support this statement. The pathogen may have reachedNhulunbuy in one or more of the following ways:(1) on roots of infected plants brought in from infested

nurseries or plantations; these plants may not haveshown any symptoms; several Phytophthora spp.,including P. cinnamomi have been isolated fromnurseries and orchards in the Northern Territory (1);

(2) in infected soil or gravel or on dirty equipment.Tracked vehicles may transport infested soil. P.cinnemomt was disseminated in this way in Vic­toria (8).

There were no symptoms near the mining and treatmentareas which are at some distance from the town (c. 22 km).A healthy and successful vegetation programme wasevident on the bauxite sites. Aborigines collected andsorted the seed of local species, the mined area was deep­ripped, overburden and topsoil were replaced andseeded. Five-and-a-half years later the resulting plantswere in seed, thus completing the cycle (4). Waste fromore treatment was ponded, and although revegetation wasmore difficult, it is apparently successful.

In a preliminary investigation (6) no pathogen wasisolated. This may have been caused by the supply of seedfrom a less susceptible lupin for baiting. The baiting failurerecorded in this paper was due to the use of seed fromLupinus a/bus L. instead of the more susceptible NewZealand blue lupin, L. angustifo/ius L. Supply of seed of L.angustifolius was not available at the time of theexperiments.

Other factors were suggested to explain the dieback,such as fire protection practices and/or a lowering of thewater table associated with use of the town lagoon. Aprevious report (1) associated disease and death in part ofthe Northern Territory with Phytophthora nieotianae.Phytophthora einnamomi and other Phytophthora specieswere also isolated from consignments of avocado andcitrus trees received from Queensland. Phytophthoranicotianae nearest var. nicottenee was established ascausing disease and death in Avicennia marina, the whitemangrove, in an uninhabited estuary on the Cape Yorkpeninsula in North Queensland (9), and was therefore apathogen in coastal vegetation at a similar latitude. Koch'spostulates have now been satisfied. The deaths anddieback observed in the forests of E. tetradonta atNhulunbuy were therefore almost certainly due to P.einnamomi either acting alone or in conjunction withenvironmental stress due to infertile soils and alternateperiods of water saturation and water stress.

I am grateful to R.N. Pitkethley (Department of PrimaryProduction, Northern Territory), to DA Hinz (Nabaleo Pty.Ltd. Nhulunbuy, Northern Territory) for assistance in thecollection of samples, and to Anne Norton (ComalcoAluminium Limited, Weipa, North Queensland) for thesupply of pathogen-free seedlings of E. tetradonta.

REFERENCES

(1) Blowes, W.M. and Pitkethley, R.N. (1981). The occurrence ofPhytophthora spp. in the Northern Territory. AustralasianPlant Pathology 10: 8-10.

(2) Chee, K. and Newhook, F.S. (1965). Improved methods foruse in studies on Phytophthora cinnamomi and otherPhytophthora species. N.Z. J. Agric. Res. 3: 88-95~

(3) Hall, N., Johnston, L. and Chippendale, G.M. (1970). Foresttrees of Australia. Aust. Govt. publicn. 3rd edition.

44

(4) Hinz, D.A. (1981). Returning land to Eucalyptus forest afterbauxite mining at Gove, NT. Paper presented to NorthernAustralian Mine Rehabilitation Workshop, Gove.

(5) Marks, G.C. and Kassaby, F.Y. (1974). Detection ofPhytophthora cinnamomi in soil. Aust. For. 36: 198-203.

(6) Pitkethley, R.M. (1982). Report on a preliminary investigationof a eucalypt dieback in the Nhulunbuy town area.Department of Primary Production, Darwin.

(7) Tsao, P.H. (1970). Selective media for isolation of pathogenicfungi. Ann. Rev. Phytopath. 8: 157-186.

(8) Weste, G. (1974). Phytophthora cinnamomi the cause ofsevere disease in certain native communities in Victoria.Aust. J. Bot. 22: 1-8.

(9) Weste, G., Cahill, D. and Stamps, J. (1982). Mangrovedieback in North Queensland, Australia. Trans. Brit.mycol. Soc. 79: 165-167.

Induced Resistance in Wheat to RustInfection

C.F. McRae' and J.F. BrownDepartment of Botany, University of New England,

Armidale, N.S.W. 2351.

IntroductionSeveral authors have reported reductions in the level of

disease in plants after previous exposure to inoculum ofrelated strains or to different species of fungi. The terms"induced resistance" and "cross protection" have beenused to describe this phenomenon. Yarwood (17) inducedlocalised resistance in bean to the bean rust fungus byprior inoculation with a non-virulent strain of the samefungus. Similar findings were reported by Littlefield (12) inflax for flax rust. He found that induced resistance waseffective for 7 days and was expressed as a reduction inthe numbers, size and rate of appearance of lesions.Johnson and Taylor (8) demonstrated that inducedresistance in wheat to stripe rust was systemic in the sensethat a non-virulent strain of Puceinia strtitormis Westinduced resistance to a virulent strain of the same funguson the opposite leaf surface. A number of workers haveshown that resistance in plants to rusts can be induced bya pathogen that does not normally infect that particularplant (9).

The experiments reported in this paper were made tocharacterize the induced resistance phenomenon in wheatto infection by the stem and leaf rust fungi. Investigationswere made to determine the time interval requiredbetween successive inoculations to induce resistance,whether induced resistance was localised or systemic,whether resistance could be induced by a non pathogen ofwheat and the means by which induced resistance wasexpressed.

Materials and MethodsSegments of primary leaves (3cm long) of the wheat

cultivars Thew and Mentana were floated on a solution ofbenzimidazole contained in petri dishes(16). Leaves wereinoculated in a chamber that enabled a uniform density of

• Present address: Centre for Irrigation Research, CSIRO,Griffith, N.SW. 2680.

Table 1. Effect of inoculating leaf segments of wheat with non-virulent strains and species of rust fungi on subsequentdevelopment by virulent strains of the wheat stem and leaf rust fungi. A period of 2 days had elapsed between successiveinoculations.

TREATMENT % Germination (G) % Appressorial formation (A)% Penetration (P) & % Germinated spores which did not

produce appressoria over stomates (G-A)Inducer/ Inducer inoculated onto Inducer inoculatedChallenge same surface as challenge onto opposite surface

strain to challenge strain

G A P G-A G A P G-AP. recondite/ 75a 81a 64a 3aP. recondita 38b 18b 14b 20bP. graminis/ C 55a 55a 53a Oa 78a 78a 78a OaP. recondita T 35b 30b 20b 3a 36b 23b 17b 14bP. helianthi/ C 60a 60a 53a Oa 75a 73a 65a 7aP. graminis T 56a 32b 16b 25b 70a 55b 21b 15bP. helianthi/ C 61a 56a 51a 7a 80a 75a 74a 5aP. recondita T 59a 48b 33b 10a 69b 57b 19b 11b

C = Control inoculation with the virulent strain onlyT = Prior treatment with the non-virulent strain• Values followed by the same lower case letter do not differ significantly (P> 0.05) for each inducer/challenge

combination.

urediospores (about 42 ureotospores/rnm-) to bedeposited onto either the adaxial or abaxial surface of theleaf segments (2). The inoculated segments wereincubated in a darkened dew chamber at 21°C for 16 hours(1) before being transferred to a growth cabinet kept at21°C and 43.5 Wm-2light intensity (12 h photoperiods).

Ten leaf segments contained in each petri dish wereinoculated with either a non-virulent strain of the leaf(Puccinia recondita Rob. ex Desm. f.sp. tritici (Eriks.)Carleton) or stem rust (Puccinia graminis Pers. f.sp. triticiEriks. & Henn.) fungus or with the sunflower rust fungus(Puccinia helianthi Schw.) which is a non pathogen ofwheat. Immediately and at daily intervals for 6 days afterthis inoculation the leaf segments were re-inoculated witha virulent strain of the wheat stem or leaf rust fungus.

Four leaf segments from within each petri dish wererandomly selected and the number of pustules producedper unit area of leaf surface was determined at dailyintervals from the onset of pustule development until 18-21days after inoculation with the virulent strain of rust. Twoleaf segments were removed at 72 and 96 h afterinoculation with the virulent rust for histological studies ofthe infection process. The segments were cleared andstained according to the method of Shipton and Brown(14). At the end of each experiment the area of 10 pustuleswas determined on the remaining two leaf segments usinga steromicroscope containing an occular micrometer.Each treatment was replicated 5 times.

ResultsSignificantly less pustules were produced on leaf

segments that had been previously inoculated on eitherthe abaxial or adaxial surface with a non-virulent strain ofthe leaf rust fungus prior to inoculating the same or theopposite surface with a virulent strain of the same fungus(Fig. 1). A similar trend was found when non-virulent andvirulent strains of the stem rust fungus was used.

Prior inoculation of leaf segments with urediospores of anon-wheat pathogen (P. helianthi) on the same or opposite

45

surface significantly reduced the number of pustulessubsequently produced by virulent strains of the leaf andstem rust fungi. (Fig. 2).

Induced resistance was evident when leaves wereinoculated with virulent rust strains immediately after theywere inoculated with non-virulent strains and it waseffective until about 4 days had elapsed betweensuccessive inoculations.

The rate of increase in pustule numbers by the leaf andstem rust fungi was significantly reduced by priorinoculation of leaf segments on the same or oppositesurface with urediospores of non-virulent strains of wheatrust fungi or P. helianthi. However, the size of pustules wasnot affected by prior inoculation with non-virulent strainsor species.

The results of histological studies on the infectionprocess are summarised in Table 1. In general, priorinoculation with a non-virulent strain resulted in a virulentstrain showing a reduction in percentage germination, areduction in the percentage of germ-tubes that producedappressoria over stomates and a reduction in thepercentage of appressoria from which penetrationoccurred. The percentage of urediospores thatgerminated but failed to produce appressoria overstomates was significantly greater on leaf segments thathad been previously inoculated with non-vir-ulent strains.

DiscussionThe data presented in this paper showed that resistance

in wheat leaf segments to the leaf and stem rust fungicould be induced by previous inoculation with non-virulentstrains of these fungi or with the non-wheat pathogen,Puccinia helianthi. The induced resistance was systemic inthe sense that it was expressed on the opposite leafsurface to that on which the inducer strain was inoculated.This finding confirms previous reports by Johnson & Allen(7) and indicates that the host plays an active role in thephenomenon.

01234560123456

TN INTERVAL BETWEEN SUCCESSIVE INOWLATIONS(MVS)

Fig. 1. Effect of inoculating the adaxial (A) and abaxial (B) leaf sur­face of wheat with an incompatible strain of P. recondita tritici onthe subsequent development of the compatible strain of the samefungus (full line, pre-inoculated with the incompatible strain andbroken line inoculated with compatible strain only).

The induced resistance response was non-specific inthat it was induced by a non-pathogen of wheat. This resultis consistent with those of other workers (18, 6, 9). Thenon-specific nature of the induced resistancephenomenon enhances its potential in the biologicalcontrol of certain diseases. We suggest that thisphenomenon could limit disease development insituations where crops are grown in mixtures such as withintercropping systems and in multiline cultivars.

One can speculate as to the extent to which weakparasites and epiphytic organisms on the rhizoplane andphylloplane might induce a resistance response in plants.

The induced resistance response occurred when thenon-virulent rust strain was inoculated onto leaf segmentsimmediately before the virulent strain. This finding differsfrom that of (3) and (9) who reported that a time period of 3to 4 days between successive inoculations was necessaryfor maximal expression of induced resistance to rusts inwheat and oats respectively. There are however, severalreports that indicate that resistance can be induced inmany host pathogen combinations by simultaneousinoculation with two fungal strains or species (13, 15, 7). Itwould seem therefore, that the time interval requiredbetween successive inoculations for induced resistance tobe expressed varies among host pathogen combinations.

Our results showed that resistance induced by non­virulent rust isolates was not apparent if the periodbetween successive inoculations exceeded 4 to 6 days.The reason for this is uncertain. Most reports in theliterature indicate that once resistance is induced in plantsit remains effective for at least 7 days. It is possible thatthis observation is an artifact related to the detached leaftechnique. The resistance factor may have either leachedinto, been diluted by or inactivated by the benzimadazolesolution. Alternatively, the host's metabolism may havebeen changed on the detached leaves with time, relative tointact leaves, in such a way as to reduce the expression ofthe induced resistance phenomenon.

Induced resistance was expressed as a reduction in thenumber of pustules produced per unit area of leaf surface.Histological studies showed that resistance induced by thenon-virulent strains or species of rust caused a reductionin the frequency of appressorial formation and penetrationfrom appressoria through stomates. Moreover, non­virulent strains of the wheat rusts, and in some instancesP. helianthi, caused a reduction in germination ofurediospores regardless of whether the inducer rust wasinoculated onto the same or opposite leaf surface to thevirulent strain. Non-virulent strains of wheat rusts tendedto affect the infection process more than P. helianthi. Thereason for this is uncertain. The results however, suggestthat a host response is induced by the inducer strain orspecies and this is consistent with the phytoalexin conceptof disease resistance (4, 10, 11,5).

REFERENCES

6

LSD r5%

42

B

Lsrr18

.1\

1 \".......... / \

v'" ......\ ,,'\\\/,'/'..............

\ I.-----/

... '.," ,\ ,

/\\ ,, ." It'--, --. , '"" ::-._.............

o6

.s>:

1234560123456

TIME INTERVAL BETWEEN SUCCESSIVE INOCULATIONS(DAYS)

A

"'" , ._........... l~DI" ..........

\ ~' \'0' \

\\

............... "/ ---.............~

LSOI51

40 lA

30

10

,.» ,.., .....~..........

","" "., I·..........20 \.,

<r:10 _.--<,J

...~ 60

~l/)...<l:~ 40...oE

c..,..... 20l/)w....::>Iii::>0..

Fig. 2. Effect of inoculating adaxial (A) and abaxial (B) leaf surfaceof wheat with a non-pathogen (P. helianthi) on the subsequentdevelopment of a compatible strain of P. graminis tritici (1) and P.recondita tritici (2) (full line inoculated with P. helianthi and brokenline inoculated with compatible strain only).

(1) Brown, J.F., Clark, D.J., and Kochman, J.K. (1974) - Atemperature controlled dew chamber to provide uniformconditions for infection by foliage pathogens. AustralianPlant Pathology Society Newsletter 3: 58.

(2) Brown, J.F., and Fittler, J.F. (1981) A quantitative method ofinoculating plants with uniform densities of fungal spores.Australian Plant Pathology 10: 51-53.

46

(3) Cheung, D.S.M., and Barber, H.N. (1972). Activation ofresistance of wheat to stem rust. Transactions BritishMycological society 58: 333-336.

(4) Cruickshank, I. (1963) Phytoalexins. Annual Review ofPhytopathology 1: 351-374.

(5) Ingham, J.L. (1972). Phytoalexins and other natural productsas factors in plant disease. The Botanical Review 38: 343.

(6) Johnson, C.O., and Huffman, M.D. (1958) Evidence of localantagonism between two cereal rust fungi.Phytopathology 48: 69-70.

(7) Johnson, R., and Allen, D.J. (1975). Induced resistance torust diseases and its possible role in the resistance ofmultiline varieties. Annals of Applied Biology 80: 359-363.

(8) Johnson, R., and Taylor, A.J. (1976). Effects of resistanceinduced by non-virulent races of Puccinia striitormis.Proceedings of the 4th European and MediterraneanCereal Rusts Conference, 1976. Interlaken/Switzerland,pp. 49-51.

(9) Kochman & Brown (1975). Studies on the mechanism ofcross protection in cereal rusts. Physiological PlantPathology 6: 19-27.

(10) Kuc, J. (1972). Phytoalexins. Annual Review ofPhytopathology. 10: 207-232.

(11) KUc, J. (1976). Phytoalexins and the specificity of plant­parasite interaction. In Specificity in Plant Disease, Ed,R.S.K. Wood and A. Graniti.

(12) Littlefield, L.J. (1969). Flax rust resistance induced by priorinoculation with an avirulent race of melampsora lini.Phytopathology 59: 1323-1328.

(13) Sabet, KA, Samra, A.S. and Mansour, I.S. (1966).Interaction between Fusarium oxysporum f. vasinfectumand Cephalosporium maydis on cotton and maize. Annalsof Applied Biology 58: 93-101.

(14) Shipton, WA and Brown, J.F. (1962). A whole-leaf clearingand staining technique to demonstrate host-pathogenrelationships of wheat stem rust. Phytopathology 52:1313.

(15) Wrather, J.A., Kuc, J. and Williams, E.B. (1972). Protectionof apple and pear fruit tissue against Fireblight withnonpathogenic bacteria. Phytopathology 63: 1074-1076.

(16) Wolfe, M.S. (1965). Physiologic specialization of Erysiphegraminis f. sp. tritici in the United Kingdom. Transactionsof the British Mycological Society 48(2): 315-326.

(17) Yarwood, C.E. (1954). Mechanisms of acquired immunity toa plant rust. Proceedings of the U.S. National Academy ofScience 40: 374-377.

(18) Yarwood, C.E. (1956). Cross Protection with two rust fungi.Phytopathology 46: 540-544.

47

Reaction of Kikuyu Cultivars and BreedingLines to Kikuyu Yellows Disease

P.T.w. Wong,Agricultural Research Centre, Tamworth,

New South Wales, 2340and

G.P.M. Wilson,Agricultural Research and Advisory Station,

Grafton, New South Wales, 2460.

Kikuyu yellows is the most serious disease of kikuyugrass (Pennisetum clandestinum Hochst. ex Chiov.)pastures and home lawns in New South Wales (2). Thedisease is caused by an undescribed phycomycete (3).The disease occurs as patches of yellowed grass, whichspread onwards at a rate of about a metre a year. Thediseased grass eventually dies and the centres of thepatches are invaded by dicotyledonous weeds and otherless desirable grass species. Pasture production issignificantly reduced. In recent years, the disease hasbecome serious in home lawns and sporting fieldsthroughout New South Wales. This has mainly resultedfrom the movement of infected kikuyu turf from the NorthCoast of New South Wales to inland areas. The diseasealso occurs in Southern Queensland but has not beenreported in other countries.

There are no satisfactory control measures against thedisease in pastures. Current recommendations include thekilling off of the diseased grass with systemic herbicidesand rotating with an immune crop for a year before re­sowing to kikuyu grass (1). To date, no fungicide hasproved effective against the disease. This has promptedthe screening of breeding material for resistance ortolerance to the disease.

In the first glasshouse experiment, kikuyu leavesinfected with the disease from the Grafton and Taree areasin New South Wales were used as inoculum. Ten gm ofdisease leaf pieces (5 mm) were added to the top 5 em ofsteam-sterilised soil in 13 cm diam. clay pots and 3cuttings of each cultivar and breeding line were plantedinto the inoculum layer. There were 3 replicates percultivar and breeding line and the pots were randomised inblocks on a bench in a glasshouse kept at 30aC. Controlpots (3 replicates) of each cultivar and breeding line wereinoculated with healthy klkuyu leaf pieces. These potswere randomised in blocks on an adjacent bench in thesame glasshouse. This was to avoid cross-contaminationby the pathogen since it is soil- and water-borne (3). Thepots were watered daily and fertilised monthly with 200 mlof a solution of "Thrive" fertiliser (Arthur Yates & Co.,Sydney). The pots were rated for disease after 3 months asfollows:

o no disease symptoms on leaves1 slight disease, less than half the pot with diseased

shoots2 moderate disease, more than half the pot with

diseased shoots but with no dead shoots.3 severe disease, the whole pot diseased and with

many dead shoots.

The breeding lines were W2, W3, W5, W6 (selectionsfrom open-pollinated Whittet kikuyu), B4, B6 (selectionsfrom open-pollinated Breakwell kikuyu), CPI 60066, anintroduction from Kenya and Camden kikuyu, a selectionfrom the Sydney University Farms, Camden, New SouthWales.