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Infection of wheat seminal roots by varieties of Phialophora radicicola and Gaeumannomyces graminis
INFECTION OF WHEAT SEMINAL ROOTS BY VARIETIES OF PHIALOPHORA RADICZCOLA AND GAEUMANNOMYCES GRAMINIS J. HOLDEN* Botany School, University of Cambridge. England (Accepted 15 August 1975) Summary-The growth of isolates of Phiulophora radicicala var. radicicaka, P. rudicicolo var. yruminicolu, Gueumannomyces yraminis var. yraminis, G. graminis var. rritici and Leprosphueria nurtizari was compared
on the coleoptiles and roots of wheat seedlings. Fungal growth was measured as the extent and density of dark runner hyphae. All except P. radicicolu var. graminicola grew on coleoptiles and all grew on roots although only G. graminis var. tritici extensively colonized the root stele. Growth rate on roots was positively correlated with that on agar, P. radicicola var. graminicaku and L. nurmuri growing at about half the rate of the other fungi; hyphal density was high for P. rudicico/a var. yruminicolu but relatively low for the other fungi. For P. radicicala var. radicicalu, P. rudicicolu var. yruminicolu and G. gruminis var. tritici growing from buried inocula, the extent and density of hyphae up roots towards the seed was similar to that down, but G. graminis var. tritici caused chocolate-brown stelar discoloration up roots only. Root invasion by P. radicicola var. radicicola, P. radicicolu var. graminicolo and G. yraminis var. rritici was described from sections. Each gave a different pattern of hyphae and host response within an inoculum layer. and progressive changes occurred away from the inoculum. Studies of the rate of penetration by eabh fungus and the rate and pattern of death’of cortical cells explained the differences between fungi. G. graminis var. tritici penetrated living cells in advance of other soil micro-organisms, and hence by hyaline hyphae inducing much lignituber formation as a host resistance reaction. P. radicicola var. yraminicola penetrated only senescent or dead cells in association with other soil microorganisms, and hence by dark hyphae, inducing little lignituber formation. P. radicicoku var. radicicala was intermediate in all these respects. The high hyphal density of P. radicicola var. yraminicola was due to the colonization of cortical cells and spaces by dark, clearly visible, rather than hyaline hyphae, which are invisible in unstained roots. Cell death in the outer cortex explained the observed progressive restriction of growth’ by all fungi to the inner cortex with increasing distance from the inoculum. Spread by G. graminis var. tritici up roots was ectotrophic relative to the stele but down roots hyphae spread rapidly within the stele. Stelar reactions suggested as resistance mechanisms occurred up roots only. Their absence down roots is attributed to infection disrupting stelar transport.
INTRODUCTION Phialophora radicicola Cain and Gaeumannomyces graminis (Sacc.) Arx & Olivier are ecologically obli-
gate parasites which grow on the roots of grasses and cereals as similar-looking dark runner hyphae. Two varieties of P. radicicola were recognized by Deacon
(1974). var. radicicola and var. graminicola Deacon. Three varieties of G. graminis were recognized by Walker (1972), var. graminis, var. tritici Walker and var. arenae (E. M. Turner) Dennis. G. graminis var. fritici. the wheat take-all fungus, has long been recognized as a cosmopolitan and serious pathogen on wheat. The host ranges and distributions of the other fungi are not yet completely known, but all are reported from wheat: e.g. Deacon (1973a 1973b, 1974) for P. radicicola var. graminicola and P. radicicola var. radicicola: Nilsson (1972) for G. graminis var. graminis: and Dennis (1944) for G. graminis var. arenae.
Deacon (1974) divided these fungi into three groups based on three aspects of their growth on wheat seed-
* Present address: Glasshouse Crops Research Institute. Littlehampton. Sussex. BN16 3PU. 1 much regret to ha\e to notify the death of Dr. John Holdrn on 10 September 1975-s. D. Gnwrr.
lings, viz: pathogenicity to roots resulting from stelar invasion, the ability to grow on stem bases. and the type of growth cessation structures formed on stem bases (hyphopodia) and within roots (pigmented cells). G. graminis var. tritici and G. graminis var. auenae are highly pathogenic to roots and grow well on stem bases. They form small unlobed hyphopodia, singly or in cushions, but no pigmented cells. G. graminis var. graminis and P. radicicola var. radicicola are rarely pathogenic to roots but grow well on stem bases. They form large lobed hyphopodia and single large pigmented cells. P. radicicola var. graminicola is non-pathogenic on roots and grows very poorly on stem bases. It forms groups of small pigmented cells. Detailed information about the progress of root infection exists only for fungi in the first group. That typically described (Fellows, 1928; Robertson, 1932; Garrett, 1934: Turner, 1940) consists in longitudinal spread by wide. dark. superficial runner hyphae with narrower. hyaline penetration hyphae growing radially into the cortex and stele. Garrett (1970) termed this ‘ectotrophic’ infection and suggested that host resistance prevented the longitudinal spread of hyphae within the root. Non-ectotrophic infection by dark
109
J. HOLLIES
110
intercellular hyphae within the cortex (Garrett. 1934) or stele (Nilsson, 1969) has been recorded well down roots first infected close to the seed. Garrett (1970) suggested that in these cases host resistance was probably reduced by stelar disruption caused by infection. The infection of root cortices by the varieties of P. radicicola has been described as identical with ectotrophic infection by G. graminis (Mckeen, 1952: Scott. 1970) but this was not borne out in my preliminary studies. The present investigation was initiated to compare in more detail the infection of wheat seedlings, and particularly of their seminal roots, by P. radicicola var. radicicoia, P. rudicicola var. graminicola and G. graminis var. tritici. G. graminis var. graminis and Leptosphaeria narmari Walker & Smith (1972), another fungus described as having a similar infection habit, were included initially for comparison.
MATERIALS
AND METHODS
Fungi
The suppliers of the fungal isolates used were Dr. J. W. Deacon (School of Agriculture, University of Edinburgh) for P. radicicola var. radicicola, P. radicicola var. graminicola and G. graminis var. tritici and Mr. J. Walker (Biological and Chemical Research Institute, Rydalmere, N.S.W., Australia) for G. graminis var. graminis and L. narmari. All agreed with the published diagnoses and remained fully infective throughout. They are listed below with the supplier’s notation, if any (in parentheses), and the host and location of origin. P. radicicola var. radicicola, PRA (G6), maize, Coggeshall, Essex, PR2 (P16). maize, Trumpington, Cambs., PR4 (P18). barley, Rothamsted, Herts.. P. radicicola var. gruminicola. PG4 and PG5, grass turf, Cambridge. PG7 (P9). grass ley. Jealott’s Hill. Berks., G. gruminis var. tritici, GTl (Gl). wheat. Trumpington, Cambs.. GT2 (G8). wheat. Jealott’s Hill, Berks., GT3 (G2), wheat. Boxworth. Cambs.. G. graminis var. graminis. GG 1 (Dar 17498) Pennisetum clundestinum, Kempsey, N.S.W. L. narmuri, LNl (Dar 20806) P. clandestinum. Cobar. N.S.W. Plant culture and inoculution
To favour infection (Garrett, 1936: Balis. 1970) and facilitate the washing out of roots, wheat plants (cv. Cappelle-Desprez) were grown in sand-soil mixture prepared by blending 3 vol dry sand with 1 vol airdried soil (2.4 mm-sieved loam from the University Botanic Garden). The mixture had a saturation capacity of 25 ml water/100 g and a pH of 7.4. Fungal cultures on 3;d (w/w) maizemeal in sand were used as inoculum. In all experiments controls were set up with the inoculum layer replaced by uninoculated sand soil mixture. Details of the soil containers and inoculum and seed positioning used in different experiments are described in their context. Incubation was at laboratory temperature (mean 20°C) and at a soil moisture content of 55-60:‘. saturation. Sctmpling methods
Plants were washed free of sand-soil mixture and preserved in 70”:, alcohol. They were examined by reflected light in a shallow dish of water over a white
tile background under the stereoscopic microscope ( x 50 and x 100). Funpal growth on roots and coleoptiles \vas assessed ( I) as the furthest extent of dark runner hyphae from the point of inoculation (Garrett. 1936) and (2) as the density of h~phne at a measured distance from the point of inoculation. assessed as follows. A line across the microscope eyepiece was rotated until it cut across the root or coleoptile at rieht angles to its axis. All dark runner hyphae thus viiible and cut by this line \vere counted to give a number referred to as the hyphal density’. Fungal penetration of roots was described from thin free-hand sections taken at measured distances from the point of inoculation. by laying roots along a microscope slide over squared paper and cutting downwards with a razor blade. They were cleared and stained by warming in 0.05”,, trypan-blue in lactophenol and mounted in glycerine jelly. This procedure stained hyaline hyphae blue thus distinguishing them from naturally dark hyphae. In describing fungal growth in the root cortex Garrett’s (1934) terminology is used with slight modification. Dark hyphae growing longitudinally are termed ‘runner hyphae’. Two sorts are recognized. ‘surface’ and ‘cortical’, the latter being those formed in intercellular spaces within the cortex. The unqualified term, ‘runner hyphae’, refers to both types. Hyaline or dark hyphae growing radially or obliquely radially within cells are termed ‘penetration hyphae’.
RESULTS
Experiment
1. Growth down root.s trntl up coleoptiles
Growth over wheat seedlings was measured for all isolates except GT3. For each. three replicate jars (6 cm dia x 12 cm) received a 5 cm layer of sand-soil mixture covered by a 0.5 cm layer of maizemeal-sand inoculum. Six presoaked wheat seeds were then sown and covered with a further 0.5 cm layer of inoculum and then with a 5 cm layer of sand-soil mixture. Plants were sampled after 15 days and measurements made of the extent of runner hyphae and of their density 7 mm from the seed. on the coleoptiles and first seminal roots (Table 1). To allow for the depth of inoculum, 5 mm was subtracted from the measured extents. All the fungi grew on roots and coleoptiles as similar-looking dark runner hyphae. The varieties of P. rtrdicicolct and G. yrctminis grew as described by Deacon (1974) in his ecological groupings. outlined above. Only isolates of G. grarninis var. tritici caused extensive stelar infection on roots with an intense chocolate-brown discoloration in the top 5lOmm and a paler discoloration below. which extended beyond the furthest extent of runner hyphae in the cortex. Growth of main and branch roots was checked only by this fungus. presumably due to infection disrupting stelar transport (Asher. 1972: Clarkson er 01.. 1973). L. rwmtrri only rarely penetrated the root stele. It grew densely on stem bases, forming circular infection cushions 300 Ltrn in diameter as described by Walker and Smith (1972). Similar structures were formed in root cortices against the endodermis. The lack of pathogenicity of L. rwmtrri to roots disagrees with the report of Walker and Smith (1972). although A.
P. raclicicoiu
and G. gruminis on wheat seminal roots
Ill
Table 1. Growth of runner hyphae on seedlings inoculated at the seed (Means of fifteen plants &S.D.) Coleoptiles
G. graminis
var. rritici
GTI GT2
G. graminis var. grrtminis P. radicicola var. rctrlicicolo
P. ra~~cicolu var. grumi~icola L. narmari
GGl PRl PR2 PR4 PG4 PGS PG7 LNl
First seminal roots
Extent (mm)
Density
Extent (mm)
Density
48.7 + 9.1 60.3 * 11.2 46.3 k 8.9
27.1 f 10.3 38.5 + 18.1 66.4 + 8.4
44.3 + 13.7 57.4 i_ 15.9 51.6 i 7.2
22.5 + 3.2 22.0 + 6.4 1.5.8 + 4.6
26.3 + IO.9 71.3 + 15.4 52.8 + 7.6 64 (8)* 4.2 (6) 3.5 (2) 8.4 + 24
27.7 f 12.1 69.1 + 17.9 58.3 f 11.4 4.3 2.1 l-1 58.3 i 18.9
53.4 & 5.0 66.4 + 20.1 57.3 * 5.4 23.2 & 4.5 21.9 & 4.8 19-1 + 5.0 18.8 * 1.4
22.3 rt 6.1 29.1 -f. 6.7 20.3 + 5.9 42.9 _+7.8 423 * 9.5 49.9 + 9.5 29.2 i 8.1
* Numbers in parentheses are coleoptiles infected out of fifteen. M. Smith (1973. personal communications agreed that severe disease is rare unless host plants are weakened or grown under aseptic conditions. Table 1 shows that the isolates of P. radicicolu var. graminicola and L. narmari gew more slowly on roots than did isolates of the other fungi. When the growth rates of the isolates on roots are plotted against their radial growth rates on potato-dextrose agar (PDA) at 20°C (mean values for five plates), they show a significant positive correlation (r = f&94, P = t3001) (Fig. 1). For most isolates of P. radicicola var. radicicola, G. graminis var. graminis and G. graminis var. tritici the extent of growth on coleoptiles was similar to that on roots. Isolate PRl was exceptional in growing less far on coleoptiles than on roots, as also did the single isolate of L. n~ri. However both PRl and LNI had high hyphal densities on coleoptiles and penetrated deeply into the tissue, in contrast to all isolates of P. radicicola var. graminicola which were obviously unable to infect coleoptiles and produced either no growth or only a few superficial hyphae. On roots there was no obvious relationship between extent and density of hyphae. All the isolates of P. radicicola var. ~aminica~a had high hyphal densities. mainly due to the presence of many cortical runner hyphae. The relatively low hyphal densities of the other varieties were due to the presence of very few or no cortical runner hyphae close to the inocu-
Ium layer. On coleoptiles, where only surface runner hyphae were formed, density and extent of hyphae amongst the isolates of each fungal variety were apparently positively correlated but there was no such relationship when isolates of several fungal varieties were compared together. Experiment
2. Growth up and down roots
If stelar disruption at the point of inoculation with G. graminis var. tritici reduces host resistance to its growth down a root, as suggested by Garrett (1970), growth up a root might be expected to be slower. This possibility was examined here in a comparison between G. graminis var. tritici and the two varieties of P. radicicola, which do not infect the stele. Wheat seeds were sown in perspex boxes with sloping detachable fronts. Each box received a 7 cm layer of sand-soil mixture, a 5 mm layer of inocuium and a further 7cm layer of sand-soil mixture. A row of five wheat seeds was then sown close to the sloping faa: so that roots grew down this and could be seen passing through the inoculum. The sloping face of the box was covered with black paper to exclude light. The iirst three seminal roots of each seedling on which observations were sub~uently made, reached the inoculum layer (7 cm below the seed) in about 5 days. Three isolates, GTl, PRl and PG5 were tested with three replicate boxes of each. Roots infected with GTl-PRl were sampled at 20 days and those with PG5 at 25 days after sowing the seed. After removing the box face, roots were picked up with a pair of forceps where they passed through the inoculum layer and cut at the top and bottom. The extent and density of runner hyphae and the kxtent of intense chocolate-brown stelar discoloration were measured up (towards the seed) and down each root from the centre of the inoculum. The results (Table 2) show that each fungus produced an equal extent and density of runner hyphae up and down roots. As in Experiment 1, hyphal densities close to the inoculum were high for P. radicicola var. graminicolu and relatively low for the other fungi, and only G. graminis var. tritici penetrated the stele. Intense chocolate-brown stelar discoloration Over poiuio-dextrose ogor occurred only in the region of the inoculum and up Fig. 1. Correlation between fungal growth rates (mm.‘24h) the root. A pale grey discoloration extended down on potato dextrose apar and wheat seminal roots. the root and beyond the furthest extent of runner (r = 1,312.r+ 1.096 (a) y = 0.676.x- 0,429 (b) r = O-94). hyphae in’the cortex.
J. HOLDEN
112 Table
2. Growth
up and down
seminal
roots
from an inoculum
Number of roots examined G. graminis var. tritici
GTl
15
P. radicicola
PRl
20
PG5
30
var. radicicola
P. radicicola var. graminicola
Fig. 2. Diagram of part of a cross section of a first seminal root of wheat showing layers of cortical cells (l-6). the endodermis (e), the pericycle (c), protoxylem (x), metaxylem (m). and phloem. consisting of groups of three cells (a). Exumination
Growth up or down
layer buried Mean extent of runner hyphae
7 cm beJoN the seed Mean extent of chocolatebrovvn stelar discoloration
(mm)
(mm)
up Down 50,; LSD up Down So/:, LSD up
576 549 9.0 426 41.0 63 31.5
48.3 0.3 5.3 0 0
Down 10 LSD 5”’
323 2.6
0
0
Mean hyphal density 7 mm from the inoculum 14.2 13.8 3.0 12.9 13.5 34 26.9
‘76 4.0
colonization and host response within the cortex. The patterns of growth of G. graminis var. rritici. P. radicicola var. radicicola and P. radicicola var. gum~inicokr up and down the root cortex in Experiment 2 are summarized in Table 3. Growth up the root cortex was identical to that down for both varieties of P. radicicola and for G. graminis var. tritici they differed only slightly (>30 mm from inoculum). The pattern of growth of all isolates of these three varieties down roots in Experiment 1 was similar to that described in Table 3 except within the top 4 mm of root below the seed which showed a more marked reaction and resistance to penetration (Table 4). Further experiments using different positioning of inoculum layers confirmed the unique reaction of this region; everywhere else penetration within an inoculum layer was as described in Table 3. The single isolate of G. graminis var. graminis in Experiment 1 had produced a similar pattern of growth to the isolates of P. radicicola var. radicicola: the isolate of L. narmari was also similar within the top 4 mm of root and outside this region similar events occurred but
of root sections
At least five first seminal
roots infected by each fungal isolate and five uninoculated roots from the controls were examined from Experiments l-2. Root structure is shown in Fig. 2; tissues are named
according to Avery (1930). The cortex, excluding the endodermis which is considered with the stele, consisted of generally six cell layers referred to by numbers l-6 where layer 1 is the epidermis. In uninoculated roots all cortical cells had thin walls and there was no blockage of cortical intercellular spaces. Xylem vessel walls had a pale yellow colour and all other walls were colourless. Some cortical cells contained bacterial colonies and, more rarely. aseptate fungal hyphae, and some cells in the outer cortex had collapsed. This suggests that these cells were dead and colonized by soil saprophytes. Roots inoculated with any of the fungi had cortical cells within the inoculum densely packed with hyphae. The density of penetration decreased with increasing distance from the inoculum until, close to the maximum extent of colonization, most cells contained no hyphae but hyphae, sometimes numerous. travelled longitudinally in the intercellular spaces. Each fungal variety gave a different
pattern
of hyphal
Fig. 3. A section taken 6mm from the seed on a root inoculated with isolate PG5 of P. dictcola var. yrumirricola. Cell layers l--5 are colonized by dark penetration hyphae and most intercellular spaces contain one or two cortical runner hyphae. ( x 350) Figs. 3-7 Cross sections of infected roots from tment I. cleared in lactophenol but not stained.
Euper-
P.
Table 3. Comparative
description
G. grarninis var. rritici GTl
Many surface and no cortical runner hyphae Penetration of cell layers l-6 by hyaline hyphae Intercellular spaces contain hyaline hyphae
No pigmented cells formed
No general thickening or browning of host cell walls Many long lignitubers (< 20 pm) in cells of all layers Many surface and no cortical runner hyphae Penetration of cell layers l-6 by hyaline hyphae
Intercellular spaces rarely contain any hyphae but these always hyaline No pigmented cells formed
No general thickening or browning of host cell walls Lignitubers becoming fewer and shorter (< 15 pm) and restricted to cell layers 2-6
rudicicolo and G.
graminis
on
wheat seminal roots
113
of typical cortical infection and host response up and down seminal roots from an inoculum layer buried 7 cm below the seed P. rodicicolu rudicicoh
var. PR 1 O-10 mm from inoculum Many surface and very few cortical runner hyphae Penetration of cell layers l-6 mainly by hyaline hyphae with a few dark hyphae Intercellular spaces contain hyaline hyphae and a very few dark (i.e. cortical runner) hyphae Occasional single large (30 x 20 pm) pigmented cells against the endodermis No general thickening or browning of host cell walls Few lignitubcrs (5-10 pm) in cells of any layer I@ 20 mm from inoculum Many surface and increasing number of cortical runner hyphae Penetration of cell layers l-6 mainly by dark with a few hyaline hyphae
P. rudicicolu gruminicolu
var. PC5
(Figs. 3. 4) Many surface and many cortical runner hyphae Penetration of cell layers l-5 by dark hyphae only. rarely of cell layer 6 Intercellular spaces contain only dark hyphae
Occasional groups of small (15 x 12 pm) pigmented cells against the endodermis (Fig. 4) No general thickening or browning of host cell walls Very few very short lignitubers (< 2 pm) in cells of any layer
Intercellular spaces contain only dark hyphae
Decrease of surface runner hyphae to none at 15-20 mm, many cortical runner hyphae Penetration of cell layers l-5 at 10-15 mm but only 2-5 at 15-20 mm by dark hyphae. rarely of cell layer 6 Intercellular spaces contain only dark hyphae
Occasional single large pigmented cells against the endodermis No general thickening or browning of host cell walls Very few lignitubers (< 3 pm) becoming restricted to cell layers 4-6
Occasional groups of small pigmented cells against the endodermis No general thickening or browning of host cell walls Very few lignitubers (< 1 pm) becoming restricted to cell layer 6
2(r30 mm from inoculum Many surface and a few cortical runner hyphae Penetration of cell layers l-4 by a few dark hyphae only, cell layers -5-6 by hyaline hyphae Intercellular spaces rarely colonized. by dark hyphae in outer cortex and hyaline hyphae in inner cortex No pigmented cells formed No general thickening or browning of host cell walls Lignitubers only in cell layers 5-6 Only cortical runner hyphae, usually between cell layers 3-4.4-5 and 5-6 Type of cell penetration differs up and down roots: up root. much penetration of cell layer 6 by hyaline hyphae and many ligmtubers formed (Fig. 7): down root. very little cell penetration. all by dark hyphae. no lignituber formation
Loss of surface runner hyphae, many cortical runner hyphae Penetration only in cell layers 56. all by dark hyphae Intercellular spaces contain many dark hyphae
Loss of runner hyphae from outer cortex, many in inner cortex Penetration rare, only in cell layers 5-6, all by dark hyphae Intercellular spaces of inner cortex only contain dark hyphae
Occasional single large pigmented cells against the endodermis No general thickening or browning of host cell walls Lignitubers only in cell layer 6
No pigmented cells formed
>30 mm from inoculum Only cortical runner hyphae, usually between cell layers 4-5 and 5-6 No cell penetration or lignituber formation
No general thickening or browning of host cell walls No lignitubers Only cortical runner hyphae, usually between cell layers 5-6 No cell penetration or lignituber formation
J. HOLDEN
114
Table 4. Comparan\e
description
of typical cortical infection and host response of the top 4 mm of >emmal roots within an inoculum layer P. ratlicicola var. rudicicola PRl PR2 PR4 (Fig. 6)
P. rtrtiic?coltr \ar. grtrrtlinrcwlo PCX PG5 PG7
Many surface and \ery few cortical runner hyphae. all between cell layers l-2 Penetration of cell laqer 1 only, always by dark hyphae
General thickening and browning of host cell wails and blockage of most inter-
Many surface and very few cortical runner hyphae, all between cell layers l-2 Penetration of cell layer 1 by dark and hyaline hyphae and cell layers 2-4. 2-S or 2-6 by hyaline hyphae Unblocked intercellular spaces contain dark hyphae between cell layers l-2 and hyaline hyphae elsewhere Very occasionally single large pigmented cells against the endodermis General thickening and browning of host cell walls and blockage of most inter-
cellular spaces
cellular spaces
Many long lignitubers (< 30 pm) in cells of any layer
Few lignitubers in cell layer 1, many long lignitubers (< 30 pm) in cell layers 2-6
G. yraminis var. rritici GTI GT2
Many surface and no cortical runner hyphae
Penetration of cell layers l-6 by hyaline hyphae
Unblocked intercellular spaces contain hyaline hyphae
No pigmented cells formed
(Fig. 5)
Intercellular spaces between cell layers l-2 contain dark hyphae. no intercellular hyphae elsewhere Groups of small pigmented cells in cell layer I No general thickening nor browning of host cell walls
nor blockage of intercellular spaces Short lignitubers ( < 10 Ltm) in cell layer 2 only
over a shorter length of root which is presumably related to its slower growth rate. Due to the basic similarity in cortical colonization by these three fungi only one, P. radicicolu var. rudicicola, was chosen for further study. Interpretation of the results in Tables 34 is simplified by distinguishing two sources of variation: (a) the differences between the fungi penetrating close to the inoculum, and (b) the progressive changes in colonization by each fungus away from the inoculum. Close to the inoculum a young cortex was invaded by each fungus at a high inoculum potential. Two factors, fungal infectivity and host resistance, appar-
ently determined the depth of invasion. Fungal infectivity was in the order G. graminis var. tritici > P. radicicola var. radicicola > P. radicicola var. graminicola, agreeing with Deacon’s (1974) assessment of their relative pathogenicities. Thus in the top 4 mm of the root (Table 4) G. graminis var. rririci penetrated the stele, P. radicicola var. radicicola was checked at the endodermis or in the inner cortex and P. radicicola var. graminicola usually at cell layer 2. Elsewhere (Table 3) G. graminis var. tritici penetrated the stele, P. radicicola var. radicicola penetrated cell layer 6 but not the endodermis and P. radicicola var. graminicola only rarely penetrated cell layer 6.
Fig. 4. A section taken IOmm from the seed on a root inoculated with isolate PG5 of P. rudic~ico/u var. qruminicolu. One ceil in layer 6 of the cortex contains a group of pigmented fungal cells. Two small lignitubcrs (I) occur
Fig. 5. A section taken 2 mm from the seed on a root inoculated with isolate PG7 P. r&cicoIc/ var. qrcminicolu. Three epidermal cells contain dark hyphae and pigmented fungal cells. Lignitubcrs (I) surround two narrow penetration hyphac under these. ( x ISOOI
P. rudicicolo and G. gruminis on wheat seminal rooi‘
Fig. 6. A section taken 2 mm down a root inoculated with isolate PRl of P. rudicicola var. rudicicolu. Cortical cel1 layers 24 or 2-5 contain hyaline penetration hyphae and long lignitubers (1). General wall thickening and browning occurs in cell layers 2-6. blocking most intercellular spaces. One cell (a) appears dark because it has been sectioned close to a thickened discoloured end wall. (x 500)
Three types of host reaction to cortical invasion were seen: wall thickening and browning both in the top 4 mm of root only (Fig. 6) and lignitubers (fingershaped wall ingrowths surrounding infection hyphae) (Figs. 47). There was no direct evidence for a role of the first two in resistance, although they occurred only in the most resistant region of the root. However there was much evidence for a major role of lignitubers. Infection hyphae within lignitubers were always markedly constricted compared with other penetration hyphae. Growth-checking of the varieties of P. radicicola was always associated with lignitubers surrounding infection hyphae passing into uninvaded cells (Figs. 4-6) and, although G. graminis var. tritici was never effectively checked in the seminal root cortex despite marked lignituber formation, effective checking in the culm bases of older plants was found to be associated with lignituber formation by Robertson (1932). Wood’s (1967) suggestion that lignitubers are only a wound response enclosing hyphae after they have been checked by some other mechanism is unlikely to be correct for wheat roots, because checked hyphae or hyphae as narrow as those within lignitubers were never seen except within lignitubers. Thus lignitubers are considered a major resistance mechanism of cortical cells. although the arguments to be used below would still apply with only slight modification if Wood’s hypothesis were true. The average length of lignitubers varied with the infectivity of the inducing fungi: they were long for G. grahis var. rritici. more variable but generally shorter for P. radicicola var. radicicola and very short for P. radicicola var. graminicola. For all fungi there was a close correlation between the length of a lignituber and the hypha within it. long lignitubers never containing short hyphae. These observations suggest that a long lignituber results from a strong fungal penetration attempt and a prolonged production of
115
lignituber materials by the host, and a short lignituber from a weak fungal penetration attempt requiring only a less prolonged host response to check its further growth. The alternative explanation for a short lignituber, that it is due to a strong penetration attempt by a fungus adapted to induce only a weak host response was discounted, because it was observed that the short lignitubers induced by P. radicicola var. graminicolu were never outgrown, all successful penetration occurring in the complete absence of wall reaction. For all fungi the colour of penetration hyphae was related to the frequency and length of lignitubers. This was well illustrated by P. radicicola var. radicicola which penetrated close to the inoculum by a mixture of hyaline and dark hyphae, the former often and the latter rarely associated with lignituber formation. The development of dark cortical runner hyphae depended on radial cortical penetration by dark hyphae in the first place. With increasing distance from the inoculum. and varying with the growth rate of each isolate and hence with the age of the cortex when colonized, progressive changes occurred in the pattern of lignituber formation and hyphal penetration (Table 3). For P. radicicola var. radicicola the frequency and length of lignitubers declined markedly within 10 mm of the inoculum and growth became almost identical with that of P. radicicola var. graminicola, with all penetration by dark hyphae and an increase in the number of cortical runner hyphae. The decline in lignituber formation was less marked for G. graminis var. tritici and no dark penetration hyphae occurred within 20 mm of the inoculum. Beyond that lignituber formation became progressively restricted to the inner cortex and this was closely paralleled by the first penetration of cells by dark hyphae and the formation of the first cortical runner hyphae by G. graminis var. tritici. This occurred up the root as well as down
Fig. 7. Cross section taken 35 mm from the inoculum towards the seed on a root infected with isolate GTI of G. grminis var. rritici in Experiment 2. Hyaline hyphae (h) associated with a group of cortical runner hyphae (c) have penetrated ceils in layer 5, layer 6 inducing lignitubers in endodermis and stele. Walls and contents of the endodermal and stelar cells are discoloured chocolate brown I x 1200)
J. HOLDEN
116 Table 5. Comparative
description of typical infection by G. graminis var. rririci and host response down seminal roots from an inoculum layer buried 7 cm below the seed
Induced up and
Down
Up (Fig. 7) In the stele. C-50 mm from inoculum
Hyphae growing radially into stele only in the 10 mm below the inoculum All tissues colonized by hyaline hyphae
Hyphae growing radially into stele along whole length (i.e. 50 mm) All tissues colonized by hyaline hyphae except for some plugged vessels and cells Little longitudinal growth of hyphae, most spread across the stele
Hyphae growing longitudinally in all tissues especially xylem vessels. no growth across the stele outside the 10 mm below the inoculum Slight wall yellowing only, in endodermis and pericycle Lignitubers in endodermis and pericycle only in 5 mm below the inoculum No blockage of protoxylem vessels nor pericycle
Intense chocolate-brown discoloration of endodermis and pericycle Lignitubers in endodermis and pericycle along whole length Most protoxylem vessels and adjacent pericycle cells blocked by yellow or brown plugs
Always by cortical runner hyphae. Penetration from these into stele induces a few lignitubers in endodermis and pericycle No hyphae in stele but xylem vessels contain yellow plugs (i.e. in advance of hyphal spread up the stele). Thus viewed from the surface, roots show yellow stelar discoloration above the maximum extent of cortical runner hyphae
t4 cm long Infection ectotrophic with surface runner hyphae, hyaline penetration hyphae and chocolate-brown stelar discoloration at the base. No root tip mycelium
cells Maximum extent of growth Usually by hyaline hyphae growing longitudinally within the protoxylem vessels of the stele and inducing no lignitubers nor vessel plugging. These stelar hyphae often far exceed the extent of cortical hyphae and cause a grey stelar discoloration when viewed from the root surface
Branch Roots Seldom >03 cm long No hyphae in the cortex, stele colonized by longitudinally growing, usually dark, hyphae often protruding as root tip mycelium
and so was not due to infection weakening the root by disrupting the stele as suggested by Garrett (1970). It occurred with cortical cell ageing. Lignituber formation in ceil layer 6, however, declined down but not up the root (Table 3, >30 mm from inoculum), presumably due to the effect of stelar infection. Eventually growth by all fungi became restricted to the inner cortex. The colonization of the outer cortical cells by other soil micro-organisms suggests that cell death might be responsible for some of these progressive changes. Stelar colonization by G. graminis var. tritici and two associated phenomena, the nature of the furthest fungal growth and the colonization of lateral roots, differed up and down roots (Table 5). Growth up roots was ectotrophic relative to the stele with spread by dark runner hyphae outside the endodermis; from these hyaline hyphae invaded the stele. The furthest growth achieved was by cortical runner hyphae and all lateral roots were colonized ectotrophicly. Down the root hyphae grew into the stele only close to the inoculum and thereafter spread was usually by hyaline stelar hyphae with no further penetration of the endodermis. The furthest growth achieved was by hyphae in the xylem vessels. Lateral roots had no cortical hyphae but were colonized by stelar hyphae growing from the stele of the main root. These were often dark and protruded as root tip mycelium as
described by Nilsson (1969). Such differences were probably due to variation in resistance of the stele according to position relative to the.inoculum layer. Three steiar reactions which are probably host-resistance mechanisms were seen up roots and within 5 mm from the inoculum but no further, down roots. These were: (a) lignitubers in the endodermis and pericycle, which formed in response to hyphae penetrating from the cortex; (b) amorphous yellow plugs filling pericycle cells and blocking xylem vessels in advance of hyphae growing up the stele; and (c) chocolate-brown discoloration of cell and vessel walls and contents. Reactions similar to (b) and (c) have been implicated in checking the spread of vascular pathogens in several hosts (Beckman, 1966). There the plugging has been attributed to the swelling of cell wall pectin and hemicellulose (Beckman and Zaroogian. 1967), and the brown coloration to oxidized phenolic substances, which increase the resistance of pectinaceous walls and plugs to rupture by fungi (Beckman et al., 1974). No chemical characterization of the plugs or brown pigments in wheat was attempted here but they are possibly similar in nature and function. In an attempt to test whether the differences between growth up and down roots were due to the effects of stelar disruption removing resistance to downwards fungal spread, the growth of isolate GTl
P. radicicola and G. gruminis on wheat seminal roots
117
Table 6. Time-course of penetration of first seminal root cortices, as shown by shift in frequency distribution cell layer penetrated in 20 sections Sample time (days) G. graminis var. rritici
CT3
P. rudicicola var. radicicola
P. rudicicola var. gruminicola
* E = endodermis,
PR1
PC5
6 11 16 21 6 11 16 21 6 11 16 21
I
of deepest
Position of deepest cell-layer penetrated 2 3 4 5 6 E*
-
-
-
-
-
-
11 1 _ 8 _
_
5 12 -
_
4 -
10 -
4 _ _ _
-
_ -
_
_
1 1 _ 1 1
8 9 8 _ 18 18
12
6
S*
_ 10 11 11 _ 1
_ _ _ -
20 20 20 _ -
1 8
_
_
-
S = stele.
from a buried inoculum layer was compared up roots severed from or still attached to the rest of the plant. The results, presented fully elsewhere (Holden, 1974), show that fungal growth and host resistance up attached roots were as described in Tables 3 and 5, whereas growth and host resistance up severed roots were identical with the growth down roots described there. It was concluded that the effects of stelar infection and root severing on host resistance and fungal growth are identical. The time course of cortical penetration
More experiments were set up to investigate further the differences between cortical colonization by G. graminis var. tritici, P. radicicola var. radicicola and P. radicicola var. graminicola. Three series of 12 jars received a 7 cm layer of sand-soil mixture and a 3 cm layer of inoculum-soil mixture (3: 1, v/v) on which four wheat seeds were sown and covered by a 1 cm layer of sand-soil mixture. Each series contained inoculum of a different fungal isolate: GT3, PRl or PG5. Three jars of each were sampled at intervals of 6. 11, 16 and 21 days. Sections were taken between l-15 cm from the seed on the first seminal roots of 10 plants per sample in each series. Two sections from each root were examined and the deepest cell layer penetrated in each of 20 sections at each sampling time was recorded (Table 6). The relative rates of cortical penetration by the three fungi agree with the previous assessment of their infectivity. The colour of penetration hyphae and the lignituber formation induced were as previously described within an inoculum layer (Table 3). The rare qf cortical cell death
Rate of cortical cell death was measured by nuclear staining (Holden. 1975) to determine how it might be affecting the pattern of fungal penetration. Plants were grown under the cultural conditions used throughout: a series of jars received a 7 cm layer of sand-soil mixture on which five presoaked seeds were sown and covered by a 1 cm layer of the same mixture. Three replicate jars were sampled at 5-day intervals from 5-30 days after sowing. The top 2 cm of each frrst seminal root with the scutellar node
attached for orientation, was stained by the Feulgen method and examined under the microscope ( x 400). Cortical nuclei were counted in three microscope fields at 2, 7 and 12 mm from the scutellar node. The proportion of cortical cells still living at each locus in the 15 roots from each time-sample was estimated by expressing the number of nuclei present as a percentage of that in the first sample at 5 days. Sections were taken at each locus and the position of the outermost nucleate cell seen in 15 sections was recorded to give an idea of the distribution of living cells. The results (Table 7) show that in most regions of the root, e.g. 7 and 12 mm from the seed, cortical cell death occurred progressively inwards and was very rapid between 5-10 days after sowing. Thereafter the rate of cell death declined and many cells in layer 6 still contained nuclei even after 30 days. However in the top 4 mm of the root, e.g. 2 mm from the seed, although the epidermal cells died, the large proportion of nuclei remaining even after 30 days indicates that little if any cell death occurred deeper in the cortex. The rate of cell death in the outer cortex in most regions of the root is rapid enough to explain the progressive restriction of lignituber formation to the inner cortex as fungi grew along the root away from an inoculum layer in Experiments l-2. Colonization of the dead cells by soil saprophytes might explain the eventual limitation of fungal growth to the inner cortex. Table 7. Rate and pattern of cortical cell death in first seminal roots of wheat. Estimated percentages of nucleate cortical cells and outermost cell layer still with nucleate cells (in parentheses) Age of plants (days) 5 10 15 20 25 30
Distance from seed (mm) 2 7 12 loo(l) 94 (1) 95 (2) 92 (2) 87 (2) 91(2)
loo(l) 40(3) 27 (5) 26 (5) 17(6) 14 (6)
100(l) 39 (4) 30 (5) 20 (5) 21 (5) 12(6)
J. HOLDES
118
A comparison of the fungal penetration within an inoculum layer previously described with the rate of cortical death found here shows that the three fungi apparently differ in their ability to penetrate living cells. Thus in the top 4 mm of the root. where only the epidermis had died, Table 4 shows that G. auraminis var. tritici penetrated all the living cell layers. P. rcldicicoln var. rudicicola penetrated some of them but P. radicicoln var. grtrminicoln penetrated only the epidermis. A comparison of Tables 6-7 shows that. between 10-15 mm down the root, the period of rapid penetration by G. grctminis var. tritici (C&6 days) preceded. whereas that by both varieties of P. radicicola (6-l 1 days) coincided with. the period of rapid cortical cell death (5-10 days). P. rudicicola var. radicicola penetrated slightly faster than P. radicicola var. graminicola, and showed a greater ability to penetrate cell layer 6 (see also Table 3) which lives longer than the rest of the cortex. These results suggest that G. graminis var. tritici is a strong parasite readily penetrating living cells, P. rudicicola var. radicicola is weaker with some ability to penetrate living cells and P. radicicola var. graminicola is very weak, penetrating cells only just before or just after death. It has been shown throughout that close to an inoculum layer G. graminis var. tritici penetrates the root cortex by hyaline hyphae, P. radicicola var. graminicola by dark hyphae and P. radicicola var. radicicola by a mixture of the two. This can be explained by postulating that when the fungi penetrate living cells they, do so by hyaline hyphae but when they penetrate senescent or dead cells the hyphae are dark in colour. I have shown (Holden, 1974) that roots inoculated with any of these fungi in sterile soil are colonized by hyaline penetration hyphae alone. This suggests that the dark colour of hyphae penetrating senescent or recently dead cells in unsterile soil is induced by other soil micro-organisms, probably allowed into the cells by hyphal disruption of their previously intact walls. DISCUSSION
of isolates of P. rudicicoltr var. yrtrmirkwltr and L. IILIVmuri (Fig. 2). This agrees with previous reports of their relative growth rates on agar (Scott. 1970: Walker and Smith. 1972: Deacon. 1974). The correlation between growth rates on PDA and wheat roots suggests that the former exhibits no differential selectivity and the latter no differential resistance to any of the fungi. Further studies of their growth on other cereals (Holden. 1974) suggests that the same is true of barley but not of rye and oats, where variety-specific and isolate-specific resistances were found. Hyphal density shows no correlation with extension growth rate. Close to an inoculum layer it was very high for P. radicicolu var. graminicoh and much lower for the other fungi. These differences depend on differing abilities of the fungi to invade living cells in a cortex where natural cell death is rapid. The most infective fungus G. graminis var. tritici. was able to penetrate the living cortical cells and their intercellular spaces well in advance of other soil micro-organisms and hence by hyaline hyphae. which are not included in hyphal density counts because they are invisible in unstained roots. The least infective fungus, P. radicicola var. graminicola could only penetrate cells when they were senescent or dead and thus it colonized cells and intercellular spaces in association with other soil micro-organisms and hence by dark hyphae. P. radicicola var. radicicola was intermediate between the other two fungi in all these respects. The similarity of G. gruminis var. yraminis and L. narmuri to P. radicicola var. radicicola in their colonization of the root cortex in Experiment 1 suggests that these two fungi also possess the intermediate level of infectivity. The question arises as to the basis of this difference in infectivity. Garrett (1970) suggested that specialization that results in a more harmonious host-parasite relationship is often characterized by a slower growth rate and a lower production of extracellular enzymes and toxins. The slow growth of P. radicicoln var. graminicola may be partly responsible for its very low infectivity but there is no obvious relationship between growth rate and infectivity among the other fungi studied. It has been claimed that cell invasion by G. graminis var. tritici is dependent on enzyme production rather than mechanical pressure (Weste. 197Oa. 1970b; Holland and Fulcher. 197 1) or toxin production (Weste. 1972) and that low virulence is related to low cellulase production in culture (Pearson, 1974). However. in culture, P. radicicola var. graminicolu has a cellulolytic ability generally greater than that of G. graminis var. tritici (Balis. 1970; Garrett, 1971. 1974) which suggests either that cellulase is not a primary determinant of infectivity in these fungi or that differences in cellulase activity in culture bear little relationship to the situation during parasitism because of enzyme suppression in host tissue. This needs further study.
Although all the fungi included in this study are obligate parasites growing on the roots of wheat (and of other cereals and grasses) as similar-looking dark runner hyphae, considerable differences occur in their infection-habits. Such differences must affect their ecology on wheat crops in the field. The growth of each isolate on roots was described here by three major characteristics: (1) ability to penetrate the stele; (2) extent of runner hyphae in the cortex: and (3) density of runner hyphae in the cortex. The fungi separate into three groups on ability to penetrate the stele. G. gruminis var. tritici always penetrated and extensively colonized the stele, P. rudicicolu var. rudicicolu, G. yruminis var. yraminis and L. nurmuri rarely penetrated and never extensively colonized the stele and P. rodicicolu var. ~~rtrminicoltr never penetrated the stele under natural conditions. wish to thank Professor S. D. GarThese are the groupings described by Deacon (1974). Acknowlrtlyenlenfs-I rett and Dr. D. S. Ingram who supervised the work. and extended here to include L. ntrnnaui. the Science Research Council for the award of a Research Growth rate of runner hyphae is positively correlated with radial growth rate on PDA; isolates of G. grumini,s var. tritici. G. gruminis var. yuminis and P. rudicicoku var. rudicicolu grew at 2-3 times the rate
Studentship. My thanks are also due to Dr. J. W. Deacon for much valuable advice and discusslon and to Mr. J. Walker and Dr. J. W. Deacon for supplying the fungal cultures used.
P. radicicolu and G. yruminis on wheat seminal roots REFERENCES
Astm~ M. J. C. (1972) Effect of Ophioholus graminis infection on the assimilation and distribution of 14C in wheat. Ann. a&. Biof. 72, 161-167. AVERYG. S. (1930) Comparative morphology of embryos and seedlings of maize oats and wheat. Bar. Guz. Ss, l-39. BALISC. (1970) A comparative study of Phialophora radicicola, an avirulent fungal root parasite of grasses and cereals. Ann. uppl. Bid. 66, 59-73. BECKMANC. H. (1966) Cell irritability and localization of vascular infections in plants. Pb~tapatho~~~ 56, 821-824. BECKMANC. H., MUELLERW. C. and MACE M. E. (1974) The stabilization of artificial and natural cell wall membranes by phenolic infusion and its relation to wilt disease resistance. Phytopatholog) 64, 1214-1220. BECKMANC. H. and ZARCXXIANG. E. (1967) Origin and composition of vascular gel in infected banana roots. Phytopatho~og~ 57, 1l-13. CLARKS~ND. T., DREW M. C. and SANDERSON J. (1973) Effect of the take-all fungus. Gaeu~nnomyces graminis, on nutrient transport by wheat. Laboratory studies. Report Agricultural Research Council Letcombe Lahorutory, 1972. 16-19. DEACONJ. W. (1973a) Phialophora radicicola and Gaeumannomyces graminis on roots of grasses and cereals. Trans. Br. mycol. Sot. 61, 471-485. DEACONJ. W. (1973b) Control of the take-all fungus by grass leys in intensive cereal cropping. Pl. Path. 22, 88-94.
DEACONJ. W. (1974) Further studies on Phiafophora rudicicola and Gaeumannom~ces graminis on roots and stem bases of grasses and cereals. Trans. Br. mycol. Sot. 63, 301-321.
DENNISR. W. G. (1944) Occurrence of OphioboQs gruminis var. avenae on wheat crops in the field. Ann. appt. Biol. 31, 100-101. FELLOWSH. (1928) Some chemical and morphological phenomena attending infection of the wheat plant by bphiobolus graminis. 2. agric. Res. 37, 647-661.-
_
GARRETTS. D. (1934)/ Factors affecting the severitv of takeall. I. The importance of micro-org&isms. J. Dep. Agric. S. Aust. 37, 664-6’74. GARRET S. D. (1936) Soil conditions and the take-all disease of wheat. Ann. appt. Biol. 23, 667-699. GARRETT S. D. (1970) Pathogenic Root-infecting _ _ Fungi. Cambridge University Press: GARRETTS. D. (1971) Effects of nitrogen level on survival of Phialophora radicicola and Coihliobolus satitus in pure culture on cellulose. Trans. Br. mycol. Sot. 57, 121-128.
II9
GARRETTS. D. (1974) Cellulose decomposition and saprophytic survival by Phialophora rudicicola. Truns. Br. mycol. Sot. 62, 622-625.
HOLDENJ. (1974) The Biology of Ph~~f~~hor~Jradicitola. an Avirulent Root Para& of Grasses and Cereals. Ph.D. Thesis. Universitv of Cambridae. HOLDENJ. (1975) Use of~nuclear stair&g to assess rates of cell death in cortices of cereal roots. Sr~il Bid. Biothem. 7, 333-334.
HOLLANDA. A. and FULCHER R. G. (1971) A study of wheat roots infected with Ophiobohts graminis Sacc. using fluorescence and electron microscopy. Aust. J. hiot. Sci. 24, 819-823.
MCKEENW. E. (1952) Phiu/ophora radicicolu Cain, a corn rootrot pathogen. Can. J. Bat. 30, 344-347. NILS~ONH. E. (1969) Studies of root and foot rot diseases of cereals and grasses. I. On resistance to Ophiobolus graminis Sacc. Lantbr Hogsk Annlr. 35, 275-807. NILWN H. E. (1972) The occurrence of lobed hyphopodia on an isolate of the take-all fungus, Ophiobolus gruminis Sad., on wheat in Sweden. Swed. J. agric. Res. 2, 105-118.
PEARSONV. (1974) Virulence and celtulolytic enzyme activity of isolates of Gueumunnomyces gruminis. Truns. Br. mycol. Sot. 63, 199-202. ROBERTWNH. T. (1932) Maturation of foot and root tissue in wheat plants in relation to penetration by Ophiobolus graminis !&cc. Scient. Agric. 12, 575-592. SCOTT P. R. (1970) Phialophora radicicoia, an avirulent parasite of wheat and grass roots. Trans. Br. mycoi. See. ss, 16>167. TURNERE. M. (1940) Ophiobofus graminis Saec. var. uwnae var. n., as the cause of take-all or whiteheads of oats in Wales. Trans. Br. mycol. Sot. 24, 269-281. WALKERJ. (1972) Type studies on Gaeumannomyces graminis and related fungi. Trans. Br. mycol. Sot. 58, 427-457.
WALKERJ. and Snnrt-r A, M. (1972) ~ptosp~eria narmari and L. korrae spp. nov., two long-spored pathogens of grasses in Australia. Trans. Br. myco2. Sot. !%I, 459-466. WES?E G. (1970a) Extracellular enzyme production by various isolates of Ophiobolus graminis and Ophiobolus graminis var. avenae. I. Enzymes produced in culture. Phytopath. Z. 67, 189-204.
Wnsm G. (1970b) Extracellular enzyme production by various isolates of Ophiobofus graminis and Ophiobo~us gr~~nis var. avenae. II. Enzymes produced within the host tissue. Phytoparh. Z. 67; 327-j36. WES‘~EG. 09721 The urocess of root infection bv Ophiobolus g&minis. Trans. Br. mycol. Sot. 59, 133-lk7.
WOODR. K. S. (1967) Physiological Plant Pathology. Blackwells, Oxford.
on the coleoptiles and roots of wheat seedlings. Fungal growth was measured as the extent and density of dark runner hyphae. All except P. radicicolu var. graminicola grew on coleoptiles and all grew on roots although only G. graminis var. tritici extensively colonized the root stele. Growth rate on roots was positively correlated with that on agar, P. radicicola var. graminicaku and L. nurmuri growing at about half the rate of the other fungi; hyphal density was high for P. rudicico/a var. yruminicolu but relatively low for the other fungi. For P. radicicala var. radicicalu, P. rudicicolu var. yruminicolu and G. gruminis var. tritici growing from buried inocula, the extent and density of hyphae up roots towards the seed was similar to that down, but G. graminis var. tritici caused chocolate-brown stelar discoloration up roots only. Root invasion by P. radicicola var. radicicola, P. radicicolu var. graminicolo and G. yraminis var. rritici was described from sections. Each gave a different pattern of hyphae and host response within an inoculum layer. and progressive changes occurred away from the inoculum. Studies of the rate of penetration by eabh fungus and the rate and pattern of death’of cortical cells explained the differences between fungi. G. graminis var. tritici penetrated living cells in advance of other soil micro-organisms, and hence by hyaline hyphae inducing much lignituber formation as a host resistance reaction. P. radicicola var. yraminicola penetrated only senescent or dead cells in association with other soil microorganisms, and hence by dark hyphae, inducing little lignituber formation. P. radicicoku var. radicicala was intermediate in all these respects. The high hyphal density of P. radicicola var. yraminicola was due to the colonization of cortical cells and spaces by dark, clearly visible, rather than hyaline hyphae, which are invisible in unstained roots. Cell death in the outer cortex explained the observed progressive restriction of growth’ by all fungi to the inner cortex with increasing distance from the inoculum. Spread by G. graminis var. tritici up roots was ectotrophic relative to the stele but down roots hyphae spread rapidly within the stele. Stelar reactions suggested as resistance mechanisms occurred up roots only. Their absence down roots is attributed to infection disrupting stelar transport.
INTRODUCTION Phialophora radicicola Cain and Gaeumannomyces graminis (Sacc.) Arx & Olivier are ecologically obli-
gate parasites which grow on the roots of grasses and cereals as similar-looking dark runner hyphae. Two varieties of P. radicicola were recognized by Deacon
(1974). var. radicicola and var. graminicola Deacon. Three varieties of G. graminis were recognized by Walker (1972), var. graminis, var. tritici Walker and var. arenae (E. M. Turner) Dennis. G. graminis var. fritici. the wheat take-all fungus, has long been recognized as a cosmopolitan and serious pathogen on wheat. The host ranges and distributions of the other fungi are not yet completely known, but all are reported from wheat: e.g. Deacon (1973a 1973b, 1974) for P. radicicola var. graminicola and P. radicicola var. radicicola: Nilsson (1972) for G. graminis var. graminis: and Dennis (1944) for G. graminis var. arenae.
Deacon (1974) divided these fungi into three groups based on three aspects of their growth on wheat seed-
* Present address: Glasshouse Crops Research Institute. Littlehampton. Sussex. BN16 3PU. 1 much regret to ha\e to notify the death of Dr. John Holdrn on 10 September 1975-s. D. Gnwrr.
lings, viz: pathogenicity to roots resulting from stelar invasion, the ability to grow on stem bases. and the type of growth cessation structures formed on stem bases (hyphopodia) and within roots (pigmented cells). G. graminis var. tritici and G. graminis var. auenae are highly pathogenic to roots and grow well on stem bases. They form small unlobed hyphopodia, singly or in cushions, but no pigmented cells. G. graminis var. graminis and P. radicicola var. radicicola are rarely pathogenic to roots but grow well on stem bases. They form large lobed hyphopodia and single large pigmented cells. P. radicicola var. graminicola is non-pathogenic on roots and grows very poorly on stem bases. It forms groups of small pigmented cells. Detailed information about the progress of root infection exists only for fungi in the first group. That typically described (Fellows, 1928; Robertson, 1932; Garrett, 1934: Turner, 1940) consists in longitudinal spread by wide. dark. superficial runner hyphae with narrower. hyaline penetration hyphae growing radially into the cortex and stele. Garrett (1970) termed this ‘ectotrophic’ infection and suggested that host resistance prevented the longitudinal spread of hyphae within the root. Non-ectotrophic infection by dark
109
J. HOLLIES
110
intercellular hyphae within the cortex (Garrett. 1934) or stele (Nilsson, 1969) has been recorded well down roots first infected close to the seed. Garrett (1970) suggested that in these cases host resistance was probably reduced by stelar disruption caused by infection. The infection of root cortices by the varieties of P. radicicola has been described as identical with ectotrophic infection by G. graminis (Mckeen, 1952: Scott. 1970) but this was not borne out in my preliminary studies. The present investigation was initiated to compare in more detail the infection of wheat seedlings, and particularly of their seminal roots, by P. radicicola var. radicicoia, P. rudicicola var. graminicola and G. graminis var. tritici. G. graminis var. graminis and Leptosphaeria narmari Walker & Smith (1972), another fungus described as having a similar infection habit, were included initially for comparison.
MATERIALS
AND METHODS
Fungi
The suppliers of the fungal isolates used were Dr. J. W. Deacon (School of Agriculture, University of Edinburgh) for P. radicicola var. radicicola, P. radicicola var. graminicola and G. graminis var. tritici and Mr. J. Walker (Biological and Chemical Research Institute, Rydalmere, N.S.W., Australia) for G. graminis var. graminis and L. narmari. All agreed with the published diagnoses and remained fully infective throughout. They are listed below with the supplier’s notation, if any (in parentheses), and the host and location of origin. P. radicicola var. radicicola, PRA (G6), maize, Coggeshall, Essex, PR2 (P16). maize, Trumpington, Cambs., PR4 (P18). barley, Rothamsted, Herts.. P. radicicola var. gruminicola. PG4 and PG5, grass turf, Cambridge. PG7 (P9). grass ley. Jealott’s Hill. Berks., G. gruminis var. tritici, GTl (Gl). wheat. Trumpington, Cambs.. GT2 (G8). wheat. Jealott’s Hill, Berks., GT3 (G2), wheat. Boxworth. Cambs.. G. graminis var. graminis. GG 1 (Dar 17498) Pennisetum clundestinum, Kempsey, N.S.W. L. narmuri, LNl (Dar 20806) P. clandestinum. Cobar. N.S.W. Plant culture and inoculution
To favour infection (Garrett, 1936: Balis. 1970) and facilitate the washing out of roots, wheat plants (cv. Cappelle-Desprez) were grown in sand-soil mixture prepared by blending 3 vol dry sand with 1 vol airdried soil (2.4 mm-sieved loam from the University Botanic Garden). The mixture had a saturation capacity of 25 ml water/100 g and a pH of 7.4. Fungal cultures on 3;d (w/w) maizemeal in sand were used as inoculum. In all experiments controls were set up with the inoculum layer replaced by uninoculated sand soil mixture. Details of the soil containers and inoculum and seed positioning used in different experiments are described in their context. Incubation was at laboratory temperature (mean 20°C) and at a soil moisture content of 55-60:‘. saturation. Sctmpling methods
Plants were washed free of sand-soil mixture and preserved in 70”:, alcohol. They were examined by reflected light in a shallow dish of water over a white
tile background under the stereoscopic microscope ( x 50 and x 100). Funpal growth on roots and coleoptiles \vas assessed ( I) as the furthest extent of dark runner hyphae from the point of inoculation (Garrett. 1936) and (2) as the density of h~phne at a measured distance from the point of inoculation. assessed as follows. A line across the microscope eyepiece was rotated until it cut across the root or coleoptile at rieht angles to its axis. All dark runner hyphae thus viiible and cut by this line \vere counted to give a number referred to as the hyphal density’. Fungal penetration of roots was described from thin free-hand sections taken at measured distances from the point of inoculation. by laying roots along a microscope slide over squared paper and cutting downwards with a razor blade. They were cleared and stained by warming in 0.05”,, trypan-blue in lactophenol and mounted in glycerine jelly. This procedure stained hyaline hyphae blue thus distinguishing them from naturally dark hyphae. In describing fungal growth in the root cortex Garrett’s (1934) terminology is used with slight modification. Dark hyphae growing longitudinally are termed ‘runner hyphae’. Two sorts are recognized. ‘surface’ and ‘cortical’, the latter being those formed in intercellular spaces within the cortex. The unqualified term, ‘runner hyphae’, refers to both types. Hyaline or dark hyphae growing radially or obliquely radially within cells are termed ‘penetration hyphae’.
RESULTS
Experiment
1. Growth down root.s trntl up coleoptiles
Growth over wheat seedlings was measured for all isolates except GT3. For each. three replicate jars (6 cm dia x 12 cm) received a 5 cm layer of sand-soil mixture covered by a 0.5 cm layer of maizemeal-sand inoculum. Six presoaked wheat seeds were then sown and covered with a further 0.5 cm layer of inoculum and then with a 5 cm layer of sand-soil mixture. Plants were sampled after 15 days and measurements made of the extent of runner hyphae and of their density 7 mm from the seed. on the coleoptiles and first seminal roots (Table 1). To allow for the depth of inoculum, 5 mm was subtracted from the measured extents. All the fungi grew on roots and coleoptiles as similar-looking dark runner hyphae. The varieties of P. rtrdicicolct and G. yrctminis grew as described by Deacon (1974) in his ecological groupings. outlined above. Only isolates of G. grarninis var. tritici caused extensive stelar infection on roots with an intense chocolate-brown discoloration in the top 5lOmm and a paler discoloration below. which extended beyond the furthest extent of runner hyphae in the cortex. Growth of main and branch roots was checked only by this fungus. presumably due to infection disrupting stelar transport (Asher. 1972: Clarkson er 01.. 1973). L. rwmtrri only rarely penetrated the root stele. It grew densely on stem bases, forming circular infection cushions 300 Ltrn in diameter as described by Walker and Smith (1972). Similar structures were formed in root cortices against the endodermis. The lack of pathogenicity of L. rwmtrri to roots disagrees with the report of Walker and Smith (1972). although A.
P. raclicicoiu
and G. gruminis on wheat seminal roots
Ill
Table 1. Growth of runner hyphae on seedlings inoculated at the seed (Means of fifteen plants &S.D.) Coleoptiles
G. graminis
var. rritici
GTI GT2
G. graminis var. grrtminis P. radicicola var. rctrlicicolo
P. ra~~cicolu var. grumi~icola L. narmari
GGl PRl PR2 PR4 PG4 PGS PG7 LNl
First seminal roots
Extent (mm)
Density
Extent (mm)
Density
48.7 + 9.1 60.3 * 11.2 46.3 k 8.9
27.1 f 10.3 38.5 + 18.1 66.4 + 8.4
44.3 + 13.7 57.4 i_ 15.9 51.6 i 7.2
22.5 + 3.2 22.0 + 6.4 1.5.8 + 4.6
26.3 + IO.9 71.3 + 15.4 52.8 + 7.6 64 (8)* 4.2 (6) 3.5 (2) 8.4 + 24
27.7 f 12.1 69.1 + 17.9 58.3 f 11.4 4.3 2.1 l-1 58.3 i 18.9
53.4 & 5.0 66.4 + 20.1 57.3 * 5.4 23.2 & 4.5 21.9 & 4.8 19-1 + 5.0 18.8 * 1.4
22.3 rt 6.1 29.1 -f. 6.7 20.3 + 5.9 42.9 _+7.8 423 * 9.5 49.9 + 9.5 29.2 i 8.1
* Numbers in parentheses are coleoptiles infected out of fifteen. M. Smith (1973. personal communications agreed that severe disease is rare unless host plants are weakened or grown under aseptic conditions. Table 1 shows that the isolates of P. radicicolu var. graminicola and L. narmari gew more slowly on roots than did isolates of the other fungi. When the growth rates of the isolates on roots are plotted against their radial growth rates on potato-dextrose agar (PDA) at 20°C (mean values for five plates), they show a significant positive correlation (r = f&94, P = t3001) (Fig. 1). For most isolates of P. radicicola var. radicicola, G. graminis var. graminis and G. graminis var. tritici the extent of growth on coleoptiles was similar to that on roots. Isolate PRl was exceptional in growing less far on coleoptiles than on roots, as also did the single isolate of L. n~ri. However both PRl and LNI had high hyphal densities on coleoptiles and penetrated deeply into the tissue, in contrast to all isolates of P. radicicola var. graminicola which were obviously unable to infect coleoptiles and produced either no growth or only a few superficial hyphae. On roots there was no obvious relationship between extent and density of hyphae. All the isolates of P. radicicola var. ~aminica~a had high hyphal densities. mainly due to the presence of many cortical runner hyphae. The relatively low hyphal densities of the other varieties were due to the presence of very few or no cortical runner hyphae close to the inocu-
Ium layer. On coleoptiles, where only surface runner hyphae were formed, density and extent of hyphae amongst the isolates of each fungal variety were apparently positively correlated but there was no such relationship when isolates of several fungal varieties were compared together. Experiment
2. Growth up and down roots
If stelar disruption at the point of inoculation with G. graminis var. tritici reduces host resistance to its growth down a root, as suggested by Garrett (1970), growth up a root might be expected to be slower. This possibility was examined here in a comparison between G. graminis var. tritici and the two varieties of P. radicicola, which do not infect the stele. Wheat seeds were sown in perspex boxes with sloping detachable fronts. Each box received a 7 cm layer of sand-soil mixture, a 5 mm layer of inocuium and a further 7cm layer of sand-soil mixture. A row of five wheat seeds was then sown close to the sloping faa: so that roots grew down this and could be seen passing through the inoculum. The sloping face of the box was covered with black paper to exclude light. The iirst three seminal roots of each seedling on which observations were sub~uently made, reached the inoculum layer (7 cm below the seed) in about 5 days. Three isolates, GTl, PRl and PG5 were tested with three replicate boxes of each. Roots infected with GTl-PRl were sampled at 20 days and those with PG5 at 25 days after sowing the seed. After removing the box face, roots were picked up with a pair of forceps where they passed through the inoculum layer and cut at the top and bottom. The extent and density of runner hyphae and the kxtent of intense chocolate-brown stelar discoloration were measured up (towards the seed) and down each root from the centre of the inoculum. The results (Table 2) show that each fungus produced an equal extent and density of runner hyphae up and down roots. As in Experiment 1, hyphal densities close to the inoculum were high for P. radicicola var. graminicolu and relatively low for the other fungi, and only G. graminis var. tritici penetrated the stele. Intense chocolate-brown stelar discoloration Over poiuio-dextrose ogor occurred only in the region of the inoculum and up Fig. 1. Correlation between fungal growth rates (mm.‘24h) the root. A pale grey discoloration extended down on potato dextrose apar and wheat seminal roots. the root and beyond the furthest extent of runner (r = 1,312.r+ 1.096 (a) y = 0.676.x- 0,429 (b) r = O-94). hyphae in’the cortex.
J. HOLDEN
112 Table
2. Growth
up and down
seminal
roots
from an inoculum
Number of roots examined G. graminis var. tritici
GTl
15
P. radicicola
PRl
20
PG5
30
var. radicicola
P. radicicola var. graminicola
Fig. 2. Diagram of part of a cross section of a first seminal root of wheat showing layers of cortical cells (l-6). the endodermis (e), the pericycle (c), protoxylem (x), metaxylem (m). and phloem. consisting of groups of three cells (a). Exumination
Growth up or down
layer buried Mean extent of runner hyphae
7 cm beJoN the seed Mean extent of chocolatebrovvn stelar discoloration
(mm)
(mm)
up Down 50,; LSD up Down So/:, LSD up
576 549 9.0 426 41.0 63 31.5
48.3 0.3 5.3 0 0
Down 10 LSD 5”’
323 2.6
0
0
Mean hyphal density 7 mm from the inoculum 14.2 13.8 3.0 12.9 13.5 34 26.9
‘76 4.0
colonization and host response within the cortex. The patterns of growth of G. graminis var. rritici. P. radicicola var. radicicola and P. radicicola var. gum~inicokr up and down the root cortex in Experiment 2 are summarized in Table 3. Growth up the root cortex was identical to that down for both varieties of P. radicicola and for G. graminis var. tritici they differed only slightly (>30 mm from inoculum). The pattern of growth of all isolates of these three varieties down roots in Experiment 1 was similar to that described in Table 3 except within the top 4 mm of root below the seed which showed a more marked reaction and resistance to penetration (Table 4). Further experiments using different positioning of inoculum layers confirmed the unique reaction of this region; everywhere else penetration within an inoculum layer was as described in Table 3. The single isolate of G. graminis var. graminis in Experiment 1 had produced a similar pattern of growth to the isolates of P. radicicola var. radicicola: the isolate of L. narmari was also similar within the top 4 mm of root and outside this region similar events occurred but
of root sections
At least five first seminal
roots infected by each fungal isolate and five uninoculated roots from the controls were examined from Experiments l-2. Root structure is shown in Fig. 2; tissues are named
according to Avery (1930). The cortex, excluding the endodermis which is considered with the stele, consisted of generally six cell layers referred to by numbers l-6 where layer 1 is the epidermis. In uninoculated roots all cortical cells had thin walls and there was no blockage of cortical intercellular spaces. Xylem vessel walls had a pale yellow colour and all other walls were colourless. Some cortical cells contained bacterial colonies and, more rarely. aseptate fungal hyphae, and some cells in the outer cortex had collapsed. This suggests that these cells were dead and colonized by soil saprophytes. Roots inoculated with any of the fungi had cortical cells within the inoculum densely packed with hyphae. The density of penetration decreased with increasing distance from the inoculum until, close to the maximum extent of colonization, most cells contained no hyphae but hyphae, sometimes numerous. travelled longitudinally in the intercellular spaces. Each fungal variety gave a different
pattern
of hyphal
Fig. 3. A section taken 6mm from the seed on a root inoculated with isolate PG5 of P. dictcola var. yrumirricola. Cell layers l--5 are colonized by dark penetration hyphae and most intercellular spaces contain one or two cortical runner hyphae. ( x 350) Figs. 3-7 Cross sections of infected roots from tment I. cleared in lactophenol but not stained.
Euper-
P.
Table 3. Comparative
description
G. grarninis var. rritici GTl
Many surface and no cortical runner hyphae Penetration of cell layers l-6 by hyaline hyphae Intercellular spaces contain hyaline hyphae
No pigmented cells formed
No general thickening or browning of host cell walls Many long lignitubers (< 20 pm) in cells of all layers Many surface and no cortical runner hyphae Penetration of cell layers l-6 by hyaline hyphae
Intercellular spaces rarely contain any hyphae but these always hyaline No pigmented cells formed
No general thickening or browning of host cell walls Lignitubers becoming fewer and shorter (< 15 pm) and restricted to cell layers 2-6
rudicicolo and G.
graminis
on
wheat seminal roots
113
of typical cortical infection and host response up and down seminal roots from an inoculum layer buried 7 cm below the seed P. rodicicolu rudicicoh
var. PR 1 O-10 mm from inoculum Many surface and very few cortical runner hyphae Penetration of cell layers l-6 mainly by hyaline hyphae with a few dark hyphae Intercellular spaces contain hyaline hyphae and a very few dark (i.e. cortical runner) hyphae Occasional single large (30 x 20 pm) pigmented cells against the endodermis No general thickening or browning of host cell walls Few lignitubcrs (5-10 pm) in cells of any layer I@ 20 mm from inoculum Many surface and increasing number of cortical runner hyphae Penetration of cell layers l-6 mainly by dark with a few hyaline hyphae
P. rudicicolu gruminicolu
var. PC5
(Figs. 3. 4) Many surface and many cortical runner hyphae Penetration of cell layers l-5 by dark hyphae only. rarely of cell layer 6 Intercellular spaces contain only dark hyphae
Occasional groups of small (15 x 12 pm) pigmented cells against the endodermis (Fig. 4) No general thickening or browning of host cell walls Very few very short lignitubers (< 2 pm) in cells of any layer
Intercellular spaces contain only dark hyphae
Decrease of surface runner hyphae to none at 15-20 mm, many cortical runner hyphae Penetration of cell layers l-5 at 10-15 mm but only 2-5 at 15-20 mm by dark hyphae. rarely of cell layer 6 Intercellular spaces contain only dark hyphae
Occasional single large pigmented cells against the endodermis No general thickening or browning of host cell walls Very few lignitubers (< 3 pm) becoming restricted to cell layers 4-6
Occasional groups of small pigmented cells against the endodermis No general thickening or browning of host cell walls Very few lignitubers (< 1 pm) becoming restricted to cell layer 6
2(r30 mm from inoculum Many surface and a few cortical runner hyphae Penetration of cell layers l-4 by a few dark hyphae only, cell layers -5-6 by hyaline hyphae Intercellular spaces rarely colonized. by dark hyphae in outer cortex and hyaline hyphae in inner cortex No pigmented cells formed No general thickening or browning of host cell walls Lignitubers only in cell layers 5-6 Only cortical runner hyphae, usually between cell layers 3-4.4-5 and 5-6 Type of cell penetration differs up and down roots: up root. much penetration of cell layer 6 by hyaline hyphae and many ligmtubers formed (Fig. 7): down root. very little cell penetration. all by dark hyphae. no lignituber formation
Loss of surface runner hyphae, many cortical runner hyphae Penetration only in cell layers 56. all by dark hyphae Intercellular spaces contain many dark hyphae
Loss of runner hyphae from outer cortex, many in inner cortex Penetration rare, only in cell layers 5-6, all by dark hyphae Intercellular spaces of inner cortex only contain dark hyphae
Occasional single large pigmented cells against the endodermis No general thickening or browning of host cell walls Lignitubers only in cell layer 6
No pigmented cells formed
>30 mm from inoculum Only cortical runner hyphae, usually between cell layers 4-5 and 5-6 No cell penetration or lignituber formation
No general thickening or browning of host cell walls No lignitubers Only cortical runner hyphae, usually between cell layers 5-6 No cell penetration or lignituber formation
J. HOLDEN
114
Table 4. Comparan\e
description
of typical cortical infection and host response of the top 4 mm of >emmal roots within an inoculum layer P. ratlicicola var. rudicicola PRl PR2 PR4 (Fig. 6)
P. rtrtiic?coltr \ar. grtrrtlinrcwlo PCX PG5 PG7
Many surface and \ery few cortical runner hyphae. all between cell layers l-2 Penetration of cell laqer 1 only, always by dark hyphae
General thickening and browning of host cell wails and blockage of most inter-
Many surface and very few cortical runner hyphae, all between cell layers l-2 Penetration of cell layer 1 by dark and hyaline hyphae and cell layers 2-4. 2-S or 2-6 by hyaline hyphae Unblocked intercellular spaces contain dark hyphae between cell layers l-2 and hyaline hyphae elsewhere Very occasionally single large pigmented cells against the endodermis General thickening and browning of host cell walls and blockage of most inter-
cellular spaces
cellular spaces
Many long lignitubers (< 30 pm) in cells of any layer
Few lignitubers in cell layer 1, many long lignitubers (< 30 pm) in cell layers 2-6
G. yraminis var. rritici GTI GT2
Many surface and no cortical runner hyphae
Penetration of cell layers l-6 by hyaline hyphae
Unblocked intercellular spaces contain hyaline hyphae
No pigmented cells formed
(Fig. 5)
Intercellular spaces between cell layers l-2 contain dark hyphae. no intercellular hyphae elsewhere Groups of small pigmented cells in cell layer I No general thickening nor browning of host cell walls
nor blockage of intercellular spaces Short lignitubers ( < 10 Ltm) in cell layer 2 only
over a shorter length of root which is presumably related to its slower growth rate. Due to the basic similarity in cortical colonization by these three fungi only one, P. radicicolu var. rudicicola, was chosen for further study. Interpretation of the results in Tables 34 is simplified by distinguishing two sources of variation: (a) the differences between the fungi penetrating close to the inoculum, and (b) the progressive changes in colonization by each fungus away from the inoculum. Close to the inoculum a young cortex was invaded by each fungus at a high inoculum potential. Two factors, fungal infectivity and host resistance, appar-
ently determined the depth of invasion. Fungal infectivity was in the order G. graminis var. tritici > P. radicicola var. radicicola > P. radicicola var. graminicola, agreeing with Deacon’s (1974) assessment of their relative pathogenicities. Thus in the top 4 mm of the root (Table 4) G. graminis var. rririci penetrated the stele, P. radicicola var. radicicola was checked at the endodermis or in the inner cortex and P. radicicola var. graminicola usually at cell layer 2. Elsewhere (Table 3) G. graminis var. tritici penetrated the stele, P. radicicola var. radicicola penetrated cell layer 6 but not the endodermis and P. radicicola var. graminicola only rarely penetrated cell layer 6.
Fig. 4. A section taken IOmm from the seed on a root inoculated with isolate PG5 of P. rudic~ico/u var. qruminicolu. One ceil in layer 6 of the cortex contains a group of pigmented fungal cells. Two small lignitubcrs (I) occur
Fig. 5. A section taken 2 mm from the seed on a root inoculated with isolate PG7 P. r&cicoIc/ var. qrcminicolu. Three epidermal cells contain dark hyphae and pigmented fungal cells. Lignitubcrs (I) surround two narrow penetration hyphac under these. ( x ISOOI
P. rudicicolo and G. gruminis on wheat seminal rooi‘
Fig. 6. A section taken 2 mm down a root inoculated with isolate PRl of P. rudicicola var. rudicicolu. Cortical cel1 layers 24 or 2-5 contain hyaline penetration hyphae and long lignitubers (1). General wall thickening and browning occurs in cell layers 2-6. blocking most intercellular spaces. One cell (a) appears dark because it has been sectioned close to a thickened discoloured end wall. (x 500)
Three types of host reaction to cortical invasion were seen: wall thickening and browning both in the top 4 mm of root only (Fig. 6) and lignitubers (fingershaped wall ingrowths surrounding infection hyphae) (Figs. 47). There was no direct evidence for a role of the first two in resistance, although they occurred only in the most resistant region of the root. However there was much evidence for a major role of lignitubers. Infection hyphae within lignitubers were always markedly constricted compared with other penetration hyphae. Growth-checking of the varieties of P. radicicola was always associated with lignitubers surrounding infection hyphae passing into uninvaded cells (Figs. 4-6) and, although G. graminis var. tritici was never effectively checked in the seminal root cortex despite marked lignituber formation, effective checking in the culm bases of older plants was found to be associated with lignituber formation by Robertson (1932). Wood’s (1967) suggestion that lignitubers are only a wound response enclosing hyphae after they have been checked by some other mechanism is unlikely to be correct for wheat roots, because checked hyphae or hyphae as narrow as those within lignitubers were never seen except within lignitubers. Thus lignitubers are considered a major resistance mechanism of cortical cells. although the arguments to be used below would still apply with only slight modification if Wood’s hypothesis were true. The average length of lignitubers varied with the infectivity of the inducing fungi: they were long for G. grahis var. rritici. more variable but generally shorter for P. radicicola var. radicicola and very short for P. radicicola var. graminicola. For all fungi there was a close correlation between the length of a lignituber and the hypha within it. long lignitubers never containing short hyphae. These observations suggest that a long lignituber results from a strong fungal penetration attempt and a prolonged production of
115
lignituber materials by the host, and a short lignituber from a weak fungal penetration attempt requiring only a less prolonged host response to check its further growth. The alternative explanation for a short lignituber, that it is due to a strong penetration attempt by a fungus adapted to induce only a weak host response was discounted, because it was observed that the short lignitubers induced by P. radicicola var. graminicolu were never outgrown, all successful penetration occurring in the complete absence of wall reaction. For all fungi the colour of penetration hyphae was related to the frequency and length of lignitubers. This was well illustrated by P. radicicola var. radicicola which penetrated close to the inoculum by a mixture of hyaline and dark hyphae, the former often and the latter rarely associated with lignituber formation. The development of dark cortical runner hyphae depended on radial cortical penetration by dark hyphae in the first place. With increasing distance from the inoculum. and varying with the growth rate of each isolate and hence with the age of the cortex when colonized, progressive changes occurred in the pattern of lignituber formation and hyphal penetration (Table 3). For P. radicicola var. radicicola the frequency and length of lignitubers declined markedly within 10 mm of the inoculum and growth became almost identical with that of P. radicicola var. graminicola, with all penetration by dark hyphae and an increase in the number of cortical runner hyphae. The decline in lignituber formation was less marked for G. graminis var. tritici and no dark penetration hyphae occurred within 20 mm of the inoculum. Beyond that lignituber formation became progressively restricted to the inner cortex and this was closely paralleled by the first penetration of cells by dark hyphae and the formation of the first cortical runner hyphae by G. graminis var. tritici. This occurred up the root as well as down
Fig. 7. Cross section taken 35 mm from the inoculum towards the seed on a root infected with isolate GTI of G. grminis var. rritici in Experiment 2. Hyaline hyphae (h) associated with a group of cortical runner hyphae (c) have penetrated ceils in layer 5, layer 6 inducing lignitubers in endodermis and stele. Walls and contents of the endodermal and stelar cells are discoloured chocolate brown I x 1200)
J. HOLDEN
116 Table 5. Comparative
description of typical infection by G. graminis var. rririci and host response down seminal roots from an inoculum layer buried 7 cm below the seed
Induced up and
Down
Up (Fig. 7) In the stele. C-50 mm from inoculum
Hyphae growing radially into stele only in the 10 mm below the inoculum All tissues colonized by hyaline hyphae
Hyphae growing radially into stele along whole length (i.e. 50 mm) All tissues colonized by hyaline hyphae except for some plugged vessels and cells Little longitudinal growth of hyphae, most spread across the stele
Hyphae growing longitudinally in all tissues especially xylem vessels. no growth across the stele outside the 10 mm below the inoculum Slight wall yellowing only, in endodermis and pericycle Lignitubers in endodermis and pericycle only in 5 mm below the inoculum No blockage of protoxylem vessels nor pericycle
Intense chocolate-brown discoloration of endodermis and pericycle Lignitubers in endodermis and pericycle along whole length Most protoxylem vessels and adjacent pericycle cells blocked by yellow or brown plugs
Always by cortical runner hyphae. Penetration from these into stele induces a few lignitubers in endodermis and pericycle No hyphae in stele but xylem vessels contain yellow plugs (i.e. in advance of hyphal spread up the stele). Thus viewed from the surface, roots show yellow stelar discoloration above the maximum extent of cortical runner hyphae
t4 cm long Infection ectotrophic with surface runner hyphae, hyaline penetration hyphae and chocolate-brown stelar discoloration at the base. No root tip mycelium
cells Maximum extent of growth Usually by hyaline hyphae growing longitudinally within the protoxylem vessels of the stele and inducing no lignitubers nor vessel plugging. These stelar hyphae often far exceed the extent of cortical hyphae and cause a grey stelar discoloration when viewed from the root surface
Branch Roots Seldom >03 cm long No hyphae in the cortex, stele colonized by longitudinally growing, usually dark, hyphae often protruding as root tip mycelium
and so was not due to infection weakening the root by disrupting the stele as suggested by Garrett (1970). It occurred with cortical cell ageing. Lignituber formation in ceil layer 6, however, declined down but not up the root (Table 3, >30 mm from inoculum), presumably due to the effect of stelar infection. Eventually growth by all fungi became restricted to the inner cortex. The colonization of the outer cortical cells by other soil micro-organisms suggests that cell death might be responsible for some of these progressive changes. Stelar colonization by G. graminis var. tritici and two associated phenomena, the nature of the furthest fungal growth and the colonization of lateral roots, differed up and down roots (Table 5). Growth up roots was ectotrophic relative to the stele with spread by dark runner hyphae outside the endodermis; from these hyaline hyphae invaded the stele. The furthest growth achieved was by cortical runner hyphae and all lateral roots were colonized ectotrophicly. Down the root hyphae grew into the stele only close to the inoculum and thereafter spread was usually by hyaline stelar hyphae with no further penetration of the endodermis. The furthest growth achieved was by hyphae in the xylem vessels. Lateral roots had no cortical hyphae but were colonized by stelar hyphae growing from the stele of the main root. These were often dark and protruded as root tip mycelium as
described by Nilsson (1969). Such differences were probably due to variation in resistance of the stele according to position relative to the.inoculum layer. Three steiar reactions which are probably host-resistance mechanisms were seen up roots and within 5 mm from the inoculum but no further, down roots. These were: (a) lignitubers in the endodermis and pericycle, which formed in response to hyphae penetrating from the cortex; (b) amorphous yellow plugs filling pericycle cells and blocking xylem vessels in advance of hyphae growing up the stele; and (c) chocolate-brown discoloration of cell and vessel walls and contents. Reactions similar to (b) and (c) have been implicated in checking the spread of vascular pathogens in several hosts (Beckman, 1966). There the plugging has been attributed to the swelling of cell wall pectin and hemicellulose (Beckman and Zaroogian. 1967), and the brown coloration to oxidized phenolic substances, which increase the resistance of pectinaceous walls and plugs to rupture by fungi (Beckman et al., 1974). No chemical characterization of the plugs or brown pigments in wheat was attempted here but they are possibly similar in nature and function. In an attempt to test whether the differences between growth up and down roots were due to the effects of stelar disruption removing resistance to downwards fungal spread, the growth of isolate GTl
P. radicicola and G. gruminis on wheat seminal roots
117
Table 6. Time-course of penetration of first seminal root cortices, as shown by shift in frequency distribution cell layer penetrated in 20 sections Sample time (days) G. graminis var. rritici
CT3
P. rudicicola var. radicicola
P. rudicicola var. gruminicola
* E = endodermis,
PR1
PC5
6 11 16 21 6 11 16 21 6 11 16 21
I
of deepest
Position of deepest cell-layer penetrated 2 3 4 5 6 E*
-
-
-
-
-
-
11 1 _ 8 _
_
5 12 -
_
4 -
10 -
4 _ _ _
-
_ -
_
_
1 1 _ 1 1
8 9 8 _ 18 18
12
6
S*
_ 10 11 11 _ 1
_ _ _ -
20 20 20 _ -
1 8
_
_
-
S = stele.
from a buried inoculum layer was compared up roots severed from or still attached to the rest of the plant. The results, presented fully elsewhere (Holden, 1974), show that fungal growth and host resistance up attached roots were as described in Tables 3 and 5, whereas growth and host resistance up severed roots were identical with the growth down roots described there. It was concluded that the effects of stelar infection and root severing on host resistance and fungal growth are identical. The time course of cortical penetration
More experiments were set up to investigate further the differences between cortical colonization by G. graminis var. tritici, P. radicicola var. radicicola and P. radicicola var. graminicola. Three series of 12 jars received a 7 cm layer of sand-soil mixture and a 3 cm layer of inoculum-soil mixture (3: 1, v/v) on which four wheat seeds were sown and covered by a 1 cm layer of sand-soil mixture. Each series contained inoculum of a different fungal isolate: GT3, PRl or PG5. Three jars of each were sampled at intervals of 6. 11, 16 and 21 days. Sections were taken between l-15 cm from the seed on the first seminal roots of 10 plants per sample in each series. Two sections from each root were examined and the deepest cell layer penetrated in each of 20 sections at each sampling time was recorded (Table 6). The relative rates of cortical penetration by the three fungi agree with the previous assessment of their infectivity. The colour of penetration hyphae and the lignituber formation induced were as previously described within an inoculum layer (Table 3). The rare qf cortical cell death
Rate of cortical cell death was measured by nuclear staining (Holden. 1975) to determine how it might be affecting the pattern of fungal penetration. Plants were grown under the cultural conditions used throughout: a series of jars received a 7 cm layer of sand-soil mixture on which five presoaked seeds were sown and covered by a 1 cm layer of the same mixture. Three replicate jars were sampled at 5-day intervals from 5-30 days after sowing. The top 2 cm of each frrst seminal root with the scutellar node
attached for orientation, was stained by the Feulgen method and examined under the microscope ( x 400). Cortical nuclei were counted in three microscope fields at 2, 7 and 12 mm from the scutellar node. The proportion of cortical cells still living at each locus in the 15 roots from each time-sample was estimated by expressing the number of nuclei present as a percentage of that in the first sample at 5 days. Sections were taken at each locus and the position of the outermost nucleate cell seen in 15 sections was recorded to give an idea of the distribution of living cells. The results (Table 7) show that in most regions of the root, e.g. 7 and 12 mm from the seed, cortical cell death occurred progressively inwards and was very rapid between 5-10 days after sowing. Thereafter the rate of cell death declined and many cells in layer 6 still contained nuclei even after 30 days. However in the top 4 mm of the root, e.g. 2 mm from the seed, although the epidermal cells died, the large proportion of nuclei remaining even after 30 days indicates that little if any cell death occurred deeper in the cortex. The rate of cell death in the outer cortex in most regions of the root is rapid enough to explain the progressive restriction of lignituber formation to the inner cortex as fungi grew along the root away from an inoculum layer in Experiments l-2. Colonization of the dead cells by soil saprophytes might explain the eventual limitation of fungal growth to the inner cortex. Table 7. Rate and pattern of cortical cell death in first seminal roots of wheat. Estimated percentages of nucleate cortical cells and outermost cell layer still with nucleate cells (in parentheses) Age of plants (days) 5 10 15 20 25 30
Distance from seed (mm) 2 7 12 loo(l) 94 (1) 95 (2) 92 (2) 87 (2) 91(2)
loo(l) 40(3) 27 (5) 26 (5) 17(6) 14 (6)
100(l) 39 (4) 30 (5) 20 (5) 21 (5) 12(6)
J. HOLDES
118
A comparison of the fungal penetration within an inoculum layer previously described with the rate of cortical death found here shows that the three fungi apparently differ in their ability to penetrate living cells. Thus in the top 4 mm of the root. where only the epidermis had died, Table 4 shows that G. auraminis var. tritici penetrated all the living cell layers. P. rcldicicoln var. rudicicola penetrated some of them but P. radicicoln var. grtrminicoln penetrated only the epidermis. A comparison of Tables 6-7 shows that. between 10-15 mm down the root, the period of rapid penetration by G. grctminis var. tritici (C&6 days) preceded. whereas that by both varieties of P. radicicola (6-l 1 days) coincided with. the period of rapid cortical cell death (5-10 days). P. rudicicola var. radicicola penetrated slightly faster than P. radicicola var. graminicola, and showed a greater ability to penetrate cell layer 6 (see also Table 3) which lives longer than the rest of the cortex. These results suggest that G. graminis var. tritici is a strong parasite readily penetrating living cells, P. rudicicola var. radicicola is weaker with some ability to penetrate living cells and P. radicicola var. graminicola is very weak, penetrating cells only just before or just after death. It has been shown throughout that close to an inoculum layer G. graminis var. tritici penetrates the root cortex by hyaline hyphae, P. radicicola var. graminicola by dark hyphae and P. radicicola var. radicicola by a mixture of the two. This can be explained by postulating that when the fungi penetrate living cells they, do so by hyaline hyphae but when they penetrate senescent or dead cells the hyphae are dark in colour. I have shown (Holden, 1974) that roots inoculated with any of these fungi in sterile soil are colonized by hyaline penetration hyphae alone. This suggests that the dark colour of hyphae penetrating senescent or recently dead cells in unsterile soil is induced by other soil micro-organisms, probably allowed into the cells by hyphal disruption of their previously intact walls. DISCUSSION
of isolates of P. rudicicoltr var. yrtrmirkwltr and L. IILIVmuri (Fig. 2). This agrees with previous reports of their relative growth rates on agar (Scott. 1970: Walker and Smith. 1972: Deacon. 1974). The correlation between growth rates on PDA and wheat roots suggests that the former exhibits no differential selectivity and the latter no differential resistance to any of the fungi. Further studies of their growth on other cereals (Holden. 1974) suggests that the same is true of barley but not of rye and oats, where variety-specific and isolate-specific resistances were found. Hyphal density shows no correlation with extension growth rate. Close to an inoculum layer it was very high for P. radicicolu var. graminicoh and much lower for the other fungi. These differences depend on differing abilities of the fungi to invade living cells in a cortex where natural cell death is rapid. The most infective fungus G. graminis var. tritici. was able to penetrate the living cortical cells and their intercellular spaces well in advance of other soil micro-organisms and hence by hyaline hyphae. which are not included in hyphal density counts because they are invisible in unstained roots. The least infective fungus, P. radicicola var. graminicola could only penetrate cells when they were senescent or dead and thus it colonized cells and intercellular spaces in association with other soil micro-organisms and hence by dark hyphae. P. radicicola var. radicicola was intermediate between the other two fungi in all these respects. The similarity of G. gruminis var. yraminis and L. narmuri to P. radicicola var. radicicola in their colonization of the root cortex in Experiment 1 suggests that these two fungi also possess the intermediate level of infectivity. The question arises as to the basis of this difference in infectivity. Garrett (1970) suggested that specialization that results in a more harmonious host-parasite relationship is often characterized by a slower growth rate and a lower production of extracellular enzymes and toxins. The slow growth of P. radicicoln var. graminicola may be partly responsible for its very low infectivity but there is no obvious relationship between growth rate and infectivity among the other fungi studied. It has been claimed that cell invasion by G. graminis var. tritici is dependent on enzyme production rather than mechanical pressure (Weste. 197Oa. 1970b; Holland and Fulcher. 197 1) or toxin production (Weste. 1972) and that low virulence is related to low cellulase production in culture (Pearson, 1974). However. in culture, P. radicicola var. graminicolu has a cellulolytic ability generally greater than that of G. graminis var. tritici (Balis. 1970; Garrett, 1971. 1974) which suggests either that cellulase is not a primary determinant of infectivity in these fungi or that differences in cellulase activity in culture bear little relationship to the situation during parasitism because of enzyme suppression in host tissue. This needs further study.
Although all the fungi included in this study are obligate parasites growing on the roots of wheat (and of other cereals and grasses) as similar-looking dark runner hyphae, considerable differences occur in their infection-habits. Such differences must affect their ecology on wheat crops in the field. The growth of each isolate on roots was described here by three major characteristics: (1) ability to penetrate the stele; (2) extent of runner hyphae in the cortex: and (3) density of runner hyphae in the cortex. The fungi separate into three groups on ability to penetrate the stele. G. gruminis var. tritici always penetrated and extensively colonized the stele, P. rudicicolu var. rudicicolu, G. yruminis var. yraminis and L. nurmuri rarely penetrated and never extensively colonized the stele and P. rodicicolu var. ~~rtrminicoltr never penetrated the stele under natural conditions. wish to thank Professor S. D. GarThese are the groupings described by Deacon (1974). Acknowlrtlyenlenfs-I rett and Dr. D. S. Ingram who supervised the work. and extended here to include L. ntrnnaui. the Science Research Council for the award of a Research Growth rate of runner hyphae is positively correlated with radial growth rate on PDA; isolates of G. grumini,s var. tritici. G. gruminis var. yuminis and P. rudicicoku var. rudicicolu grew at 2-3 times the rate
Studentship. My thanks are also due to Dr. J. W. Deacon for much valuable advice and discusslon and to Mr. J. Walker and Dr. J. W. Deacon for supplying the fungal cultures used.
P. radicicolu and G. yruminis on wheat seminal roots REFERENCES
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