Mucoromycotina ‘fine root endophytes’ form globally widespread, nutritional mutualisms with plants

Mucoromycotina ‘fine root endophytes’ (MFRE), referred to previously as Glomus tenue (Greenall) or more recently Planticonsortium tenue (Walker et al., 2018), are a globally distributed group of soil fungi (Orchard et al., 2017a) that form endosymbioses with plants from across most of the land plant phylogeny (Hoysted et al., 2018, 2019; Rimington et al., 2019). Despite much progress having been made in characterizing plant–MFRE symbioses in the last decade, significant challenges remain. Here, we mark out these challenges and discuss future directions for promoting research in this rapidly developing field.

MFRE, within Endogonales (Mucoromycotina, Mucoromycota), are recognized as phylogenetically (Bidartondo et al., 2011; Spatafora et al., 2016; Orchard et al., 2017b) and functionally (Field et al., 2015, 2019; Hoysted et al., 2019) distinct from the more commonly studied arbuscular mycorrhizal fungi (AMF), which belong to the Glomeromycotina (or Glomeromycota) (Spatafora et al., 2016). Research using isotope tracers has shown that MFRE exchange both phosphorus and nitrogen for plant-fixed carbon when in association with liverworts (Field et al., 2015, 2016, 2019) and with the vascular plant Lycopodiella inundata (Hoysted et al., 2019, 2021b), while a cryo-scanning electron microscopy (SEM) and X-ray microanalysis study suggests MFRE may play a role in phosphorus assimilation in Trifolium subterraneum (Albornoz et al., 2020). Where it has been measured, MFRE have been shown to transfer a significant amount of nitrogen to their host plant (Field et al., 2016, 2019; Hoysted et al., 2019, 2021a), suggesting that there may be a complementary role for these fungal symbionts alongside AMF. In contrast to their well-established role in plant phosphorus nutrition, the extent to which AMF contribute directly to host plant nitrogen nutrition has been subject to some debate (Smith & Smith, 2011; Hodge & Storer, 2015; Thirkell et al., 2016) which is now pertinent given the widespread misidentification of fungal endosymbionts, including MFRE, as AMF (Orchard et al., 2017a; Field et al., 2019). A meta-analysis of the literature on MFRE revealed that many past studies have neglected to focus on MFRE due to difficulties in distinguishing between MFRE and AMF morphologies (Orchard et al., 2017a), the challenge of isolating MFRE, and the absence of MFRE from plant specimens as a result of degradation brought about by sample storage conditions and duration (Orchard et al., 2017c). As the importance of MFRE in plant nutrition is increasingly recognized, further research into their form and function has become critical for understanding of the flows of carbon and nutrients through plant and soil communities. Such findings may have potentially important implications for applications of mycorrhizal fungi in sustainable agriculture (Thirkell et al., 2017).

The choice of plant host for MFRE in experiments represents a critical consideration for researchers, particularly given that relatively little is known about compatibility and variability in function of MFRE symbionts across plant clades. To date, the majority of experiments have been conducted using a relatively limited range of plant hosts, focusing on species where MFRE but not AMF have been detected molecularly across multiple wild populations (e.g. Lycopodiella inundata and some Haplomitriopsida liverwort species), or those which are readily colonized by MFRE in soil-based inocula (e.g. Trifolium spp.). The breadth of host range for MFRE symbionts, inclusive of compatibility, structure and function of plant–MFRE associations, warrants further investigation (Sinanaj et al., 2020). Experiments involving the use of plants, particularly those where genomes are available, that might be considered as models for symbiosis research (e.g. Medicago, Lotus) would be especially valuable in unpicking the molecular and physiological mechanisms underpinning the symbiosis.

Using light microscopy, MFRE are generally recognizable by their fine hyphae (< 1.5 µm diameter) with small intercalary and terminal swellings and ‘fan-like’ branching structures (Thippayarugs et al., 1999). These contrast with the relatively coarse hyphae (> 3 µm diameter) of AMF (or ‘coarse root endophytes’) (Field & Pressel, 2018). Arbuscules (highly branched intracellular fungal structures) are characteristic of plant–AMF symbioses; however, their occurrence and appearance in MFRE symbioses across host plants and even plant lifecycles (Hoysted et al., 2021a), is variable (Orchard et al., 2017b; Hoysted et al., 2019). Morphological plasticity has also been noted in transmission and scanning electron micrographs of the ultrastructure of symbioses in plants where only MFRE were detected (Field et al., 2015; Hoysted et al., 2019), making it challenging to distinguish them in planta in co-colonizations with AMF (Field et al., 2016). In contrast with the generally very well-characterized AMF spores, those of MFRE are poorly documented. Brief descriptions of their appearance and size occur but are unaccompanied by images (Hall, 1977; McGee, 1987); in fact, only a single unvalidated image of an Endogonales MFRE spore has been published to date (Orchard et al., 2017a).

The prevailing symbiotic scenario among mycorrhiza-forming vascular plants is colonization by multiple fungal symbionts (Hoysted et al., 2019; Teste et al., 2020). Over the years, techniques for the detection and characterization of mycorrhizal fungi have been refined, including molecular detection methods using fungal-specific primers that target marker genes (White et al., 1990), the MaarjAM curated database dedicated to AMF sequences (Öpik et al., 2010), and inoculation methods using either axenic fungal cultures (Mugnier & Mosse, 1987) or fungal spores extracted from soil (Gerdemann & Nicolson, 1963) to generate plants colonized exclusively by specific species of mycorrhizal fungi. This approach is particularly challenging for MFRE, as their spores are poorly characterized and difficult to isolate, and available fungal isolates are few (Field et al., 2015). This represents perhaps the most pressing obstacle to MFRE research progress, highlighting the need for MFRE–plant experimental systems that allow researchers greater control over biotic and abiotic factors that may influence form and function of MFRE symbioses. Here, we discuss the three state-of-the-art approaches currently available to investigate these associations, including use of soil sieving, wild plants and axenic fungal isolates, together with the caveats that should be considered where each method is employed.

中文翻译：

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毛霉菌“细根内生菌”面临的关键研究挑战

毛霉菌“细根内生菌”与植物形成全球广泛的营养共生关系

毛霉菌“细根内生菌”(MFRE)，以前称为Glomus tenue (Greenall) 或更近的称为 Planticonsortium tenue (Walker et al ., 2018 )，是全球分布的土壤真菌群 (Orchard et al ., 2017a )与来自大部分陆地植物系统发育的植物形成内共生 (Hoysted et al ., 2018 , 2019 ; Rimington et al ., 2019）。尽管在过去十年中在表征植物-MFRE 共生方面取得了很大进展，但仍然存在重大挑战。在这里，我们指出了这些挑战，并讨论了在这个快速发展的领域促进研究的未来方向。

MFRE，内Endogonales（Mucoromycotina，Mucoromycota），被识别为系统发育（Bidartondo等人，。2011 ;斯帕塔福拉等人，。2016 ;果园等人，。2017b）和功能（场等人，。2015，2019 ; Hoysted等al ., 2019 ) 不同于更常研究的丛枝菌根真菌 (AMF)，后者属于Glomeromycotina (或Glomeromycota) (Spatafora et al ., 2016)）。使用同位素示踪剂的研究表明，当与苔类植物（Field等人，2015 年，2016 年，2019 年）和维管植物Lycopodiella inundata（Hoysted等人，2019 年，2021b )，而低温扫描电子显微镜 (SEM) 和 X 射线微量分析研究表明 MFRE 可能在三叶草地下的磷同化中发挥作用（Albornoz等人，2020）。在已测量的地方，MFRE 已被证明可将大量氮转移到寄主植物中（Field等人，2016 年，2019 年；Hoysted等人，2019 年，2021a），这表明MFRE可能具有互补作用这些真菌共生体与 AMF 一起存在。与它们在植物磷营养中的公认作用相反，AMF 对寄主植物氮营养的直接贡献程度一直存在争议（Smith & Smith，2011 年；Hodge & Storer，2015 年；Thirkell等，2016 年）) 考虑到真菌内共生体（包括 MFRE）被广泛错误地识别为 AMF（Orchard等人，2017a；Field等人，2019 年），这现在是相关的。对 MFRE 文献的荟萃分析表明，由于难以区分 MFRE 和 AMF 形态（Orchard et al ., 2017a）、隔离 MFRE 的挑战以及缺乏 MFRE ，许多过去的研究忽略了关注MFRE由于样品储存条件和持续时间引起的降解而从植物标本中分离出来（Orchard等人，2017c）。随着人们越来越认识到 MFRE 在植物营养中的重要性，对其形式和功能的进一步研究对于了解碳和养分通过植物和土壤群落的流动变得至关重要。这些发现可能对菌根真菌在可持续农业中的应用具有潜在的重要意义（Thirkell et al ., 2017）。

在实验中为 MFRE 选择植物宿主代表了研究人员的一个重要考虑因素，特别是考虑到对跨植物进化枝的 MFRE 共生体功能的兼容性和可变性知之甚少。迄今为止，大多数实验是使用相对有限范围的植物宿主进行的，重点是在多个野生种群中分子检测到 MFRE 但未检测到 AMF 的物种（例如Lycopodiella inundata和一些 Haplomitriopsida livewort 物种），或那些MFRE 容易在基于土壤的接种物中定殖（例如Trifolium spp.）。MFRE 共生体宿主范围的广度，包括植物-MFRE 关联的兼容性、结构和功能，值得进一步调查（Sinanaj等人，2020 年）。涉及使用植物的实验，特别是那些有基因组可用的实验，可能被认为是共生研究的模型（例如紫花苜蓿、莲花）对于揭示支持共生的分子和生理机制特别有价值。

使用光学显微镜，MFRE 通常可以通过其细小的菌丝（< 1.5 µm 直径）与小的中间和末端肿胀以及“扇形”分枝结构来识别（Thippayarugs等人，1999 年）。这些与 AMF（或“粗根内生菌”）相对较粗的菌丝（> 3 µm 直径）形成对比（Field & Pressel，2018 年）。丛枝（高度分支的细胞内真菌结构）是植物-AMF 共生的特征；然而，它们在宿主植物甚至植物生命周期的 MFRE 共生中的发生和出现（Hoysted等人，2021a）是可变的（Orchard等人，2017b；Hoysted等人，2019 年））。形态可塑性也已经在仅检测到MFRE共生的植物中的超微结构的发送和扫描电子显微照片指出（场等人。，2015 ; Hoysted等人，。2019），使之成为具有挑战性的区分它们在植物中的共同-AMF 的殖民化（Field等人，2016 年）。与普遍特征很好的 AMF 孢子相比，MFRE 孢子的记录很少。出现了对其外观和大小的简要描述，但没有图像（Hall，1977；McGee，1987); 事实上，迄今为止，仅发布了一张未经验证的内单胞菌 MFRE 孢子图像（Orchard等，2017a）。

形成菌根的维管植物中普遍存在的共生情景是多种真菌共生体的定植（Hoysted等人，2019 年；Teste等人，2020 年）。多年来，菌根真菌的检测和表征技术得到了改进，包括使用靶向标记基因的真菌特异性引物的分子检测方法（White et al ., 1990），Maarj AM策划的 AMF 序列数据库（Öpik等人，2010 年)，以及使用无菌真菌培养物的接种方法 (Mugnier & Mosse, 1987) 或从土壤中提取的真菌孢子 (Gerdemann & Nicolson, 1963 ) 以生成专门由特定菌根真菌物种定殖的植物。这种方法对 MFRE 尤其具有挑战性，因为它们的孢子特征不佳且难以分离，可用的真菌分离物很少（Field等，2015）。这可能代表了 MFRE 研究进展的最紧迫障碍，突出了对 MFRE 植物实验系统的需求，该系统允许研究人员更好地控制可能影响 MFRE 共生形式和功能的生物和非生物因素。在这里，我们讨论了目前可用于研究这些关联的三种最先进的方法，包括使用土壤筛分、野生植物和无菌真菌分离物，以及在使用每种方法时应考虑的注意事项。