Characteristics of anthracnose resistance induced by localized infection of cucumber with tobacco necrosis virus

Physiological

Plant Pathology

(1980)

17,81-91

Characteristics of anthracnose resistance induced by localized infection of cucumber with tobacco necrosis virus ANNE E. JENNS and Department (AcceQtedfor

of Plant Pathology, Publication

Kui:

JOSEPH

January

Universi~

of Kentucky,

Lexington,

Kentucky

40546,

U.S.A.

1980)

The resistance to anthracnose in cucumber, induced systemically by a localized infection with tobacco necrosis virus (TNV), resembled that induced by Collctotrichum lagenariwn and Pseudomonas lachtymuns. Systemic resistance to anthracnose was first detectable at the time Increased resistance to anthracnose in the when symptoms caused by TNV appeared. second leafwas detected when eight necrotic lesions were produced on the first leafby infection with TNV, and increasing the number of TNV lesions on the first leaf increased anthracnose resistance in the second leaf, C. lagenarium penetrated uninfected leaves of TNV-infected plants less frequently than leaves of uninfected plants. Systemic infection of cucumber with tobacco ringspot virus but not with tomato ringspot virus induced resistance to anthracnose. Localized infection of cucumber cotyledons with tobacco mosaic virus did not induce the resistance of true leaves to anthracnose. The resistance induced by C. lugenuriam, P. Luh?ymans and TNV appears dependent upon the activation of more than one mechanism. It is unlikely to be unique to the infectious agent but rather is dependent upon a common function of the infectious agents.

INTRODUCTION Inoculation of one leaf of a cucumber plant with Colletotrichum lagenarium [6], tobacco necrosis virus (TNV) [4j, or Pseudomonaslachrymans [2] induced systemic resistance to anthracnose caused by Colletotrichum Lagenarium. Similarity in the characteristics of the resistance induced by the three pathogens would imply a common mechanism. The anthracnose resistance of plants inoculated with TNV was compared with that of plants inoculated with C. lagenarium [5, 6, IO] and P. lachrymans [2]. To investigate the specificity of virus infection on the induced resistance phenomenon in cucumber, the effect of infection with tobacco mosaic virus (TMV), tobacco ringspot, and tomato ringspot viruses on anthracnose was also tested. All pathogens previously shown to induce anthracnose resistance in cucumber caused local necrotic symptoms. TMV, however, is localized in cucumber cotyledons but does not produce necrotic lesions [9]. MATERIALS AND METHODS Culture of hosts and pathogens Colletotrichum lagenarium (Pass.) Ell. & Halst. race 1 was maintained on green bean agar at 24 “C, in the dark. Tobacco necrosis virus (TNV) was obtained from R. W. Fulton (University of Wisconsin, Madison, Wisconsin 53706), tobacco ringspot virus (TRSV) isolate NY63-110 and tomato ringspot virus (TMRSV) isolate NY-K67-140 from R. Providenti (Cornell University, Geneva,, New York 14456) and tobacco 004~4059/80/040081+

11 $02.00/O

Q 1980 Academic

Press Inc.

(London)

Limited

A. E. Jenns and J. KuC

82

mosaic virus (TMV) from T. P. Piione (University ofKentucky, Lexington, Kentucky 40546). Viruses were maintained in cucumber cultivar Wisconsin SMR-58, except TMV which was maintained in tobacco cultivar Kentucky 16. Cucumbers (Cucumis sativus L. cultivars Wisconsin SMR-58, Marketer, Ashley, Straight 8) were grown in 9 or 10 cm plastic pots containing Pro-Mix Bx (Premier Peat Moss Corporation Marketing, New York, New York 10036). A nutrient solution (Ra-Pid-Gro, Dansville, New York 14437) was applied twice weekly after seedling emergence. Plants were grown in a glasshouse at 23-31 “C under daylight supplemented during the winter months with 14 h of fluorescent and incandescent light. Inoculations

Conidial suspensions of C. lagenarium were prepared by flooding 4 to 7 day cultures with distilled water, agitating the agar surface with a rubber policeman and filtering the suspension through two layers of cheesecloth. Conidial concentration was determined with a haemocytometer. Conidial suspensions were either sprayed onto leaves or were applied in 40, 5 ~1 drops per leaf with a 100 ~1 hypodermic syringe. Plants were incubated in humidity chambers at 22-25 “C; after 24 h the chambers were opened, and after 48 h the plants were returned to the glasshouse. In some experiments the intercellular spaces ofleaves were infiltrated with conidial suspensions using a 100 fl hypodermic syringe. Infiltrated plants were not placed in humidity chambers. Plants were inoculated with TNV by rubbing Carborundum-dusted leaves with a cheesecloth pad soaked in either a crude homogenate of TNV-infected cucumber tissue in 0.05 M phosphate buffer, pH 7, or a suspension of purified TNV in water. Inoculations with TRSV, TMRSV and TMV were made by rubbing carborundumdusted cotyledons with a crude homogenate of TRSV-or TMRSV-infected cucumber tissue or TMV-infected tobacco tissue in O-05 M phosphate buffer, pH 7. Purijication

of TNV

Purification was based on the method described by Uyemoto et al. [15]. The virus was increased in cucumber cultivar Wisconsin SMR-58 and infected leaves were harvested 4 to 6 days after inoculation and homogenized (1 : 1, w : v) in O-005 M phosphate buffer, pH 7.6, containing 0.2% mercaptoethanol. The homogenate was filtered through two layers of cheesecloth and stored at -15 “C until further purification. To the thawed extract, n-butanol (9% by volume) was added dropwise with constant stirring. After standing for 15 h at 3 “C, the extracts were clarified by low speed centrihrgation at 0 “C at 9000 rev mind1 for 10 min in a Damon IEC B20-A centrifuge. Solid ammonium sulphate was added to obtain a final concentration of l-75 M and the mixture was held at 3 “C for 3 h during which time a foamy layer, containing the virus and other precipitated materials, rose to the surface. This layer was collected and centrifuged at low speed, which separated the contents of the tube into a top layer (mostly butanol) and a layer of precipitate floating on top of the ammonium sulphate solution. The precipitate was transferred to dialysis tubing and dialysed against cold distilled water until all visible insoluble material sank to the bottom of the tube. The contents of the dialysis tube were centrifuged at

Characteristics of anthracnose resistance

83

low speed and the supernatant, which contained the virus, was centrifuged at 4 “C at 27 000 rev min -1 for 90 min in a Beckman model L centrifiige, using a Spinco 30 rotor. The virus pellet was resuspended in distilled water and the suspension centrifuged at 9000 rev min -1 for 10 min. Virus concentration was determined by optical density measurements using a Per-kin-Elmer model 124 double beam spectrophotometer. The extinction coefficient of 5.5 OD mg -l ml -r at 260 nm was used [ 151. Penetration studies The procedures used were based on those of Richmond et al. [lo]. Half leaves of control plants and plants with TNV-infected cotyledons were inoculated with 30, 5 pl drops of a suspension of C. lagenarium spores (5 x 1OSml -‘) ; on the opposite half leaves four 5 ~1 drops of the suspension were placed between two felt pen marks. The leaf tissue between the felt pen marks was harvested 57 to 58 h after inoculation and placed in small vials containing saturated chloral hydrate for clearing. After heating the vials to dissolve crystals of chloral hydrate, the strips of tissue were placed on glass slides, the inoculated surfaces uppermost, and lactophenol was added. Slides were heated, the lactophenol replaced by cotton-blue stain (four drops of 0.2% cotton blue stain in 15 ml of lactophenol), and the slides reheated. After 20 min, excess stain was removed and the tissue was mounted in lactophenol. Appressoria were viewed at 1000 x magnification and the presence of penetration hyphae determined. Visualization ofstarch lesions The method described by Noordam [9] was used. Six days after inoculation of cucumber cotyledons of cultivar SMR-58 with TMV, the plants were placed in darkness overnight . Next day, the cotyledons were placed in 70% ethanol and heated to 80 “C on a water bath. The ethanol was decanted and the cotyledons stained in iodine KI-lactic acid (one part 2% iodine 6% KI solution to 20 parts lactic acid) for 15 min then rinsed in water. Cotyledons infected with TMV showed small dark blue spots. RESULTS Time requiredfor induction of systemicresistance Cotyledons of cucumber cultivar SMR-58 were inoculated with TNV and the first true leaves inoculated with C. lagenarium 12,24,48, 72 or 96 h later. The number of anthracnose lesions produced by the second inoculation was significantly smaller in TNV-infected plants than in untreated plants if the time between inoculations was at least 48 h (Table 1). Similarly, when cotyledons or first leaves were removed 1, 2, 3, 4 or 5 days after inoculation with TNV, and the leaves above were inoculated with C. lagenarium on the sixth day, increased resistance to anthracnose was detected only when TNV-infected tissue had been removed 2 or more days after inoculation (Tables 2 and 3). Maximum induced resistance was attained if TNV-infected leaves remained on the plant for only 48 h (Table 3). Removal of TNV-infected cotyledons at the time of inoculation with C. lagenarium did not affect the resistance of the leaves (Table 4). Resistance to anthracnose in leaves is still effective 12 days after inoculation of cotyledons with TNV (Table 4).

84

A. E. Jenns TABLE Effect tobacco

of vatying necrosis

Trial 1 2

number

of 0 h’

TNV inoculation Untreated TNV inoculation Untreated

1

the interval between inocukation of cuMtmber cultivar viras aad the first tm.s leaves with Colletotrichum resistance to anthracrwse Mean

Treatment cotyledons

37&O-60* 37*0-78

of lesions on first C. hgenmium 24h 12 h

30&2*16 28hl.67

SMR-58 cotyledons with lagenarium on induced

true leaf 7 days after inoculation (10’ spores per ml) 48 h 72 h

29k1.60 29Al.18 25hl.73 28&l-92

15fl-98*c 22&l-85 13f148** 25hl.25

7f1,89** 18i1.97 6+1.25*** 2Oh2.88

LI Time after inoculation of cotyledons with TNV when first true leaves with C. lagenarium. * The data represent mean values for 10 plants in each trial&standard c Values followed by asterisks (+, l *, or ***) were significantly different for untreated controls at the O-05,0*01 or 0.001 levels.

TABLE Effect of excising

Cotyledon

tobacco necrosis virus-infected resistance

treatment

Time cotyledons removed after inoculation 04

TNV

and J. KuC

with 96 h

7&l-16*** 14&1*03 8f2.lo*** 33-&1*61

were

inoculated

errors. from the values

2

cotyledons of cucumber to anthracnose

cultivas

SMR-58

on induced

Mean number of lesions on leaves 6 days after inoculation with Colletotrichum lagenarium (10’ spores per ml) a First leaf Second leaf 23b

30

24

28

18*=

20***

22

29

14**

12***

19

27

11***

12***

21

28

24 Untreated

48 Untreated

72 Untreated

96 Untreated

lo***

TNV

7*+*

120 Untreated

21

28

E First and second true leaves were inoculated with C. lageaariam 6 days after treatment of cotyledons. * The data represent mean values for 27 plants in three trials. Standard errors of means were: leaf 1 = l-17; leaf2 = l-37. c Values followed by asterisks (*, **, or ***) were significantly different from the values for untreated controls at the 0.05, O-01 or O*OOl levels.

Characteristics of anthracnose resistance

85

Resistanceinduced by increasing concentrationsqf TNV Inoculation of first leaves of cucumber cultivar SMR-58 with increasing concentrations of purified TNV resulted in increasing numbers of necrotic lesions and increasing resistance to anthracnose in second leaves (Table 5). The coefficient of the correlation between the number of TNV lesions on the first leaf and the number of anthracnose lesions on the second leaf was -0.54, which is significantly different from zero at the O-001 level. TABLE Effect of excising

Time removed First

3

the tobacco necrosis virus-infected jirst true leaf of cucumber anthracmse resistance in the second leaf

leaf treatment

first leaf after inoc. (4

cultivar

SMR-58

on

Mean number of lesions on second leaf 7 days after inoculation with CoUetotrichm lagenarim (10’ spores per ml)a 20f2*168

24 Untreated

21 f2.07 4f

1*79*++c

48 Untreated

20&3-25 3&o-92+** 72 24hl.64

Untreated

O&0-31*** 96 Untreated

31&2*40

TNV

1*0*48++* 120

Untreated

26&2-72

a Second leaf was inoculated 6 days after inoculation of first leaf. b The data represent the mean values for 10 plants in one trial&standard e Values followed by asterisks (***) were significantly different from appropriate untreated controls at the O-001 level.

errors. the values

for

the

Induction of resistancein severalcucumbercultivars Inoculating the cotyledons of three susceptible cucumber cultivars, Ashley, Marketer, and Straight 8, with TNV induced resistance in the first two leaves. Resistance was expressed as a reduction in the number and diameter of lesions produced by inoculation with C. lagenarium (Table 6). The penetration of C. Iagenarium

into Leaveswith induced resistance

Germination and formation of appressoria by C. lagenarium was the same on control leaves and those in which systemic resistance was induced. On control leaves, about 25% of the appressoria produced penetration hyphae, but on leaves with induced

A. E. Jenns and J. KuC

86

resistance only about 3% produced penetration hyphae (Table 7). Leaves with induced resistance to anthracnose may be resistant only to penetration of the pathogen or may have other resistance mechaniims, such as the ability to inhibit hyphal growth. When inoculum is infiltrated into the intercellular spaces of leaves, penetration of the epidermis is unnecessary for lesion formation. Infiltrating C. lagenarium conidia into leaves of cucumber plants, previously infected with TNV or C. lagenarium, produced significantly fewer lesions than infiltrating into uninfiected plants (Table 8). There was a smaller reduction in lesion number in TNV-infected plants compared with untreated plants when the challenge inoculum was infiltrated than when the

Effect

Treatment

of excising tobacco necrosis virus-infected &edons cultivar SMR-58 arc inocufuted with C. lagenarium

of cotyledona on day 0

at the time the Jirst leaves of cucwnbcr on induced resiskque to anthracnose

Mean number after inoculation

Day of inoculation of first leaf and removal of cotyledons

of lesions on 6rst leaf 7 days with Colletotrichum lapnariwn (10’ spores per ml) removed Cotyledons not removed

Cotyledons 8f

146***4

12 f 1*57+‘+

3 Untreated

25hl.65

24k1.96

13& 1*59***

lOf2*09*+*

34*0*99

35+1*41

6 Untreated

9*1*39***

10-&2*35***

9 23h2.15

Untreated

23h2.81

5f0.86***

6f

1.23**+

12 Untreated

30*1*91

28*2.27

a The data represent the mean valuer for 10 plants in one trial&standard errors. Values followed by asterisks (***) were significantly different from the values for untreated controls at the O@l level. TABLE Efjcct

5

of inoculating

Jirst leaves of cucwnber cultivar SMR-58 with increasing concentrations tobacco necrosis t&as on the resistance of the second leaves to anthracnose

TNV concentration on first leaf (J&g ml -1) 0 0.23 2.3 23.0 230.0

Mean number of lesions on6rstleaf3days after inoculation 0 Ab 4; Bc 74 D 176 E

Mean number of leaions on second after inoculation with Colletotrichum (106sporesperml)’

leaf 7 days lageaarium

39 A 27 B 14 c 12 c 5D

8 Second leaf war inoculated 7 days after the inoculation of first leaf. b The data represent the mean valuer for 10 plants in one trial. Values followed by different lettera arc significantly different at the 0.05 level.

in each

of

column

Characteristics of anthracnose resistance

87

challenge inoculum was applied as drops (Table 8). Lesion diameter, however, was significantly reduced in protected plants when the challenge inoculum was infiltrated, which implies that inhibition of epidermal penetration was not the only resistance mechanism in such plants. The reduction in lesion diameter in TNV-infected plants was not a result of TABLE 6 Effect of

inoculating co@dons

of three cwxmber cultivars with tobacco necrosis of the leaves to anthramosc

Symptoms on leaves 7 days after inoculation with Colletotrichum lagenarium (10s spores per ml)a Mean lesion diameter in mm Mean number of lesions * I\ r---w First leaf Second leaf First leaf Second leaf

Cotyledon treatment

Cultivar

virus on the resistunce

TNV

5&0*93****

4f0*95**+

1~2fo~ll**+

0.6f0.09*+*

37hO.95

2*7&0*11

3*1*0*07

14&1*38***

1.5+0.23***

0.7f0.42+*+

4Ohl.38

3.3 &-0.20

3.3 f0.39

1.8&0*22+*+

0.5&0.27+**

2*9&0*18

3.0 f0.22

Ashle? Untreated

33&O-93 6&O-97***

Marketef Untreated

33&O-97 6fl.15***

8% l-18**+

Straight 8’ Untreated

34f I.15

38hl.18

a Leaves were inoculated 7 days after the inoculation of cotyledons. b Three trials were performed with this cultivar. c Two trials were performed with these cultivars. dThe data represent mean values for 11 plants in each trial-J+andard errors. Values followed by asterisks (***) were significantly different from the values for the appropriate untreated controls at the O*OOl level. TABLE 7 Effect

of imadding

cotyledons of cucumber cultivar SMR-58 with tobacco necrosis penetration of the jirst leaf by Colletotrichum lagenarium

Cotyledons inoculated with TNV c

Trial 1” 2 3

Total appressoria examined

total

4253 5867 6512

126 162 100

Cotyledons untreated *

I

------Gz

virus on the

percent

number of lesions on opposite half-leafb

Total appressoria examined

total

percent

Mean number of lesions on opposite half-leaf

3 3 2

19 15 14

4313 7027 6661

1341 1251 1626

31 18 24

30 30 30

Penetrationsa

Penetrations

a Penetration from appressoria was determined by examination at 1000 x magnification of cleared and stained tissue, inoculated 57 hours previously with C. lagcnarium (5 x 10s spores/ml). b Half-leaves opposite to those to be examined microscopically were inoculated simultaneously with 30,5 pl drops of C. lugeaarium (5 x 10s spores/ml) and lesions were counted 7 days later. 5 Six plants were used per treatment in each trial.

,

88

A. E. Jenns and J. KuC

.

x P

Characteristics of anthracnose resistance

89

exposure to a smaller volume of inocuhun, since equal areas of water-soaking were produced by infiltrating leaves of TNV-infected and untreated plants. Moreover, there was no correlation’between the diameter of the water-soaked panels and the diameter of lesions subsequently produced at the site of infiltration. Effect of TJW-inoculation on stem height and leaf length of cuMcmbercultivar SMR-58 After measuring the length of cucumber plants from soil line to plant apex, the first leaves were either inoculated with 230 pg ml-r TNV; abraded and rubbed with a water-soaked pad; damaged in 20 places with dry ice; or left untreated. Plant length was measured four more times at intervals. Six days after the treatment of first leaves, second leaves were inoculated with C. lagenarium (lo5 spores/ml). There were no significant differences in the lengths of plants treated in the different ways. In another experiment, the lengths of second leaves were measured before, and at seven P-day intervals after treatment of the first leaves. No significant variation in the length of second leaves resulted from treating first leaves in the ways described above. EJect of infection with several viruses on the resistance of cucumber cultivar SMR-58 to anthraznose When cotyledons of very small cucumber seedlings (planted 7 days before inoculation) were inoculated with TMRSV, the cotyledons and first two leaves developed chlorotic lesions, some ofwhich eventually became necrotic, but the third leaves were symptomless. When similar plants were inoculated with TRSV, necrotic lesions were formed on the inoculated cotyledons and the iirst two leaves; the plants were severely stunted but the third leaves, although abnormally small and dark green, did not develop lesions. Infective virus was recoverable from third leaves of plants inoculated on the cotyledons with either virus. Twenty-four days after the inoculation of the cotyledons with TMRSV or TRSV, or rubbing cotyledons with sap from uninfected plants, the third leaves were inoculated with C. fagenariwn (1 x lo6 spores per ml). Significantly fewer anthracnose lesions were produced on plants infected with TRSV than on plants infected with TMRSV or uninfected. The lesions produced on the first and second leaves of cucumbers with a localized TMV-infection in the cotyledons were neither fewer nor smaller in diameter than those on uninfected plants. DISCUSSION Resistance to anthracnose was induced systemically in cucumber plants by infection with TNV as well as by localized infection with C. lagenarium [6] and P. lachtymans [2]. The appearance of necrotic lesions and the development of systemic resistance to anthracnose occurred between 24 and 48 h after TN-V-inoculation. The resistance was no better if cotyledons infected with TNV remained on the plant during infection of leaves with C. lagenarium than if they were removed. Resistance to anthracnose induced by C. lagenarium and P. lachrymans was not detected until about 96 h after inoculation with these pathogens [2,5] which is also when symptoms of anthracnose and angular leaf spot first appeared. Therefore, with the three inducing pathogens,

90

A. E. Jenns and J. KuC

induced resistance was observed at the time of symptom appearance, and all three pathogens produced necrotic lesions. Increasing the amount of C. lagenatium or P. lachynans inoculum applied to the first leaf increased the anthracnose resistance of the second leaf [2, 51. Similarly, when increasing concentrations of TNV were applied to the first leaf and produced between 8 and 206 lesions, there was a significant negative correlation between the number of TNV lesions on the iirst leaf and the number of anthracnose lesions on the second leaf. Systemic resistance to anthracnose was induced by fewer than 10 TN’V lesions on the first leaf, as it was by a single anthracnose lesion on the first leaf [.5]. Resistance to anthracnose was induced in several cucumber cultivars by TNV as it was by C. lagenarium [5] and P. 1achrynan.s[2]. Uninfected leaves on cucumber plants infected with either C. lagenarium [IO] or TNV were less frequently penetrated by C. lagenarium than leaves on uninfected plants. The numbers and sizes of lesions produced by inoculating TNV-infected or control plants with a range of concentrations of C. lagenarium suggested that there were fewer penetrations into infected than into control plants [4]. However, fewer lesions were produced by inoculating a TNV-infected plant with a given concentration of C. lagenarium spores than by inoculating a control plant with a lo-fold lower concentration [4j. On TNV-infected cucumber plants inoculated with a given concentration of C. lagenarium spores, the mean lesion diameter was the same as that on untreated plants inoculated with a 50-fold lower concentration of spores [4]. Since the reduction in the number of penetrations in TNV-infected plants is less than lo-fold, inhibition of penetration by the fungus is probably not the sole mechanism for induced resistance. Consistent with this hypothesis was the observation that when the C. lagenarium challenge inoculum was infiltrated into leaves, which obviated the need for epidermal penetration by the pathogen, resistance was still expressed in plants infected by TNV or C. lagenarium. More lesions were produced in infected plants when the challenge inoculum was infiltrated than when it was applied as drops, but this would be expected if part of the resistance mechanism were by-passed. Stunting is a common symptom of virus infections [8]. Increased auxin levels have often been reported in virus-infected plants [14] and the stunting caused in cucumbers by infection with cucumber mosaic virus and in barley by infection with barley yellow dwarfvirus is associated with a reduction in the endogenous gibberellin content [I, 131. A reduction of stem height or leaf size in plants infected with TNV on the first leaf would suggest the involvement of a plant hormone in the induced resistance. A reduction in height or size, however, was not observed in TNVinfected plants. Stem-inoculation of tobacco with Peronosfioratabacina, which induced foliar resistance to blue mould, also reduced the number and size of leaves under certain nutrient conditions [3]. In Samsun NN tobacco, systemic resistance to TMV was induced by infections with TNV or TRSV which caused necrotic lesions, but not by infection with alfalfa mosaic virus, potato virus X, or potato virus Y, which did not cause necrotic lesions [II]. Similarly in cucumber, resistance to anthracnose was induced by TNV, which caused local necrotic lesions and did not infect systemically, and by TRSV which caused necrotic lesions and did infect systemically. Anthracnose resistance was not

Characteristics

of anthracnose

resistance

91

observed in cucumbers infected systemically with TMRSV which caused initially chlorotic lesions which eventually became necrotic. The mechanism of local anthracnose resistance in systemically virus-infected plants, however, may not be the same as that of systemic resistance in locally virus-infected plants. Localized infections of cucumber cotyledons with TMV, which caused starch lesions but no necrosis, did not induce systemic resistance to anthracnose. Loebenstein [7] was unable to demonstrate systemic resistance to virus in cucumber cotyledons with starch lesions. The data in this paper are consistent with the presence of common mechanisms for the resistance induced by C. lagenarium, P. lachrymans and TNV, and are remarkably similar to those reported by Ross and his colleagues for resistance to local lesion viruses in plants infected with local lesion viruses [12]. The induced resistance described, therefore, is unlikely to be unique to the infectious agent but rather is dependent upon a common function of the infectious agents. Journal Paper No. 79-11-148 of the Kentucky Agricultural Experiment Station, Lexington, Kentucky 40546. The research reported in this paper has been supported in part by a grant from the Ciba Geigy Corporation. REFERENCES 1. BAILIS, K. W. (1974). The relationship of gibberellin content to cucumber mosaic virus infection of cucumber. Physiological Plant Pathology 4, 73-79. 2. CARUSO, F. L. & Ku&, J. (1979). Induced resistance of cucumber to anthracnose and angular leaf spot by Pseua%monas lachrymans and Colletotrichum lagenarium. Physiological Plant Patholagy 14, 191-201. 3. CRUICKSHANK, I. A. M. & MANDRYK, M. (1960). The effect of stem infection of tobacco with Peronospora tabacina (Adam) on foliage reaction to blue mould. Joumul of the Australian Znstitutc of Agricultural Science 26,369-372. 4. JENNS, A. E. & Kud, J. (1977). Localized infection with tobacco necrosis virus protects cucumber against Colletotrichtan lagenarium. Physiological Plant Pathology 11, 207-212. 5. Ku&, J. & RICHMOND, S. (1977). Aspects of the protection of cucumber agninst Colletotrichum lagenarium by Collctotrichum lagenarium. PhytOpathology 67,533-536. 6. Ku&, J., SHOCKLEY, G. & KEARNEY, K. (1975). Protection of cucumber against Colletotrichum lagenarium by Colletotrichum lagenarium. Physiological Plant Pathology 7, 195-199. 7. LOEBENSTEIN, G. (1972). Localization and induced resistance in virus infected plants. Annual Review of Phytopathology 10, 177-206. 8. MATIWEWS, R. E. F. (1970). Plant Virology. Academic Press, New York. 9. NOORDAM, D. (1973). Identification of Plant Viruses, Wageningen, Centre for Agricultural Publishing and Documentation. 10. RICHMOND, S., KuC, J. & ELLISTON, J. E. (1979). P enetration of cucumber leaves by Colletotrichum lagenarium is reduced in plants systemically protected by previous inoculations with the pathogen. Physiological Plant Pathology 14, 329-338. 11. Ross, A. F. (1961). Systemic acquired resistance induced by localized virus infections in plants. Virology 14, 340-358. 12. Ross, A. F. (1966). Systemic effects of local lesion formation. In: Viruses of F’hnts, Ed. by A. B. R. Beemster &J. Dijkstra, pp. 127-150, North Holland Publishing Co., Amsterdam. 13. RUSSEL, S. L. & K~MINS, W. C. (1971). Growth regulators and the effect of barley yellow dwarf virus on barley (Hordeum vulgare L.): Annals of Bo&ny 35, 1037-1043. 14. SEQUEIRA, L. (1963). Growth Regulators in Plant Disease. Annual Review of Phyt@athologv 1,5-30. 15. UYEMOTO, J. K., GROWN, R. G. & WAKEMAN, J. R. (1968). Selective activation of satellite virus strains by strains of tobacco necrosis virus. Virology 34,410-418.