid with the HO gene deleted and the 93 external isolates were genetically manipulated and made homozygous before sequencing.

Lotus Base also offers Lotus-related expression data sourced from various studies. The first dataset was derived from the L. japonicus gene expression atlas (LjGEA) project10 (link), which combined expression data from additional studies33 (link)34 (link)35 (link)36 (link)37 (link). The whole LjGEA dataset consists of 81 conditions sourced from 6 independent published studies10 (link), such as the investigation of draught responses33 (link), effect of mycorrhizal and symbionts inoculation34 (link)35 (link), transcriptome changes in symbiosis defective mutants35 (link), effect of salt and nitrate treatment35 (link)36 (link)37 (link), and transcriptome regulation in various plant organs10 (link). We have mapped probe identifiers from the LjGEA dataset against the annotated proteins of L. japonicus genome v3.0 by performing BLAST alignments of LjGEA probe set against the predicted transcripts from L. japonicus genome v3.0 and selecting for hits with the lowest E-value(s). In addition to the LjGEA dataset, we have also integrated expression data from Lotus roots in response to germinating spore exudates from arbuscular mycorrhiza5 (link), containing 3 conditions.

The primary outcome will be complete healing at 3 months follow-up: (yes/no), where complete healing is defined as complete epithelialisation maintained for at least 2 weeks and the time (in days) between the start of the study and complete wound healing.
Secondary variables are defined as complete healing at 6 months follow-up (yes/no), the degree of healing (Resvech 2. 0) which consists of 6 dimensions (depth, size, edges, wound bed, exudate and signs and symptoms of infection) with ascending scoring scales based on the severity of the dimension studied and a total score ranging from 0 to 35 [26 ]; the ulcer area (cm2), measured by digital photography and subsequent image processing using the open source Java image processing program “The ImageJ ecosystem” [27 (link)]; the VAS scale for perceived pain [28 ]; the level of adherence to the “Active Legs” intervention measured by the combined variable number of steps and time by means of the pedometer record (Yamax PZ270) and patients’ self-reported information on the home exercise program by means of the activity diary and the health-related quality of life measured with the CCVUQ-e, a questionnaire which in addition to an overall synthetic quality of life score, assesses the following dimensions: pain, depression, social relationships, impairment in performing activities of daily living and body image, with a score of 22 to 112 [29 ].
Variables related to the healing process are defined as Body Mass Index (kg/cm2); baseline pathology, diabetes mellitus (yes/no); last glycosylated haemoglobin (HbA1) value in the EHR; ABI score; tobacco and alcohol consumption; topical treatments used in existing ulcers; systemic treatments; adherence to multilayer compression therapy (yes/no); and physical activity level measured by the Minnesota Leisure Time Physical Activity Questionnaire, short version [30 (link)] and type of daily ambulation.
Prognostic variables are defined as location of the ulcer at the time of the study, number of ulcers at the time of the study, time (in days) of evolution of the venous ulcers before inclusion in the study and whether they are recurrent ulcers (yes/no).
Variables related to recurrence (measured at 6 months follow-up) are defined as occurrence of recurrence (yes/no), use of compression stockings (yes/no), light/normal/strong compression and hydration of the legs (yes/no).
Additionally, data such as age, sex, living alone (yes/no), employment status and level of education are collected.

Microcosms with two chambers, one root chamber for plant growth (3 × 10 × 15 cm³) and one hyphal chamber for hyphal growth (7 × 10 × 15 cm³), were constructed (Fig. 1). The two chambers were separated by a 30-μm or a 0.45-μm mesh that allowed or prevented AMF hyphae access to the hyphal chamber. In all cases, the roots were not allowed to access the hyphal chamber.Fig. 1

Flow chart of the experiments. The study comprised several experiments: two pot experiments, in vitro experiments, and an inoculation experiment. The two pot experiments (pot expts 1 and 2) tested whether the proliferation of AMF into microsites of residues may reduce N₂O emissions and disassemble the regulation pathway. We also tested the abundance of the microbiome in the hyphosphere; the in vitro experiment assessed the importance of P. fluorescens co-colonization with AMF for reduced N₂O emissions. We isolated denitrifiers and identified the key components of hyphal exudates. We then examined the chemotaxis, growth, N₂O emission, and denitrifying gene expression of P. fluorescens in response to AMF exudates and key compounds under in vitro culture conditions; finally, the inoculation experiment validated the effects of AMF or citrate exuded by AMF on N₂O emissions and nosZ gene expression of P. fluorescens in pot culture. A conceptual model is used to illustrate the pathways by which AMF interact with P. fluorescens to mitigate N₂O production

In each hyphal chamber, we introduced a patch with a 30-μm pore nylon mesh bag (4 × 7.5 cm², 5 cm high) that could be filled with residues. A gas probe was inserted into the patch to collect gas samples to measure the N₂O concentration as an indicator of N₂O production in the patch (Fig. 1).

A sealed serum bottle assay was conducted to examine the effects of hyphal exudates and major compounds on net N₂O production by P. fluorescens JL1. Hyphal exudate was applied as one treatment. Fructose, trehalose, citrate, malate, glutamine, or glutamic acid was selected as the carbon source treatment because these compounds were detected at high concentrations in hyphal exudates. Glucose was used as the control. There were 8 treatments in total. The same liquid MSR medium [17 (link)] as that used for the collection of hyphal exudates (see Supplementary Information) was used to dissolved specific carbon source. The carbon and nitrogen contents in the medium were adjusted to the same level as those in the hyphal exudate solutions (7.16 mM C and 2.35 mM N). The hyphal exudate medium and specific compound medium were supplemented with 10% FeNaEDTA (relative to MSR medium) to ensure denitrification. The medium pH was then adjusted to 7.2, and the medium was filtered through an Acrodisc syringe filter (0.22-μm Super Membrane, Pall Corporation, Port Washington, NY) to obtain carbon-based medium (CB medium). The CB medium was supplemented with 92.84 mM glucose to reach an initial C concentration of 100 mM. NO₃⁻-N was supplemented to reach a level of 10 mM to ensure denitrification. The pellet obtained from the centrifugation of 1 mL P. fluorescens JL1 suspension was re-suspended in 10 mL modified CB medium and transferred to a 120-mL anaerobic serum bottle. All serum bottles were shaken at 180 rpm and maintained at 30 °C. The gas was measured after 0.5, 1, 2, 3, 6, 8, 10, and 12 h. Each treatment was set up in triplicate. Details are shown in the Supplementary Information.