Phialophora zeicola sp.nov., and its role in the root rot-stalk rot complex of maize

[ 247 ] Trans. Br. mycol. Soc. 81 (2) 247-262 (1983) Printed in Great Britain

PHIALOPHORA ZEICOLA SP.NOV., AND ITS ROLE IN THE ROOT ROT-STALK ROT COMPLEX OF MAIZE By J. W. DEACON Microbiology Department, School of Agriculture, West Mains Road, Edinburgh, EH9 3JG, Scotland AND D. B. SCOTT Plant Protection Research Institute, Private Bag X 134, Pretoria,

0001,

South Africa

Phialophora zeicola sp.nov, is described from collections on maize roots in South Africa and France. It is different from a lobed hyphopodiate Phialophora sp. from maize in France, and the relationship of both to Phialophora radicicola is discussed. P . zeicola seems to be common and widespread as a root parasite of maize. It is suggested to have a significant role in the root rot-stalk rot complex, firstly by damaging some of the root laterals and thereby predisposing the plants to environmental stress and secondly by rotting the root tissues as they begin to senesce.

basal stalk tissues senesee as a result of carbohydrate deficiency if the plant is unable easily to meet the carbohydrate demands of the developing ears, and environmental stresses operate directly or indirectly on this situation (Dodd, 1980). In South Africa several different fungi are associated with stalk rot, but the fungus here described as Phialophora zeicola is frequently a cause of root rot. We found it in 14 out of 22 randomly selected maize crops in a limited survey in 1981, at a time (Jan.-Feb.) when none of the crops was showing evidence of root rot-stalk rot. These crops were distributed between Pietermaritzburg in Natal and the Springbok Flats, north of Pretoria in the Transvaal, i.e. over a distance of several hundred km; they included several in the main maize-growing region of the country, in south-eastern Transvaal near Bethal and Volksrust. THE DISEASE In 1980 P. zeicola was the main fungal pathogen Symptoms associated with infection of maize by causing root rot of plants that subsequently died Phialophora zeicola sp.nov. in South Africa from stalk rot on a 600 ha farm at Bronkhorstpruit, Root rot and stalk rot are important diseases of north-east of Pretoria (Fig. 1). In this case the maize in South Africa, where the conditions disease was associated with drought stress in the associated with them are similar to those reported critical period of grain filling. The same farm was elsewhere (e.g. Dodd, 1980). In particular, they are studied intensively in 1981 but there was no sign often associated with drought stress during the of root rot or stalk rot at a time when the plants had period of grain filling, and they are most severe on been dead in the previous year. P. zeicola was individual plants or cuitivars with the highest present abundantly on the roots of the crop and on potential yields (D . B. Scott, unpubl.). Root rot is the stubble remains of the previous crop, but in generally considered to precede stalk rot (Whitney 1981 the period of drought stress occurred before & Mortimore, 1957) but there is still much anthesis, when it has least effect on plant vigour . uncertainty as to the importance of any single Observational evidence of this type, coupled with microorganism in this disease complex, because the the results of inoculations to be described later, disease is considered to be one of senescence (Dodd, leads to the conclusion that P. zeicola is a weak 1980). The suggestion is made that the roots and pathogen that causes severe disease only when the

In addition to a range of previously reported fungi, a Phialophora sp. is frequently associated with root rot of maize (Zea mays L.) in South Africa. The same fungus has been isolated from maize crops in France and is probably common and widespread as a parasite of maize. It is described here as Phialophora zeicola sp.nov. The symptoms of infection in field and laboratory conditions are recorded, and the taxonomic relationships between P. zeicola and similar fungi are discussed with special reference to the confusion surrounding the name Phialophora radicicola Cain. A second type of Phialophora (with deeply lobed hyphopodia) which occurs on maize in France and Britain is compared with P. zeicola in this paper but not formally named .

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J. W. Deacon and D. B. Scott

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plant is predisposed to damage . Nevertheless the are roughly 20 pm diam and thin-walled as fungus may help to bring about this predisposition evidenced by their tendency to collapse in slide preparations (F ig. 10). Hyaline or darkly pigmented by its early parasitic activities . In severely damaged plants the roots are infection hyphae can be seen crossing the root blackened and rotted a few centimetres below their cortex from the surface runner hyphae in transverse points of emergence (Figs 2, 3) and the plants are sections. In some cases also the dark hyphae are easily pulled from the ground. The adventitious seen in xylem vessels or in the surrounding stelar roots show pronounced dark streaks just above or tissues (Fig. 8) but we do not know if this occurs just below ground level (Fig. 4) and these are a before or after the root dies. It is notable that the conspicuous feature on the stubble remains . They large dark vesicle-like fungal cells are not seen on are composed of fungal pseudoparenchyma which roots that bear the conspicuous black streaks of completely fills the host cortical cells (F ig. 5). In pseudoparenchyma (Figs 4, 5); rather they tend to transverse sections of roots this fungal tissue is seen be associated with browning of the root cortex. to be largely confined to the epidermis or These two cell types thus seem to represent sub-epidermal layer (Fig. 6) but it can sometimes be different phases of the disease. P. z eicola can cause limited necrotic flecking of seen also at the endodermis. At an earlier stage of infection, and in less severely damaged plants, the the maize stem base, especially at the points where root axes show little evidence of lateral branching. adventitious roots emerge. But it does not form a On close inspection, however, the root laterals are conspicuous network of dark hyphae on the stem seen to have been rotted; they are present as stumps base. It seems to grow poorly within the stalk tissues, a few millimetres long and show pronounced so any stalk rot phase of the disease is caused by cortical and vascular discolouration (F ig. 7) which other, secondary, invaders. Nevertheless, we have ends quite abruptly where the vascular system of occasionally seen P . zeicola within the stalk tissues the lateral joins that of the root axis. Some of the of mature plants, a few centimetres above soil xylem vessels in the root axes are also partly or level. It then forms dark hyphae within the pith and completely occluded by yellow-brown gum-like it forms pseudoparenchyma in or around the material (Figs 6, 8). The lack of well-developed vascular bundles (Figs 12, 13). lateral branching in infected roots is considered to be important because it is likely to reduce the Artificial inoculations of wheat and maize seedlings efficiency of water and mineral nutrient uptake. Thus the fungus might predispose the host to P. zeicola has been isolated from several samples of drought stress (or otherwise reduce the rate of diseased material plated onto potato-dextrose agar photosynthesis)and thereby hasten the development containing novobiocin, and the cultures have been of the main phase of the root rot-stalk rot complex used in artificial inoculations to confirm and extend by inducing early senescence of the basal regions . observations on field material. For routine work the Roots cleared in warm KOH (Phillips & inoculations were done in sand,perliteor vermiculite Hayman, 1970) are seen to bear a sparse network by placing pre-soaked germinating grains over agar of anastomosing dark runner hyphae, accompanied inoculum discs; the plants were incubated at by small amounts of darkly pigmented pseudopar- 20--25 °C in humid chambers and sampled after 3-4 enchyma which can sometimes be seen to have weeks. For more critical comparative studies developed from fascicles of branches (Figs '}-11). involving French isolates the fungi were grown as Occurring separately or closely associated with the maizemeal-sand cultures (3 %, w jw) for 28 days at pseudoparenchyma are large, rounded, darkly 25° and these were mixed into soil at a rate of 3 % pigmented cells, some of which have an obvious (w jw). The soil in this case was a commercial soil 'pore ' (Figs 10, 11). These vesicle-like fungal cells mixture (John Innes Number 3 Compost) brought Fig . 1. Maize killed by root rot-stalk rot , Bronkhorstpruit, Transvaal, South Africa, 1980; type locality of Phialophora zeicola. Figs 2, 3. Maize stem bases from site in F ig. 1, showing root rot caused by P. zeicola. Fig. 4. Maize roots from site in Fig . 1, showing dark streaks caused by P. zeicola. Fig. 5. Detail of dark streaks in Fig . 4, composed of pseudoparenchyma of P . zeicola. Figs 6-13 . Maize grown to maturity in a glasshouse in soil from the site in Fig . 1. Fig. 6. Transverse section of maize root, showing pseudoparenchyma in the epidermis and sub-epidermal cells, and occlusion of some xylem vessels by gum-like material. F ig. 7. As Fig. 6, showing dark vascular discolouration of a root lateral emerging from the root axis.

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Fig. 8. As Figs 6 and 7. showing xylem vessel partly occluded by gum-like material and containing dark hypha of P. zeicola. Fig . 9. Surface view of maize root axis. showing dark runner hyphae and dark vesicle-like cells of P. zeicola. Figs 10, 11. Detail of Fig. 9, showing limited development of pseudoparenchyma from fascicles of hyphae, and dark vesicle-like cells, some of which are collapsed. some showing obvious 'pores '. Figs 12, 13. Squash preparation of pith from maize stalk about 10 em above soil level, showing dark hyphae of P. zeicola in the pith and pseudoparenchyma associated with vascular bundles.

15

21 Figs 14-26. Artificial inoculations of maize and wheat seedlings with P. zeicola in perlite or sand in laboratory conditions. Fig. 14. Root system of 17 day old maize inoculated with S. African isolate Zi , showing death of many roots which failed to grow through the inoculum disc placed below the grain. Fig. 15. As Fig. 14, but French isolate SMH

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Fig. 16. Necrosis of maize root tip (outlined) caused by S. African isolate Z8. Fig. 17. S. African isolate Z61 (= PREM 45754, type) on maize root, showing dark runner hyphae and vesicle-like cells. Figs 18, 19. S. African isolate Z8 on maize roots, showing simple hyphopodia and vesicle-like cells. Fig.

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S. African isolate Z61 (type), showing hyphopodia on maize root.

Fig. 21. French isolate CF 78E on maize root, showing vesicle-like cells and associated groups of smaller cells (compare with Figs 10, 11).

Phialophora zeicola sp.nov. on maize initially to 60 % saturation, and the containers were restored regularly to their original weights by adding water. Cereal grains used in the tests were surface-sterilized (Deacon & Henry, 1978a ). In all tests, P. zeicola caused little or no reduction in shoot growth despite its good growth on the roots. In several cases the roots of wheat and maize failed to emerge from the inoculum discs placed below the grains (Fig. 14); these roots had been killed by the fungus and their tips were necrotic. The roots that did emerge or that grew round the inoculum discs often showed pronounced browning of the cortex and they were colonized by numerous dark runner hyphae, but they seldom showed damage to the stelar tissues in the root axes themselves . The root laterals showed both cortical and vascular browning near their points of emergence from the root axes (Fig. 15); this was reminiscent of the damage to laterals seen in field material (Fig. 7). It was seen in the case of both wheat and maize. In addition, some of the tips of the root laterals in maize (but not wheat ) were necrotic just behind the meristem (Fig. 16) and this necrosis was intimately associated with dark hyphae on the root surface and within the tip itself. In regions where there was little evidence of damage to the plant roots, P. zeicola had formed numerous dark runner hyphae and these were accompanied by the formation of dark, vesicle-like fungal cells in the cortex (Figs 17-19). There was an interesting difference between wheat and maize in this respect: in wheat these cells were always formed in the inner cortex near the stele, whereas in maize they occurred in the outer cortex, predominantly in the epidermis. In maize also, though not in wheat, P . zeicola formed small, club-shaped, grey or brown hyphopodia on the root surface (Figs 18-20). These structures are termed , simple hyphopodia ' by Walker (1972, 1980, 1981). As in the case of field-grown roots, the dark vesicle-like structures in artificially inoculated roots occurred either singly or in small clusters and sometimes they were closely associated with small amounts of pseudoparenchyma (Figs 21, 22, cf. Figs 10, 11). Beneath the vesicle-like cells there was sometimes a proliferation of lignitubers (Fig. 23) indicating that the underlying host cells were alive and resisting invasion . In maize the Iignitubers often gave the roots a distinct yellow colour which turned to brown near the inoculum discs . The roots seldom bore pronounced streaks of pseudoparenchyma like those seen in field material (F ig. 4) but occasionally the host cortical cells were filled with similar pseudoparenchyma within the inoculum discs (Fig. 24). P. zeicola grew poorly on wheat and maize stem bases but it sometimes colonized the senescing

maize coleoptiles , In such cases it caused limited necrotic streaking (the brown colour being closely associated with lignitubers) and it formed small club-shaped hyphopodia (Fig. 25), small aggregations of pseudoparenchyma (Fig. 26) and a few large vesicle-like cells similar to those in roots (Fig. 26). P. zeicola never formed a dark mycelial network on the first leaf sheath (under the coleoptile). It grew well on the seed coats of both wheat and maize, forming black sclerotium-like bodies similar to those formed in culture (see below).

THE PATHOGEN

Cultural characteristics Colonies of P. zeicola closely resemble those of the wheat take-all fungus Gaeumannomyces graminis (Sacc.) Arx & Olivier. On agar at 25° they increase radially at 5-9 (usually about 7) mm/24 h. They are white at first, becoming grey or greyish brown and with a dense felt of aerial mycelium which is never more than 1-2 mm high (Fig. 27). The colony margin is somewhat irregular, due partly to the tendency of the leading hyphae to curl back and partly to the fact that the hyphal branches form fascicles. Some isolates degenerate in culture and then tend to form arachnoid colonies. Normally, as the colonies age they develop black, subglobose, sclerotium-like bodies within the agar or projecting from its surface. These bodies are 1-2 mm diam and are composed of isodiametric cells with thickened walls, black towards the outside of the body but hyaline near the centre (Fig. 28). Two types of conidia are formed in culture. One type is narrow and strongly curved and has never been seen to germinate (Fig. 29). Such conidia are formed by all isolates as the colonies age and the ability to form them does not seem to be lost on prolonged storage of isolates. They develop from variously shaped phialides borne on the colony surface or within the aerial mycelium, sometimes on multiple-branched conidiophores (Figs 30-"33). The other type of conidium is larger, rounded and germinates readily, often at the tips of the phialides (Figs 34-36). These conidia are formed from phialides immersed in the agar or in the water film at the agar surface . They are seen most readily just behind the colony margin, before they have had time to germinate (when the germ tubes anastomose). They occur mainly in young fresh isolates, and the ability to produce them seems to be lost on repeated sub-culturing. Some isolates that fail to produce them on agar can be induced to do so by flooding the agar with sterile water (Deacon, 1976) but some isolates that used to form them no longer do so under any conditions that we have tried.

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value was discovered by chance when some infected roots from the field were left in Petri dishes filled with water on the laboratory bench. P. zeicola grew out from the roots as dark hyphae and formed conidiophores in the water (Fig. 37). The conidiophores were repeatedly branched and bore narrow, non-germinating conidia. In subsequent tests this worked repeatedly if white, little-damaged roots were used but it was never successful either with black heavily rotted roots or with roots bearing pronounced dark streaks (Fig. 4). In the case of white roots the fungus grew from the inner cortical tissues at the ends of the root pieces, rather than from the root surface where dark runner hyphae could be seen. A fringe of hyphae of saprophytes always developed from the root surface and apparently suppressed the outgrowth of P. zeicola. This test for the presence of P. zeicola, though subject to some difficulties as mentioned above, might be developed as a simple confirmatory procedure. It has also worked well in the case of air-dried roots on the few occasions when we tried them. Taxonomy The Phialophora from maize roots clearly belongs to the group of dark mycelial parasites related to the take-all fungus and discussed in detail by Walker (1972, 1980, 1981). The presence of narrow, strongly curved conidia suggests that it is closely related if not identical to Phialophora radicicola Cain (1952). However, there are strong arguments against using the name P. radicicola, as discussed fully by Walker (1980, 1981). Two points are especially relevant in this respect. Firstly, the information given in the original description (Cain, 1952) could apply to several different fungi that have since been discovered, and recourse to the type material or to the living type culture does not help to resolve this problem (Walker, 1980, 1981). Secondly, and resulting mainly from the first point, the name P. radicicola has been used in several different senses by subsequent workers with the result that it has become a persistent source of error (Walker, 1980, 1981). Article 69 of the International Code ofBotanical Nomenclature (Stafleu et al., 1978) enables such a name to be rejected if it has been widely and persistently used for a taxon not including its type. Walker (1980) recommends that the name P. radicicola be used only for the fungus originally described by Cain (1952). The name should not be used for any material other than the type, according to Walker's recommendation based on Article 69 (Stafleu et al., 1972). We accept Walker's (1980) arguments in general and, for the most part, in detail. Moreover, if we

were to assign the Phialophora in this paper to P. radicicola then we would be using this name for yet another fungus, different from those that the name has been used for to date (except perhaps the type). This would engender further confusion even if it were correct, and we therefore feel that it would be counter-productive. For these reasons, and to give plant pathologists asatisfactory, clear nomenclature, we propose the name Phialophora zeicola for the fungus from maize roots in South Africa. Phialophora zeicola sp.nov. (Figs 1-39). Coloniae celeriter crescentes, primo albae, deinde canae vel cano-brunneae, reverso fusco-brunneae vel nigrae, cum mycelis aerio brevi velutino, sclerotia erumpentia nigra subglobosa hebent. Mycelium statu jevenili hyalinum, deinde brunneum vel olivaceo-brunneum, inter dum ex hyphis latis fuscatis et ramis hyphalibus hyalinus angustatis constans; hyphae primariae ad marginem coloniae cum inclinatione retrocurvata crescentes. Sclerotia 1-2 rom diam, ex cellulis isodiametris 8-21 pm (plerumque 12 pm) diam, parietibus griseis vel atris. Conidiophora ex hyphis aeriis evoluta, a hyphis vegetativis non semper distinguenda, sed interdum brunnea, septa, ad 1 rom longa, 3-5 pm lata, monopodiale vel dichotome prope apices repetite ramificata, phiales ad positiones dissimiles habentia, ita capitulum inordinatum formantia. Phiales hyalinae vel pallido-brunneae, irregulares, saepe curvatae, interdum sigmoideae, divergentes, parum obclavata ad vel infra medium latissimae (ad 3-4 pm latae), 1
Colonies growing rapidly, white at first, becoming grey or greyish brown, with a short felt of aerial mycelium and producing erumpent black, sub-

J. W. Deacon and D. B. Scott

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Fig. 39. Germinable conidia of French isolates produced in response to flooding of agar colonies: (a) and (b) isolates CF78D and SGM77 of Phialophora sp. (lobed hyphopodia); (c}-(f) isolates SMH77, CF78C, CF78E and CF78H of P. zeicola. Bars = 10 pm.

globose sclerotium-like bodies; colony reverse dark brown to black. Mycelium hyaline when young, becoming brown to olivaceous brown, sometimes consisting of wide darkly pigmented hyphae and narrower hyaline branch hyphae, the leading hyphae tending to curl back at the colony margin. Sclerotium-like bodies 1-2 mm diam, composed of isodiametric cells 8-21 p,m (usually about 12 p,m) diam, with grey or black walls. Conidiophores produced in the aerial mycelium, not always distinguishable from vegetative hyphae but sometimes brown, septate, up to 1 mmlong,3-5 p,mwide, with repeated monopodial or dichotomous branching near the apex and bearing phialides at different levels so that an irregular head is formed. Phialides hyaline to light brown, irregular, often curved, sometimes sigmoid, diverging or forming a narrow angle with their bearers (the latter if formed in complex heads), broadest (3-4 p,m) at or below the mid-point, 10--22 p,m long, borne in clusters of up to twelve on metulae or occurring directly on aerial or immersed hyphae; collarettes inconspicuous to 9

distinctly funnel-shaped, up to 2"5 p,m wide. Phialidic conidia hyaline, uninucleate, single-celled,

of two types: (1) variable but usually ovoid or cylindrical with rounded ends, sometimes with a constriction at one end, never strongly curved, 6-20 x 1"5-6 p,m, developing from phialides on immersedhyphae in young cultures and germinating readily; (2) narrow, non-germinating, often strongly curved, falcate or semicircular, 5--9x 1-1'5 p,m, developing from phialides on aerial hyphae. Superficial hyphae common on maize roots but scarce or absent on leaf sheaths, brown, 4-6 p,m wide, bearing hyphopodia. Hyphopodia simple, clubshaped, 10--14x 5-8 p,m, hyaline to greyish brown. Darkly pigmented cells in host roots of two types: (1) large, round or oval, 17-22 x 14-19 p,m, grey to light brown, thin-walled, occurring singly or in small clusters; (2) isodiametric, 10--13 p,m diameter, appearing angular, thick-walled, grey to dark brown, aggregated into pseudoparenchyma which often fills host cortical cells and is seen as conspicuous streaks on the roots or stubbles. MYC 81

Phialophora zeicola sp. nou . on maize Table 1. French isolates of Phialophora zeicola and Phialophora sp , (lobed hyphopodia) used in comparative tests Provenance of isolates Mons-en-Chaussee, Northern France (maize/ wheat rotation on loam)

P. zeicola Mons 77/16

St Geours-de-Maremne, S.W, France (maize monocropping on sandy soil)

SGM 77/20 (DAR 35785)

St Martin-de-Hinx, S.W. France (maize monocropping on loam)

SMH 76/1 SMH 76/2 SMH77A SMH77 SMH78

Clermont-Ferrand, Central France

CF78C C F78E CF78G CF78H

Castelnaudary, Southern France (maize/ wheat/sorghum rotation)

Phialophora sp. (lobed)

Mons 14 Mons 77B Mons 77/13 Mons 77/15 SGM77 SGM 77B SGM 77/01

(DAR 35787) (DAR 35789) (DAR 35788) (DAR 35784)

(DAR 35782) (DAR 35783) (DAR 35786) (DAR 35780) (DAR 35781)

CF78D CF78F

(DAR 35779)

CAL 77C

Specimens examined : In roots of Zea mays L. , Bronkhorstpruit, near Pretoria, Transvaal, South Africa, 1980, D . B. Scott, PREM 45754, holotype ; duplicate as DAR 41950, DAR 41951 and DAR 41952, isotype ; PREM 45707 from Krugersdorp, Transvaal ; PREM 45708 from the same locality ; PREM 45709 from Ventersdorp, Transvaal; PREM 45716, 45717, from Heilbron, Orange Free State; PREM 45718, 45720, from Settlers, Transvaal; PREM45938 fromBethal, Transvaal; PREM 45939 from Delmas, Transvaal. All on agar media and on roots of Zea mays. Living cultures from maize and maize-wheat cropping in France are deposited as DAR 35780, DAR 35781, DAR 35782, DAR 35783, DAR 35785 and DAR 35786. COMPARISON WITH SIMILAR FUNGI

Comparative studies on French Phialophora spp. Before P. zeicola had been discovered in South Africa, 21 isolates of Phialophora from maize and maize-wheat rotations in France had been supplied by Dr C. M. Messiaen as •P. radicicola'. Their origins are given in Table 1. In detailed comparative studies, 12 of these isolates were found to be identical to P . zeicola as now described from South Africa . The other 9 were different in many respects ; they produced deeply lobed hyphopodia in culture and on host coleoptiles, and closely resembled the lobed hyphopodiate isolates from Britain studied by Deacon (1974). Full details of the comparative studies will not be given but a summary is appropriate in view of the fact that two distinct types of Phialophora occur on maize in France and maybe elsewhere. The lobed

hyphopodiate type has not been named (see discussion), French lobed hyphopodiate isolates grew at the same rate as unlobed ones (P. zeicola ) on PDA at 25° (means 7'24 and 7'18 mm/24 h respectively). Both types grew poorly at 10° (mean 29 %, range 24-39 % of their rates at 25°). They behaved differently from one another at 30°: the lobed ones grew at 30 % (range 14-52 % ) and the unlobed ones at 91 % (range 71-104 % ) of their rates at 25°. For comparison, British lobed isolates in these tests grew at 10 % and South African unlobed isolates (P . zeicola ) grew at 85 % (range 81-87 %) of their rates at 25°. Therefore, irrespective of origin, the lobed hyphopodiate isolates grew poorly at 30° whereas P. zeicola grew well. The French isolates were again clearly separable on colony morphology. The lobed hyphopodiate ones formed profuse fluffy aerial mycelium on PDA plates (containing 2 % dextrose, 15 ml agar medium per 9 cm Petri dish); this was usually several milIimetres high and often reached the Petri dish lid if the plates were incubated in a humid environment. The isolates of P. zeicola formed a much shorter felt of aerial mycelium (Fig. 38). The lobed hyphopodiate isolates formed hyphopodia against the Petri dish base, though not frequently enough for this to be used as a reliable distinguishing character in culture. The two types ofisolate could not be distinguished satisfactorily by the following criteria. (1) Sclerotium-like bodies. These were produced by both types of isolate in ageing agar cultures; they were

J. W. Deacon and D. B. Scott

259

Table 2 . Dimensions of the swollen vesicle-like cells (growth-cessation scructures)formed by French and South African isolates ofP. zeicola and Phialophora sp, (lobed hyphopodia) in roots of wheat and maize in artificial inoculations Maize

Wheat

Cell length (pm)

Mean* Range

Cell width (pm)

Mean* Range

Length :breadth ratio Volume (pm 3)t

Mean* Range

No. isolates tested

Mean* Range

P . zeicola (French) 18,8 17'0-21 '4

16'4 15'3- 18 '7 1'14 : 1 1'04-1 '33: 1 2807 2173-3682 12

Phialophora sp. (lobed, French)

30'5 26'9-33 '2 21'1 16'8-24'7 1'45: 1 1'3 1- 1'75: 1 8215 5278-11919 9

P. zeicola (French) 18,8 17'3-20'5 15.8 14'3-17'5

1'19: 1 1'14-1 '29 : 1 2667 2005-3399 10

Phialophora sp, P. zeicola (lobed, French) (S. Africa):!:

1'47: 1 1'37-1 '63: 1 5569 4533--6432

20'1 17'8-22'3 16'7 16'1-17'4 1'20:1 1'11-1 '29 : 1 3198 2531-39 23

5

5

27'0 25'1-28'1 18'5 17'2-19'8

* Means for all isolates tested; for each isolate the values were based on at least 10 cells. Calculated by assuming that each cell is a cylinder with hemispherical ends. :I: Values for isolate Z61 (type) in maize in field material were: length, 19'9 ; width, 17'0; volume 3231 p.m3 •

t

often, but not invariably, larger in the case of P. zeicola. (2) Non-germinating conidia. These narrow, strongly curved conidia were formed by some isolates of both types. The arrangement ofthe phialides on the conidiophores (where present) and the complexity of branching of the conidiophores were variable; often the isolates of P. zeicola showed the most pronounced and complicated arrangement of conidiophores, but this was not invariably so. (3) Larger, germinating conidia. Some isolates of both types formed these spores in response to flooding (Deacon, 1976). The spores were variable in shape (Fig. 39) and sometimes (not always) larger in the case of lobed hyphopodiate isolates than in the case of P . zeicola. Pathogenicity tests The two types of Phialophora from France showed basic similarities in their growth on wheat and maize seedlings, but with some important differences. The similarities were in growth by dark runner hyphae, the marked cortical browning of infected roots and the presence of dark vascular discolouration of the root laterals where they joined the root axes. Neither type of isolate caused noticeable reductions in shoot growth, so both were considered to be weakly pathogenic. The differences in behaviour between types of isolate were as follows, (1) P. zeicola caused necrosis of the root tips of both cereals, such that several roots failed to emerge from the inoculum discs or were damaged at the tips

from dispersed inoculum in the soil. The lobed hyphopodiate isolates never caused root tip necrosis. (2) P. zeicola grew well onto the seed coats from the roots whereas the lobed hyphopodiate isolates seldom did so. (3) P. zeicola colonized the stem bases very poorly; the lobed hyphopodiate isolates grew very well on the stem bases in humid conditions. This difference is reminiscent of the difference in production of aerial mycelium in agar culture and is possibly related to it. (4) P. zeicola formed simple club-shaped hyphopodia on coleoptiles (especially of maize) and sometimes formed larger vesicle-like cells on coleoptiles, similar to those formed in roots. The lobed hyphopodiate isolates formed deeply lobed hyphopodia from networks of dark hyphae that covered the seedling coleoptiles and that were present also on the first leaf sheaths. (5) Both types of isolate formed large, swollen, darkly pigmented vesicle-like cells in roots, but the cells were consistently larger in the case ofthe lobed hyphopodiate isolates (T able 2; see also Figs 6, 7 in Deacon, 1981). The results in Table 2 are based on measurements ofat least ten cells for each isolate when the roots were viewed from the surface at high magnification. The cell volumes were calculated on the assumption that each cell is a cylinder with hemispherical ends. It is seen that the cells of the lobed hyphopodiate isolates were both longer and wider than those of P. zeicola; their length: breadth ratio was also greater, reflecting the fact that their

260

Phialophora zeicola sp.nov. on maize

width was limited by the width of the host cells that contained them. There was no overlap between the types of isolate in their cell volumes in either wheat or maize (Table 2). However, it is interesting that the cell volumes of the lobed hyphopodiate isolates were consistently much larger in wheat than in maize. The reasons for this are unknown. Size of the vesicle-like cells seems to be a useful diagnostic feature, as discussed previously by Deacon (1981). Indeed, it was on this basis that we first recognized that South African isolates of P. zeicola were similar to the French isolates (Deacon, 1981) and this has now been reinforced in several ways - not least by further measurements ofthe cell sizes of South African isolates, given in Table 2. DISCUSSION

Work in this paper has shown that two types of Phialophora occur on maize. Both types seem to be common and widely distributed. The lobed hyphopodiate type is found in several parts of France (Table 1) and in Britain (Deacon, 1974; Speakman, Garrod & Lewis, 1978; Hornby, 1978). P. zeicola also occurs in several parts of France (Table 1) and South Africa. It is notable that both types occur in the same regions of France, so perhaps the fungi themselves or the symptoms caused by them have been confused in the past. Nevertheless the types are distinguishable in several ways, and we found no intermediates between themamong the French isolates. Moreover, single-conidium isolates always resembled the parent isolates from which they were derived. We did not try crosses between the isolates, but all available evidence suggests that the two types of isolate are taxonomically distinct. In view of their differences in behaviour it would be surprising if the types did not differ in some aspects of their ecologies, but there is no information on this point. The darkly pigmented swollen cells in roots could be used reliably to distinguish between the two types of Phialophora, so this work has confirmed and extended previous reports on the diagnostic value of these structures (Scott, 1970; Deacon, 1973, 1974, 1976, 1981; Slope et al., 1978). Walker (1980) stressed the need to examine them on several hosts with a world-wide range of isolates before they could be used for taxonomic purposes. However, we feel that this is over-cautious and that these structures (termed growth-cessation structures by Deacon, 1976) merit at least as much attention as do hyphopodia as taxonomic characters. They show as much variation and as clear-cut differences between fungi in the Gaeumannomyces graminis - Phialophora complex as do hyphopodia. The only qualification to be made in this respect is

that most or all of the fungi can form pseudoparenchyma as well as the more characteristic structures, so pseudoparenchyma alone has little or no diagnostic value. This is no different from the situation with hyphopodia, because the fungi that form complex lobed hyphopodia also form simple ones (Walker, 1972, 1980, 1981). Taxonomically P. zeicola seems very close to P. radicicola Cain and is possibly identical to it. We therefore considered referring our isolates to P. radicicola and perhaps emending the original description on the basis that the type material and the description are at present consistent with several closely related fungi in Phialophora. But we rejected these possibilities because (a) we could never show with certainty that the fungus on which Cain (1952) based his orginal description of P. radicicola was identical to the fungus here described as P. zeicola, and (b) even if we were to show the likelihood of this, the use of the name P. radicicola for yet another fungus, different from those listed by Walker (1980, 1981), would add further to the potential confusion surrounding this name. The name P. radicicola has been used in the past to refer to (1) the type material (TRTC 23360, examined in detail by Walker, 1980) and living type culture (CBS 296.53, examined in detail by Deacon, 1974); (2) a lobed hyphopodiate fungus from France (Messiaen, Lafon & Molot, 1959) and Britain (Deacon, 1974 and subsequent British publications in which the name P. radicicola var. radicicola is used); (3) the imperfect state of the take-all fungus (Lemaire & Ponchet, 1963; Simonsen, 1971); (4) the Phialophora common in British grasslands, subsequently named P. radicicola var. graminicola and now P. graminicola (Deacon) Walker. Fuller accounts of these misapplications of the name P. radicicola are given in Walker (1980, 1981) and Wong & Walker (1975). Full reasons for rejecting the name P. radicicola for any fungus other than that on which Cain's description was based are given in Walker (1980, pp. 86-90). We accept these arguments in general, though our experience suggests that McKeen (1952) could have been describing the behaviour of a fungus like P. zeicola and is unlikely to have been describing the behaviour of P. graminicola as Walker (1980, pp. 89-90) suggests. A consequence of Walker's recommendation to restrict usage of the name P. radicicola (see above) is that the lobed hyphopodiate Phialophora previously called P. radicicola var. radicicola by British workers is now un-named. Indeed, Walker (1980, 1981) recommends that it should not be named at present but should be called Phialophora sp. (lobed hyphopodia), pending further attempts to find the sexual stage. This argument is based on the fact that

J. W. Deacon and D. B. Scott lobed hyphopodiate Phialophora isolates closely resemble G. graminis var. graminis and may be identical to it. Nevertheless there seem to be some important behavioural and ecological differences between them. G. graminis var. graminis is reported mainly from rhizomatous/stoloniferous grasses like Cynodon dactylon L. and Pennisetum clandestinum Hochst. ex Chiov. and from rice (Oryza sativa L.); to our knowledge it is not reported from maize. This is consistent with our observations in South Africa, where G. graminis var. graminis is found commonly on the stolons of grasses in irrigated turf (though not in non-irrigated turf) but where we have seen it on maize in only one locality; in this exceptional case it was present on the stem bases of a few plants grown in experimental plots in humid, sub-tropical conditions, and we considered that the grass turf between the plots had provided the inoculum source. Holden (1980) showed that lobed hyphopodiate Phialophora isolates from Britain are very sensitive to the preformed inhibitors in root and shoot extracts of oats (Avena sativa L.) whereas isolates of G. graminis var. graminis (from Australia) were insensitive to the inhibitors. This difference was reflected in the abilities of the isolates to grow on oat seedlings (Holden, 1980). Additionally, British Phialophora isolates with lobed hyphopodia were very susceptible to mycoparasitism by Pythium oligandrum Drechsler and Pythium acanthicum Drechsler, whereas G. graminis var. graminis (from Australia) was resistant to the mycoparasites (Deacon & Henry, 1978b). The fungi differ further in temperaturerequirementsandotherphysiological characteristics (Deacon, 1974) but as yet it is unknown if all these differences are merely strain differences, reflecting the different origins of the isolates used. The apparently common occurrence of the lobed hyphopodiate Phialophora on maize in Europe will, understandably, create pressure for a satisfactory binomial for this fungus. Indeed its characteristics are now very well known. At present, however, we do not propose to name it. The contribution of P. zeicola (or of the lobed hyphopodiate Phialophora) to the maize root rot-stalk rot complex is difficult to determine. Dodd (1980) tends to discount the fungal pathogens as being important in stalk rot, stating that the host-environment interactions are the pertinent variables (any of several fungi being able to cause rotting of the senescing tissues, and senescence being brought about by host-environment interactions). In large part, our results could be used to support this view. The laboratory and glasshouse tests suggest that P. zeicola is a weak pathogen that can damage maize root tips and can cause some vascular damage to the laterals but is normally restricted to the outer cortex of the maize root axes.

261

Our results place P. zeicola in the moderately invasive category of root parasites (Deacon, 1974, 1976, 1981), so the fungus would be expected to benefit from a reduction in host resistance during senescence of the root cortex. The positions of growth-cessation structures in the roots of laboratory-grown plants underline this point. The structures were present in the inner cortex of wheat roots, which is consistent with the fact that the wheat seminal root cortex dies early from natural (non-pathogenic) causes in the course of plant development (Holden, 1975, 1976; Henry & Deacon, 1981). The structures were present mainly in the outer cortex or even in the epidermis of maize roots, and there was a pronounced development of lignitubers beneath them (Fig. 23) indicating that the underlying cells were alive and resisting invasion at the time of attempted penetration of maize. Despite these comments, P. zeicola could playa significant role in the root rot-stalk rot complex by causing damage to root laterals in the field. This would reduce the efficiency of water and mineral nutrient uptake, with the result that the plants might suffer drought stress in conditions that normal healthy plants could tolerate, or it might similarly reduce the efficiency of photosynthesis. In other words, substantial early levels of infection by P. zeicola may predispose the plants to environmental stresses. Any such role of P. zeicola might go unrecognized because the main phase of the disease would still be associated with obvious environmental stresses. Moreover, once the root and stalk tissues had begun to senesce as a result of these stresses then they would be colonized by P. zeicola and similar weak pathogens, the presence of which might (mistakenly) be ascribed solely to the reduction in host vigour . We suggest, therefore, that weak pathogens like P. zeicola might have a dual role in the root rot-stalk rot complex, firstly by reducing the plant's tolerance of environmental stresses and secondly by rotting the tissues that senesce as a result of environmental stresses. Further studies on P. zeicola may be especially useful in this regard, because the presence and the distribution of the fungus on the root system can be determined accurately by means of the dark runner hyphae and darkly pigmented growthcessation structures. Therefore the role of the fungus in causing early damage to plants in the field might be assessed, without the need to isolate fungi from the roots and introduce possible artifacts in this way. Part of this work was done while J. W. Deacon held a Research Fellowship from the Department of Agriculture and Fisheries, Republic of South

262

Phialophora zeicola sp.nov. on maize

Africa. This support is gratefully acknowledged. We are especially pleased to thank Dr G . C. A. van der Westhuizen for helpful discussion and suggestions, Dr I. H. Wiese for providing facilities at Pretoria, Dr C. M . Messiaen for supplying French isolates of Phialophora and Dr A. Bennell for supplying the Latin diagnosis.

REFERENCES

CAIN, R. G. (1952). Studies of Fungi Imperfecti. I. Phialophora. Canadian Journal of Botany 30, 338-343· DEACON, J. W. (1973). Phialophora radicicola and Gaeumannomyces graminis on roots of grasses and cereals. Transactions of the British Mycological Society 61, 471-4 85. DEACON, ]. W. (1974). Further studies on Phialophora radicicola and Gaeumannomyces graminis on roots and stem bases of grasses and cereals. Transactions of the British Mycological Society 63, 307-327. DEACON, J. W. (1976). Biology of the Gaeumannomyces graminis Arx & Olivier I Phialophora radicicola Cain .complex on roots of the Gramineae. EPPO Bulletin 6, 349-3 63. DEACON, J. W. (1981). Ecological relationships with other fungi : competitors and hyperparasites. In Biology and Control of Take-all (ed. M. J. C. Asher & P. J. Shipton), pp. 75-101. London: Academic Press. DEACON, ]. W. & HENRY, C. M. (1978a). Studies on virulence of the take-all fungus, Gaeumannomyces graminis, with reference to methodology. Annals of Applied Biology 89, 401-409. DEACON,] . W.&HENRY,C . M. (1978b). Mycoparasitism by Pythium oligandrum and P. acanthicum . Soil Biology and Biochemistry 10,409-415. DODD, ] . L. (1980) . The role of plant stresses in development of com stalk rots. Plant Disease 64, 533-537· HENRY, C. M. & DEACON, J. W. (1981). Natural (nonpathogenic) death of the cortex of wheat and barley seminal roots, as evidenced by nuclear staining with acridine orange. Plant and Soil 00, 255-274. HOLDEN, J. (1975). Use of nuclear staining to assess rates of cell death in cortices of cereal roots. Soil Biology and Biochemistry 7, 333-334· HOLDEN, J. (1976). Infection of wheat seminal roots by varieties of Phialophora radicicola and Gaeumannomyces graminis. Soil Biology and Biochemistry 8, 109-119. HOLDEN, J. (1980). Relationship between pre-formed inhibitors in oats and infection by Gaeumannomyces graminis and Phialophora radicicola. Transactions of the British Mycological Society 7S, 97-1°5. HORNBY, D. (1978). Gaeumannomyces - Phialophora complex: an early isolate of P . radicicola var. radicicola. Rothamsted Experimental Station, Report for 1977, Part 1,216.

LEMAIRE, J. M. & PONCHET, J. (1963). Phialophora radicicola Cain, forme conidienne du Linocarpon cariceti B. et Br. Compte rendu hebdomadoire des seances de rAcademie d' Agriculture de France 49, 1067-1069 . McKEEN, W. W. (1952). Phialophora radicicola Cain, a com root rot pathogen. Canadian Journal of Botany ]0, 344-347· ). MESSIAEN, C. M ., LAFON, R . & M OLOT, P . ( 1959. Necroses de racines, pourritures de tiges et verse parasitaire du mais. Annales des Epiphytes 4, 441-474. PHILLIPS, J. M. & HAYMAN, D. S. (1970). Improved procedure for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessmentofinfection. Transactions ofthe British Mycological Society 55, 158-161. SCOTT, P. R. (1970). Phialophora radicicola, an avirulent parasite of wheat and grass roots. Transactions of the British Mycological Society 55, 163-167. SIMONSEN, J. (1971). Phialophora radicicola Cain, the conidial stage of Gaeumannomyces graminis in Denmark. Friesia 9, 361-368. SLOPE, D. B., SALT, G. A., BROOM, E. W. & GUTTERIDGE, R. J. (1978). Occurrence of Phialophora radicicola var. graminicola and Gaeumannomyces graminis var. tritici on roots of wheat in field crops. Annals of Applied Biology 88, 239-246. SPEAKMAN, J. B., GARROD, B. & LEWIS, B. G. (1978). Limitation of Gaeumannomyces graminis by wheat root responses to Phialophora radicicola. New Pltytologist 80, 373-380. STAI'LI!U, F. A. et al. (ed.) (1972). International Code of Botanical Nomenclature. Regnum Vegetabile 8z, 1-4%6. STAl'LEU, F . A. et al. (ed.) (1978). International Code of Botanical Nomenclature. Regnum Vegetabile 97, 1-457 · WALKER, J. (1972). Type studies on Gaeumannomyces graminis and related fungi. Transactions of the British Mycological Society 58,4%7-457. WALKER J. (1980). Gaeumannomyces, Linocarpon, Ophiobolusand several other genera of scolecospored Ascomycetes and Phialophora conidial states, with a note on hyphopodia. Mycotaxon 11, 1-129. WALKER, J. (1981). Taxonomy of take-all fungi and related genera and species. In Biology and Control of Take-all (ed. M . J. C. Asher & P. J. Shipton), pp. 15-74. London: Academic Press. WHITNEY, N . J. & MORTIMORE, C. G. (1957). Root and stalk rot of field com in southwestern Ontario. I. Sequence of infection and incidence of the disease in relation to maturation of inbred lines. Canadian Journal of Plant Science 37, 342-346. WONG, P. T . W. & WALKER, ]. (1975). Germinating phialidic conidia of Gaeumannomyces graminis and Phialophora-like fungi from Gramineae. Transactions of the British Mycological Society 65, 41-47.

(R eceiv ed j or publication 22 October 19 82)