Pseudoparenchyma produced by Cercosporella herpotrichoides in culture

[ 537 ] Trans. Br, mycol. Soc. 60 (3),537-545 (1973) Printed in Great Britain

PSEUDOPARENCHYMA PRODUCED BY CERCOSPORELLA HERPOTRICHOIDES IN CULTURE By J. W. DEACON Botany School, University ofCambridge (With Plate 48 and

I

Text-figure)

Cercosporella herpotrichoides Fron formed pseudoparenchyma in agar culture and liquid culture when growth was halted by either mechanical or nutritional means, but full development of this tissue only occurred if an adequate energy source was available. Development proceeded by the repeated cessation of growth of hyphal branch apices, followed by sub-terminal branching, to produce a compact mass of uninucleate isodiametric cells with thickened, pigmented walls. The morphology, sequence ofdevelopment and factors influencing the development of this tissue are compared with those of the 'melanized' tissues produced by some other fungi.

Cercosporella herpotrichoides Fron, the cause of eyespot lodging disease of cereals, produces both vegetative and stromatic mycelia. The latter, according to Sprague's (1931) original description, is characterized as medium to heavily walled and frequently consisting of polygonal cells that form charred masses on the bases of wheat culms. Several authors (Schaffnit, 1933; Sprague & Fellows, 1934; Lange-de la Camp, 1966; Defosse, 1967) have subsequently discussed the abundance of this tissue, both as flat plates of cells between successive leaf-sheaths and as smaller aggregates of cells in the lumina of sclerenchyma cells of the leaf-sheaths and culms. It has most frequently been called 'stroma', but this term implies subsequent spore production (Ainsworth, 1971). In this paper it will be termed 'pseudoparenchyma' to avoid unintended comparison with tissues of known function produced by other fungi. A morphologically similar tissue produced by C. herpotrichoides in culture (Dickens, 1964; Berger, 1965;) was used in survival studies on this fungus (Deacon, 1971). As a preliminary to this, its structure and development were studied in hanging-drop cultures and factors determining its production in culture were examined experimentally. As the results of this work have wider implications in morphogenesis of fungal resting structures, they are presented here. MATERIALS AND METHODS

Pathogenic isolates of C. herpotrichoides from wheat crops in Southern England were used. Development of pseudoparenchyma was followed, using drops of potato dextrose agar and water agar suspended on microscope coverslips over van-Tieghem cells. It formed exclusively in contact with the coverslip surface. When the agar was removed, pseudoparenchyma, together with

538 a(O h)

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c~

d(l ' ~

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I (9 h) ._--~

Fig. 1 . Stages in the d evel opment of pseudoparenchyma, in culture, by Cercosporella herpotrichaides, Arrows denot e structures to which sp ecific referen ce is made in the text.

Cercosporella pseudoparenchyma. J. W. Deacon

539 .~.

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q, (34h)

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r (44 h)

10 JlIl1 L.-J

Fig.

35

I.

(cont.) For legend see opposite.

MYC 60

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much of the associated mycelial network, was left attached to the coverslips. Nuclear distribution in these tissues was examined, using Giemsa's stain after hydrolysis with hot, normal hydrochloric acid for up to 60 min. In nutritional experiments on development of pseudoparenchyma, the basal medium used was: K 2H P0 4, 1 g; MgS0 4. 7H20, 0'5 g; KCI, 0'5 g; FeS04.7H20, 0'01 g; thiamin hydrochloride, o- 1 mg; agar, 20 g; distilled water, 1 1. This was supplemented with dextrose and nitrogen (as sodium nitrate) in different combinations and 17 ml agar was poured into each Petri dish. Plates were inoculated with water-agar inoculum disks and incubated in darkness at 21 °C. RESULTS

Structure and development ofpseudoparenchyma The tissue formed in culture consists ofa mass ofisodiametric, polygonal cells (6-10 JIm diam), with thickened and heavily pigmented walls, intimately associated with short lengths of the hyaline hyphae (about 3 JIm diam) from which they were formed (Pl. 48, fig. 1). On nutrient-poor media, small tissue masses are formed, often interconnected by narrow ( 1- 2 JIm), anastomosing hyphae that radiate from them and later lack cytoplasm throughout much of their length (Pl. 48, fig. 2). On richer media, however, pseudoparenchymatous groups of cells may develop abundantly and coalesce to form extensive plates of tissue. Pseudoparenchyma formed in eyespot lesions (Pl. 48, figs. 3,4) is morphologically similar to that produced in culture. Stages in the development of pseudoparenchyma in culture are shown in Fig. 1 a-rl' Development begins with cessation of growth of a main hypha, followed by branch-initiation a short distance behind the hyphal tip (Fig. 1 a-c ). Branches thus formed are of limited growth and new branches are then formed either from these branches or from the parent hypha. Repeated several times, this process results in a mass of tissue composed of short branches (Fig. 1 d-n). When a branch stops growing it swells immediately behind the hyphal tip, as described by Robertson (1958) for hyphae of Fusarium oxysporum Schlecht. Regrowth from these tips sometimes occurs later, but many remain as enlarged, angular tips of hyphae that have much shorter interseptal distances than those in actively growing hyphae, so that a mass of polygonal, isodiametric cells is formed (Fig. 1 n-r l ) . This stage is often completed within 24-48 h of initiation, by which time growth has been renewed by thinner, anastomosing hyphae, produced from the swollen, extreme tips of some of the branches that had earlier stopped growing (Pl. 48, fig. 2; arrows in Fig. 1 j, m), similar to that described for F. osysporum (Robertson , 1958). These anastomosing hyphae radiate and interconnect discrete pseudoparenchymatous clumps formed from different primary hyphae. Similar hyphae, of more limited growth, bridge individual cells of the developing tissue and give rise to further enlarged, isodiametric cells, the cytoplasm of which presumably comes from the parent cells (Fig. 1 p-rl ) . The processes of wall thickening and pigmentation proceed over several days, so that development is not complete until up to 9 days after initiation.

Cercosporella pseudoparenchyma. J. W. Deacon

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By this time, the narrow, radiating, anastomosing hyphae have formed isolated isodiametric cells, similar to those of the main body of tissue, by enlargement of short lateral branches at intervals along their lengths. The cytoplasm for these comes from the narrow hyphae, considerable lengths of which completely lose their contents.

Nuclear arrangement in pseudoparenchyma Each isodiametric cell of the pseudoparenchyma and each cell of the parent hypha that formed this tissue characteristically contained a single spherical nucleus (PI. 48, figs. 5,6). Each cell of the narrower, anastomosing hyphae also contained one nucleus, although this was often very elongated through constriction from the hyphal walls. No evidence of nuclear transfer across hyphal bridges was found, but nuclei were frequently positioned near to a bridging hypha in one of the cells involved in each fusion and they often had protrusions into these bridging hyphae. This could be explained by assuming that cytoplasmic transfer between the hyphae had occurred, the nuclei being carried passively in the direction of cytoplasmic flow until they reached a barrier. In most of the hyphal fusions observed, an obvious barrier to nuclear transfer was provided by the incomplete breakdown of end-walls of the anastomosing hyphae. Factors influencing development if pseudoparenchyma

General observations. Pseudoparenchyma was always formed in contact with a solid surface: in agar culture, where it formed in the bottom of the agar against the base of the Petri dish; in filter-paper culture, when the filter paper was adjacent to a glass surface; and at the meniscus ofliquid culture, against the containing wall. The effects of other types of solid surface were tested by inoculating the fungus on to membrane filters and nylon gauze lying on top of an agar medium that favoured development of pseudoparenchyma. It formed on both of these surfaces and, in the latter case, microscopical observation revealed that it formed exclusively in contact with the nylon threads and not at all in the interstices. However, solid surfaces, per se, were not stimulatory, because no development of pseudoparenchyma occurred when these tests were repeated using a range of common agar media. Cutting the agar surface either in advance of, or behind, the margins of growing colonies did not consistently induce pseudoparenchyma formation. However, when colonies on different nutrient agars were cut with a cork-borer behind their margins and the resulting disks left in position, a localized development of this tissue sometimes occurred, both on the isolated disks and on the main body of agar next to the cut surfaces, but only where partial drying caused the disks to shrink away from the rest of the agar. On those cut surfaces that remained touching no development occurred. On nutrient-rich media, pseudoparenchyma frequently formed, at the bottom of the agar, in a ring coincident with the limit of the central zone of aerial mycelium characteristic of this fungus. Often, a localized zone of development also occurred immediately beneath the inoculum disk, but 35-2

Transactions British Mycological Society Table I. N umbers of variously-supplemented agarplates (maximum 5) bearing pseudoparenchyma of Cercosporella herpotrichoides after incubation Supplementary nitrogen (% ) No. of weeks Supplementary of incu ba tion dextrose (%)

3·5

I

2 4 6

I

2 4

7

I

2 4

,

0

0 '01

5 5 5 5 5 5 5 5 5

0 0 0

0 '02

0 '04

0 '08

0'16

0

0 0 0

0

0

0 0 0

0 0

0

4

5

5

5

3

5

0

4

0

0 0

0 0

0 0

5 5 5

5 5

5

5

5

4 0

2

0

0

0

4

this only became noticeable when the outer, marginal zone began to appear. On nutrient-deficient media, the main zone of development of pseudoparenchyma, when present, was close to the inoculum disk but not immediately below it. Nutritional experiments. The foregoing observations suggested that nutritional factors might influence the initiation or de velopment of pseudoparenchyma. One isolate was, therefore, grown on mineral nutrient agar supplemented factorially with nitrogen, as sodium nitrate, at the levels 0, 0'01, 0'02, 0 '04, 0'08 and 0'16% and dextrose at the levels I, 2 and 4 %. Growth rate was similar on all media but colony morphology differed, the diameter of the central zone of aerial mycelium being decreased with progressively lower nitrogen concentrations and colony colour ranging from cream, with both nitrogen and dextrose, to grey-green, without nitrogen. After 3'5, 6 and 7 weeks, colonies were scored for pseudoparenchyma on a presence or absence basis (T a ble I ). Pseudoparenchyma initially formed only on colonies growing without supplementary nitrogen. Later, however, it developed (at the bottom of the agar) in a zone at or just behind the margins of colonies growing on more nutrient-rich media, the distance between this zone and the point of inoculation increasing with progressively higher nutrient levels. Thus, Table I shows that this tissue developed on more colonies with increasing time of incubation, as either dextrose or nitrogen presumably became depleted. After 7 weeks, only colonies growing with both 4 % dextrose and high nitrogen levels had, assumably, still not depleted either of these nutrients and, therefore, had not formed pseudoparenchyma. As before, the zone of pseudoparenchyma was coincident with the limit of aerial mycelium on the colonies, near or complete exhaustion of either nitrogen or dextrose presumably causing both effects. The above results were confirmed in a similar experiment, using a different isolate and different nutrient levels, including a series without supplementary dextrose. Pseudoparenchyma did not de velop in the series without supplementary dextrose, but a zone of less noticeable 'initials'

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or poorly developed groups of cells was found instead. The lighter colour of this isolate also enabled a large number of similar initials to be seen, scattered thinly over the under surfaces of most colonies irrespective of the presence or absence of definite zones of well-developed pseudoparenchyma. DISCUSSION

A tissue morphologically similar to that of C. herpotrichoides described here is commonly formed by many fungi in agar culture. It cannot be regarded as a laboratory artifact, however, because it is formed in profusion by C. herpotrichoides in the disease lesion and a similar 'platemycelium' often extends for several centimetres up stems of wheat plants severely infected by Gaeumannomyces graminis (Sacc.) Arx & Olivier, a root-pathogen. The groups of dark-walled cells produced by Phialophora radicicola Cain in the cortex of wheat and grass roots (Cain, 1952; Scott, 1969) and the pre-penetration tissues formed by many fungi on their hosts bear some resemblance to this tissue. In culture, Sclerotinia species also produce a tissue morphologically similar to that of C. herpotrichoides (Willetts & Wong, 1971), but this differs developmentally in that it is formed by repeated dichotomous branching of a hyphal apex. The mode of development of the tissues of G. graminis and P. radicicola mentioned above have not been studied but, as with sclerotia (Townsend & Willetts, 1954), a number of different modes of development will probably be found. Willetts & Wong suggest that the tissue of Sclerotinia represents sclerotial initials that have failed to develop normally. As C. herpotrichoides does not produce sclerotia such a morphogenetic comparison cannot be made. However, despite its persistence in soil for long periods of time, pseudoparenchyma of C. herpotrichoides is not functionally equivalent to sclerotia (Deacon, 1971). Results presented here indicate that pseudoparenchyma of C. herpotrichoides is produced in response to a near or complete exhaustion of either nitrogen or dextrose. The non-specificity of this nutritional effect suggests that a more basic underlying mechanism (mediated through nutritional, and perhaps other, factors) governs production of pseudoparenchyma. All of the observations are consistent with the hypothesis that either initiation or maturation of this tissue is stimulated when extension growth is halted, but that further development depends upon a supply ofdextrose. Thus, exhaustion of either nitrogen or dextrose induced pseudoparenchyma formation, but the tissue only developed abundantly in the former case, using the remaining dextrose that was no longer needed for extension growth. A halt in growth imposed by trauma due to cutting the mycelium would explain the presence of a ring of pseudoparenchyma immediately beneath the inoculum disks on agar plates, whereas physical restriction could explain its production against solid surfaces and in the positions it occupies on the host. The localized development of pseudoparenchyma where shrinkage of the agar occurred around cuts is more difficult to explain. However, Smith & Griffin (1971) have shown that very low partial pressures of oxygen exist at the bottoms of agar colonies. Increased activity of C. herpotrichoides, resulting in a localized nutrient

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depletion, might, therefore, have occurred in these tests, following exposure of cut surfaces to the air. The growth-check hypothesis is supported by the observation (Deacon, 197 I) that both C. herpotrichoides and G. graminis formed pseudoparenchyma when their growth into fresh areas of buried coleoptiles was halted by the activities of the soil microflora. It also follows naturally from a study of the development of pseudoparenchyma, involving a series of stoppages of growth followed by branching. Furthermore, it helps to explain the observation that tips of hyphae associated with pseudoparenchyma, like every other cell, were uninucleate, whereas Davies & Jones (1970) found that hyphal tips of C. herpotrichoides normally contain three nuclei. Thus, a shortage of nitrogen, or perhaps other nutrients, might limit the rate of nuclear division before it affects the rate of hyphal extension, the cytoplasm for which is supplied largely by subapical regions (Zalokar, 1959), so that nuclear number in the hyphal tip is progressively diminished until it becomes uninucleate. The above hypothesis to explain development of pseudoparenchyma was first proposed by Garrett (1970) to explain his earlier results (1949) on the effects of different carbon/nitrogen ratios on sclerotium production by Helicobasidium purpureum Pat. It is possibly applicable to resting bodies of many other fungi, because these structures are characteristically formed near the onset of unfavourable conditions and often when an energy source is still plentiful. However, other workers, e.g. Townsend (1957) and Henis, Chet & Avizohar-Hershenzon (1965), who have discussed the relationship between mycelial growth and production of sclerotia by different fungi, have not reached this conclusion, perhaps because other factors are also involved in sclerotium production by these fungi (Chet, Henis & Mitchell, 1966). I wish to thank Professor S. D. Garrett and Dr H. J. Hudson for helpful discussion, and the Science Research Council for the award of a Research Studentship, during the tenure of which this work was performed. REFERENCES

AINSWORTH, G. C. (1971). Ainsworth and Bisby's dictionary of thefungi, sixth edition. Commonwealth Mycological Institute, Kew. BERGER, R. G. (1965). Sporulation and stroma development of Cercosporella herpotrichoides. Phytopathology 55, 127 (Abstract). CAIN, R. F. (1952). Studies of fungi imperfecti. 1. Phialophora. Canadian Journal if Botany 30, 33 8-343. CHET, 1., HENIS, Y. & MITCHELL, R. (1966). The morphogenetic effect of sulphurcontaining amino-acids, glutathione and iodoacetic acid on Sclerotium rolfsii Sacco Journal of General Microbiology 45, 541-546. DAVIES, J. M. L. & JONES, D. G. (1970). The origin of a diploid 'hybrid' of Cercosporella herpotrichoides. Heredity 25, 137-139. DEACON, J. W. (1971). Survival of the eyespot fungus iCercosporella herpotrichoides and other cereal foot rot fungi on infected wheat stubble. Ph.D. Thesis, University of Cambridge. DEFOSSE, L. (1967). Etude, en conditions experimentales, des facteurs qui regissent l'inoculation et l'infection du froment par Cercosporella herpotrichoides Fron. Bulletin des Recherches Agronomiques de Gembloux N.S. II, 38-5 I.

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DICKENS, L. E. (1964). Eyespot footrot of winter wheat caused by Cercosporella her'potrichoides. Memoirs, Cornell University Agricultural Experiment Station no. 390. GARRETT, S. D. (1949). A study of violet root rot. II. Effect of substratum on survival of Helicobasidium purpureum colonies in soil. Transactions of the British Mycological Society 32, 2 I 7-223. GARRETT, S. D. (1970). Pathogenic root-infecting fungi, Cambridge University Press. HENIS, Y., CHET, I. & AVIZOHAR-HERSHENZON, Z. (1965). Nutritional and mechanical factors involved in mycelial growth and production of sclerotia by Sclerotium rolfsii in artificial medium and amended soil. Phytopathology 55, 87-91. LANGE-DE LA CAMP, M. (1966). Die Wirkungsweise von Cercosporella herpotrichoides Fron, dem Erreger der Halmbruchkrankheit des Getreides. I. Festellung der Krankheit Beschaffenheit und Infektionsweise ihres Erregers. Phytopathologische Zeitschrift 55, 34-3 6. ROBERTSON, N. F. (1958). Observations on the effect of water on hyphal apices of Fusarium oxysporum. Annals of Botany, N.S. 22, 159-174. SCHAFFNIT, E. (1933). Cercosporella herpotrichoides Fron als U rsache der Halmbruchkrankheit des Getreides. Phytopathologische Zeitschrift 5, 493-503. SCOTT, P. R. (1969). Effects of nitrogen and glucose on saprophytic survival of Ophiobolus graminis in buried straw. Annals of Applied Biology 63, 27-35. SMITH, A. M. & GRIFFIN, D. M. (1971). Oxygen and the ecology of Armillariella elegans Heim. Australian Journal if Biological Sciences 11:4,231-262. SPRAGUE, R. A. (193 I). Cercosporella herpotrichoides Fron, the cause of the Columbia Basin footrot of winter wheat. Science, New York 74,51-53. SPRAGUE, R. A. & FELLOWS, H. (1934). Cercosporella footrot of cereals. United States Department if Agriculture, Technical Bulletin no. 428. TOWNSEND, B. B. (1957). Nutritional factors influencing the production of sclerotia by certain fungi. Annals if Botany, N.S. 21, 153-166. TOWNSEND, B. B. & WILLETTS, H. J. (1954). The development of sclerotia of certain fungi. Transactions if the British Mycological Society 37, 2 I 3-22 I. WILLETTS, H.J. & WONG, A. L. (1971). Ontogenetic diversity of sclerotia of Sclerotinia sclerotiorum and related species. Transactions of the British Mycological Society 57, 5 15-524. ZALOKAR, M. (1959). Growth and differentiation of Neurospora hyphae. American Journal if Botany 46, 602-610. EXPLANATION OF PLATE

Fig. Fig. Fig. Fig. Fig. Fig.

I. 2.

3. 4. 5. 6.

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Pseudoparenchyma of Cercosporella herpotrichoides Pseudoparenchyma in agar culture (x 870). Pseudoparenchyma in agar culture (x 340). Portion of wheat leaf-sheath bearing pseudoparenchyma (x 87o). Portion of wheat leaf-sheath bearing pseudoparenchyma (x 340). Nuclei in young pseudoparenchyma from agar culture (x 870). Nuclei in young pseudoparenchyma from agar culture (x 870).

(Acceptedfor publication

12

November 1972)