An Investigation of Thismia rodwayi F. Muell. and its Associated Fungus

Transactions of the Royal Society of New Zealand : Botany, Volume 3, Issue 14, 23 August 1968, Page 209

An Investigation of Thismia rodwayi F. Muell. and its Associated Fungus

Ella O. Campbell

[Received by the Editor, April 13, 1967i]

Abstract

Thismia rodwayi in New Zealand grows in association with a fungus which is also to be found on the roots of tawa and of conifers. The morphology and embryology of Thismia rodwayi are considered. The root structure of tawa is described.

Introduction

In the early New Zealand literature any references to Thismia rodwayi will be found under the name of Bagnisia hillii Cheeseman. Both Pfeffer (1914) and McLennan (1958) consider Bagnisia hillii to be identical with the Australian Thismia rodwayi. The North American T. americana Pfeffer is allied but differs in the shape and colour of the flower and in the root system. The main concentration of Thismia species is from Ceylon through the Malaysian-Indonesian region to New Guinea.

Until comparatively recently Thismia was known in New Zealand only from Opepe Forest, near Taupo, growing in gloomy glens on mounds of humus at the base of Podocarpus dacrydioides and other trees (Cheeseman, 1925). But colonies have now been found in tawa-podocarp forest on Mount Pirongia, in kauri forest at Trounson Park, and under Podocarpus spicatus at Taurewa, in Tongariro National Park (Steele, 1966). The plant is readily recognised by the small flowers which appear from late October until February, although these are not easily found, since many fail to reach the surface of the forest floor. Only when exposed do they develop to the full their very deep salmon-pink colour.

Collection and Treatment of Material

Material was collected from November until January in two localities. One was on Mount Pirongia in an area of mixed forest where several podocarp and other trees had been felled leaving many chips of wood and broken twigs amongst the leaf litter accumulated on the forest floor. The only standing trees were of Beilschmiedia tawa (A. Cunn.) Benth. et Hook. f. ex Kirk. There was no undergrowth. The other locality was in Trounson Park where Beilschmiedia tawa and B. tarairi (A. Cunn.) Benth. et Hook. f. ex Kirk were present along with tall trees of kauri, Agathis australis Salisb., and a variety of shrubs and ferns, and where large flakes of kauri bark added to the litter derived from fallen leaves on the forest

floor. Throughout the litter layer in both localities grew the interwoven roots of tawa, and in Trounson Park there were the nodulated roots of kauri and the roots of other plants besides. Also amongst the litter there occurred the roots of Thismia and coarse, white fungal rhizomorphs which fanned out into a soft, white felt on pieces of bark or twig. Some material was killed and fixed in formalin-acetic-alcohol in the field and later embedded in paraffin, then sectioned and stained. Turves cut out of the litter were brought back to the laboratory and either carefully dissected out immediately or kept for a year in large, unglazed porcelain pots with plastic covers. In the following October flowers were produced, but fruits did not form and the new portions of root were thin; the fungus was still growing amongst the pieces of kauri bark in the turves. Methods used in culturing the fungus are described later in the paper.

Morphology of the Shoot and Root

Since the morphology of the shoot and root of Thismia rodwayi has been described by several writers (von Mueller, 1890; Cheeseman, 1908; McLennan, 1958), only a few points are noted here. The plant is of an unusual type. The fleshy roots, whitish when young and pale brownish when older, grow more or less horizontally underground. Small roughened patches on their surface indicate sites of old fungal infections. At intervals buds arise endogenously and normally grow into short, white, erect shoots carrying a few colourless scale-leaves and a terminal flower but under unfavourable conditions the buds may remain minute. Simultaneously with the development of the shoot a new root arises at its base, as described and illustrated for T. americana (Pfeffer, 1914), and grows so rapidly that the shoot later appears to lie in an axillary position to this new root (Fig. 1), There are, however, no additional adventitious roots as found at the base of the shoot of T. americana. The horizontally growing, underground structures are certainly roots, for each has a root cap and a radially constructed, conducting region. The flowering stem has a cylinder of collateral vascular bundles encircling a pith. In both cases an endodermis with Casparian bands is present as well as a cortex and a hyaline epidermis. The xylem with its vessels lignified in either an annular, a helical or a reticulate pattern is regarded as protoxylem following the nomenclature of the International Association of Wood Anatomists (Committee on Nomenclature, 1957). The phloem likewise is only protophloem and so is difficult to distinguish from adjacent cells. It has been stated that sieve plates are lacking in both T. rodwayi (McLennan, 1958) and T. americana (Pfeffer, 1914). However, careful examination leaves no doubt as to the identity of the phloem. Its conducting elements are elongated, extremely narrow, and lack the nucleus and starch grains which are present in adjacent cells. When sections of 4/x thickness are stained with either aniline blue or Delafield’s haematoxylin and viewed under oil immersion, sieve areas on the lateral walls of the sieve elements and sieve plates on the end walls are faintly visible. A further distinctive feature of the sieve elements is the presence of callose which was determined by fluorescence. In longitudinal sections of thickness 4ft, stained for one hour in dilute, aqueous, aniline blue and mounted in levulose syrup, fluorescence was definite and confined to the walls of the sieve elements. The cortex of both stems and roots consists of parenchyma, initially storing starch which gives a reddish colour with iodine, except in isolated mucilage-cells containing calcium oxalate as a bundle of raphides, and in its outermost layer (exocortex) which may lack starch. The epidermis lacks starch and in the root is papillate.

Morphology of the Flower and Fruit

The gross morphology of the flower has been described previously (Cheeseman, 1908; 1925) and corresponds for the most part with the fuller description given of

T. americana (Pfeffer, 1914). The inferior ovary is unilocular with very numerous anatropous ovules arranged on the three placentas which at first are parietal but later become free from the lateral walls. The morphology of the ovule shows an unusual feature in that with the close crowding on the placenta many ovules have the very long funicle which is found also in other Thismia species (Bernard and Ernst, 1911). In transverse sections of flower buds the megaspore mother cell becomes obvious by its size and hypodermal position at the stage when the inner integument of the ovule is apparent (Fig. 5) ; later the megaspore mother cell divides to form a linear tetrad of which only the cell farthest from the micropyle is functional as a megaspore. By the time the flower is open this has enlarged into an embryo sac with the usual seven nuclei but as in other Thismia species the antipodal cells are inconspicuous (Bernard and Ernst, 1911). The nucellus corresponds with that of other Thismia species (Bernard and Ernst, 1911) in that it is well developed at the chalazal end of the ovule but elsewhere consists of one layer of cells (Fig. 6). There are the usual two integuments.

Since the fruit is fleshy and indehiscent, it must be classed as a berry (Fig. 1). The white lateral wall of the fruit is 11-13 cells wide. As the fruit matures, the top portion tends to become thin and translucent, then slowly to disintegrate so exposing the brown seeds, but there is no particular place of opening. Soon the whole fruit, followed shortly afterwards by the flowering stem, rots away. In some other species of Thismia the fruit is reported to be a capsule dehiscent by an operculum (Ridley, 1895; Groom, 1895). At least in T. clandestina the capsule is fleshy (Smith, 1911).

The seed has a two-layered testa of thin-walled cells and a thick-walled cellular endosperm surrounding the embryo. As in other members of the Burmanniaceae the embryo is not fully differentiated by the time the seed is ripe. In no case did more than one embryo occur in a seed but in each fruit it was found that a considerable number of the ovules had failed to develop an embryo even although a walled tissue had formed from the earlier nuclear endosperm.

Embryology

Development of the embryo was found to correspond with the early development described for Luzula forsteri (Soueges, 1923) except in the matter of the precocious formation of periclinal walls in the latter, and so belongs to the Onagrad type (Johansen, 1945). The first division of the fertilised egg is transverse resulting in a basal cell ch and a terminal cell ca (Fig. 7). The next division in the basal cell is transverse giving two superposed cells ci and m, and in the terminal cell is vertical producing two juxtaposed cells which again divide vertically, but at right angles to the plane of the previous division so giving the quadrants q (Fig. 8). A transverse division of each of the quadrants results in an octant stage which consists of two tiers of cells, I and I 1 (Figs. 9, 11) . Meanwhile ci has divided transversely into two superposed cells n 1 and n. Cell n 1 usually divides again transversely giving the two cells, o and p, of the very short suspensor (Figs. 10, 12). In the cells constituting the octants a set of periclinal walls appears followed by the commencement of a second set, whereas cell m divides by two intersecting, median, vertical walls producing four circumaxial cells, and then by further vertical walls which separate peripheral cells from inner cells (Figs. 12, 13). The fate of these regions could not be determined as the most developed stage that was found was as illustrated in Fig. 14, a stage similar to that figured for T. clandestina (Bernard and Ernst, 1911) and more advanced than that figured for T. americana (Pfeffer, 1914) and for other members of the Burmanniaceae.

The Roots of Beilschmiedia tavoa

The root system of tawa, based on the examination of plants growing on Mount Pirongia and in the Tiritea Valley near Palmerston North, consists of long,

pioneer roots of indefinite extension in length and indefinite expansion in width together with numerous, shorter, brown, feeding roots of a remarkably uniform diameter of 0.5-I.Omm. Although there is no sharp line of demarcation between the two types, the general pattern is one of long and short roots. The short roots tend to interweave into a close, compact felt in the upper layers of the soil, and in clay soils may occupy a depth of no more than Bcm; they are either unbranched or branched racemosely at a wide angle to produce a pyramidal outline; many die off and are replaced functionally by new ones arising from the long roots. When dug up from dry soil the short roots are light brown in colour due to the reflection of light from the intercellular air spaces in the cortex, but when placed in water they are dark brown. As growth recommences in autumn and in spring the new part of the root of lighter colour is evident as it extends beyond the old sheath.

In sections the short roots in some respects show a peculiar structure. The small stele built of primary tissues is surrounded by an endodermis in which the cells develop a suberin lamella on all their walls except for groups of one to seven passage cells situated externally to the protoxylem poles. The passage cells have a Casparian band stainable with phloroglucin followed by strong hydrochloric acid but usually showing up as only a very narrow band, if at all, with Sudan 111. The parenchymatous part of the cortex, 9 to 12 cells wide, in the absence of fungal infection has scattered volatile oil cells and tannin cells amongst ones containing starch; it shows small intercellular air spaces throughout. In old roots U-shaped sclereids may also be present. The cortex is bounded outwards by the exodermis of radially elongated cells, most of which have suberised walls becoming pitted with age, but isolated passage cells have a protoplast with a large nucleus and when young contain volatile oil. On the surface of the root there are no root hairs but an outer zone, three to seven cells in width overlying the epidermis, where the cells are all alike and have pitted, brown walls (Fig. 16) and contents of tannin. Longitudinal sections of the root apex show that these represent persisting cells of the root cap Which, instead of sloughing off, elongate in the extension region and develop firm walls. Only very gradually are the outermost cells disjoined. The epidermis itself develops thickened, pitted walls as the root ages and contains tannin.

When the short roots of the tawa from the Tiritea Valley were examined by means of transverse and longitudinal sections, it was found that a non-septate, mycorrhizal fungus was present. The fungus is little evident on the outside of the root but as hyphae of diameter 1.7 to 3.4/x it grows abundantly between the cells of the outer zone. It penetrates through the epidermis and the passage cells of the exodermis into the cortex where it is largely intercellular but in addition appears within the cells and there shows much-forked branching and gives rise to arbuscules. The short roots in shape resemble outsize mycorrhizal roots of Fagus and other forest trees but in these the fungus, usually one of the Basidiomycetes, is septate and forms a superficial mantle.

The long roots differ from the short roots in that secondary growth from the vascular cambium adds to the vascular tissue and a phellogen arises in the pericycle. As a result the long roots increase in girth as well as in length.

The Roots of Agathis australis

The root system of kauri consists of long roots, on the finer branches of which are numerous root-nodules which harbour mycorrhizal fungi (Yeates, 1925). The nodule has a cortex which for some time is renewed annually from within by the meristematic activity of the pericycle (Baylis, McNabb and Morrison, 1963). The old, dead cortical tissue remains as an outer sheath around the base of the nodule.

The Fungus Associated with Thismia

The fungus occurs in the litter as white, rhizomorphic strands up to o.lmm in width which build up into a white felt on decaying stems and roots and attach themselves at a few points to the roots of Thismia, tawa, kauri, and of other plants as well. In the kauri forest the strands tend to follow along the surfaces of the flakes of kauri bark which accumulate on the forest floor and to run upwards through the

bark persisting on any dead kauri trees which are still standing. From the rhizomorphs in the litter there grow out single hyphae up to 7jx in diameter which form a fine, cobweb-like growth over the Thismia roots and lie close against them in any depressions of the surface. These hyphae are septate and without clamp connections. Some of them have thick walls. The strand consists of thin-walled hyphae, the outer loosely interwoven, the inner more compactly arranged (Figs. 3 and 4), and when viewed from the outside under the microscope presents a shaggy appearance due to the numerous projecting hyphae. Some of the strands were traced to a sporophore (Fig. 2) growing out of the fallen, much-decayed trunk of a kauri tree. Further details regarding this fungus are given below.

Head of diameter 3cm, lavender-brown, fleshy, sunken at the centre and rolled downwards at the edge. Hyphae of the head more or less parallel in a radial direction, the lower ones 1.7-1 1.9 min diameter and without clamp connections, the upper ones 1.7-3.4 m in diameter with some clamp connections and appressed. Hyphae of the surface layer appressed and pigmented, a few with inflated, pigmented, erect or prostrate endings. Stalk fleshy, hollow, 6.5 cm high, o.scm in diameter at the base but narrower towards the top, white on the outside. Veil lacking. Gills sinuate, white, sometimes forked, thin, 0.3-o.4mm broad at the base but narrower towards the tip. Hyphae of the trama 1.7-13.5 min diameter, intermixed to subregular, slightly inflated, constricted at the septa, not showing clamp connections except for a few near the subhymenium. Hyphae of the subhymenium of diameter 1.7—2.5 m, loosely arranged and with frequent clamp connections. Basidia club-shaped, very long and thin, 30.5-39.0 X 1.7—5.0 m, with clamps at the base. Sterigmata 4. Spores oval or somewhat ovate, 4.2-5.1 X 2.5-3 .4 m, hyaline, smooth. Rhizomorphs few, white, long, up to I.omm in diameter with two kinds of hyphae, the outermost sometimes clamped, rather thin (1.7—3.6 min diameter), loosely interwoven, slightly swollen, constricted at the septa, the inner ones more appressed, not clamped, 1.7—7.0 m (mostly 3.5—5.0 m) in diameter, arranged in a more regular bundle.

Reaction with KOH—nil.

Reaction with Melzer reagent—nil.

Reaction with cresyl blue—metachromatic reaction in hyphal walls of the pileus and the hymenophoral trama and in the endosporium of the spore.

Carminophilous granulation absent.

Infection of the Thismia

Fungal hyphae are abundantly present in the cortex of Thismia roots except close to the growing apex. They are not found in the stems. The behaviour of the fungus in the roots of Thismia in Australia, with infection occurring from single hyphae, has been described in detail (McLennan, 1958). In New Zealand the fungus gains entry either by single hyphae or by rhizomorphs.

Entry by single hyphae takes place from branches of the thick-walled hyphae lying in depressions on the Thismia roots or from branch hyphae coming off adjacent rhizomorphic strands and follows a course similar to that found in Thismia in Australia. At a few points hyphae which eventually are thick-walled penetrate through the epidermis into the subepidermal layer (exocortex) where they spread tangentially, branching and forming coils which almost completely occupy the cell cavities as they proceed. The host cells enlarge in a radial direction up to three times their former diameter but there is no development of collar-like thickenings where the fungus penetrates the walls and for some time the nucleus and cytoplasm persist. The hyphae in the exocortex are unseptated; some are thin-walled and of diameter 3.5/*,, while others enlarge to a diameter of up to 7/x and have densely staining contents. Later the wide hyphae develop thick walls and remain as transporting hyphae while the fine hyphae disappear. Branches from the tangentially spreading hyphae extend radially into the cell layer beneath and form coils there. They may spread laterally but the cells themselves do not enlarge and the hyphae remain thin-walled and of diameter 2.0-3.5 jx. Later they appear shrunken and empty. Hyphae extend more deeply into the root into what may be called the digestive zone Which may reach as far as, but does not include, the endodermis.

Already for the Australian plants the process of digestion has been described in full and it has been shown that food material is transferred from the hyphae to the Thismia plant (McLennan, 1958).

But infection of the Thismia may also take place by rhizomorphs (Fig. 15). In this event the Thismia roots are lying very close to short roots of tawa or to the roots of other plants, the two being connected by rhizomorphs up to 42//, in diameter. In these rhizomorphs the hyphae are compactly arranged and there may be an outer zone with brown, firm walls. The rhizomorph is either attached to the surface of the Thismia root as a disk up to 0.35 mm in diameter or may occupy an epidermal cell and even branch within it giving off slender strands up to 10//, in diameter which penetrate in a tangential direction through some nine cells of the exocortex. In this way it obtains a secure hold. On the outside of the root there radiate from the edge of the infection disk narrow, thin-walled hyphae as well as wider, thick-walled hyphae of diameter 7//, and slender rhizomorphic strands of diameter 14//,. All of these follow the root surface closely and either the strands or the thick-walled hyphae may give rise to secondary infections at a short distance from the original one. Other epidermal cells are not directly affected. Inside the root, hyphae originating from the attachment disk penetrate deeply and behave as in the single-hyphal infection except that they are more vigorous. Wide, densely staining hyphae up to IQ//, in diameter, or occasionally as much as 14//, together with narrower hyphae, 3.5//, in diameter, occupy the cells of the exocortex. Sometimes the wide hyphae run lengthwise from cell to cell in the root and do not form coils. They later become thick-walled and may show occasional transverse septa. In the layer below the exocortex there may be some wide hyphae amongst the narrow ones. In still deeper cells a digestive process takes place.

In some parts of the root the tips of wide hyphae in the exocortex, or more usually in somewhat deeper cells, enlarge to form spherical or deeply lobed structures, as found also in the Australian plants where they have been termed vesicles (McLennan, 1958). Each almost fills a cell and later becomes surrounded by a thick wall. Most commonly they occur near the base of the flowering shoots; they seem to be caused by some check to the forward growth of the fungus, for they contain dense cytoplasm and numerous nuclei as do the adjacent hyphae during their formation.

Infection of the Tawa

Rhizomorphs attach themselves on to the outside of the short roots of tawa and put out branches which either grow externally for a short distance and then produce secondary attachments or force their way between the outer layers of cells and gain a firm hold internally in the root (Fig. 16). Single hyphae growing out from the rhizomorphs penetrate between the cells of the outer zone in both a tangential and a radial direction, then pass through exodermal cells to the cortical parenchyma beneath. Some of them become thick-walled. In the cortex the hyphae are 3.5/x in diameter, and appear to grow slowly, since for a time many cells are by-passed. Once a hypha penetrates a cell it grows towards the nucleus; it may branch or even form a coil in the cytoplasm but does not give rise to a densely coiling mesh-work nor does it form arbuscules as does the mycorrhizal fungus. The nucleus of the infected cell enlarges to twice its former size and remains spherical, then loses its affinity for stains and disappears. The starch grains for a time lie clumped together, then their identity is lost and they gradually disappear along with the cytoplasm. The hyphae develop thick walls and persist, while their growing tips move on into adjacent cells. For the most part the infection is confined to the cortex but in roots which had died the fungus was also present in the vascular tissues.

Infection of the Kauri

In the nodules on the kauri roots single, thick-walled hyphae of diameter up to 7/x, as well as rhizomorphs up to 20/*, in diameter, may be present in the old cortical layers. The rhizomorphs are especially significant, since hyphae alone would be difficult to distinguish from those of the mycorrhizal fungus associated with the kauri roots. In most cases infection is confined to the old cortex, but in those nodules which are dead the hyphae are present in the vascular tissues also. It was not established whether the hyphae entered the vascular tissue before or after the death of the nodules.

Experimental Culture of the Fungus

In an attempt to culture the fungus several turves of soil 0.3 cubic metres in volume were dug up from the forest in November and in January and transported in polythene bags to the laboratory where they were carefully dissected out. Promising pieces of root and of rhizomorph were washed by shaking for four hours in sterile distilled water in McCartney tubes, or in larger glass jars, on an end-over-end shaker (40r.p.m.) the water being changed at 30-minute intervals. In a few cases the washing was carried out for a period of 24 hours. In still other cases the roots or rhizomorphs, after being washed for either stated time, were surfacesterilised in chlorogene at a concentration of one tablespoonful to one pint of distilled water for periods of three seconds, five minutes or 10 minutes, then washed again by shaking for 30 minutes in four changes of sterile distilled water.

Following these treatments the material was cut into pieces 2cm long in a sterile chamber. One larger root after four hours of washing was stripped of its surface layers at the cambium and the remaining central part was cut into lengths. In all cases single pieces were placed on malt agar in petri dishes, a drop of sterile 5 percent lactic acid was added in order to depress the growth of bacteria, and the cultures were kept in an incubator at 22°G.

Although some plates out of the 600 inoculated produced no growth and others produced rapidly growing saprophytic fungi, 4 percent after six days showed rhizomorphs growing out from the inoculum. These appeared from short lengths of tawa root, from pieces of rhizomorph near the sporophore or near the Thismia plants and from the central portion of the larger root. None grew from pieces of Thismia root or from pieces of kauri root.

Best results were obtained when the material was placed on top of the agar rather than pressed into it. Washing for four hours was found to be just as satisfactory as washing for a longer period. Chlorogene was too drastic in its effect on hyphae and yet did not kill spores; it proved satisfactory only when used for five minutes on the larger root, the surface of which was later removed. It was concluded that the condition of the fungus was of the greatest importance and that only when it was in an actively growing state did rhizomorphs result.

Hyphae from the rhizomorphs produced in culture were transferred to fresh media in McCartney tubes. When malt agar was used, no significant growth resulted. A satisfactory medium consisted of:

Dextrose 20.0 gm Ferric chloride 2.omg per litre Potassium phosphate l.Ogm Thiamine 0.25 mg per litre Magnesium sulphate o.sgm Agar 20.0 gm Peptone 6.ogm Water t 1,000.0 ml

Malt peptone agar, with or without dextrose, and other media were also tried. Transfers were later made to 250 ml flasks containing Modess’ medium (Modess, 1941); or the technique of Tamblyn and Da Costa (1958) was followed using sawdust from tawa, rimu and Finns radiata in the proportions of 3:3:4 and blocks of Finns radiata newly felled.

Although the fungus has been kept growing, so far no sporophores have been obtained in culture. Rhizomorphs only projected from the pine blocks used according to the method described by Tamblyn and Da Costa.

Discussion

Thismia rodwayi in its morphology corresponds essentially with what has been recorded for other species of Thismia and shows no features which are unusual for this genus. Like the others it, too, lives in association with a fungus.

In New Zealand this fungus is largely an inhabitant of the dead parenchyma cells amongst layers of corky bark. Kauri and those podocarps with a habit of shedding corky bark from the trunk and renewing the cortex in the root nodules provide it with a favourable substrate on which to become established. Tawa roots, too, provide a suitable substrate. But at both Pirongia and Trounson Park the fungus also penetrates into the cortex of the short roots of tawa, where it appears to behave as a weak parasite, for many roots near the fungus are dead. That the roots of tawa are dead is not immediately obvious for the firm, outer zone and the exodermis persist. Only close examination shows 'that the whole central region has disintegrated and by this time other fungi and a variety of other organisms have arrived on the scene. Many kauri nodules near the fungus at Trounson Park are also dead and here, too, the outer layers tend to persist after the death of the root so that the form of the nodule is retained. At Taurewa, where kauri and tawa are absent, other plants must be involved.

Certainly the short roots of tawa have a limited life and this applies to the kauri nodules also. Undoubtedly many factors are involved in their death. But the proportion of dead roots near the fungus is much greater than elsewhere. That the fungus can penetrate living tissue is proved by its presence in the roots of Thismia but here its morphology is altered for it is unseptated, and as well it is held in check and eventually deprived of its substance.

The Thismia plants have been found only where there is much accumulation of litter on the forest floor. It is considered that the large amount of debris left after the felling of timber or accumulated by the shedding of bark has given the fungus an opportunity to build up sufficiently to support the Thismia plants for always where undisturbed these latter occur in colonies. The fungus then is considered to be mainly a saprophyte but at times it is a weak parasite on the roots of trees. The Thismia is considered to be parasitic on the fungus.

Acknowledgments

The writer wishes to thank Mr Reg. Bell, who discovered Thismia on Mount Pirongia, for assistance in finding the Thismia plants used in this investigation, also Mr L. A. McLean for permission to collect plants in Trounson Park, Mr F. H. Wood and Miss D. J. Scott for taking the photographs and the University Grants Committee for a research grant.

References

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Ella O. Campbell, Massey University, Palmerston North, New Zealand.