Plant Soil DOI 10.1007/s11104-017-3220-2

REGULAR ARTICLE

The rhizosheath – a potential trait for future agricultural sustainability occurs in orders throughout the angiosperms

Lawrie K. Brown & Timothy S. George & Konrad Neugebauer & Philip J. White

Received: 21 December 2016 /Accepted: 6 March 2017 # Springer International Publishing Switzerland 2017

Abstract when root hairs were extremely short, but the presence Aims The rhizosheath is defined as the weight of soil of root hairs was required for rhizosheath formation. adhering strongly to roots on excavation, and current Conclusions The rhizosheath is present in species from interest in this trait as a potential tolerance mechanism to many angiosperm orders. Potential to enhance the trait is abiotic stress has prompted us to explore the extent of its likely to exist in a range of crop species and could help occurrence throughout the angiosperm phylogeny. contribute to future agricultural sustainability. Methods Here we describe a robust, novel method which was used to screen species for the presence/absence and Keywords Rhizosheath . Angiosperm phylogeny. strength of a rhizosheath. We correlate the latter with root Root hairs . Agricultural sustainability. Abiotic stress hair length to provide insight into some of the factors tolerance . Second green revolution affecting its formation. We go on to compare experimen- tal data with previous observations in the literature. Results Results of a glasshouse screen demonstrate that rhizosheaths exist in species from many angiosperm Introduction orders, and the frequency of their occurrence and their strength and size are related. No correlation between It is now well over 100 years since the root feature, root hair length and rhizosheath size was found, except known today as the rhizosheath, appeared in the litera- ture (Volkens 1887) with early research into its form being conducted on samples of desert grasses collected by botanists of the time, including Arthur Tansley (Price Responsible Editor: Hans Lambers. 1911). Until recently, the limited research into this be- lowground trait has focused mainly on its structure, Electronic supplementary material The online version of this article (doi:10.1007/s11104-017-3220-2) contains supplementary formation and function. material, which is available to authorized users. Originally described as a Bpeculiar sheath, composed : : : of agglutinated particles of sand^ (Volkens 1887), today L. K. Brown T. S. George (*) K. Neugebauer it is defined operationally as the weight of soil that P. J. White The James Hutton Institute, Dundee DD2 5DA, UK adheres strongly to roots on excavation (George et al. e-mail: [email protected] 2014). Its formation and composition is complex and associated with many factors, including root hair length, P. J. White density and morphology (Haling et al. 2010), root and King Saud University, Riyadh 12372, Saudi Arabia microbial mucilage (Watt et al. 1993; McCully 1999; K. Neugebauer Barre and Hallet 2009; Albalasmeh and Ghezzehei University of Nottingham, Loughborough LE12 5RD, UK 2014), soil water content (Watt et al. 1994; Ghezzehei Plant Soil and Albalasmeh 2015), soil texture (Haling et al. 2014), population, and the rhizosheath could have an important mycorrhizal fungi (Moreno-Espindola et al. 2007)and role in a Second Green Revolution (Lynch 2007). free-living bacteria (Unno et al. 2005). While there has been considerable research into the Various functions of the rhizosheath have been sug- rhizosheaths of desert grasses, in which they were first gested. Price (1911) proposed rhizosheaths to be a observed (Price 1911;Buckley1982; Goodchild and mechanism for improving drought resistance and the Myers 1987;Danin1996a;Othmanetal.2004), and protection of roots under arid conditions, and more some research in other grass species (Duell and Peacock recent research supports this hypothesis (Pate and 1985;BaileyandScholes1997; Bergmann et al. 2009), Dixon 1996; Shane et al. 2010;Benardetal.2016). In the restriction of rhizosheath research to species within addition to its role in protecting the root from drought the Poales has led to the misconception that rhizosheaths and heat stress, the rhizosheath has potential importance are confined to this order (Duell and Peacock 1985). in coping with other abiotic stresses. These include There has been some research into the rhizosheath in nutrient deficiencies like phosphorus (Brown et al. cereal species including wheat, barley and maize (Watt 2012; Haling et al. 2013), zinc (Nambiar 1976) and et al. 1994; Young 1995; Czarnes et al. 2000;Haling nitrogen (Wullstein 1991;Danin,1996a;Othmanetal. et al. 2013, 2014; Delhaize et al. 2012;Georgeetal. 2004), and stresses associated with soil acidity (Haling 2014) and other monocotyledons, such as rushes (Shane et al. 2010; Delhaize et al. 2012) and soil strength et al. 2009, 2010, 2011), but only a few studies have (Haling et al. 2013, 2014; Albalasmeh and Ghezzehei been performed on a wider range of plants. These in- 2014). Rhizosheaths are effective in alleviating such clude studies on some eudicotyledonous species such as stresses because they provide and maintain good contact cacti (McCully 1999), soybean (Sprent 1975), lupin at the interface of the root and the soil which is essential (Unno et al. 2005) and fig (North and Nobel 1997). for nutrient and water uptake (North and Nobel 1997). McCully (1999) acknowledges the presence of Root hairs, which are tubular shaped cells specialised rhizosheaths on the fine roots of some legumes and a for nutrient uptake (Jungk 2001), enmesh soil particles few eudicotyledonous crop plants, but these have not around the root (Bristow et al. 1985)andareessential been investigated further. The literature suggests that the for the formation of rhizosheaths (Haling et al. 2010; rhizosheath is limited to a discrete range of species Brown et al. 2012). Correlations have been found be- which are adapted to stressful environments. This study tween root hair length and rhizosheath weight, but they will challenge this preconception. vary in strength depending on plant species. In wheat, In a recent study, Smith et al. (2011) performed a global root hair length and rhizosheath weight are strongly survey of the rhizosheath trait in a phylogenetic context correlated (Delhaize et al. 2012), while in barley there using field-grown and herbarium specimens, to produce a is a weaker correlation (George et al. 2014), suggesting phylogenetic map of the trait in the Haemodoraceae fam- that factors other than root hair length are important in ily (Commelinales order). The authors concluded that the determining rhizosheath weight. rhizosheath is probably an ancestral trait for the Recent studies of the rhizosheath have attempted to Haemodoraceae family which is associated with a large gain an understanding of the genetics of the trait in crop degree of phylogenetic conservatism and some secondary plants and quantitative trait loci (QTL) for rhizosheath loss. While such detailed information is helpful in build- weight and relative rhizosheath weight have been iden- ing a picture of the heritability of the trait at family level, tified in barley (George et al. 2014) and wheat (Delhaize no studies to date have attempted to investigate the extent et al. 2015; James et al. 2016). Many of the major of the occurrence of the rhizosheath trait across the whole cereals (wheat, maize, barley, oats, rye and sorghum) angiosperm phylogeny. The bias towards the study of arid form rhizosheaths (Duell and Peacock 1985). Under- grasses and the patchy information on other species means standing the genetics behind this belowground trait we do not have a clear picture of the extent of this trait may provide plant breeders with genetic markers to among angiosperms. improve the ability of other species of crop plants to In addition, there is no consensus methodology for deal with environmental variability and abiotic stress. determining the presence, or measuring the strength, of This understanding has importance for improved agri- rhizosheaths. Much of the historical data pertaining to cultural sustainability in the context of future climate the rhizosheath are based on subjective observation and change, finite resources, and an increasing global anecdotal evidence. The subjective interpretation of the Plant Soil term Brhizosheath^ means that the extent of the research methodology and broaden the extent of the study. The into this trait may be underestimated. For example, combined data have then been mapped onto an abridged many studies into the microbiology of the rhizosphere phylogenetic tree at order level. might actually be studies of the rhizosheath (Bulgarelli et al. 2012). Baily and Scholes (1997)alsonotethatthe strength of bonding of the rhizosheath differs between Materials and methods species, with the rhizosheaths of some species being more easily removed than those of other species, sug- Glasshouse screen gesting that variation exists in rhizosheath type or form. Here we describe a simple, quick and environmen- An objective method for assaying the presence/ tally robust method for screening a broad range of absence and strength of the rhizosheath was devel- agriculturally and horticulturally relevant species to es- oped for glasshouse-grown plants and used to score tablish the frequency (presence/absence) and strength of 58 species in 19 orders of angiosperms (Table 1). the rhizosheath across the angiosperm phylogeny. In Four replicates of each species were pre-germinated addition, measurements of root hair length for the same on distilled water agar before being transferred to species were recorded to provide an insight into the individual containers of 175 cm3 (12cmdeep× importance of root hairs in determining rhizosheath 4 cm × 4 cm tapering), (Deep Rootrainers™, presence and weight. Data derived from this screen have Ronaash Ltd., Kersquarter, Kelso, UK) (Fig. 1a). been compared with reports of rhizosheaths in the liter- Each Deep Rootrainer™ was filled with 170 g of ature and the two datasets synthesised to verify the soil collected from James Hutton Institute land (NO

Table 1 Orders and species included in the glasshouse screen

Order ID Order Number of species Species

1Asparagales6 Allium porrum, Allium schoenoprasum, Allium cepa, Allium cepa Aggregatum Grp, Asparagus officinalis, Iris pseudocorus 2 Commelinales 1 Commelina coelestis 3Dioscoreales1 Dioscorea japonica 4Poales11 Agrostis canina, Agrostis stolonifera, Cymbopogon citratus, Dactylis glomerata, Festuca rubra, Hordeum vulgare, Juncus effusus, Oryza sativa, Pennisetum villosum, Sorghum sp., Zea mays 5Apiales4 Apium graveolens, Coriandrum sativum, Daucus carota, Pastinaca sativa 6Asterales2 Eupatorium cannabinum, Helianthus annuus 7Boraginales1 Myosotis arvensis 8 Brassicales 4 Brassica rapa, Cardamine pratensis, Lunaria annua, Raphanus sativus 9 Caryophyllales 4 Beta vulgaris, Rumex acetosa, Stellaria media, Silene latifolia 10 Dipsacales 3 Dipsacus fullonum, Scabiosa atropurpurea, Valeriana officinalis 11 Fabales 12 Anthyllis vulnerania, Glycine max, Lathyrus odoratus, Lotus corniculatus, Medicago sativa, Onobrychis sativa, Phaseolus vulgaris,Pisum sativum, Trifolium repens, Vicia cracca, Vicia faba, Vicia sativa 12 Geraniales 1 Geranium pratense 13 Lamiales 1 Mentha × piperita 14 Malpighiales 1 Viola cornuta 15 Malvales 1 Malva sylvestris 16 Myrtales 2 Lythrum salicaria, Oenothera biennis 17 Rosales 1 Urtica urens 18 Saxifragales 1 Heuchera sp. 19 Solanales 1 Solanum lycopersicum Plant Soil

Fig. 1 a Rootrainer™ set-up used in the glasshouse screen. b Harvested replicates of two species with contrasting rhizosheaths. Dioscorea japonica – rhizosheath score 1 (left), Malva sylvestris – rhizosheath score 4 (right)

299330) and sieved to 4 mm. This soil was a typical 0 = No soil attached to roots East of Scotland agricultural soil defined as Garvock 1 = Only a few particles of soil attached to the roots, series, a sandy loam (Sand 54%, Silt 40%, Clay 6%) all soil shakes off with an organic carbon content of 1.97% and pH of 2 = Soil attached to roots, most soil shakes off 6.3 (in water), 6.0 (in calcium chloride). Containers 3 = Soil attached to roots remains after shaking, were watered daily with distilled water to maintain a most is removed after 5 mins in sonic bath moisture content equivalent to 80% Field Capacity 4 = Soil attached to roots remains attached after (FC) to standardise for soil moisture content across shaking and 5 mins in sonic bath all samples. Day length was maintained at 16 h with supplemental lighting allowing a minimum light in- Species with rhizosheath strength scores of ≥2were tensity of 200 μmol quanta m−2 s−1.Temperature taken to represent Btrue^ rhizosheaths. was allowed to fluctuate between 25 and 15 °C. After excavation from root trainers, roots were sepa- Species replicates were harvested at the 2–4 leaf rated from the shoots and weighed with soil attached growth stage (26 days post emergence on average) by prior to washing. Rhizosheaths of category 0, 1 and 2 disassembling the individual containers and shaking could be shaken or washed off. Rhizosheaths of catego- each plant by hand until no more bulk soil became ry 3 could be removed by placing roots in individual detached. The remaining soil attached to the roots was 50 ml tubes filled with tap water and standing these considered to be the rhizosheath (Fig.1b). Rhizosheath tubes in a sonic bath with a 2 L capacity, 75 W Output strength was categorised using the following scoring power at 35–45 kHz (Decon FS100b, East Sussex, UK) method: for 5 min. Rhizosheaths of category 4 remained attached After excavation from root trainers to roots after sonication. Plant Soil

All roots were washed clean over a 0.2 mm sieve using soil collected from a site near the James Hutton Institute, tap water with due care taken to avoid any damage to Dundee, Scotland (NO 456265). This soil was defined roots and root hairs. They were then systematically dried as Carpow series, a silt loam (Sand 33%, Silt 58%, Clay with absorbent tissue paper and reweighed to establish the 9%) with an organic carbon content of 2.6% and pH of rhizosheath mass (i.e. the difference in weight between 6.2 (in water) and 5.5 (in calcium chloride), which was roots with soil attached and clean roots = rhizosheath sieved to 4 mm. size). Means of specific rhizosheath weight of species were calculated and expressed on a root fresh weight Survey of published data basis. Root fresh weight was measured prior to storage for root hair measurements. It is acknowledged that the An extensive literature search for all relevant publica- root fresh / dry weight quotient of species might differ. tions was performed. This began by searching for the Roots were stored in 50% (v/v) ethanol until root hair term Brhizosheath^ in all databases of The Web of length could be determined using a compound light Science, which revealed 53 articles. These articles were microscope (Leica MZF111, Leica Microsystems, Ban- sourced and read and any articles cited in these articles nockburn, IL, USA) at 5× magnification with camera likely to include data on the occurrence of rhizosheaths attachment (Leica DC480) to image the roots and mea- in angiosperms were again sourced and read. This pro- surement of the root hair length was aided by ImageJ cess was repeated until no further papers referring to software (U.S. National Institutes of Health, Bethesda, rhizosheath were found. The literature search revealed a Maryland, USA). Observation under the microscope total of 88 non-redundant articles reporting relevant data confirmed there was no quantifiable damage to root on rhizosheaths (Supplementary Material Table S1 and hairs resulting from the processing and storage of the List S1) covering a total of 341 species across 21 orders. roots prior to imaging. The rhizosheath trait was scored as being present or Representative roots from four replicates of each absent. A report of a Bloose sheath^ was recorded as species were imaged and a total of 10 individual root the presence of a rhizosheath. A report that a rhizosheath hair measurements were taken from the root hair zones was Boccasionally present^ or Bsparse^ was recorded as of each replicate. The mean was calculated for each absent. The number of species and genera surveyed in species replicate and the species mean and standard each order was determined and the number of species errors were based on the means of the four replicates. with rhizosheaths and genera containing species with Where the number of replicates was less than 4, due to rhizosheaths was calculated. The nomenclature of all failed plant growth (n = 7 species), means and standard species followed that of The Plant List (http://www. errors were calculated accordingly. theplantlist.org/; March 2017). In order to establish the reproducibility of the rhizosheath scoring method, a further screen of a subset Phylogeny of the rhizosheath trait of species was carried out in a second soil type (Table 2). This screen was carried out for species exhibiting a Data on the number of species and genera surveyed range of scores from 0 to 4 in the initial screen using a in each order, and the number of species with

Table 2 Comparison of data from rhizosheath screening in different soil types

Species Order Rhizosheath weight g g-1 Rhizosheath weight g g-1 Rhizosheath Rhizosheath freshrootweightSoil1 fresh root weight Soil 2 score Soil 1 score Soil 2

Scabiosa atropurpurea Dipsacales 6.37 2.55 1 1 Trifolium repens Fabales 33.23 6.57 4 3 Viola cornuta Malpighiales 26.93 9.35 2 2 Dactylis glomerata Poales 100.63 23.50 4 4 Myosotis arvensis Boraginales 75.19 24.90 3 4 Stellaria media Caryophyllales 24.51 32.09 3 3 Allium cepa Asparagales 0.00 0.01 0 0 Plant Soil rhizosheaths and genera containing species with and specific rhizosheath weight varied from 0 to rhizosheaths from the glasshouse screen and the 142.3 g g−1 root fresh weight (Fig. 2). Within the survey of published data were plotted both monocots there was a clear difference in rhizosheath independently, and as a combined dataset, onto an weight between the commelinid group, which in- abridged phylogenetic tree of the angiosperms that cludes the Commelinales (order 2) and Poales orders included only the orders represented in the (order 4), in which specific rhizosheath weight glasshouse screen or literature survey. The rangedfrom6.5to101.8gg−1 root fresh weight Angiosperm Phylogeny Group IV (2016) provided withameanof28.5gg−1 root fresh weight (n =12), the phylogenetic relationships between angiosperm and the non-commelinid group, which includes the orders. Asparagales (order 1) and Dioscoreales (order 3), in which specific rhizosheath weight ranged from 0 to 1.1 g g−1 root fresh weight with a mean of 0.3 g g−1 Results root fresh weight (n = 7). The rhizosheath weight in eudicot species (n = 39) ranged from 0.4 to 142.3 g − − Glasshouse screen g 1 root fresh weight with a mean of 19.6 g g 1 root fresh weight. Specific rhizosheath weight was much Rhizosheaths were found in both monocotyledonous more variable both between and within eudicot (monocot) and eudicotyledonous (eudicot) species orders.

200

150

100

50 Specific rhizosheath weight g g-1 root fresh weight

0 Zea mays Vicia faba Vicia Vicia sava Allium cepa Allium Sorghum sp Vicia cracca Glycine max Urca urens Urca Heuchera sp. Beta vulgaris Beta Viola cornuta Viola Brassica rapa Festuca rubra Silene lafolia Juncus effusus Juncus Allium porrum Allium Lunaria annua Pisum savum Pisum Stellaria media Rumex acetosa Agross canina Agross Malva sylvestris Iris pseudacorus Iris Trifolium repens Pasnaca sava Medicago sava Medicago Lythrum salicaria Lythrum Hordeum vulgare Hordeum Raphanus savus Raphanus Onobrychis sava Myosos arvensis Myosos Apium graveolens Apium Mentha × piperita × Mentha Lotus corniculatus Lotus Lathyrus odoratus Lathyrus Dipsacus fullonum Dipsacus Oenothera biennis Oenothera Phaseolus vulgaris Phaseolus Helianthus annuus Oryza sava (bala) sava Oryza Dactylis glomerata Dactylis Dioscorea japonica Dioscorea Geranium pratense Agross stolonifera Anthyllis vulnerania Anthyllis Valeriana officinalis Valeriana Coriandrum savum Coriandrum Commelina coeless Pennisetum villosum Pennisetum Cardamine pratensis Cardamine Asparagus officinalis Asparagus Cymbopogon citratus Solanum lycopersicum Solanum Allium schoenoprasum Allium Scabiosa atropurpurea Scabiosa Eupatorium cannabinum Eupatorium Daucus carota (subsp sava) (subsp carota Daucus Allium cepa Aggregatum Grp Aggregatum cepa Allium 1234 5678910 111213141516171819 Fig. 2 Specific rhizosheath weight (g g−1 root fresh weight) of species within order groups in a glasshouse screen. Shaded bars represent monocot species and block colour bars eudicot species. Species groupings are order numbers referred to in Table 1 Plant Soil

4.0

3.0

2.0 Rhizosheath strength strength Rhizosheath score

1.0

0.0 Zea mays Vicia faba Vicia Vicia sava Allium cepa Allium Sorghum sp Vicia cracca Vicia Glycine max Urca urens Urca Heuchera sp. Heuchera Beta vulgaris Beta Viola cornuta Viola Brassica rapa Festuca rubra Festuca Silene lafolia Juncus effusus Juncus Allium porrum Allium Lunaria annua Pisum savum Pisum Stellaria media Rumex acetosa Agross canina Agross Malva sylvestris Malva Iris pseudacorus Iris Trifolium repens Trifolium Pasnaca sava Pasnaca Medicago sava Medicago Lythrum salicaria Lythrum Hordeum vulgare Hordeum Raphanus savus Raphanus Onobrychis sava Myosos arvensis Myosos Apium graveolens Apium Mentha × piperita × Mentha Lotus corniculatus Lotus Lathyrus odoratus Lathyrus Dipsacus fullonum Dipsacus Oenothera biennis Oenothera Phaseolus vulgaris Phaseolus Helianthus annuus Oryza sava (bala) Dactylis glomerata Dactylis Dioscorea japonica Dioscorea Geranium pratense Geranium Agross stolonifera Anthyllis vulnerania Anthyllis Valeriana officinalis Coriandrum savum Coriandrum Commelina coeless Commelina Pennisetum villosum Pennisetum Cardamine pratensis Cardamine Asparagus officinalis Asparagus Cymbopogon citratus Cymbopogon Solanum lycopersicum Solanum Allium schoenoprasum Allium Scabiosa atropurpurea Scabiosa Eupatorium cannabinum Eupatorium Daucus carota (subsp sava) (subsp carota Daucus Allium cepa Aggregatum Grp cepa Aggregatum Allium 1 2 3 4 5 6 7 8 9 10 11 12131415 16 171819

Fig. 3 Rhizosheath strength score by species within order groups line at strength score 2 are defined as rhizosheaths. Shaded bars in a glasshouse screen. Strength score 0 = no rhizosheath to represent monocot species and block colour bars eudicot species. 4 = strong rhizosheath. All points on and above the red horizontal Species groupings are order numbers referred to in Table 1

Species exhibiting Btrue^ rhizosheaths, defined as throughout the angiosperm phylogeny and the having a strength score ≥ 2(Fig.3), were distributed rhizosheath trait occurred in 81% of the 58 species and

Fig. 4 Plot of average rhizosheath strength against rhizosheath frequency of 4.0 15,19 occurrence for all orders in 4 11 glasshouse screen. Red triangles represent monocots where n >1 8 3.0 7,9,12,17 and red + symbols monocot species where n =1.Blue squares 6 eudicots where n >1andblueX 2.0 symbols eudicots where n =1. 2,13,14,16,18

Numbers refer to order groupings 5 in Table 1 Rhizosheath strength score 1.0 3,10

1

0.0 020406080100 Frequency % Plant Soil

89% of the 19 orders studied. No Btrue^ rhizosheaths occurrence and included the Dioscoreales (order 3) and were observed in species from the Dioscoreales (order the Dipsacales (order 10). The Poales (order 4) and 3) or Dipsacales (order 10) orders. Within the monocots, Fabales (order 11) are notable examples of orders with the rhizosheath strength of non-commelinid species strong rhizosheaths that occur frequently. No orders had ranged from scores of 0 to 2 with a mean of 0.6, while strong rhizosheaths with a small frequency of occurrence. the rhizosheath strength of commelinid species ranged The commelinid (Poales (order 4) and Commelinales from 2 to 4 with a mean of 3.6. For eudicot species, (order 2) and non-commelinid Asparagales (order 1) scores for rhizosheath strength ranged from 1 to 4 with a and Dioscoreales (order 3)) monocot orders fell into mean of 2.8. distinct groups: the commelinids exhibit strong A plot of rhizosheath strength against rhizosheath rhizosheaths that occur frequently and the non- frequency (Fig. 4) highlights groupings of the orders commelinids exhibit weak rhizosheaths that occur fre- screened. Frequency was defined as rhizosheath occur- quently or infrequently depending upon the order. rence as a percentage of the total number of species Mean root hair length (Fig. 5)variedfrom0mm studied in a given order. Orders with weak rhizosheaths (species with no root hairs) to 1.5 mm in Hordeum and a low frequency of rhizosheath occurrence (11% of vulgare and Silene latifolia. The mean root hair length orders studied) included the Asparagales (order 1) and for non-commelinid species ranged from 0 to 0.4 mm Apiales (order 5). Of the 19 orders studied, 11% demon- with a mean of 0.1 mm, while commelinid species strated weak rhizosheaths with a large frequency of ranged from 0.1 to 1.5 mm with a mean of 0.6 mm.

1.80

1.60

1.40

1.20

1.00

0.80

Mean root hair length mm mm length hair root Mean 0.60

0.40

0.20

0.00 Zea mays Vicia faba Vicia sava Allium cepa Allium Sorghum sp Vicia cracca Vicia Glycine max Urca urens Heuchera sp. Beta vulgaris Beta Viola cornuta Viola Brassica rapa Brassica Festuca rubra Silene lafolia Juncus effusus Juncus Allium porrum Allium Lunaria annua Pisum savum Pisum Stellaria media Rumex acetosa Agross canina Agross Malva sylvestris Malva Iris pseudacorus Iris Trifolium repens Trifolium Pasnaca sava Pasnaca Medicago sava Medicago Lythrum salicaria Lythrum Hordeum vulgare Hordeum Raphanus savus Raphanus Onobrychis sava Myosos arvensis Myosos Apium graveolens Apium Mentha × piperita × Mentha Lotus corniculatus Lotus Lathyrus odoratus Lathyrus Dipsacus fullonum Oenothera biennis Oenothera Phaseolus vulgaris Phaseolus Helianthus annuus Oryza sava (bala) Dactylis glomerata Dactylis Dioscorea japonica Dioscorea Geranium pratense Geranium Agross stolonifera Anthyllis vulnerania Anthyllis Valeriana officinalis Coriandrum savum Coriandrum Commelina coeless Commelina Pennisetum villosum Pennisetum Cardamine pratensis Cardamine Asparagus officinalis Asparagus Cymbopogon citratus Cymbopogon Solanum lycopersicum Solanum Allium schoenoprasum Allium Scabiosa atropurpurea Scabiosa Eupatorium cannabinum Eupatorium Daucus carota (subsp sava) (subsp carota Daucus Allium cepa Aggregatum Grp Aggregatum cepa Allium 1 2 3 4 5 6 7 8 9 10 11 12131415 16 171819

Fig. 5 Plot of mean root hair length (mm) of species within order groups in a glasshouse screen. Shaded bars represent monocot species and block colour bars eudicot species. Species groupings are order numbers referred to in Table 1 Plant Soil

Fig. 6 Correlation of specific 140 −1 rhizosheath weight g g root y = 75.833x - 3.3806 120 fresh weight against mean root r² = 0.422 hair length (mm) of all species in 100 glasshouse screen. Dotted line is root fresh weight fresh root 1 - best fit line for all species 80 r2 = 0.146 (not significant). Solid line is best fit line for species with y = 31.463x + 5.4932 60 r² = 0.145 root hair lengths of 0.28 mm or 2 less r = 0.422 (weak positive 40 correlation P <0.05) 20 Specific rhizosheath weight g g weight rhizosheath Specific 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Mean root hair length mm

Similarly, the mean root hair length of eudicot species Reproducibility in different soils ranged from 0.1 to 1.5 mm, but with a mean of 0.4 mm. No relationship was found between specific rhizosheath In general, scores for both the presence and strength of weight and mean root hair length (Fig. 6); however, rhizosheaths were consistent between soils, with species there was a weak, but significant positive correlation showing mostly the same scores in the two soil types (r2 = 0.422, P < 0.05) between specific rhizosheath studied (Table 2). However, rhizosheath weight was, on weight and root hair length for species with mean root average, 3 fold greater when assayed in soil 1 than when hair lengths of 0.28 mm and less (n =21). assayed in soil 2, although there was a strong correlation

Literature Screen Combined (Literature+Screen) Genera Species Species Genera Species N+RN+RN+RN+RN+R 2233113344 95 88 292 254 11 11 97 89 305 252 75966295136 1111 1111 101010 2020 2020 2020113131 96117121213111815 2131112142 2030 2030 3030113141 909044124134 1121223343 1020112131 111111 21 12 23 12 4 4 22 15 26 16 1010 1010 1111 1111 17 7 18 7 2 2 18 8 18 8 1010425252 303030 1111112222 4353115464 5262115262

Fig. 7 Abridged phylogenetic tree of the angiosperms showing Numbers in brackets next to orders correspond to order number the prevalence of the rhizosheath trait. Data are from both the ID’sinTable1. N is number of species or genera and +R is the glasshouse screen and the literature survey. Numbers on lines number of species or genera where rhizosheath is present represent the percentage frequency of rhizosheath in a given order. Plant Soil

(r2 = 0.92) between data obtained on both soils for all Discussion species with the exception of Stellaria media. The rhizosheath trait has potential to improve future agricultural sustainability by enhancing stress tolerance Combined analysis in a variety of crop plants. The present study has re- vealed that the rhizosheath trait is present throughout the The combined data from both glasshouse and literature angiosperm phylogeny (Fig. 2) and dismisses the pre- studies (Fig. 7) illustrate the distribution and frequency conception that rhizosheaths are only a feature of a small of occurrence of the rhizosheath trait throughout the number of species from extreme environments. In addi- angiosperm phylogeny. Data for four broad groupings tion, it describes, a robust methodology for screening a of orders were compiled. In the first group, the mono- range of plant species for the presence of this trait. cotyledonous orders, the trait was prevalent among The importance of an objective, quantitative method- species in the Poales (252/305 species), Commelinales ology for defining and measuring the rhizosheath is (4/4 species) and Liliales (1/1 species), occasionally apparent when attempting to screen for a trait both observed in species from the Asparagales (6/13 spe- within and across species. It is clear from the published cies) and was not observed in the only Dioscoreales literature, that the current ambiguity around what de- studied, Dioscorea japonica. The second group com- fines a rhizosheath means that the full extent of infor- prised the Ranunculales in which the trait was not mation regarding this trait is not being captured. In observed in either of the two species studied. The third addition, the fact that much of the research interest to group, the superrosids, included species in the Rosales, date has focused on the Poales, in particular those spe- Fabales, Malpighiales, Zygophyllales, Malvales, cies of arid climates means that the presence, structure Brassicales, Myrtales, Geraniales and Saxifragales. In and formation of rhizosheaths may have been these orders the proportion of species with a overlooked in a wider range of species. rhizosheath varied greatly, and ranged from the The methodology reported here allows comparisons Fabales (15/18 species) and Myrtales (3/4 species), in to be made for all terrestrial angiosperm species and which the occurrence of rhizosheaths was common, to measures rhizosheath size and strength in an objective the Zygophyllales (0/1 species), Malvales (1/4 spe- way. It also allows many plants to be screened in a short cies), Geraniales (1/3 species), Brassicales (4/13 spe- period of time and was found to be robust for the cies) and Rosales (1/3 species), in which the presence different soil types in this study. Previous methods to of a rhizosheath was rarely observed. The fourth establish a standard measurement method for measuring group, the superasterids, included the Caryophyllales, rhizosheath strength were described by Watt et al. 1994 Cornales, Ericales, Lamiales, Solanales, Boraginales, in maize, however this method was not suitable for the Asterales, Dipsacales and Apiales. Again, the propor- higher throughput of samples achieved in our screen. tion of species with a rhizosheath varied greatly be- While many reliable, non-invasive methods for studying tween these orders. A rhizosheath was commonly ob- roots have been developed recently (Gregory et al. served in species from the Ericales (1/1 species), 2009; White et al. 2013; Koebernick et al. 2014; Solanales (2/2 species), Lamiales (4/6 species) and Downie et al. 2015) the rate at which plants can be Caryophyllales (16/26 species), but was not observed screened using such techniques is often limited and the in the species of Cornales (0/3 species) or Dipsacales detail provided by these methods is not a prerequisite for (0/1 species) studied. determining the size and strength of the rhizosheath. For A comparison of data from the glasshouse screen the purposes of this study the rhizosheath has been with previous literature showed broad concordance with defined as the soil attached to the root after excavation, the following exceptions: (1) rhizosheaths were ob- whatever components are involved in its formation. This served in the glasshouse screen but had not been report- contrasts with Danin (1996b) who made a distinction ed previously in the literature for species in the Rosales, betweenrhizosheathsformedfrommucilageandroot Malvales, Brassicales and Geraniales and (2) the fre- hairs together and particles enmeshed by root hairs quency of occurrence of the rhizosheath trait among the alone. Fabales and Caryophyllales was greater in the glass- The standardisation of the growth conditions allows house screen than had been reported in the literature. the elimination of many of the environmental factors Plant Soil that are known to affect rhizosheath formation, such as A focus of this study was on the contribution of root soil moisture, temperature, soil type, soil pH and nutri- hairs in the formation of rhizosheath and was conducted ent availability. While the effects of plant development in Blive^ soil, meaning that the effects of the presence of on rhizosheath formation were standardised by harvest- microorganisms and fungi on rhizosheath formation ing plants at a given growth stage (2–4 leaf stage), it is cannot be eliminated (Bulgarelli et al. 2013). Further acknowledged that comparisons between different spe- screening of species using methods such as those de- cies could be confounded by their differing develop- scribed by Watt et al. (1993), may provide valuable mental habits. The standardisation of the harvesting information on the relative contribution of microbial method, which minimises root damage, ensures all versus plant mucilage and the subsequent effects on plants have been extracted from the soil in the same rhizosheath formation. way. Smith et al. (2011), who found herbarium samples The abridged phylogenetic tree, which covers one of various species exhibited no rhizosheaths, whilst they third of the angiosperm orders (Fig. 7), illustrates the exhibited rhizosheaths when carefully excavated in the distribution and frequency of occurrence of the field, cautioned against sampling procedures that might rhizosheath trait. The data demonstrate that the shear away the rhizosheath. This might explain some of rhizosheath trait occurs throughout the angiosperms the differences identified in our study when comparing and is not restricted to a few orders. The rhizosheath trait the data from the glasshouse experiment with those in is commonly observed in the commelinid monocot or- the literature (Fig. 7). ders Poales (252/305 species) and Commelinales (4/4 By performing the screen in a second soil type for a species), but occurs less frequently in the non- subset of species exhibiting contrasting rhizosheath commelinid monocot orders Asparagales (6/13 species) strengths from the first screen, we were able to confirm and Dioscoreales (0/1 species). Among the eudicots, the the method to be robust for scoring the presence and trait is observed frequently in the Fabales (15/18 spe- strength of rhizosheaths (Table 2). Although there was, cies), but is less frequent in the Caryophyllales (16/26 on average, a 3 fold difference in rhizosheath weight species), Asterales (8/18 species) and Brassicales (4/13 between soils, there was a strong correlation in species), and was not observed in species of the rhizosheath weight between the two soils for all species Ranunculales, Zygophyllales, Cornales and Dipsacales with the exception of Stellaria media. These weight that were studied. However, it should be noted that many differences may be explained by variations in a range angiosperm orders are represented by only a few species of properties between the two soils resulting in smaller and so the data should be interpreted with some caution. or larger rhizosheaths being formed dependent on soil Future work should extend the number of species studied type. Observations at harvest suggest Stellaria media to provide better representation both within and across may be employing a different strategy in rhizosheath angiosperm orders and include all functional groups. formation and seems to trap soil in a net of fine roots The current data do not provide a detailed understand- rather than soil being attached through interactions of ing of the evolution of the rhizosheath trait in the angio- root hairs and mucilage. These results suggest that sperms. Nevertheless, it is likely that, since the preva- rhizosheath screens which take into account lence of the trait differs markedly between closely related rhizosheath strength as well as size are more robust orders, this trait evolved independently in different than those which screen only for size. Baily and branches of the angiosperm phylogeny in response to Scholes (1997) observed that rhizosheaths of some plant contrasting environmental stresses (Davies et al. 2004). species are more easily detached than those of others, Further understanding of the individual traits involved in which suggests that different mechanisms may be in- rhizosheath formation might be gathered from compari- volved in rhizosheath formation. Our study corroborates sons with other phylogenetic studies, for example, root the observations of Baily and Scholes (1997) and is the morphology and architecture, mycorrhizal colonisation first to present data that quantify differences in (Bennett et al. 2013) and plant metabolism (discussed rhizosheath strength using a systematic scoring method. later). In addition, more comparative studies at the family We hypothesise that different plant species form differ- level may provide a more detailed understanding of the ent types of rhizosheath, which in turn affects their evolution of the trait (Smith et al. 2011). strength and potential benefit to the plant in stress A Btrue^ rhizosheath was present in 81% of the orders tolerance. studied and throughout the angiosperm phylogeny Plant Soil

(Fig. 3). The rhizosheaths of species from orders with a others (data not presented), implying the presence of greater proportion of species with Btrue^ rhizosheaths some form of water-resistant coating. This corroborates exhibit greater strength than those of species from orders the findings of Carminati et al. (2010) who found in which the occurrence of rhizosheath is Brare^ among rhizosheath soil to be wetter than bulk soil during drying species. There is no order in which rhizosheaths are and that it remained dry temporarily on rewetting. This observed rarely, and exhibit strong rhizosheaths (Fig.4). was explained by the hypothesis that mucilage becomes It has been established that root hairs are required for hydrophobic as it shrinks upon soil drying due to water formation of rhizosheaths (Moreno-Espindola et al. uptake by roots. In the context of the current study this 2007; Shane et al. 2011) and our data (Figs 5, 6) sub- would suggest variation in mucilage production/ stantiates this. In the 58 species studied in the glasshouse composition across different species. experiment the rhizosheath was absent in all species that Differences in rhizosheaths between species might lacked root hairs. However, attempts to find a correlation also be attributed to differences in the metabolism of plant between rhizosheath size and root hair length produced species. For example, Harris and Trethewey (2010)in- contrasting results to those of Delhaize et al. (2012)who vestigated the distribution of the presence of ferulic acid demonstrated a strong correlation between rhizosheath in cell walls across the angiosperms and identified its specific weight and root hair length among wheat presence in the commelinid monocotyledons such as genotypes. George et al. (2014) found only a weak Hordeum vulgare and Zea mays and the core relationship between rhizosheath weight and root hair Caryophyllales such as Beta vulgaris. This is indicative length in barley. George et al. (2014) suggested this of a unique metabolism in these species, and, interesting- inconsistency may be species specific or it might reflect ly, this corresponds with the species with the strongest, the wider range and maximum length of root hairs and largest and most frequently occurring rhizosheaths in our range and maximum weight of rhizosheath than was study, i.e. the Poales, Commelinales and species within found in the wheat material of Delhaize et al. (2012). the core Caryophyllales, Beta vulgaris, Stellaria media This further suggests other components are important in and Silene latifolia. It is also of interest that the largest the formation of rhizosheaths. These hypotheses also rhizosheaths are found in two eudicot species, namely provide possible explanations for the lack of a significant Myosotis arvensis and Stellaria media. Collectively, this correlation among the 58 species for rhizosheath weight provides further evidence that the rhizosheath trait is and root hair length in our study (Fig. 6). It appears that present in a wide range of angiosperms. when root hair length exceeds 0.28 mm other factors The multi-trait nature of the rhizosheath suggests that have increasing importance in rhizosheath formation and no single gene determines its size and strength. Indeed, that multiple traits are involved. These may still be George et al. (2014) observed quantitative trait loci for the related to other features of root hairs, for example greater rhizosheath trait on four different chromosomes of barley, root hair distortions, including bent or hooked forms that which suggests that the character of the rhizosheath com- may help trap more soil, or differences in root hair prises a suite of traits that can be utilised collectively to density, but mucilage also plays an important role. Root produce large, strong rhizosheaths, which can benefit the and microbial mucilage stabilises and aggregates the soil ability of plants to tolerate abiotic stresses. particles round the root, providing a cohesive compound This is the first study to establish the presence and in the formation of rhizosheaths (McCully 1999). While extent of the rhizosheath trait among angiosperms. While there is evidence that these compounds allow it is acknowledged that the angiosperm phylogeny is not rhizosheaths to develop beyond the extent of the root completely represented, it provides sufficient coverage to hairs (McCully 1999) our study demonstrates that demonstrate the presence of a rhizosheath in many orders rhizosheaths only develop where root hairs are present of angiosperms and, importantly, in many orders contain- and suggest that any exudation of mucilage alone is not ing crop species. In addition, we describe a robust, novel sufficient for rhizosheath formation. However, the diver- methodology that allows the comparison of rhizosheath sity of compounds present in root exudates may impact strength and size across a diverse range of species. Re- the ability of different species to bind soil around their sults from assaying of the rhizosheath of many angio- roots. For example, observations made during this study sperm species suggest the multi-trait nature of its pres- when measuring rhizosheath strength suggested that the ence and structure and screens for size and strength could roots of some species were much more hydrophobic than provide useful parameters in screening for some of these Plant Soil traits. In addition, identification of molecular markers for Schmelzer E et al (2012) Revealing structure and assembly these traits and their heritability might provide useful cues for Arabidopsis root-inhabiting bacterial microbiota. Nature 488:91–95 information for breeders whoaresearchingforsuitable Carminati A, Moradi AB, Vetterlein D, Vantobel P, Lehmann E, root traits to include in future crop genotypes. Enhancing Weller U, Vogel H-J, Oswald SE (2010) Dynamics of soil rhizosheath traits across a range of crop species is cer- water content in the rhizosphere. 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