Cross-protection against the wheat and oat take-all fungi by Gaeumannomyces graminis var. graminis

Smf Biol. B~,~hum. Vol

7. pp. 1X9-194. Pergamon

Press 1975. Prmted

,n Great Bntain.

CROSS-PROTECTION AGAINST THE WHEAT AND OAT TAKE-ALL FUNGI BY GAEUMANNOMYCES GRAMINIS VAR. GRAMINIS P. T. Biological

and Chemical

Research

W.

Institute,

(Acceptrrl

WON@

P.M.B.

10, Rydalmere.

I Orroher

N.S.W. 21 16, Australia

1974)

Summary-Glasshouse experiments have shown that the prior colonisation of wheat roots by GUYIIIIUIIIIOIU_VXSgrumir~is var. yrminis, a fungus closely related to the wheat and oat take-all fungi but non-patho-

genic to temperate cereals, reduced take-all infection along the roots. Cross-protected wheat plants produced grain yields significantly greater than those of unprotected plants but not significantly different to those of healthy wheat plants. A Phialophora-like fungus from grass roots did not confer the same degree of protection. There is some evidence that the cross-protection mechanism may be a specific host response induced

by var. gramiuis. The possible use of var. gruminis

in the biological

INTRODUCTION

Research

of take-all

is discussed.

EXPERIMEKT4L

The phenomenon of cross-protection against fungal plant pathogens by closely-related or other fungi has been demonstrated in a number of diseases (Yarwood, 1956; Johnson and Huffman. 1958; Schnathorst and Mathre, 1966; Davis, 1967, 1968; Deverall et al., 1968; Littlefield, 1969; Cheung and Barber, 1972; Skipp and Deverall, 1972). In the United Kingdom, a fungus resembling Phialophora radicicola Cain was found to restrict the rate of colonisation of (I) the wheat take-all fungus (Gaeumannomyces yrarninis (Sacc.) von Arx and Olivier var. tritici Walker) on wheat roots when the fungus was established on the roots prior to their encountering the pathogen (Balis, 1970; Scott, 1970) and (2) the oat take-all fungus (G. gruminis (Sacc.) von Arx and Olivier var. ULV~IX(E. M. Turner) Dennis) on grass roots (Deacon, 1973~). Deacon (1973a. 1973b) h& shown the possibility of controlling take-all by encouraging a high population of P. rudicicola in grass leys before cropping with cereals. In Australia, G. yranlinis (Sacc.) von Arx and Olivier var. granllnzs, a close relative of the wheat and oat take-all fungi, occurs commonly as a parasite on the roots of many grasses and less commonly on healthy cereals. With the exception of rice, of which it causes Crown Sheath Rot disease, it is non-pathogenic to cereals or grasses (Walker. 1972). Since it occupies the same ecological niche as the take-all fungi, a study was made on the possible used of this fungus to cross-protect against the wheat and oat take-all fungi with a view to the biological control of take-all. * Present address: Agricultural worth, N.S.W. 2340, Australia.

control

Centre.

Tam189

Fungi All the fungi used (Tdblc 1) were isolated from 0.5 cm root pieces, surface-sterilised for l-2 min in equal volumes of 957, ethanol and IOU/, NaOCl. rinsed in sterile water and plated onto one-quarter strength potato dextrose agar (QPDA), containing 100 parts/lo” of sodium novobiocin. The fungi were maintained at 25’C on QPDA and potato carrot agar slopes.

Soil

The soil was an alkaline self-mulching Black Earth (pH 7.6) from Warialda, New South Wales. It was taken from a field which had take-all in a wheat crop 2 years previously. but which has since not shown take-all. The soil was air-dried and passed through a 6 mm sieve before use. Either the natural soil or a mixture of 2 vol of pasteurised sand and 1 vol of soil were used. The lighter texture of the latter soil was used to favour the development of take-all (Garrett, 1934. 1937).

Inoculurn

In early experiments, agar plugs of G. yraminis var. (hereafter called var. yrarninis) were used as ‘inoculum to establish the fungus on wheat roots. Subsequently, a finely-ground oat grain inoculum of var. gvarninis was mixed into the soil at a rate of 5 g kg- ’ of air-dried soil. The take-all inoculum was added in the latter form in all experiments. qrurninis

190

P. T.

Table

w.

I. Fungz used in cross-protection

Fungus

Isolate

WONC;

experiments Locality in Australia

Host Prairie

grass

(Crrurochlouunioloidcs H.B.K.) G2 and

Kikuyu

grass

Turramurra. N.S.W.

(Pemiactum c~/urdrstinu~~

G3

Weeping

Hochst.) grass

Newport,

N.S.W.

Plains grass (Sfipu uri,ti~q/umisF. Muell.)

Warialda.

N.S.W.

Kikuyu

Taree. N.S.W.

(Microlwtm

.sripoidrs

R.Br.)

grass

Wheat (Ejricn,n

Seaside bentgrass (A6qrosti.s pulustri.\

ue.sriau,n L.)

OF PRIOR COLONISATION

ROOTS BY VAR. GRAMlNlS SPREAD

OK

OF WHEAT INFECTION

OF THE WHEAT TAKE-ALL FUNGUS

Two series of five plastic pots (15 cm dia.) were halffilled with the soil:sand mixture containing 0.5 per cent of wheat take-all inoculum (isolate Tl). Another series of five pots contained soil with 0.5 per cent of ground oat grain colonised by a Mucor sp. The latter acted as controls and were preferable to uncolonised killed oat grains (Balis, 1970). A 6 cm layer of uninoculated soil was placed above this and six wheat seeds (cv. Gabo) were sown, each placed directly above a 6 mm agar disc of either var. grarninis (isolate G 1) or the Mucor sp. The seeds were covered by 1.5 cm of uninoculated soil. There were three treatments: (a) agar discs of var. paminis over var. tritici inoculum, (b) agar discs of var. yraminis over Mucor inoculum and (c) agar discs of Mucor sp over var. tvifici inoculum. The pots were randomised in a growth chamber ( 14 h day

Perth. W.A.

Concord.

yratnirli.7 Mwor

Distance

(mm)

14.3

sp.

OF WHE-ZT ROOTS BY ISOLATES

OF VAR. GRAMl.VIS AND A, PH14LOPHORA-LIKE’ FUNGUS

FROM INFECTIOY TAKE-ALL

var. qrcminis

5” 0 1.s.d.

32.0

8.9

N.S.W.

at 2O’C and 10 h night at 12 C) and kept watered to above field capacity by placing in shallow plastic saucers containing 0.5 cm of water. On seedling emergence, the plants were thinned to four seedlings per pot. After 4 weeks, the seedling roots were washed free of soil and observed under the dissecting microscope. The distance between the seed and the nearest vascular discolouration due to var. tritici was measured for the three eldest seminal roots on each of 20 seedlings, giving a total of 60 measurements per treatment (Table 2). The series without take-all inoculum showed no vascular discolouration and was omitted from the results. These roots. however, were heavilv colonised by the dark runner hyphae of var. qtw~~~i.s. The difference between the means of the other two treatments was significant (5 per cent level) and indicates that the prior colonisation of var. yrarnirzis retarded the rate of infection spread by var. rritici along wheat roots. PROTECTION

Table 2. Mean distance between seed and nearest take-all lesion on seminal roots. pre-colonised by Mucor sp or var.

Carnamah, Western Australia (W.A.)

Huds.)

Seaside bentgrass

EFFECT

Rydalmerc. New South Wales (N.S.W.)

BY THE OAT

Fl’NGLS

An isolate of the oat take-all fungus (isolate Al), known to be pathogenic to wheat. was used in this experiment. Four isolates of var. grtrrninis and a P&J/Ophoru-like fungus closely resembling var. grmninis.

Biological control of take-all of wheat and oats Table 3. Effect of prior colonisation of wheat roots by isolates of var. gramink development by var. urenar Mean number of crown roots plant- ’ Extensively diseased Healthy

Protecting fungus

1.4 2.5 2.5 3.2 3.3 2.2 0

var. gramini.5 (Cl) var. grarnirlis (G2) var. gruminis (G3) var. graminis (G4) Phialophoru-like fungus (Pl) Mucor sp Healthy control 5”0 1.s.d.

were used to investigate the ability of these fungi to protect wheat roots against the oat take-all fungus. The experiment was set up as in the previous one. except that a 4 cm layer of uninoculated soil separated the two inocula. There were four replicates with six seedlings per pot, giving a total of 24 seedlings per treatment. The pots were randomised in a glasshouse (1525°C) and watered as before. The experiment was terminated at 5 weeks when the seedlings started to show signs of N deficiency due to competition. The root systems were examined for the extent of vascular discolouration and the dry weights of the tops were determined after drying at 80°C for 48 h (Table 3). Seminal roots were so severely diseased by the oat take-all fungus that there were no obvious differences between the different treatments and the results are omitted. The crown roots, however, showed marked differences and were rated as extensively diseased when the vascular discolouration reached the crown of the plant. Crown roots were considered healthy when the 3 cm of root closest to the crown were free of vascular discolouration. The dry weights of wheat plants inoculated with the four isolates of var. grarnirzis were significantly greater (5 per cent level) than those inoculated with the Mucor sp but were not comparable to the healthy controls (Table 3). The latter was due to the severity of the takeall. The Phialophora-like fungus did not give as good protection as var. yrarnirris. The dry weights of the plants correlated inversely with the number of extensively diseased crown roots per plant and was reflected even better by the number of healthy roots per plant. EFFECT

OF DEPTH

OF VAR. GRAMINIS

LAYER ON PROTECTION

AGAIlVST VAR.

TRlTlCI

In this experiment, var. grarnirzis (isolate G 1) was incorporated into the soil:sand mixture by blending in 0.5 per cent by weight offinely-ground oat grain inoculum. There were two series: (1) a 4 cm layer of var. graruinis over an 8 cm layer of var. tritici (isolate T2) and (2) an 8 cm layer of var. gruminis over a 4 cm layer of

3.2 2.4 2.0 2.2 I.6 I.1 7.2

191

and a Phiulophoru-like fungus on take-all

Mean dry weight of tops (mg plant ‘) 147.3 133.4 130.5 1416 106.6 74.2 303.9 37.8

var. tritici. Controls contained either the Mucor sp or no inoculum. There were four treatments: (1) var. grarthis over var. tritici (cross-protected), (2) Mucor sp over var. tritici (unprotected), (3) var. grunzinis over Mucor sp (var. grurninis control) and (4) uninoculated. Owing to insufficient inoculum, treatment (3) was omitted from the second series. Eight wheat seeds (cv. Gabo) were sown on top of the var. grmuinis layer and covered with 1.5 cm of uninoculated soil. There were four replicates per treatment. The pots were randomised in a glasshouse (15 25-C) and watered as before. On seedling emergence, the plants were thinned to six seedlings per pot. In the first series, the unprotected seedlings showed severe chlorosis and necrosis after 3 weeks and many seedlings were dead when the experiment was terminated at 5 weeks. The dry weights of the seedlings are given in Table 4. The protected seedlings showed significantly greater growth compared to the unprotected seedlings, but they were not comparable to the controls. The growth of the control seedlings inoculated with var. gruminis was not significantly different to the uninoculated controls, showing that var. graminis had no adverse effect on growth. The second series showed less take-all because of the larger volume of soil without take-ah inoculum. This series was harvested at 7 weeks, when the root systems were scored using the same criteria as before. The dry weights of the tops were also obtained (Table 5). All the seminal roots and most of the crown roots of the unprotected plants were extensively diseased. In contrast, few seminal roots and none of the crown roots of the protected plants were extensively diseased. The crown roots were colonised by dark runner hyphde of var. grarninis but were free of vascular discolouration for at least 3 cm from the crown. The dry weights of the protected plants were significantly greater than those of unprotected plants (Table 5). In the uninoculated controls, although no take-all inoculum was added to the soil, there were take-all lesions in the root systems. This was most likely due to a low residual take-all population in the soil since the soil was taken from a former take-all field. It is

192

I’. T. W. WANG Table 4. Dry weights of tops of variously

inoculated

wheat plants

Mean dry weight of tops (mg plant ‘) Table 5. Comparison

of take-all

development

Health) Unprotected Cross-protected Uninoculated control

in unprotected.

Mean number Seminal Diseased

0.0 4.8 4.5

OF WHEAT ROOTS IN FKOM

A WHEAT FIELD

In the preceding experiments, the isolates of var. gra~t~ini.swere not from wheat areas and the soil: sand mixTable 6. Comparison

of disease symptoms No. of plants killed (out of 18)

Unprotected Cross-protected Control 5”I, 1.s.d. I”,, 1.s.d.

4 0 0

of roots plant--

’

wheat plants

Healthy

Diseased

Mean dry weight of tops (mg plant- ‘)

0.5 9.7 12.0

4.5 0.0 0.0

283 1046 1617

Crown

ture caused such severe take-all that the cross-protected plants were not comparable in vigour to uninoculated controls. To overcome these deficiencies, the Warialda soil was used undiluted with sand and an isolate of var. graminis (isolate G5) from the roots of Plains grass (Sripu uristighk F. Muell) growing in a wheat field at Warialda was used. The pots were prepared as in the previous experiment, with an 8 cm layer of soil inoculated with var. grutnir~is or Mucor sp over a 4cm layer of var. tritici (isolate Tl) or Mucor sp to give three treatments: (I) var. grunkis over var. trifici (cross-protected), (2) Mucor sp over var. tritici (unprotected) and (3) var. gruminis over Mucor sp (control). There were six replicates with three plants per pot. The pots were randomised in a growth chamber (set as before) for 6weeks, after which they were transferred to a glasshouse (I 5 25°C) till the grain ripened. The plants were fertilised weekly with a modified Hoagland’s solution. From about the fourth week in the growth chamber, unprotected plants were noticeably stunted and chlorotic. A few eventually died (Table 6). In contrast. crossprotected plants were nearly as vigorous as the controls, but because of greater tillering, individual tillers were not as tall as the controls. In pots where one or two plants had died, the remaining plants usually compensated in growth and tillering, so that a measure of the mean grain yield pru plant was misleading. The variability in growth of in-

NATCRAL WHEAT SOIL BY AN ISOLATE OF VAR. GRAMhvIS

and uninoculated

5.5 0.4 I.1

noteworthy that there were more extensively diseased seminal roots in control plants than in protected plants, showing that without the protective influence of var. qruIni~li.s even a small level of take-all inoculum can cause considerable disease in seminal roots. Though none of the crown roots in the controls was extensively diseased, there were cortical take-all lesions on some of the crown roots. These lesions, however. did not extend to the vascular system, and the crown roots were probably wholly functional. Thus, the significantly greater growth in the uninoculated controls compared to the protected plants was likely to be due to the larger number of healthy crown roots (at least two more) per plant. The crown roots of the protected plants were completely free of take-all lesions. These roots were extensively colonised by the runner hyphae of var. gmr~~inis, which also extended up the lower leaf sheaths. The characteristic lobed hyphopodia of var. grarninis (Walker, 1972) were observed in large numbers on the leaf sheaths and slightly less lobed ones were present on the roots. CKOSS PROTECTIOY

cross-protected

and mean grain yields of unprotected,

Total no. of heads treatment81 95 99

’

cross-protected Total no. of unfilled heads or whiteheads 51 21 8

and control

wheat plants

Mean grain yield (mg pot I.34 4.63 5.0 1

Im 2.38

I)

Biological

control

of take-all

fected plants in the same pot has been observed by other workers (Gerlagh, 1968; MacNish, 1973). The grain yields in this experiment are therefore expressed as mean grain yield per pot, indicating the grain yields from equal volumes of soil. The mean grain yield per pot of the cross-protected and control wheat plants was significantly greater (1 per cent level) than that of unprotected plants while the grain yields of the controls and cross-protected plants were not significantly different (5 per cent level). In spite of the greater tillering, or because of it, the crossprotected plants had fewer tillers with filled heads, compared to the controls. Unprotected plants produced numerous whiteheads (Table 6). The root systems were washed and examined, but owing to extensive rotting and therefore incomplete recovery of the roots, the dry weights were not determined. All the crown roots of unprotected plants were extensively diseased. while the majority of roots of the other two treatments were healthy or only slightly diseased. As in the previous experiment, some roots of the uninoculated controls showed slight take-all infection due to the low residual population of take-all in the soil. DISCUSSION

Glasshouse and growth chamber studies have shown that, while G. grarninis var. grurhis has no adverse effect on wheat growth, it can cross-protect wheat roots against the wheat and oat take-all fungi. Both the dry weights and grain yields of cross-protected plants were significantly greater than those of unprotected plants and suggest a promising role for var. graminis in the biological control of take-all. It is not known at this stage whether this is possible in the field, but experiments are continuing to investigate this. The mechanism for the cross-protection phenomenon is not clear. On present evidence, the restriction of var. tritici from progressing towards the crown does not appear to be entirely due to its physical exclusion from areas of the root pre-colonised by var. gru~~inis. Ectotrophic spread of var. tririci may be curtailed. but it is difficult to explain why vascular spread was restricted, since var. graminis rarely invades vascular tissues, It is possible that a specific host response stimulated by the penetration of cortical tissues by var. gramirlis may be responsible for containing var. rritici. Four isolates of var. gruminis gave comparably high levels of protection, while a Phialophoru-like fungus in the same experiment did not provide a high degree of protection (Table 3). However, this does not explain the findings of Balis (1970) Scott (1970) and Deacon (1973a. 1973b. 1973~) who obtained good control of take-all fungi with P. rudicicola. Further work is required with other Phialophoru-like fungi to investigate the above hypothesis, especially as the taxonomy of this group of fungi is still under review (Deacon, in press; Wong and Walker, unpublished). The efficiency

of wheat and oats

193

of the protective influence will depend on the intensity of take-all attack and a large amount of take-all inoculum may over-ride the acquired resistance. as was seen in the oat take-all experiment. The success of this form of biological control will rest on at least two factors. One is the rapid colonisation of wheat roots by var. gruminis so that the proximal portions of the seminal roots and later the subcrown internode and crown roots are colonised before they come in contact with take-all inoculum. Secondly. var. grumir~is has to persist in the soil at a high level in order to inoculate future crops of cereals. The first of these may conceivably be achieved by having a period of pasture with pasture species known to carry var. grur~inis so that a high population of the fungus will build up to ensure a rapid colonisation of the wheat roots. At least eight pasture species are known to consistently yield var. gvnmirlis from their roots (Wong. unpublished data). and it is interesting that Australian farmers have observed that the first cereal crop after a pasture phase is seldom severely attacked by take-all. except where the pasture has had a large component of barley grass (Hordeum leporiwm Link.), a notorious carrier of take-all. This appears also to be the experience in Europe, where a low incidence of take-all has been reported after a ley period (Lewis, c’r al., 1960; Gerlagh, 1968; Scott, 1970). It is possible that different grass species will differentially harbour the three varieties of G. gruminis and the encouragement of pasture species which carry high populations of var. gruminis should favour this form of biological control. A more direct way of ensuring that emerging wheat roots are colonised by var. gruminis is by pelletting wheat seeds with a mycelial or conidial suspension of the fungus. A new type of phialidic conidium, different to the normal lunate microconidium. is known in var. arurhis (Wong and Walker. unpublished). These contdia germinate readily on agar and on sterile wheat roots to form normal mycelium. and there are early indications that these conidia may serve to introduce var. grumirzis to roots by seed pelletting. There is little information on the persistence of var. gruminis during wheat cultivation. It is possible that var. gwmirzis may not persist in soil beyond several years of wheat cultivation since it is more commonly associated with grasses than with healthy cereals. If this proves to be the case, the fungus will have to be introduced with each wheat crop by seed pelletting, or else a system of cultivation different to the present practice may have to be adopted. In contrast to the British situation. where short-term leys are common and where a high population of P. radicicolu develops under a ley of several months to a year (Deacon, 1973a, 1973b), wheat management in Australia often requires permanent pastures of 337 years for the regeneration of soil fertility. Thus, for biological control to operate in this situation a high population of the protective fungus will have to be maintained during wheat cultivation, once it has been established in the soil. Brooks and Dawson (1968) showed in Britain that take-all was

w. less severe when wheat was drilled directly into pastures than when drilled in ploughed pastures, and though they offered no explanations at the time, it is tempting to suggest that the cause may have, in part. been a natural form of biological control mediated bq Phiulophoru-like fungi or fungi similar to var. yrur~inis. It is possible that var. yraminisand other avirulent parasites of grass roots may be favoured more by the relatively undisturbed pasture situation (direct-drilling) than in ploughed soils. and so provide a greater protective influence. There is considerable interest. at present, in direct-drilling and other minimum tillage methods in wheat cultivation in Southern New South Wales and Victoria. following yield results comparable to those of conventional ploughing (Reeves and Ellington. 1974; McNeill, 1974). and this should add scope to investigations into the possible use of var. gra~ninis to biologically control take-all. Ack,~or~letlyr,,~rr~rs-I wish to thank my colleagues Dr. A. M. Smith and Mr. K. J. Moore for helpful criticisms and especially Mr. J. Walker for his keen interest and invaluable advice. I wish also to thank Professor S. D. Garrett, University of Cambridge. for the stimulating discussions we had, while he was a visitor at our Institute.

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WONG

J. W (1973~) Factors affecting occurrence of the ophiobolus patch disease of turf and its control by Phialophoru rtrdicicolu. PI. Pd. 22, 149-155. DLACOX J. W. (1974) Further studies on Phiuhphoru rccdicico/u and Gururnunnont~~~~.~ qrumiCs on roots and stem bases of grasses and cereals. Trclns. BP. rnrcol. Sot. 63, (in press). DEVERALL B. J.. SMITH I. M. and MAKKIS S. (1968) Disease resistance in Vicicl firhu and Plzusrolus culgurk. Nrtk. J. PI. Path. 22, 88-94. GARRETT S. D. (1934) Factors afiecting the severity of takeall. I. The importance of micro-organisms. J. Dep. A~qric. S. Amt. 37, 664-674. GAKRETT S. D. (1937) Soil conditions and the take-all disease of wheat. II. The relation between soil reaction and soil aeration. Ann. uppl. Bid. 24, 747- 75 I. GEKLAGH M. ( 1968) Introduction of Ophioholu.~ qrurnitlis into new poldcrs and its decline. !Vcrk. J. PI. Path. 74 suppl. 2, 97 pp. JOFIX?.OYC. 0. and HUF~MAN M. D. (195X) Evidence of local antagonism between two cereal rust fungi. Ph~topatholo A. E. M. (1960) A comparison of ley and arable farming systems. J. trgric. Sci. 54, 310 317. LITrLf:FIbLu L. J. (1969) Flax rust resistance induced by prior inoculation with an avirulent race of Mdtr~ysora /i/Ii. Ph~toputholoyy 59, 13% 1328. MA(.NISH G. C. (1973) Effect of mixing and sieving on incidence of GIC,LII~I(II~II~~III.(.CC qramini\ var. trltici in field soil. .-lust. J. hiol. Sci. 26, 1277 1283. McNr~u A. A. (1974) Modified combine point for direct seeding wheat. Acgr~c’.Guz. h’.S.W 85, 47. Rrcvr-s T. G. and ELLINC;~~N A. ( 1974)Direct drilling experiments with wheat. Amt. J. c~xp. Ayric. A/lirn. Hush. 14, 231 240. SCHNATHORSTW. C. and MATHKI. D. E. (1966) Cross-protection in cotton with strains of Vwticillium ulho-atrum Phytopddoyy 56, 1204 1209. Scar-r P. R. (1970) Phialophoru rud~%~h. an avirulcnt parasite of wheat and grass roots. 7i~11.s. Br. IIIJ,CO/.Sot. 55, 163-167. SKIPP R. A. and D~V~KAL.LB. J. (1972) Relationship between fungal growth and host changes visible by light microscopy during infection of bean hypocotyls (P/~a.srolu.s vulqcwis) susceptible and resistant to physiological races of C‘o/I~,~tof~ickl,fil /i,lt/o,llrfhiurlL,111.Ph,vsiul. PI. Put/z. 2, 357374. WAt.KkKJ. (1972) Type studies on Gu~u~~~unr~o~t~~cr.sgr~(rnirl~s and related fungi. 7?una. Br. /II~CO~.Sot. 58, 427 457. YAKWOOI) C. E. (1956) Cross-protection with two rust fungi. Ph!,toptrtlloloU~ 46, 540 544. DCACON