Do Chlorophyllous Orchids Heterotrophically Use Mycorrhizal Fungal Carbon?

Spotlight

Do chlorophyllous orchids heterotrophically use mycorrhizal fungal carbon?

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Marc-Andre´ Selosse and Florent Martos

Muse´ um national d’Histoire naturelle, De´ partement Syste´ matique et Evolution (UMR 7205 ISYEB), CP 50, 45 rue Buffon, 75005

Paris, France

School of Life Sciences, University of KwaZulu-Natal, Private Bag X01 Scottsville, Pietermaritzburg 3209, South Africa

The roots of orchids associate with mycorrhizal fungi, intermediate between those of achlorophyllous orchids

the rhizoctonias, which are considered to exchange min- and autotrophic plants [3], which is expected because they

eral nutrients against plant carbon. The recent discovery mix both nutritional strategies.

that rhizoctonias grow endophytically in non-orchid Beyond these exceptions, however, the vast majority of

plants raises the possibility that they provide carbon orchids associate with rhizoctonias and, in adulthood, dis-

to orchids, explaining why some orchids differ in isoto- play a C abundance almost similar to that of autotrophic

pic abundances from autotrophic plants. plants (Figure 1) [1,3], in spite of some small deviations (see

below). It is thus currently assumed that rhizoctonia-asso-

Orchids display several evolutionary innovations, including ciated orchids are autotrophic in adulthood, and do not use

a mycorrhizal symbiosis with particular fungal lineages. rhizoctonias C after germination (e.g., [4]).

These fungi, hitherto considered to be soil saprotrophs, are But these assumptions are challenged by two observa-

collectively called the rhizoctonias [1]. One characteristic tions. First, rhizoctonia-associated orchids often display

feature of this symbiosis is symbiotic germination, where unusually high nitrogen (N) and N content compared to

rhizoctonias colonize the reserveless orchid seeds and pro- other autotrophic plants [3,5]. These features are observed

vide carbon (C) to the seedlings that grow heterotrophically to a greater extent in the fully or partially heterotrophic

[1]. After the development of green leaves, adult orchids orchids that obtain organic matter from their mycorrhizal

acquire autotrophy, and mycorrhizal fungi are thought to fungi [3,4], likely because (i) their pathway for gaining N

receive C as a reward for providing mineral nutrients [2], as differs from that in autotrophic plants and (ii) the fungi have

in all other mycorrhizal symbioses [1]. However, recent high N and N contents. A gain of fungal organic matter in

works suggest the reverse mechanism might also take rhizoctonia-associated orchids could thus explain their high

place, and that fungal C could actually be provided by the N and N content. Second, a recent paper reports that

fungi to adult chlorophyllous orchids. seedlings of two rhizoctonia-associated orchids, which het-

erotrophically recover C from their rhizoctonias, display

Orchids that use and abuse mycorrhizal fungal C lower C abundances than mycoheterotrophic orchids, al-

A dependence on fungal C in adult orchids has been shown though these abundances are somewhat above those of

for a small number of forest species that are partially or fully autotrophic plants [5]. Thus, C transferred from rhizocto-

photosynthetic and display two particular features. First, nias to orchid protocorms is isotopically closer to C acquired

their mycorrhizal partners have changed from the usual through photosynthesis, and small C transfers may be

rhizoctonias to other fungal lineages: these orchids now difﬁcult to detect. It is still ignored whether this reﬂects a

associate with fungi, mostly from Basidiomycota, that either speciﬁc fractionation during rhizoctonia–to–orchid transfer,

form mycorrhizae on surrounding trees (the so-called ecto- or if, as for fully or partially heterotrophic orchids mentioned

mycorrhizal fungi) [3] or live as litter- or wood-decaying previously, this simply reﬂects the C abundance of the

saprotrophs [4]. Second, these orchids display much higher fungal source without fractionation. We speculate that the

C abundance than the C3 plants that surround them. The second explanation is likely, because an overlooked ecologi-

achlorophyllous orchids that obtain their entire C from their cal niche of rhizoctonias indeed predicts C abundance close

mycorrhizal fungi (mycoheterotrophic orchids) have a C to that of autotrophic plants.

abundance identical to that of the saprotrophic or ectomy-

corrhizal fungi that feed them (Figure 1), which are them- Rhizoctonias revisited

selves C-enriched compared with autotrophic plants There is evidence that rhizoctonias do not exclusively

[3,4]. Moreover, the green orchids that are partially depend on orchid mycorrhizae for their C nutrition, but

heterotrophic (mixotrophic orchids) have a C content have their own, independent nutritional niche. Their

saprotrophy is only indirectly suggested by the observation

of in vitro growth on dead and sometimes complex organic

Corresponding author: Selosse, M.-A. ([email protected]).

matter [1], but there is no direct evidence under natural

Keywords: root endophytic fungi; mixotrophy; mycoheterotrophy; orchid mycorrhizal 13

conditions. Strikingly, the C abundance observed in ger-

symbiosis; stable isotope abundances; rhizoctonias.

minating seedlings [5] does not ﬁt the high C abundance

1360-1385/

the real ecological niche of rhizoctonias?

Trends in Plant Science, November 2014, Vol. 19, No. 11 683

Spotlight Trends in Plant Science November 2014, Vol. 19, No. 11

(A) Green orchids Orchids associated with ectomycorrhizal fungi: Orchids associated associated with with parally heterotrophic fully heterotrophic saprotrophic fungi rhizoctonias (green, mixotrophic) (achlorophyllous) (achlorophyllous)

? δ 13C

(B) C3 photosynthates

Endophyc fungi, including rhizoctonias, Ectomycorrhizal Saprotrophic under the hypotheses of the present paper fungi fungi

Key: 1 ‰ in δ13C

TRENDS in Plant Science

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Figure 1. Relative spontaneous C abundance for the main nutritional types of orchids (A) and their respective mycorrhizal fungi (B), distributed along increasing d C

values. The vertical grey lines indicate trophic links, that is, exploitation of a C source: orchids may either rely on a single source or mix two sources. Fully heterotrophic

(mycoheterotrophic) orchids are largely enriched in C: they rely on C provided either by fungi that are ectomycorrhizal with surrounding tree roots (light brown) or by

saprotrophic fungi (brown) that are even more enriched. Mixotrophic orchids (in yellow green) display intermediate enrichments: they use both C from ectomycorrhizal

fungi and from their own photosynthesis. On the very left, green orchids (green) associated with rhizoctonias include the vast majority of species and are often considered

purely autotrophic in adulthood: they display C enrichments close to those of C3 autotrophs (blue-green range), but sometimes with a small excess or deficit. We propose

that the newly emerging ecological niche for rhizoctonias (blue) predicts similar C enrichments in their biomass: thus, a partial use of C from rhizoctonias (question mark)

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would explain the large range of C abundances in rhizoctonia-associated orchids. Absolute values of C enrichments are not given on the diagram, because they vary

according to environmental conditions, but an order of magnitude of the relative difference (expressed in %) is given in the key.

Rhizoctonias belong to three independent Basidiomy- Hidden C ﬂow from rhizoctonias to green adult orchids?

cota lineages, Tulasnellaceae [1] and Ceratobasidiaceae [6] It is difﬁcult to sample mycelia of endophytic fungi to

(two families of the order Cantharellales which contains assess their C abundance. However, fungi producing

the chanterelle mushrooms), and Sebacinales [7]. Endo- ﬂeshy fruitbodies, which allow analysis of isotopic abun-

phytism, that is, diffuse growth within living plant tissues, dances, recently turned out to have endophytic abilities,

without apparent infection symptoms or symbiotic organs such as for instance in the genus Hygrocybe [8]. Interest-

such as mycorrhizae, is common among fungi. Endophytic ingly, these fruitbodies reveal variable C abundances,

growth in the roots of non-orchid plants now turns out to be which are rather close (equivalent, slightly lower or slight-

a frequent and ancestral ability of Ceratobasidiaceae [6] ly higher) to those of autotrophic plants (Figure 1; see [8]

and Sebacinales [7]. Tulasnellaceae, the most common and references therein). At ﬁrst glance, this may seem

orchid mycorrhizal lineage, is often missing in molecular unexpected because the ectomycorrhizal fungi, which re-

studies of fungal communities, which usually rely on bar- ceive C from tree photosynthesis, are enriched in C

coding with the fungal ribosomal DNA (rDNA): because of (Figure 1), likely thanks to a fractionation at the root–

their unusual rDNA sequences, Tulasnellaceae cannot all fungus interface. However, another mycorrhizal fungal

be detected by general fungal-speciﬁc PCR primers. More group, the strictly biotrophic Glomeromycota forming

environmental studies focusing on the overlooked Tulas- arbuscular mycorrhizae in many land plants [1], displays

nellaceae are needed, for example, using the available variable C abundances that are close to or even below

Tulasnellaceae-speciﬁc rDNA primers (e.g., [4,5]), to sup- those of host plants [9]. Thus, not all plant symbiotic fungi

port that they grow endophytically in non-orchid plants undergo the C enrichment characterizing ectomycorrhi-

like the other rhizoctonias. Endophytism among rhizocto- zal symbioses, perhaps due to a different biochemical

nias does not preclude partial saprotrophy under natural pathway for C transfer at the plant-fungal interface. More-

conditions, for example, in soil, because these fungi can over, among rhizoctonias, C abundance may well vary

also grow on organic matter [6,7], but this awaits rigorous depending on their respective levels of partial saprotrophy,

investigation. an input that would result in C-enrichment (Figure 1).

It is assumed that in many fungal lineages, such as the Thus, C transfer from rhizoctonias to orchids may entail

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orchids’ rhizoctonias, mycorrhizal ability evolved among slight C depletion or C enrichment in receiving plants.

saprotrophic fungi that ﬁrst became root endophytes, Small enrichments are indeed reported in some green

because endophytism acted as a symbiotic ‘waiting room’ rhizoctonia-associated orchids compared to neighbouring

predisposing to the evolution of tighter mycorrhizal mu- C3 plants [3]; some green orchids in the tribes Orchideae

tualism [7]. Interestingly, recent publications suggest and Cranichideae even display an intriguing small, but

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that endophytism may predict low C abundances for consistent, C depletion [3,10], which are hitherto unex-

rhizoctonias. plained. We speculate that their associated rhizoctonias

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Spotlight Trends in Plant Science November 2014, Vol. 19, No. 11

may provide them with C-depleted C. In all, the gain of C Are the fungi providing C parasitized, or do they gain some

from rhizoctonias would explain the unusually ﬂexible C other rewards? And what about the ﬁtness of the C-donating

abundances reported for rhizoctonia-associated orchids endophyted plants?

(Figure 1). Such a hidden C ﬂow would be congruent with To further support our speculation of mixotrophy in

two observations (not to mention the high N and N rhizoctonia-associated orchids, new tools are required to

contents reported above): ﬁrst, rhizoctonias can provide track C transfers. First, we call for a better understanding

C to any orchid, as evidenced during germination of the ecology of rhizoctonias, especially the Tulasnella-

[1,5,10]. Second, an in vitro labelling experiment with ceae, and their C sources. Second, in situ labelling experi-

the green Goodyera repens (which, interestingly, belongs ments may test the gain of fungal C: following our

to Cranichideae), associated with Ceratobasidiaceae, hypothesis on the endophytic ability of most rhizoctonias,

showed reciprocal C exchange between the green plant labelling the photosynthates of surrounding plants in situ

and its rhizoctonia [2], although the net ﬂow was in favour may be one way of tracking C transfers toward green

of the fungus in these in vitro experimental conditions. orchids.

The fact that no fully heterotrophic orchid associates

with rhizoctonias has been puzzling; although full hetero- Acknowledgements

trophy evolved more than 30 times independently in orch- The authors warmly thank Mariangela Girlanda and Myriam Gaudeul

for critically reading the manuscript. F. Martos is funded by the Claude

ids [3], it is always associated with a change to

Leon Foundation of South Africa.

ectomycorrhizal or saprotrophic, non-rhizoctonia fungi

(Figure 1). Although rhizoctonias can support any germi-

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