Aerial nodules in Casuarina cunninghamiana

Vol. 57, No. 3 APPLIEDANDENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 871-874

0099-2240/91/030871-04$02.00/0

Aerial

Nodules in

Casuarina

cunninghamiana

Y.

PRIN,1*

E. DUHOUX,l H. G. DIEM,' Y. ROEDERER,2 AND Y. R. DOMMERGUES'

Biotechnologie des Symbioses Forestieres Tropicales (CTFT-Centre de Cooperation Internationale enRecherche AgronomiquepourleDeveloppement

[CIRAD]IORSTOM),

45bisAv. dela

BelleGabrielle, 94736 Nogent-sur-Marne

Cedex,1

and CTFT-CIRAD/Office National desForets, 7 Chemin de l'IRAT, Ligne Paradis, 97410St.-Pierre, France

Received 15 August1990/Accepted 10 December1990

A complete survey of La Reunion Island showed that, in 40- to 50-year-old Casuarina cunninghamiana

plantationslocatedin the northeastatanaltitude above 400m, sometreesboreaerial nodulesashighas6to

7mupthe trunk. The nodules exhibiteda significant specificacetylene reduction by the ARA method (0.77

,umol of

C2H4

per

h/g

[dry weight]

of

nodule)

atthe timeof

sampling

(June

1990).

Aerial noduleswere also foundon aCasuarinaglauca trunk. Preliminary observations show thatanatomically aerial and underground nodules donotdiffer significantly. In additiontohostplant genetic determinants, aerial nodule formation is

assumedtorequire sufficientrainfall,anabundance ofFrankiaspp.in thesoil and air, and rhytidomeonthe

treetrunk.

On leguminous plants, N2-fixing nodules are generally formed on the roots and function in the soil. Aerial (stem) nodules also exist and have been described on the stems of speciesbelonging to the genera Sesbania (7), Aeschynomene (1, 2, 14), and Cassia andParkinsonia (13). Walter and Bien (12) reported that another type of aerial nodule had been discovered on the adventitious roots ofPentaclethra mac-roloba, a tropical leguminous tree that grows in swamps in Costa Rica, but that these aerial nodules are located on the adventitious roots at some distance from the trunk and, consequently, do not adhere to it.

Untilnow, noaerial nodules on the trunks of actinorhizal trees had been reported, which explains why the recent discoveryby one ofus(Y.R.D.)of profuse aerial nodulation on the trunk of a number of Casuarina cunninghamiana trees in 40- to 50-year-old plantations in LaRdunion Island was unexpected.

Thisnotedescribesourfirst results of field and laboratory investigations on this new and unusual type of N2-fixing nodules and suggests different hypotheses to explain their development.

LaReunion Island islocated in the IndianOcean, to the

east ofMadagascar. In June 1990 a survey was conducted throughout the island with afocuson five stations: Station A, Hautsde Moka(altitude [alt.], 800 m); Station B, Hauts de Moka(alt., 750m); Station C, Hautsde Moka(alt., 700 m); StationD, Hauts de Moka(alt., 670m); and Station F, Hautsde la Ressource (alt., 800m).

Atfour stationstwotypes of noduleswerecollected from C. cunninghamiana: (i) aerialnodules,collectedat different heightsonthetrunk,and(ii) underground nodules, collected fromthe rootsofhomologoustrees.Aerial noduleswerealso collected from a C. glauca tree trunk at Station D. The sampleswere immediately incubated in situ under C2H2to assess their nitrogenase activity by using the acetylene reductionmethod(ARA) (9). Eachnodulesample, 1.0to 1.2

g, wasplaced ina serumvialcontaining 10% acetylenein air and incubated for 45 min at20 to 22°C. Gas

samples

were

collected in triplicate in air-rinsed 5-ml

Venoject

tubes (Polylabo, Strasbourg, France) and taken to the

laboratory

* Correspondingauthor.

foranalysis. Ethylene levels in each tubewereestimatedby using a Delsi C330 gas chromatograph (Delsi Instruments, Suresnes, France). After each assay, the nodules were air dried and thenweighed. Comparative ARAestimationswere

madefrom a nodule-free piece of C. cunninghamiana bark (StationA) and fromunderground nodules of Acacia decur-rens (Station A).

Scanning electron microscope examination of the aerial nodules was done on an ISI 60 microscope (International Scientific Instruments, Paris, France) after the nodules had beenfixed in 3% glutaraldehyde(Agar Aids, Evry, France) in 0.1 M sodium cacodylatebuffer(Merck, Nogent, France) and dehydrated by using a critical point drying apparatus (PolaronEquipment Limited, Watford, UnitedKingdom).

C. cunninghamiana that bore aerial nodules were only found inplantationsin the northeast of LaRdunionIsland,at analtitudeofover400 m,generallyless than 10 km from the

sea. In this area, the climate is characterized by relatively high air humidity (the only dry months are August and September) and by heavy though erratic rains (cyclonic regimen), with an annual rainfall of upto 3,000mm, which

causeabundantstemflow.Aerial nodules seemedto

develop

only on the trunks of less than 10% ofthe trees and were

generallylocated alonga large vertical

strip

onthat side of the trunk which conveyed most of the stemflow. Whether the percentage of nodulated trees varied with

sampling

stationorwithin thestation has notbeen

explored.

Ontrees

with aerialnodules,nodules werefoundat a

height

ofup to

6to7 mandcovered about 5to20%of the lowest2mof the trunk(wherethe noduleswere morenumerous).

At oneof the sites where C. cunninghamiana withaerial nodules was found

(Station

D), we found that a C.

glauca

treealso bore aerialnodules;thisfindingindicated that aerial noduleformationwasnot restrictedtoC. cunninghamiana. Aerial nodulesareuneven,roughlycircular

protuberances

of the tree's

rhytidome (the

deadoutertissueof the bark in

a mature tree [10]), 20to 30 cm in diameter and 4 to 8cm

thick. Theprotuberancesadhere

tightly

tothetrunk

(Fig.

1A andB) andare

composed

of dead

rhytidomal

tissues embed-ding adventitious roots intermixed with

typical

coralloid (Fig. 1C),

dichotomously

branched actinorhizal nodules whose lobes bear a nodular root at their apex.

During

the

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APPL.ENVIRON. MICROBIOL.

FIG. 1. (A) Trunk of C. cunninghamiana with profuseaerial nodulationat 130to 150cmabove theground. Bar, 10cm. (B) Detailed photograph of aerial nodules, whichappearasroughly circular protuberancesthat adhere tightlytothetrunk. Bar,3cm.(C)Aerialnodule detached from the trunk. Bar,1cm.Inpanel C,uppernodule,theouterface made ofrhytidomal tissues;inthe lower nodule is the innerface, showingthetypical yellowactinorhizal lobes(L)withadventitious roots(R)embeddedinrhytidomaltissues. Thepink spots (X)aresections oftissues, including xylem, thatconnectthe nodule with the trunk.

seasonofheavy rains (DecembertoFebruary), the lobes of the actinorhizal nodules are yellow and healthy looking,

whereas in the drier, cooler season (June) largeportions of the nodules are desiccated or even decayed and thus con-tributetothe accumulationof deadorganicmatterinside the

protuberance. Under the right environmental conditions,

new nodule tissues are expected toform within the

protu-berance; inotherwords,thereshouldbeanannualturnover of thenodule lobes inside theprotuberance.

SpecificARAvalues of aerial nodulesatdifferent heights andunderground nodulesare shown in Table 1. In the four

stations wheretheyweresampled, aerial nodules hada mean

specificARAvalue of0.77 ,umol of C2H4perh/g(dry weight) ofnodule, whereas the mean specific ARAvalue of under-ground noduleswas2.88 P,molofC2H4perh/g (dry weight) ofnodule. The specific ARA value of C. cunninghamiana nodule-freebark samples was 0, while that ofunderground

nodules ofayoungA.decurrenswas15.4.Thespecific ARA ofaerialnoduleswaslower thanthat ofunderground nodules forthefollowingreasons. (i)Specific ARA estimationswere

made inJune, when hygrometry and temperature werelow

and most of the nodule lobes were senescent. Nodule

activity was probably far from maximum since the aerial

locationmakes the nodules particularly sensitivetodrought

and low temperatures. Underground nodules enjoy a more

buffered environment. (ii) Specific ARA estimations were made on samples in which fresh nodule lobes were mixed withdead tissue that couldnotbe discarded before

incuba-tion. Since underground nodules are easier to clean, their

specific ARA is higher. As a typical highland species, A.

decurrensmayhavehigh specific ARA values because itis well adapted to the low ambient temperature prevailing at

thetimeofsampling (June).

The absence of ARA in the C. cunninghamiana

nodule-TABLE 1. SpecificARA valuesof aerial andunderground nodulesofC. cunninghamiana and C. glauca thatwere

sampled in June 1990atLaRdunionIslanda

Nodule type Station(s) Specific

Aerial nodules of C. cunninghamiana at aht aboveground of:

40cm A 1.2 115cm A 0.6 130cm B, C 0.7 160 cm A, F 0.5 280 cm A 0.9 450cm A 1.6

AerialnodulesofC.glaucac D 0.7

Underground nodules ofC. cunning- A, B,C,F 2.8 hamiana

Nodule-freebark ofC.cunninghamianac E 0 Underground nodulesof A. decurrens A 15.4

aComparativespecificARAestimationsmadefrom nodule-free bark ofC.

cunninghamianaand fromundergroundnodulesof A. decurrens.

bMicromolesofC2H4 perhour per gram(dryweight) ofnodule.

c130 cmabovetheground.

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NOTES 873

FIG. 2. (A)Scanningelectron micrograph of an aerial nodule section showing the central vascular system (VS) and numerous parenchymal Frankia-infectedcells. Bar, 200 ,um. (B) High magnification of an infected cell showing a characteristic cluster of endophyte hyphae. Bar, 10 ,um.

free bark sample indicates that N2 fixation occurred only in the aerial nodules, not in the bark itself.

Histological examinations of sections of aerial nodules revealed the presence of numerous infected cells in the parenchyma (Fig. 2A). The infecting organism had the aspectofafilamentousbacterium and was not differentiable from typicalFrankia-infected cells (4). Preliminary observa-tions do not show significant differences in the anatomy of aerial and underground nodules. Scanning electron micro-scope observations(Fig. 2B) clearly confirmed the

filamen-tous appearanceof the infecting bacteria and the absence of typicalvesicles. More investigations are obviously required

toelucidate the taxonomic position, physiology, and ecology ofthe Frankia strain(s) involvedinthis aerial nodule sym-biosis.

The following factors are probably ofparamount

impor-tance in inducing aerial nodulation. (i) Sufficient rainfall is distributed through a relatively long rainy season, and air humidity is high throughout the year; this climate is found onlyin the northeast portion ofthe island, which explains why C. cunninghamiana there has aerial nodules. (ii) Frankia cells are present several meters up the tree trunk, which implies thatFrankia cells are present notonly in the soilbut alsointheair. In field surveysthroughouttheisland, Frankia cells were found in all ofthe soils, including soils formed in 1977 from the lava emitted from the Pitonde la Fournaise. Frankia cells probablyarewind borne and

trans-ported to the trees as sporesin dust or are transported up

tree trunks by insects orothervectors. (iii) Arhytidome is present. Nodules wereobservedonlyonthe trunksof older trees,which hadwell-developed

rhytidome,

which isaniche withspecific chemical, physical, andbiological characteris-tics that stimulate the formation of adventitious roots, facil-itate their infection by Frankia cells, and thus favor the subsequent development of the nodules. The

rhytidome

functions in the same manner as a sponge retaining water

andis amicrosite where chemical stimuli, possibly synthe-sizedbyfungiorotherorganisms that thrive in the bark, are producedandinducetheemission of adventitious roots. (iv) The host plantis able to produce aerial nodules. The fact that, regardless of site, only a small proportion of the trees bore aerial nodulesstrongly suggests that host genetic deter-minantsaffecttheestablishment of the symbiosis.

Theinfection sites for stem-nodulated legumes (7) and for C. cunninghamiana (and C. glauca) are similar, viz., the lateral adventitious roots. But there are four major differ-ences between stem nodules of legumes and actinorhizal nodules. (i) The adventitious roots of stem-nodulated

leg-umes are preformed as latent meristematic primordia; the adventitious rootsofCasuarina spp. arenotpreformed. (ii)

In Casuarina spp., Frankia cells, rather than Rhizobium cells, are the infection agents. (iii) For Casuarina spp. to

develop aerial nodules requires the existence ofthe niche created by the rhytidome, the role of which is speculated uponabove.(iv) Withintheniche formedbytherhytidome, the Casuarina nodule lobes grow for some time on the adventitious roots, dryordecay in the course ofthe (rela-tively) dry and cold season, and resume growth when environmental conditions are favorableagain; this suggests successiveturnover of nodule lobes.

Likestemnodulationinlegumes (3, 5, 6),aerial nodulation inC.cunninghamiana (and inC.

glauca) probably

confersto

thehostplant(i) highN2-fixing potentialand(ii) highlevels of actualN2fixation in the field because of its

independence

from soil constraints, especially acidity and an excess of combined N. Since their

N2-fixing ability

would be reduced only slightly by soil combinedN, trees with aerial nodules

are assumedtofix N2for mostof their life and to be very efficient soilimprovers,whereastreeswith

only

rootnodules (underground nodules) usually fix N2

only

during

the first yearsafterbeing

planted

in the

field,

untilthesoilNexceeds

acertain threshold.

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APPL. ENVIRON. MICROBIOL.

Aerial nodulation of members of the familyCasuarinaceae

should be produced artificially in the near future since preliminary laboratory experiments have shown that adven-titious roots can be induced easily on the stems of C. cutininghamiana and Casuarina equisetifolia. Once the Frankia strains from La Reunion aerial nodules have been isolated, further research should lead to the development of a device to simulate the rhytidomial niche on the stem of young treesand to select (11) and vegetativelypropagate(8) Casuarina clones with the capacity to bear aerial nodules.

This work was supported by ORSTOM (Institut

Frangais

de Recherche Scientifique pour leDdveloppement en Cooperation) and by the Centre deCooperationInternationale en Recherche Agrono-mique pour leDdveloppement.

Wethank M. Bord6re and other members of the Office National des Forets de LaR6union for field support and B. de Palmas, who sograciously received us in his plantation.

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8. Duhoux, E.,B. Sougoufara,and Y. Dommergues. 1986. Propa-gation ofCasuarina equisetifolia through axillary buds of im-maturefemaleinflorescences cultured in vitro. PlantCell Rep. 3:161-164.

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