Effects of nitrogen level on survival of Phialophora radicicola and Cochliobolus sativus in pure culture on cellulose

[ 121 ] Trans. Br. mycol. Soc. 57 (I), 121-128 (1971) Printed in Great Britain

EFFECTS OF NITROGEN LEVEL ON SURVIVAL OF PHIALOPHORA RADICICOLA AND COCHLIOBOLUS SATIVUS IN PURE CULTURE ON CELLULOSE By S. D. GARRETT

Botany School, University (With

1

ofCambridge

Text-figure)

At nitrogen levels below the optimum for survival in pure culture on filterpaper cellulose, both Phialophora radicicola and Cochliobolus sativus survived much better than Ophiobolus graminis had done in an earlier experiment by the same technique. The better survival of these two species at low nitrogen levels is ascribed to their higher values for the cellulolysis adequacy index (CAl), which expresses the cellulolysis rate of a species in relation to its general metabolic rate. The adverse effect of excess nitrogen on saprophytic survival of these two species in colonized wheat straws buried in soil is attributed to earlier exhaustion of the substrate when nitrogen supply is not limiting rate of decomposition. This effect could be reproduced only partially with P. radicicola in pure culture on cellulose, and so the larger part of the effect is ascribed to decomposition of the straw tissue by other micro-organisms invading the substrate from the surrounding soil. Species with a high CAl value, such as P. radicicola and C. sativus, liberate sugars by cellulolysis in excessoftheir immediate metabolic needs; when nitrogen and other mineral nutrients are provided by the soil, then nutritional requirements for invasion of the substrate by other micro-organisms are met in full, and so decomposition and ultimate exhaustion of the substrate are thereby accelerated.

Fungi infecting roots and tiller-bases of cereals have been found to show a wide variety ofresponse to nitrogenous nutrients in the longevity of their saprophytic survival in colonized wheat-straw tissue buried in soil; if supplementary nitrogen is added to a nitrogen-poor soil, the longevity of some fungi is increased whereas that of others is decreased, and in yet others there is no response either way (Garrett, 1970, p. 149). To explain these differences in behaviour, I suggested that they depended on the cellulolysis rate of individual hyphae in relation to the general metabolic rate of the particular fungal species (Garrett, 1966). Thus, other things being equal, a fast-growing fungus has a higher metabolic rate and therefore requires more soluble carbohydrate, to maintain respiration and support growth, than has a slow-growing one. Because the active life of any particular length offungal hypha in cellulolysis is oflimited duration, the fungus must slowly extend its mycelial network in the straw tissue, so as to be able to hydrolyse fresh areas of plant cell-wall. For this reason, a continued supply of soluble nitrogen, and of some other mineral nutrients too, is often essential both for the continuation of cellulolysis and for

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survival of the cellulose-decomposing fungus. This essential supply of nitrogen is provided by inwards diffusion from the surrounding soil, though whether it is sufficient for the needs of the fungus depends on the nitrogen-supplying power of the particular soil; for maximum longevity, Ophiobolus graminis (Sacc.) Sacco requires about 0'5 g NjIOO g air-dry wheat straw. But we have also found that this dose of nitrogen is equally effective in promoting maximum longevity of O. graminis if it is supplied to the wheat-straw pieces in the pure-culture flasks before inoculation with the fungus (Garrett, 1944; Scott, 1969). Scott has shown that about fourfifths of the nitrogen thus supplied is taken up as mycelial nitrogen during the pure-culture incubation period of one month, and hence must be available for further development of the fungal colony within the straw tissue. This need for nitrogen to secure prolonged survival of the fungal colony, however, is known to vary widely amongst the species of cereal foot-rot fungi so far studied. Thus for a weak cellulose-decomposer, the low cellulolysis-rate of individual hyphae must be compensated by a sufficient supply of nitrogen to permit mycelial extension at a rate adequate for survival of the colony. For a strong cellulose-decomposer, the higher cellulolysis-rate of individual hyphae is sufficient to keep the colony alive at a lower rate of mycelial extension, and hence at a lower rate of nitrogen supply. To epitomize these relationships, I proposed the cellulolysis adequacy index (CAl). The CAl expresses the ratio of cellulolysis rate to linear growth rate, the latter having been selected as a convenient parameter of general metabolic rate. Cellulolysis rate has been expressed as percentage loss in dry weight of filter-paper cultures after 7 weeks at 22'5 cC, and linear growth rate in mmj24 h at the same temperature. We have found that if the CAl value is < 0'5, as for Ophiobolus graminis, then longevity of the fungus is much increased by supplementary nitrogen. But if the CAl value exceeds 1'5, as for Cochliobolus sativus (Ito & Kuribay.) Drechsl. ex Dastur and for Phialophora radicicola Cain, then supplementary nitrogen has the converse effect and sharply reduces longevity. It is easy to understand why a fungus with a low CAl value has a substantial need for nitrogen if its colony is to survive; nitrogen is the mineral nutrient required in the highest proportion for synthesis of microbial protoplasm, and is therefore the one most likely to be in short supply in the soil. I was able to demonstrate this critical need for an adequate level of nitrogen supply by pure cultures of O. graminis growing on filter-paper as sole carbon source (Garrett, 1967). But the mechanism of the converse effect, whereby excess nitrogen sharply reduces the longevity of both C. sativus and P. radicicola in colonized wheat straws buried in the soil, is at present more debatable. It seems likely that this effect operates through a hastening of substrate exhaustion, both by the primary fungal colonizer and by other cellulose-decomposers that invade the substrate after burial in the soil; both invasion and decomposition are accelerated by a freely available supply of soluble nitrogen. The question now at issue is the relative importance of these two effects. The experiments to be described hereunder were an attempt to get further evidence on this question. For this purpose, I repeated with P.

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radicicola and C. sativus the experiment I had earlier conducted on the survival of O. graminis in pure culture on filter-paper at different nitrogen levels, so as to evaluate the contribution of the primary fungal colonizer to substrate exhaustion at high nitrogen levels. C. sativus is a well known cause of cereal foot rot in North America and Australia. The occurrence of P. radicicola in this country was first recorded by Scott (1970) ; it is an avirulent root parasite of grasses and cereals, with an ectotrophic infection habit closely similar to that of O. graminis, though it causes no necrosis of the vascular cylinder and at most only a local and faint discoloration of the root cortex. Although this species resembles O. graminis in many ways, it contrasts strongly with it in its response to nitrogen in saprophytic longevity, and so was a suitable choice for thepresent study. Balis (1970) determined the CAl value of his isolate as I '66, which is within the range of variation shown by six isolates of C. sativus (Garrett, 1970, p. 155). In accordance with prediction from its CAl value, Balis found a strongly negative response to nitrogen in its saprophytic longevity. Thus in wheat straws originally colonized by the fungus in pure culture, in absence of nitrogen or any other nutritional supplement, and then buried in a nitrogen-poor soil (total N = 0'1 % dry matter), maximum curtailment of survival followed addition to the soil of calcium nitrate at the rate of 0·6 g NllOo g air-dry straw. But a significant decline in survival also followed a nitrate addition at only one-quarter of the above rate. EXPERIMENTAL WORK

Survival of Phialophora radicicola in pure culture on filter-paper The isolate of P. radicicola employed for this experiment was that used by Balis (1970) and originally isolated by Scott (1970) from the roots of old grassland at the Botany School Field Station. Its survival in pure culture on filter-paper at different nitrogen levels was tested by the technique I had devised earlier for O. graminis, but with certain modifications. For the present experiment the unit substrate was a single circle (7 ern diam) of Whatman no. 2 filter-paper, which was placed in a 250 ml conical flask over 150 g dry sand, previously acid-treated and then well washed. Filterpapers and sand were then brought to a moisture content of 90 % saturation by adding 31 ml nutrient solution. For the experiment with O. graminis, the standard nutrient solution (designated'S ') had contained 0'33 gil sodium nitrate; this provided nitrogen at the rate of 0'235 g N I 100 g air-dry filter-paper, and has proved to be the optimum nitrogen level for survival of this fungus. In the present experiment, the object of using a lighter grade of filter-paper had been to reduce the survival period of the fungus. The mean weight of a no. 2 paper was determined as 0'364 g and that of a no. 3 paper as 0'709 g; in consequence, concentrations of nitrogen and other nutrients were reduced by a factor of 0'513 from those earlier employed for O. graminis on single circles of no. 3 paper. So the S concentration of nutrients employed for the present experiment contained (per litre) the following: NaNOa, 0'171 g; K 2HP04 , 0'138 g; MgS0 4 • 7H20, 0'069 g; FeCl a, 0'14 mg; thiamin, 14 flg; biotin, 1'4 flg. In all treatment-series, the concentrations of all mineral nutrients except

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nitrogen were kept constant at the above levels, which were designed to be adequate for nutrition of the fungus at the highest nitrogen level. In the five treatment-series, nitrogen alone was varied by a factor of two, namely S/4, S/2, S, 2S and 4S. Sufficient replicate flasks were set up for each treatment-series to permit sampling in triplicate after 18, 24 and 32 weeks from inoculation. After filling, all flasks were autoclaved for 30 min at 123°, and the filter-paper circles were later inoculated at the perimeter with a single 11 mm disk cut from the growing margin of a colony of P. radicicola on potato-dextrose agar. Thereafter all flasks were incubated at 22'5°. The method of testing for survival of P. radicicola was similar to that employed for O. graminis, and involved the planting of twelve presoaked wheat seeds equidistantly over each filter-paper circle, which was placed on moist soil (50 % saturated) in a glass tumbler. The soil was made up of I vol. Field Station soil to 3 vol. quartz sand, which gives optimum conditions for infection by both P. radicicola and O. graminis (Balis, 1970), After filling and planting, each tumbler was weighed (to permit daily restoration of soil moisture content), and the fifteen tumblers (5 nitrogen levels x 3 replicates) were placed in a water bath held at 22'5° and stood against a north window, After 14 days from planting, wheat seedlings were washed free of soil and filterpaper, and the proportion of infected roots on each one was recorded. The most reliable criterion for infection by P. radicicola is the presence of the ectotrophically spreading, dark-coloured runner hyphae on the root surface. So each root was scanned for a sufficient distance from its point of origin under the x 50 magnification of the stereomicroscope. The figures for percentage roots infected in each treatment-series at each sampling time are based on the examination of not less than 180 roots from 36 seedlings (Table I, to be considered in the Discussion) , Table

I.

Percentage roots infected by Phialophora radicicola Time of sampling (weeks) Nitrogen series S/4 S/2 S 2S 4S

,

18

24

32

93 100 100 100 100

89 96 100 100 100

44

75 90 97 87

Standard error of means = 2"71. Least difference for significance at 5 % level = 8.

Survival

if Cochliobolus sativus

in pure culture onfilter-paper

Insufficient replicates were prepared for this experiment to permit sampling beyond 25 weeks from inoculation, because I had not anticipated that C. sativus would survive for so long with almost undiminished vigour. In preliminary trials, cultures of C. sativus had been found to sporulate profusely on the upper surface of the filter-paper, and it had proved impossible to remove all the conidia therefrom; if these conidia had been allowed to remain, estimation of saprophytic survival by the

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mycelium would have been confounded by dormant survival through the conidia. This difficulty was evaded by the use of two superimposed circles of Whatman no. 3 filter-paper (7 em diam); at sampling, the upper circle of each pair was removed and discarded. In other respects, the technique was as described above for P. radicicola, except that concentrations of nitrogen and other mineral nutrients were double those originally employed for O. graminis, in which one circle only of Whatman no. 3 paper had been employed as the unit substrate. Flask cultures were again incubated at 22'5°. The method of testing for viability was that devised by Butler (1959); each filter-paper to be tested was incubated for 1 week at 22'5° in a sand plate (50 g sand + 10 ml water in a 9 em Petri dish) to induce sporulation. By means of a mechanical stage on the stereomicroscope (x 50 magnification), 100 fields of view of 2 mm diam and spaced 2 mm apart were scanned for freshly sporulating conidiophores; the presence of one or more of these in any field was taken to indicate viability of C. sativus within that area. Results were recorded as percentage of microscope fields in which C. sativus was still viable, but only those for the final sampling, at 25 weeks from inoculation, are shown in Fig. 1. Each point on the graph for C. sativus in Fig. 1 is derived from a total of 200 fields scanned, i.e, 100 on each of two replicate papers. Table

2.

CAl values offungal isolates tested in survival experiments Loss in fil ter- paper dry wt, (%) after 7 weeks at 22'5°

Linear growth rate of fungus (mm/aq h) at 22'5°

CAl value

7,6 2'5

Ophiobolus graminis Phialophora radicicola Cochliobolus satious

3'5 DISCUSSION

For a period of up to 25 weeks, data are now available for a comparison of survival in pure culture between Ophiobolus graminis (after 23t weeks, from Garrett, 1967), Phialophora radicicola (after 24 weeks) and Cochliobolus sativus (after 25 weeks). In all three experiments the temperature of incubation was 22'5°; the results are presented in Fig. 1. In Table 2 are given the relevant data for calculation of CAl values for the particular isolates of the three fungi used in the respective survival experiments. Fig. 1 shows that both P. radicicola and C. sativus survived much better at the two lowest nitrogen levels, 5/2 and 5/4, than did O. graminis; this behaviour agrees with expectation from their CAl values, as outlined in the Introduction. It explains why O. graminis needs a higher level of supplementary nitrogen for survival in colonized wheat straws buried in soil than does either of the other two species, But if we compare the graph of O. graminis in Fig. 1 with the figures for percentage survival of P. radicicola after 32 weeks in Table 1, we see that the optimum nitrogen level for survival by P. radicicola is actually higher

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than that for O. graminis. Excess nitrogen (at the 4S level) has not much curtailed the survival of P. radicicola in pure culture, whereas even a low dose of supplementary nitrogen was found by Balis (1970) to shorten its survival in colonized wheat straws buried in a nitrogen-poor soil. It seems, therefore, that the direct effect of excess nitrogen upon rate of substrate decomposition by this primary fungal colonizer makes a smaller contribution to eventual exhaustion of the substrate than does the indirect effect, whereby nitrogen accelerates invasion and decomposition of the substrate by other cellulose-decomposers from the surrounding soil.

100

.----r

5/4

.-

5/2

_.----- -----.

5 Nitrogen levels

25

45

Fig. 1. Percentage survival, after 25 weeks, of Ophiobolus graminis, Phialophora radicicola and Cochliobolus sativus at different nitrogen levels in pure culture on filter-paper cellulose.

This conclusion with respect to the behaviour of P. radicicola suggests a reappraisal of my earlier conclusion from the results of a different type of experiment with Cochliobolus satiuus (Garrett, 1966). In this experiment, nitrogen was supplied, at the rate of 0'45 g Nlloo g air-dry straw, in two ways: (I) to the wheat straws in the pure-culture flasks, before inoculation; (2) to the soil after burial of the colonized straws. I had argued that the first mode of application should enhance operation of the direct effect, because most of the nitrogen would be taken up by the mycelium of C. satiuus during incubation of the straws in pure culture for 33 days at 23°. There can be little doubt that nitrogen applied in this way would have operated almost entirely by the direct effect. Thus Scott (1969) found that O. graminis took up 0'4 g NlloO g air-dry straw as mycelial nitrogen during an incubation period of I month; from Table 2 we can see that over the first 7 weeks, C. satiuus decomposes cellulose more than twice as fast as does O. graminis, so it must take up nitrate nitrogen more quickly, and may be assumed to have absorbed the whole dose of nitrogen during the pure-culture period. Both the isolates ofC. satiuus tested in this experiment died out more quickly from the straws when the nitrogen was given

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to the pure-culture flasks; under the particular conditions ofthisexperiment, therefore, the direct effect of nitrogen on C. sativus was greater than its indirect effect through the mediation of other soil micro-organisms. But the design of this experiment was biased in favour of the direct effect, because nitrogen given to the culture flasks before inoculation had 33 days longer in which to work than nitrogen given to the soil after straw burial. Table 3 shows the periods to 50 % survival of the two isolates of C. sativus in the pure-culture nitrogen and soil nitrogen series, respectively. Table 3 shows that, although pure-culture nitrogen had 33 days longer in which to act, it reduced survival of the two isolates below that in the soil nitrogen series by only 9 and 14 days, respectively. From this calculation it now seems likely that the direct effect of nitrogen is in reality smaller than the indirect effect. This is certainly the conclusion that is compatible with the fresh evidence now reported in this paper. Table 3. Effects of mode and time of nitrogen application on saprophytic survival if Cochliobolus sativus (from data ofGarrett, 1966) Time to 50 % survival (days) A

Pureculture nitrogen Isolate poi Isolate B.S. 790

Soil nitrogen

81

go

94

108

This conclusion that the indirect effect of nitrogen is the more important of the two prompts a question as to its mechanism. Is there some characteristic of a high CAl value that makes fungal species possessing it more sensitive to the indirect effect of excess nitrogen than are species with a low CAl value? The most probable answer is that species with a high CAl value are more sensitive to competition precisely because they have a high CAl value, and so release sugars by cellulolysis in excess of their immediate metabolic needs; when mineral nutrients, and especially nitrogen, are also provided by the environment, then a complete nutrient solution is available to other micro-organisms, both cellulose-decomposers and commensals, attempting to invade the straw tissue from the surrounding soil. This release of sugars into the immediate environment of cellulose-decomposing fungi has been elegantly demonstrated by Tribe (1966); he showed, by means of oospore counts, that several species of Pythium, although themselves unable to decompose cellulose film, could live on it as commensals in association with various species of cellulose decomposing fungi. So the principal conclusion emerging from discussion of the data presented in this paper is that the cellulolysis adequacy index now seems capable of explaining the negative as well as the positive response to nitrogen by these fungi in their longevity of saprophytic survival. I am indebted to Mr G. Purss, of the Queensland Wheat Research Institute, for supplying me with the isolate of Cochliobolus sativus.

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Transactions British Mycological Society REFERENCES

BALIS, C. (1970). A comparative study of Phialophora radicicola, an avirulent fungal root parasite of grasses and cereals. Annalsof AppliedBiology 66, 59-73. BUTLER, F. C. (1959). Saprophytic behaviour of some cereal root-rot fungi. IV. Saprophytic survival in soils of high and low fertility. Annals of AppliedBiology 47, 28-36. GARRETT, S. D. (1944). Soil conditions and the take-all disease of wheat. VIII. Further experiments on the survival of Ophiobolus graminis in infected wheat stubble. Annals of AppliedBiology 31, 186-191. GARRETT, S. D. (1966). Cellulose-decomposing ability of some cereal foot-rot fungi in relation to their saprophytic survival. Transactions of the British Mycological Society 49,57-68. GARRETT, S. D. (1967). Effect of nitrogen level on survival of Ophiobolus graminis in pure culture on cellulose. Transactions oftheBritish Mycological Society, 50, 519-524. GARRETT, S. D. (1970). Pathogenic root-infecting fungi. Cambridge University Press. SCOTT, P. R. (1969). Effects of nitrogen and glucose on saprophytic survival of Ophiobolus graminis in buried straw. Annalsof AppliedBiology 63,27-36. SCOTT, P. R. (1970). Phialophora radicicola, an avirulent parasite of wheat and grass roots. Transactions of the British Mycological Society, 55, 163-167. TRIBE, H. T. (1966). Interactions of soil fungi on cellulose film. Transactions of theBritish Mycological Society 49, 457-466.

(Accepted for publication 29 March 1971)