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Induction of localized and systemic protection against Phytophthora parasitica var. nicotianae by tobacco mosaic virus infection of tobacco hypersensitive to the virus
Physiolugical Plant Pathology (1979)
15,32
l-330
Induction of localized and systemic protection against
Phytophthora parasitica var. nicotianae by tobacco mosaic virus infection of tobacco hypersensitive to the virus JOHN
L.
MCINTYRE
and J. ALLAN DODDS
Defiartmcnt of Plant Pathology, The ConnecticutAgricuItural Ex@rkent Station, P.O. Box 1106, New Haven, Connecticut06504, U.S.A. (Accepedfor publication May 1979) Within 3 days of inoculating expanded leaves of NicotMM tubacum cv. Windsor Shade 117 with tobacco mosaic virus (TMV), viral lesions were apparent and, in younger uninoculated leaves, systemic induced protection was detected against race 3 of Phyfophthora @arasitica var. nicotianue. This protection occurred on all leaves above the TMV-inoculated leaves for at least 22 more days. The level of protection usually approached absolute (less than one fungal lesion per leaf as compared to 3 to 14 fungal lesions per leaf on unprotected plants). As few as 12 TMV local lesions per inoculated leaf were sufficient to induce significant levels of systemic protection. Localized protection was also induced within 3 days of inoculating leaves with TMV, but 7 days were required to induce signiticant levels of protection in halfleaves opposite TMV-inoculated half-leaves. TMV-inoculated plants flooded with a zoospore suspension or transplanted to infested soil developed fewer black shank infections and were less extensively damaged than control plants.
INTRODUCTION Interactions between viral and fungal pathogens of plants have been observed but the consequence of the interaction for any one system is difficult to predict. The virus infection may predispose the plants to greater [l-2, 3, 7, 13, 40, 41, 45, 511 or lesser [9, 15,17, 19,21, 22, 24,33,54] susceptibility to the fungal pathogen, or have no effect [al, 45, 481. In the three reports [17, 22, 541 where localized virus infections decreased susceptibility, induced or acquired resistance may have been involved. This type of interaction is also observed between viruses [27, 46, 471, fungi [5, 8, II, 16, 25, 26, 34, 351 or bacteria [4, 12, 29, 31, 37, 38-J. In some of these systems only localized protection was detected [4, 11, 12, 16, 29, 35, 371, but in others systemic resistance developed in host tissues remote from those treated with the inducing agent [S, 8, 25, 26, 31, 34, 38, 46, 471. We report here on a new system for studying such interactions: the induction of localized, opposite half-leaf and systemic leaf, stem and root protection of tobacco against Phytophthora pamsitica var. nicotianae by localized leaf infection with tobacco mosaic virus, MATERIALS Plan&
AND METHODS
The host plant used was Ncotiana tabacum cv. Windsor Shade 117 (WS 117), a cultivar hypersensitive to tobacco mosaic virus (TMV) and highly susceptible to Phytophthora pamritica Dast. var. nicotianae (Breda de Haan) Tucker ( = Phytophthma 0048459/79/06032
1+ 11$02.00/0
@ 1979 Academic
Press Inc.
(London)
Limited
3!2!?
J. L. McIntyre and J. A. Dodds
nicotianae van Breda de Haan var. nicotianac). In preliminary studies N. tabacum cvs Burley 21 and Samsun NN were also used. These cultivars are also susceptible to P. @arasitiGavar. nicotianae and hypersensitive to TMV. Tobacco seed was germinated in the greenhouse on a mixture of soil : peat : sand (1 : 1 : 1) and then transplanted at the two leaf stage to 350 ml Styrofoam cups containing the same soil mixture. Plants were maintained in a greenhouse at 26 f 5 “C until they reached the four to six leaf stage. Virus Tobacco type, strain (U-l) of tobacco mosaic virus (TMV) was maintained in and purified from N. tabacum cv. White Burley. Infected tissue was homogenized in 0.02 M KsHPO,, 1o/o P-mercaptoethanol and strained through cheesecloth. The extract was stirred for 15 min with 8% butanol then clarified by centrifugation (10 OOOgx 15 min). Clarified extracts were adjusted to 0.1 M NaCl and TMV was precipitated with 4% polyethylene glycol (mol. wt = SOOO),collected by centrifugation (10 000 g x 30 min) and resuspended in 0.01 M sodium citrate buffer, pH 8.0. Further purification of TMV was by differential centrifugation (80 000 g x 90 min, 10 000 g x 15 min) and sucrose density gradient centrifugation (10 to 40% sucrose, Beckman SW27 rotor, 131 000 g x 3-O h, 5 “C) in 0.01 M sodium citrate buffer, pH 8.0. An E&x = 3.0 was used to estimate the concentration of TMV in the final preparation. Polyacrylamide gel electrophoresis of viral RNA or viral protein [IO] indicated that the virus preparation used in this study was not detectably contaminated with host components. Fungus Race 3 of P. parasitica var. nicotianae (Ppn) (isolate M-15T = isolate A7Y) [36] was maintained on P-l medium [18] in the dark at 25 “C. Single mycelial plugs, cut with a no. 2 cork borer from ‘I-day-old cultures, were placed into 125 ml Erlenmeyer flasks containing 25 ml of lima bean broth (Difco Laboratories, Detroit, MI 48232, U.S.A.). After 5 days’ growth in the dark at 25 “C, mycelia from three flasks were harvested by filtration onto single sterile pieces of Miracloth (Chicopee Mills, Inc., Milltown, NJ 08850, U.S.A.) in a sterile Buchner funnel. To induce zoosporangial formation, mycelia were washed with sterile water and transferred on the Miracloth pieces to Petri plates containing 3% water agar. These cultures were incubated for 5 to 7 days in the dark at 25 “C, zoospores were released and the concentration adjusted to lo6 zoospores per ml as previously described [ 391. Virus inoculations to induceprotection The adaxial surface of entire tobacco leaves (the youngest expanded leaf and up to the three next oldest leaves) or a lateral half of each leaf (tobacco leaves are divided into two halves by the midvein) was rub-inoculated with cotton gauze soaked with TMV (1, 10, 25, 50, 100 or 200 pg ml -1) or inactivated TMV (25 pg ml-l, boiled for 25 min) in 0.01 M potassium phosphate buffer, pH 7.2, containing 1% of w/v celite as an abrasive. Uninoculated control plants were abraded with celite in buffer only. Plants were then maintained in a greenhouse at 25 & 5 “C.
Induction
of localised
and systemic protection
Three days after inoculation TMV prior to the fungal inoculation. Fungus inoculation
to test protection
323
local lesions were visible and they were counted
(challenge
inoculation)
The adaxial surface of leaves to be challenged wa:s abraded by rubbing with a cotton gauze soaked in buffer with celite and immediately sprayed to run off with the zoospore suspension using a hand-pump sprayer. Plants were maintained at 25 + 2 “C in closed plastic bags containing a moistened hand towel and subjected to a 12 h photoperiod under fluorescent lights (260 PE rnT2 s-l PAR at plant height). Two days after the leaves were inoculated with zoospores, the black shank lesions were counted. Leaf abrasion prior to inoculation with the zoospore suspension was necessary for the development of maximum lesion numbers. Protection studies
Protection was studied on entire or half-leaves inoculated with TMV (localized leaf protection), on half-leaves opposite to those inoculated with TMV (half-leaf protection), on leaves above those inoculated with TMV (systemic leaf protection), and by flooding TMV-inoculated plants with a zoospore suspension or transplanting TMV-inoculated plants into soil infested with the black shank pathogen (root and stem systemic protection). Phytophthora parasitica var. nicotianae is a soil-borne pathogen, and roots and underground portions of the stem are the normal site of infection [32]. The fungus inoculations to test leaf protection were made immediately after the virus inoculations or at intervals up to 25 days later. Root and stem protection was tested starting 10 days after virus inoculation. AI: that time 2 ml of a zoospore suspension was pipetted around the stem at the soil line. The zoospores were washed into the soil by immediately flooding with 100 ml of distilled water. In another experiment, and again 10 days after virus inoculation, the plants’ root ball with soil was cut to a manageable size (diam. = 2 cm), and then two or three plants were transplanted into single 5 1 plastic containers containing field soil naturally infested with race 3 of Ppn. In a second experiment, the naturally infested soil was reused and also diluted 1 : 3 and 1 : 8 with Pro Mix (Premier Peat Moss Corporation Marketing, New York, NY 10036, U.S.A.). Plants were maintained in the greenhouse (26 + 5 “C) and observed daily for black shank stem lesions or wilting. Several days after all of the control plants in naturally infested soil had black shank lesions, control and TMV inoculated plants were removed .from the soil, washed free of debris, and the black shank lesions on the exterior surface of roots and stems were measured. Then roots and stems of each plant were split longitudinally and the length of pith necrosis and destruction was measured. All experiments had a minimum of five plants per treatment and were performed at least twice, and always celite-abraded plants were included as controls. Data are presented as averages for all observations and results were analyzed by Student’s standardized t-test or Duncan’s new multiple range test. RESULTS
Tobacco cv. WS 117 exhibiting the hypersensitive response to TMV was protected against Ppn and this protection has been observed i.n over 250 plants (Plate 1).
324
J. L. McIntyre
and J. A. Dodds
Similar results were obtained in preliminary studies using tobacco cvs Burley 21 and Samsun NN. Black shank lesions were evident 2 days after inoculating WS 117 plants with the black shank pathogen, and the number did not increase on subsequent days. These lesions enlarged at about the same rate on control and protected plants, and within several days leaves with lesions were entirely necrotic. Two leaves with 101 or more TMV lesions per leaf provided the maximum protection level for all leaves (Table 1). However, as few as 12 to 50 TMV lesions per leaf provided significant protection. A virus concentration of 25 pg ml-r provided 229 + 34 (2 + se.3 lesions per leaf and was used in most experiments. Inactivated TMV at this concentration did not cause the hypersensitive interaction and did not induce protection to Ppn (Table 2). Localized protection against Ppn was detected when leaves were challenged as early as 3 days after they were inoculated with TMV (Table 3). When challenge followed TMV inoculations by 7 days, TMV-treated leaves averaged 98% fewer black shank lesions than did control leaves. TABLE 1 Effect of number of tobacco mosaic virus (TMV) 1esions on the number of lesions caused by Phytophthora parasitica Dar. nicotianae (Ppn) on tobacco leaves2
Average TMV
no. of lesions
per
leaf
( f s.e.z)r
PPn ll*2+1.1az 2*9&0*7b 2*2*0.6b 1*5+0.4bc 1.0 f 0.4bc 0.3 + 0.2c 0.5 * 0*2c
0 12-50 51-100 101-200 201-300 301-490 401-500
z The adaxial surface of the youngest fully-expanded leaf and one leaf below it on tobacco plants at the six leaf stage was inoculated by rubbing with 1, 10, 25, 50, 100 or 200 pg ml-’ TMV. Plants were challenged 7 days later by spraying all leaves to run-off with a suspension containing 10s zoospores per ml of P@n. r Values represent averages for two leaves per plant for TMV and six leaves per plant for P/m. S Numbers followed by the same letter are not significantly different at PGO.01.
Effect
TABLE 2 of tobacco mosaic virus (TMV) and heat-inactivated tobacco mosak virus on the number lesions on tobacco Ieaves caused by Phytophthora parasitica var. nicotianae (Ppn)”
Treatment None Inactivated TMV
Average
TMV
no. of Ppn lesions
of
per leaf (+ s.e.;)r
12.5 k 2.0aS 9-7 *343a 0.6kO.lb
0 The adaxial surface of the youngest fully-expanded leaf and one leaf below it on tobacco plants at the six leaf stage was inoculated by rubbing with 25Fg ml-r TMV or inactivated TMV (25 Pg ml-’ TMV; boiled for 25 min). Plants were challenged 7 days later by spraying all leaves to run-off with a suspension containing 10s zoospores per ml of Ppn. s Values represent averages for six leaves per plant. * Numbers followed by the same letter are not significantly different at P
PLATE 1. Black shank lesions on tobacco leaves 2 days afte:r challenging with zoospores of Phytophthora pamsitica var. nicotianae. Leaves from tobacco mosaic virus-inoculated plant (left) and control plant (right). Local and opposite half-leaf protection. LL = TW local lesion. BSL = black shank lesion.
8*5+0*9 5.3 + l-1 l-3+0*5 0.8 + 0.3 3-l &O-6 1*4+1-l 0*2+0*1
TMV ‘2.17* 0.97 3*01*+ 4.OO** 7*45*+ Z-71** g-35**
t value’
Average
var. nicotianae
l-7*0-5 3.2 &- l-3 4*9+ l-6 2.5kO.8 1-I f0.7 Z-5+ l-4 1*3&O-5
TMV
6y
( + s.e.,)v
O-88 l-53 1.47 l-86 l-52 l-37 2*96**
t value
leaf
(Ppn)
4-65 l-3 z-5+ 1.2 7*4* 2.0 3.9+ 1.8 3-450-8 13.5+3*3 8.9+2.0 4-o_+ 1.1 13.9k3.5 9.8+1-O
Untreated
5*5_+ 1.3 3*1+ 1.2 Z-3+ 1-l 0.42 o-3 0.840-Z 0*5+0-Z O*Z~O*l O*l+O*l 2-75 l-1 0*7+0.2
TMV
Systemic
tobacco mosait virus (TMV)*
half
leaf
(half-leaf)
or younger
leaves
(systemic).
below it on tobacc- v p!auts at the six to eight leaf stage was inocuor up to 25 days later by spraying leaves to run off with a sus-
2.3_+0-4 1*2*0-z 7*5+0-7 5*9* l-9 Z-7+0.8 4-9&1-l 4.7 * I.3
Untreated
no. of Ppn lesions per Half leaf
3 parasitica
fullysxpauded leaf and up to three leaves TMV. Plants were chzl!enged immediately per ml of Pfm. lesions on the same leaf (local), opposite or P
so_+ l-4 7-l + l-5 13.4p4.0 8-4 + O-8 14.8+0*2 6*1+1*3 ll*l+ 1.8
Untreated
e The adaxial surface of the youngest lated by rubbing with 25 pg ml-1 pension containing !OL zouspores s Values represent averages for Pfin BValues significant at PdO.05 (*)
1:
0 2 3 4 5 6
Daye after TMV that challenge was applied
Local
Local, hof leafr and pstemic protection of tobacco@ants against
TABLE Phytophthora
o-47 O-36 Z-25** Z-34** 4.63*+ 3.91.2 6*07** 3.60”’ 3*05** g-99+*
tvalue
-_
5 v a g 5 =
3 0
iii 0. % Q
328
J. L. McIntyre and J. A. Dodds
One-half leaf protection against Ppn occurred when challenge followed TMV inoculation by 7 days, at which time half-leaves opposite virus inoculated halfleaves averaged 73% fewer black shank lesions than did control half-leaves. No significant protection was detected earlier than this (Table 3). Systemic protection also occurred since leaves younger than TMV-treated leaves were protected against the black shank pathogen (Table 3). Significant protection occurred when plants were challenged 3 days after inoculation with TMV, and protection was still evident when challenge followed TMV-inoculation by 25 days. All younger leaves, regardless of position above TMV-inoculated leaves, were protected at each of the challenge times. This is demonstrated by data for a challenge inoculation occurring 7 days after TMV inoculation (Table 4). Reductions in fungal lesion numbers on progressively younger leaves were due primarily to reduction in leaf area of these expanding leaves. At the latest challenge date the youngest leaves inoculated with Ppn were eight to nine leaf positions above the youngest TMV-inoculated leaf.
Dirtribution
of vs&mic
Leafno.* 1 2 3 4
TABLET protcctiun of tobacco leaves against Phytophthora tianae (Ppn) by tobacco mosaic virus (TMV)@ Average Untreated 10~0+3~1 4.3k2.5 2.0-11.3 1*0&0.6
no. of Ppn lesions TMV 0*8+0*3 0 0.1 &O-l 0
per
parasitica
leaf
var. nico-
(+ se.,) t value 4.69** 2~83~. 3*29** 2*39*
a The adaxial surface of the youngest fully-expanded leaf and one leaf below it on plants at the six leaf stage was inoculated by rubbing with 25 pg ml-s TMV. Plants were challenged 7 days later by spraying to run-off with 10s zoospores per ml of Ppn. r Leaf 1 is the first leaf above the leaves inoculated with TMV. l Values significant at PGO.05 (*) and PsO.01 (**).
Protection of plants against Pbn also occurred when plants with TMV-treated leaves were transplanted to soil infested with the black shank pathogen (Fig. 1). Similar results were obtained when plants were flooded with a zoospore suspension, and 21 days after inoculation 8 of 10 control plants had black shank lesions while only 2 of 10 plants with TMV-treated leaves had symptoms. A dilution of infested soil did not result in increased levels of protection. In protected plants, the length of external and internal lesions due to the disease was reduced significantly at all soil dilutions tested (Table 5). Also observed, in protected plants only, were small lesions that were not enlarging, indicating an apparent arrest of the infection. DISCUSSION
Our results show local, opposite half leaf, and systemic protection of tobacco against P/m by prior inoculation with TMV. The high level of protection achieved and its longevity and systemic nature indicate that this system may be useful for studies
induction
327
of localised and systemic protection
Tme (days)
Fro. 1. Incidence of black shank lesions on stems of tobacco plants growing naturally infested with Phytophthora parasitica var. nicotiaaae. Leaves of plants were (0) or inoculated with tobacco mosaic virus (0) 7 days prior to transplanting infested soil. TABLE 5 caused by Phytophthora parasitica var. nicotianae inoculated with tobacco mosaic Z&W.J (TAG’) w
Root and stem damage
(Ppn)
Length Experiment
Soil dilution=
Stem necrosis
Untreated
1
0
Exterior Interior”
14.8&2-3 18.8k l-6
2
0
Exterior Interior
1:3
Exterior Interior
1:8
Exterior Interior
in soil abraded them to
in plants
of necrosis
(cm)
TMV
t values
4.3 + 2.0 6.9 f 2.3
3.35** 4*19**
7.4kO.6 6.2kO.7
2.6+0-7 1*5+0*5
5*03** 5.19;’
5.3 f o-9 4*4* 1.0
0.7kO.4 0*4+0-3
4.69** 3.94**
6.6k2.0 5*7+ 1.7
3.12** 4.33**
14*2* 14.5*
1.6 1.1
W The adaxial surface of the youngest fully expanded leaf amcl one leaf below it of plants at the six leaf stage was inoculated by rubbing with 25 pg ml-’ TMV. Plants were transplanted 7 days later to soil naturally infested with Pp. e Soil was diluted as indicated with Pro Mix. svalues significant at P
on the physiological and biochemical changes that result in induced protection. The root and stem protection suggests that by further understanding and then exploiting this system, control of black shank in field situations may be achieved by induced resistance. Two observations indicated that a host response was required for protection to occur. A time period was necessary between TMV inoculation and the onset of protection, and a direct interaction between viru.s and fungus did not occur 22
J. L. McIntyre and J. A. Dodds
328
since protection
was systemic and virus particles are localized in the TMV
lesions
[201The level of protection in induced plants was routinely high, and often was absolute. The reduction in lesion numbers on leaves of protected as compared to control plants was 25 + ‘I-fold (5 f s.e.r.). This equalled or exceeded protection levels for other host-parasite systems where lesion numbers were used to assay protection [5, 6, 8, 17, 19, 22, 25, 34, 46, 47, 541. Local and systemic leaf protection occurred 3 days after TMV inoculation, coincident with the appearance of TMV lesions, whereas opposite half-leaf protection occurred 7 days after TMV inoculation. If phloem transport of the inducing principle or protectant is assumed, materials would translocate throughout the induced leaf half and to the remainder of the plant prior to their appearance in the opposite leaf half [52]. Ross [46, 471 demonstrated that tobacco plants hypersensitive to TMV developed protection against several different viruses following inoculation with TMV. Results of the present study and those of Ross show similar kinetics for local, opposite half leaf, and systemic protection. Protection of tobacco against bacteria and other fungi has also been induced by TMV [17, 301. In those studies only local protection was reported, but methods of assay may have precluded the observation of systemic protection. Resistance of tobacco to fungi, bacteria, and viruses can also be induced in ways other than by the use of TMV [6, 8, 14, 17, 23, 27-29, 31, 34, 35, 42-44, 50, 531. Sequeira & Hill [49] have noted that it seems obvious that all of these protection phenomena cannot be due to the presence of a single inhibitor or the result of a single protective response. However, our results in combination with the numerous reports cited above of methods to induce protection of tobacco against a wide array of micro-organisms, suggests that it is unlikely that tobacco plants are capable of responding specifically to the varied stresses that induce protection, We therefore propose a generalized stress response to these many different stimuli. This stress response may in turn initiate several different protection mechanisms. However, a specific stress or challenge inoculation may favour the activity of a certain protection mechanism. The technical assistance of M. Reisner and M. Tiffany
is gratefully
acknowledged.
REFERENCES 1. BATEMAN, D. F. (1961). Synergism between cucumber mosaic virus and Rhizoctoaia in relation to Rhizoctonia damping-off of cucumber. Phytopathology 51, 574-575 (Abstr.). 2. BEUTE, M. K. (1970). Effect of virus infection on susceptibility to certain fungus diseases and yield of Gladiolus. Phytopathology 60, 1809-1813. 3. BEUTE, M. K. & LOCKWOOD, J. L. (1967). Mechanism of increased susceptibility to root rots in virus-infected pea. Phytofiathologv 57, 804 (Abstr.). 4. CARROLL, R. B. & LUKEZIC, F. L. (1972). Induced resistance in alfalfa to Copmebacterium insidiosum by prior treatment with avirulent cells. Phytopathology 62, 555-564. 5. CARUSO, F. L. & Kuc, J. (1977). Protection of watermelon and muskmelon against Collctotrichum lagenarium by Colletotrichum lagenarium. Phytopathology 67, 1285-1289. 6. COHEN, Y., REUVENI, M. & KENNETH, R. G; (1975). Resistance to powdery mildew in tobacco induced by Peronospora tabacina. Phytopathology 65, 1313-1315. 7. CRANE, G. L. & CALPOUZOS, L. (1969). Synergism of Cercospora beticola and beet yellows virus in Idling sugar beet leaves. Phytopatholog)r 59, 1338-1339.
lnductlon
8.
of locallsed
CRUKXSHANK, Peronos$ora
820
and systemic protection
I. A. M. & MANDRYK, M. (1960). The tabacina on foliage reaction to Blue Mould.
effect of stem infection Journal of ths Australian
of tobacco with Institute of Agri-
cultural.%ience 26, 369-372. 9. DIAZ-POLAN~~, C., Surrrr, S. H. & H~~cocx, J. G. (1969). Effect of virus infection on stem rot of squash caused by Fusarium solani f. sp. -bitae. Phytopathology 59, 18-22. 10. DODDS, J. A. & ELTON, R. I. (1974). Masking of the RNA genome of tobacco mosaic virus virology 59, 418-427. by the protein of barley stripe mosaic virus in doubly infected barley. 11, ELLISTON, J., KIJC, J. & WILLLUS, E. (197 1). Induced resistance to bean anthracnose at a distance from the site of the inducing interaction. Phyto~atholo~ 61, I1 10-l 112. 12. ERCOLANI, G. L. (1970). Bacterial canker of tomato. IV. The interaction between virulent and avirulent strains of Coqwbacterium michiganense (E. F. Sm.) Jens. in vivo. Phytopathologia MediterfUfleU 9, 151-159. 13. FARLEY, J. D. & LOCKWOOD, J. L. (1964). Increased susceptibility to root rots in virus-infected peas. Phytojathology 54, 1279-1280. 14. GLWNAZZI, S. & -19, B. (1974). Virus resistance induced in plants by._ polyacrylic acid. Jownal of General Virology 23,‘1-9.. 15. GOHEEN. A. C. & SCHNATHORST W. C. 11961). Resistance to uowderv mildew in leaf roll-affected ’ grap&Gnes. Plant Disease Re#orter 45; 641L643. [ T\ A 16. ~R~CHMIDT, R., ACRES, S. & Kuc J. (1976). Protection of cucumber against Col[ctotrihum lagenarium and Claa@nium cucumerinum. Phytopathology 790-794. 17. Hxcnrr, E. I. & BATEMAN, D. F. (1964). Nonspecific acquire% resistance to pathogens resulting from localized infections by Thhiclaoiopsis basicola or viruses in; tobacco leaves. Phytobathology
54, 523530. 18. HOI-IL, H. R. (1975). Levels of nutritional complexity in PhytoOp?iwra: lipids, nitrogen sources and growth factors. Phytofiathologische
330
J. L. McIntyre and J. A. Dodds
36. MCINTYRE, 37. 38. 39. 46. 41. 42. 43. 44. 45. 46.
47. 48. 49.
50. 51. 52.
53. 54.
J. L. & TAYLOR, G. S. (1978). Race 3 of Phytophthora parasitica var. nicotianae. Phytopathology 68, 35-38. MCINTYRE, J. L., KUC, J. & WILLIAMS, E. B. (1973). Protection of pear against tire blight by bacteria and bacterial sonicates. Phytopathalogy 63, 872-877. MCINTYRE, J. L., KUC, J. & WILLIAMS, E. B. (1975). Protection of Bartlett pear against 6re blight with deoxyribonucleic acid from virulent and avirulent Erwinia amylovora. Physiological Plant Pathology 7, 153-170. MILLER, P. M. & MCINTYRE, J. L. (1976). Oxamyl-treated soil protects tobacco against black shank. Phytopathology 66, 221-224. MWANZA, W. P. & WILLIAMS, L. E. (1966). Viruses as predisposing factors in the susceptibility of corn and wheat plants to other pathogens. Phytopathology 56, 892 (Abstr.). NITZANY, F. E. (1966). Synergism between Pythium uitimum and cucumber mosaic virus. Phytopatholoo 56, 1386-1389. NOVACKY, A. (1972). Suppression of the bacterially induced hypersensitive reaction by cytokinins. Physiological Plant Pathology 2, 101-104. NOVACKY, A. & HANCHEY, P. (1976). Effect of internal leaf injury on bacterial hypersensitivity. Acta Phytnpatlwlogica Acaakmiae Scientiarum Hungaricae 11, 217-222. NOVACKY, A., ACEDO, G. & GOODMAN, R. N. (1973). Prevention of bacterially induced hypersensitive reaction by living bacteria. Physiological Plant Pathology 3, 133-136. RAJU, D. G., SILL, W. H., JR & BRO~XR, L. E. (1969). The combined effects of two viral diseases and leaf rust on wheat. Phytopathology 59, 1488-1492. Ross, A. F. (1961). Localized acquired resistance to plant virus infection in hypersensitive hosts. Virology 14, 329-339. Ross, A. F. (1961). Systemic acquired resistance induced by localized virus infections in plants. Virology 14, 340558. RUSSELL, G. E. (1966). Some effects of inoculation with yellowing viruses on the susceptibility of sugar beet to fungal pathogens. Transactions of ti Britih Mycological So&& 49, 611619. SEQ~~IRA, L. & HILL, L. M. (1974). Induced resistance in tobacco leaves: the growth of Pseudomonas solanacearum in protected tissues. Physiologkal Plant Pathology 4, 447-455. SEQUUEIRA, L., Arsr, S. & AINSLEE, V. (1972). Prevention of the hypersensitive reaction in tobacco by proteinaceous constituents of Pseudomonas solanacearum. Phytopathology 62, 536-541. SMITH, H. C. (1962). Is barley yellow dwarf virus a predisposing factor in the common root rot disease of wheat in Canada? Canadian Plant Disease Survey 42, 143-148. THAINE, R., OVENDEN, S. L., & TURNER, J. S. (1959). Translocation of labelled assimilates in the soybean. Australian 3oumai of Biological S&rue 12, 349-372. WACEK, T. J. & SEQUEIRA, L. (1973). The peptidoglycan of Pseudomonas solanaceanun: chemical composition and biological activity in relation to the hypersensitive reaction in tobacco. Physiological Plant Pathologv 3, 363-369. WILSON, E. M. (1958). Rust-TMV cross-protection and necrotic-ring reaction in bean. Phytepathology 48, 228-231.
15,32
l-330
Induction of localized and systemic protection against
Phytophthora parasitica var. nicotianae by tobacco mosaic virus infection of tobacco hypersensitive to the virus JOHN
L.
MCINTYRE
and J. ALLAN DODDS
Defiartmcnt of Plant Pathology, The ConnecticutAgricuItural Ex@rkent Station, P.O. Box 1106, New Haven, Connecticut06504, U.S.A. (Accepedfor publication May 1979) Within 3 days of inoculating expanded leaves of NicotMM tubacum cv. Windsor Shade 117 with tobacco mosaic virus (TMV), viral lesions were apparent and, in younger uninoculated leaves, systemic induced protection was detected against race 3 of Phyfophthora @arasitica var. nicotianue. This protection occurred on all leaves above the TMV-inoculated leaves for at least 22 more days. The level of protection usually approached absolute (less than one fungal lesion per leaf as compared to 3 to 14 fungal lesions per leaf on unprotected plants). As few as 12 TMV local lesions per inoculated leaf were sufficient to induce significant levels of systemic protection. Localized protection was also induced within 3 days of inoculating leaves with TMV, but 7 days were required to induce signiticant levels of protection in halfleaves opposite TMV-inoculated half-leaves. TMV-inoculated plants flooded with a zoospore suspension or transplanted to infested soil developed fewer black shank infections and were less extensively damaged than control plants.
INTRODUCTION Interactions between viral and fungal pathogens of plants have been observed but the consequence of the interaction for any one system is difficult to predict. The virus infection may predispose the plants to greater [l-2, 3, 7, 13, 40, 41, 45, 511 or lesser [9, 15,17, 19,21, 22, 24,33,54] susceptibility to the fungal pathogen, or have no effect [al, 45, 481. In the three reports [17, 22, 541 where localized virus infections decreased susceptibility, induced or acquired resistance may have been involved. This type of interaction is also observed between viruses [27, 46, 471, fungi [5, 8, II, 16, 25, 26, 34, 351 or bacteria [4, 12, 29, 31, 37, 38-J. In some of these systems only localized protection was detected [4, 11, 12, 16, 29, 35, 371, but in others systemic resistance developed in host tissues remote from those treated with the inducing agent [S, 8, 25, 26, 31, 34, 38, 46, 471. We report here on a new system for studying such interactions: the induction of localized, opposite half-leaf and systemic leaf, stem and root protection of tobacco against Phytophthora pamsitica var. nicotianae by localized leaf infection with tobacco mosaic virus, MATERIALS Plan&
AND METHODS
The host plant used was Ncotiana tabacum cv. Windsor Shade 117 (WS 117), a cultivar hypersensitive to tobacco mosaic virus (TMV) and highly susceptible to Phytophthora pamritica Dast. var. nicotianae (Breda de Haan) Tucker ( = Phytophthma 0048459/79/06032
1+ 11$02.00/0
@ 1979 Academic
Press Inc.
(London)
Limited
3!2!?
J. L. McIntyre and J. A. Dodds
nicotianae van Breda de Haan var. nicotianac). In preliminary studies N. tabacum cvs Burley 21 and Samsun NN were also used. These cultivars are also susceptible to P. @arasitiGavar. nicotianae and hypersensitive to TMV. Tobacco seed was germinated in the greenhouse on a mixture of soil : peat : sand (1 : 1 : 1) and then transplanted at the two leaf stage to 350 ml Styrofoam cups containing the same soil mixture. Plants were maintained in a greenhouse at 26 f 5 “C until they reached the four to six leaf stage. Virus Tobacco type, strain (U-l) of tobacco mosaic virus (TMV) was maintained in and purified from N. tabacum cv. White Burley. Infected tissue was homogenized in 0.02 M KsHPO,, 1o/o P-mercaptoethanol and strained through cheesecloth. The extract was stirred for 15 min with 8% butanol then clarified by centrifugation (10 OOOgx 15 min). Clarified extracts were adjusted to 0.1 M NaCl and TMV was precipitated with 4% polyethylene glycol (mol. wt = SOOO),collected by centrifugation (10 000 g x 30 min) and resuspended in 0.01 M sodium citrate buffer, pH 8.0. Further purification of TMV was by differential centrifugation (80 000 g x 90 min, 10 000 g x 15 min) and sucrose density gradient centrifugation (10 to 40% sucrose, Beckman SW27 rotor, 131 000 g x 3-O h, 5 “C) in 0.01 M sodium citrate buffer, pH 8.0. An E&x = 3.0 was used to estimate the concentration of TMV in the final preparation. Polyacrylamide gel electrophoresis of viral RNA or viral protein [IO] indicated that the virus preparation used in this study was not detectably contaminated with host components. Fungus Race 3 of P. parasitica var. nicotianae (Ppn) (isolate M-15T = isolate A7Y) [36] was maintained on P-l medium [18] in the dark at 25 “C. Single mycelial plugs, cut with a no. 2 cork borer from ‘I-day-old cultures, were placed into 125 ml Erlenmeyer flasks containing 25 ml of lima bean broth (Difco Laboratories, Detroit, MI 48232, U.S.A.). After 5 days’ growth in the dark at 25 “C, mycelia from three flasks were harvested by filtration onto single sterile pieces of Miracloth (Chicopee Mills, Inc., Milltown, NJ 08850, U.S.A.) in a sterile Buchner funnel. To induce zoosporangial formation, mycelia were washed with sterile water and transferred on the Miracloth pieces to Petri plates containing 3% water agar. These cultures were incubated for 5 to 7 days in the dark at 25 “C, zoospores were released and the concentration adjusted to lo6 zoospores per ml as previously described [ 391. Virus inoculations to induceprotection The adaxial surface of entire tobacco leaves (the youngest expanded leaf and up to the three next oldest leaves) or a lateral half of each leaf (tobacco leaves are divided into two halves by the midvein) was rub-inoculated with cotton gauze soaked with TMV (1, 10, 25, 50, 100 or 200 pg ml -1) or inactivated TMV (25 pg ml-l, boiled for 25 min) in 0.01 M potassium phosphate buffer, pH 7.2, containing 1% of w/v celite as an abrasive. Uninoculated control plants were abraded with celite in buffer only. Plants were then maintained in a greenhouse at 25 & 5 “C.
Induction
of localised
and systemic protection
Three days after inoculation TMV prior to the fungal inoculation. Fungus inoculation
to test protection
323
local lesions were visible and they were counted
(challenge
inoculation)
The adaxial surface of leaves to be challenged wa:s abraded by rubbing with a cotton gauze soaked in buffer with celite and immediately sprayed to run off with the zoospore suspension using a hand-pump sprayer. Plants were maintained at 25 + 2 “C in closed plastic bags containing a moistened hand towel and subjected to a 12 h photoperiod under fluorescent lights (260 PE rnT2 s-l PAR at plant height). Two days after the leaves were inoculated with zoospores, the black shank lesions were counted. Leaf abrasion prior to inoculation with the zoospore suspension was necessary for the development of maximum lesion numbers. Protection studies
Protection was studied on entire or half-leaves inoculated with TMV (localized leaf protection), on half-leaves opposite to those inoculated with TMV (half-leaf protection), on leaves above those inoculated with TMV (systemic leaf protection), and by flooding TMV-inoculated plants with a zoospore suspension or transplanting TMV-inoculated plants into soil infested with the black shank pathogen (root and stem systemic protection). Phytophthora parasitica var. nicotianae is a soil-borne pathogen, and roots and underground portions of the stem are the normal site of infection [32]. The fungus inoculations to test leaf protection were made immediately after the virus inoculations or at intervals up to 25 days later. Root and stem protection was tested starting 10 days after virus inoculation. AI: that time 2 ml of a zoospore suspension was pipetted around the stem at the soil line. The zoospores were washed into the soil by immediately flooding with 100 ml of distilled water. In another experiment, and again 10 days after virus inoculation, the plants’ root ball with soil was cut to a manageable size (diam. = 2 cm), and then two or three plants were transplanted into single 5 1 plastic containers containing field soil naturally infested with race 3 of Ppn. In a second experiment, the naturally infested soil was reused and also diluted 1 : 3 and 1 : 8 with Pro Mix (Premier Peat Moss Corporation Marketing, New York, NY 10036, U.S.A.). Plants were maintained in the greenhouse (26 + 5 “C) and observed daily for black shank stem lesions or wilting. Several days after all of the control plants in naturally infested soil had black shank lesions, control and TMV inoculated plants were removed .from the soil, washed free of debris, and the black shank lesions on the exterior surface of roots and stems were measured. Then roots and stems of each plant were split longitudinally and the length of pith necrosis and destruction was measured. All experiments had a minimum of five plants per treatment and were performed at least twice, and always celite-abraded plants were included as controls. Data are presented as averages for all observations and results were analyzed by Student’s standardized t-test or Duncan’s new multiple range test. RESULTS
Tobacco cv. WS 117 exhibiting the hypersensitive response to TMV was protected against Ppn and this protection has been observed i.n over 250 plants (Plate 1).
324
J. L. McIntyre
and J. A. Dodds
Similar results were obtained in preliminary studies using tobacco cvs Burley 21 and Samsun NN. Black shank lesions were evident 2 days after inoculating WS 117 plants with the black shank pathogen, and the number did not increase on subsequent days. These lesions enlarged at about the same rate on control and protected plants, and within several days leaves with lesions were entirely necrotic. Two leaves with 101 or more TMV lesions per leaf provided the maximum protection level for all leaves (Table 1). However, as few as 12 to 50 TMV lesions per leaf provided significant protection. A virus concentration of 25 pg ml-r provided 229 + 34 (2 + se.3 lesions per leaf and was used in most experiments. Inactivated TMV at this concentration did not cause the hypersensitive interaction and did not induce protection to Ppn (Table 2). Localized protection against Ppn was detected when leaves were challenged as early as 3 days after they were inoculated with TMV (Table 3). When challenge followed TMV inoculations by 7 days, TMV-treated leaves averaged 98% fewer black shank lesions than did control leaves. TABLE 1 Effect of number of tobacco mosaic virus (TMV) 1esions on the number of lesions caused by Phytophthora parasitica Dar. nicotianae (Ppn) on tobacco leaves2
Average TMV
no. of lesions
per
leaf
( f s.e.z)r
PPn ll*2+1.1az 2*9&0*7b 2*2*0.6b 1*5+0.4bc 1.0 f 0.4bc 0.3 + 0.2c 0.5 * 0*2c
0 12-50 51-100 101-200 201-300 301-490 401-500
z The adaxial surface of the youngest fully-expanded leaf and one leaf below it on tobacco plants at the six leaf stage was inoculated by rubbing with 1, 10, 25, 50, 100 or 200 pg ml-’ TMV. Plants were challenged 7 days later by spraying all leaves to run-off with a suspension containing 10s zoospores per ml of P@n. r Values represent averages for two leaves per plant for TMV and six leaves per plant for P/m. S Numbers followed by the same letter are not significantly different at PGO.01.
Effect
TABLE 2 of tobacco mosaic virus (TMV) and heat-inactivated tobacco mosak virus on the number lesions on tobacco Ieaves caused by Phytophthora parasitica var. nicotianae (Ppn)”
Treatment None Inactivated TMV
Average
TMV
no. of Ppn lesions
of
per leaf (+ s.e.;)r
12.5 k 2.0aS 9-7 *343a 0.6kO.lb
0 The adaxial surface of the youngest fully-expanded leaf and one leaf below it on tobacco plants at the six leaf stage was inoculated by rubbing with 25Fg ml-r TMV or inactivated TMV (25 Pg ml-’ TMV; boiled for 25 min). Plants were challenged 7 days later by spraying all leaves to run-off with a suspension containing 10s zoospores per ml of Ppn. s Values represent averages for six leaves per plant. * Numbers followed by the same letter are not significantly different at P
PLATE 1. Black shank lesions on tobacco leaves 2 days afte:r challenging with zoospores of Phytophthora pamsitica var. nicotianae. Leaves from tobacco mosaic virus-inoculated plant (left) and control plant (right). Local and opposite half-leaf protection. LL = TW local lesion. BSL = black shank lesion.
8*5+0*9 5.3 + l-1 l-3+0*5 0.8 + 0.3 3-l &O-6 1*4+1-l 0*2+0*1
TMV ‘2.17* 0.97 3*01*+ 4.OO** 7*45*+ Z-71** g-35**
t value’
Average
var. nicotianae
l-7*0-5 3.2 &- l-3 4*9+ l-6 2.5kO.8 1-I f0.7 Z-5+ l-4 1*3&O-5
TMV
6y
( + s.e.,)v
O-88 l-53 1.47 l-86 l-52 l-37 2*96**
t value
leaf
(Ppn)
4-65 l-3 z-5+ 1.2 7*4* 2.0 3.9+ 1.8 3-450-8 13.5+3*3 8.9+2.0 4-o_+ 1.1 13.9k3.5 9.8+1-O
Untreated
5*5_+ 1.3 3*1+ 1.2 Z-3+ 1-l 0.42 o-3 0.840-Z 0*5+0-Z O*Z~O*l O*l+O*l 2-75 l-1 0*7+0.2
TMV
Systemic
tobacco mosait virus (TMV)*
half
leaf
(half-leaf)
or younger
leaves
(systemic).
below it on tobacc- v p!auts at the six to eight leaf stage was inocuor up to 25 days later by spraying leaves to run off with a sus-
2.3_+0-4 1*2*0-z 7*5+0-7 5*9* l-9 Z-7+0.8 4-9&1-l 4.7 * I.3
Untreated
no. of Ppn lesions per Half leaf
3 parasitica
fullysxpauded leaf and up to three leaves TMV. Plants were chzl!enged immediately per ml of Pfm. lesions on the same leaf (local), opposite or P
so_+ l-4 7-l + l-5 13.4p4.0 8-4 + O-8 14.8+0*2 6*1+1*3 ll*l+ 1.8
Untreated
e The adaxial surface of the youngest lated by rubbing with 25 pg ml-1 pension containing !OL zouspores s Values represent averages for Pfin BValues significant at PdO.05 (*)
1:
0 2 3 4 5 6
Daye after TMV that challenge was applied
Local
Local, hof leafr and pstemic protection of tobacco@ants against
TABLE Phytophthora
o-47 O-36 Z-25** Z-34** 4.63*+ 3.91.2 6*07** 3.60”’ 3*05** g-99+*
tvalue
-_
5 v a g 5 =
3 0
iii 0. % Q
328
J. L. McIntyre and J. A. Dodds
One-half leaf protection against Ppn occurred when challenge followed TMV inoculation by 7 days, at which time half-leaves opposite virus inoculated halfleaves averaged 73% fewer black shank lesions than did control half-leaves. No significant protection was detected earlier than this (Table 3). Systemic protection also occurred since leaves younger than TMV-treated leaves were protected against the black shank pathogen (Table 3). Significant protection occurred when plants were challenged 3 days after inoculation with TMV, and protection was still evident when challenge followed TMV-inoculation by 25 days. All younger leaves, regardless of position above TMV-inoculated leaves, were protected at each of the challenge times. This is demonstrated by data for a challenge inoculation occurring 7 days after TMV inoculation (Table 4). Reductions in fungal lesion numbers on progressively younger leaves were due primarily to reduction in leaf area of these expanding leaves. At the latest challenge date the youngest leaves inoculated with Ppn were eight to nine leaf positions above the youngest TMV-inoculated leaf.
Dirtribution
of vs&mic
Leafno.* 1 2 3 4
TABLET protcctiun of tobacco leaves against Phytophthora tianae (Ppn) by tobacco mosaic virus (TMV)@ Average Untreated 10~0+3~1 4.3k2.5 2.0-11.3 1*0&0.6
no. of Ppn lesions TMV 0*8+0*3 0 0.1 &O-l 0
per
parasitica
leaf
var. nico-
(+ se.,) t value 4.69** 2~83~. 3*29** 2*39*
a The adaxial surface of the youngest fully-expanded leaf and one leaf below it on plants at the six leaf stage was inoculated by rubbing with 25 pg ml-s TMV. Plants were challenged 7 days later by spraying to run-off with 10s zoospores per ml of Ppn. r Leaf 1 is the first leaf above the leaves inoculated with TMV. l Values significant at PGO.05 (*) and PsO.01 (**).
Protection of plants against Pbn also occurred when plants with TMV-treated leaves were transplanted to soil infested with the black shank pathogen (Fig. 1). Similar results were obtained when plants were flooded with a zoospore suspension, and 21 days after inoculation 8 of 10 control plants had black shank lesions while only 2 of 10 plants with TMV-treated leaves had symptoms. A dilution of infested soil did not result in increased levels of protection. In protected plants, the length of external and internal lesions due to the disease was reduced significantly at all soil dilutions tested (Table 5). Also observed, in protected plants only, were small lesions that were not enlarging, indicating an apparent arrest of the infection. DISCUSSION
Our results show local, opposite half leaf, and systemic protection of tobacco against P/m by prior inoculation with TMV. The high level of protection achieved and its longevity and systemic nature indicate that this system may be useful for studies
induction
327
of localised and systemic protection
Tme (days)
Fro. 1. Incidence of black shank lesions on stems of tobacco plants growing naturally infested with Phytophthora parasitica var. nicotiaaae. Leaves of plants were (0) or inoculated with tobacco mosaic virus (0) 7 days prior to transplanting infested soil. TABLE 5 caused by Phytophthora parasitica var. nicotianae inoculated with tobacco mosaic Z&W.J (TAG’) w
Root and stem damage
(Ppn)
Length Experiment
Soil dilution=
Stem necrosis
Untreated
1
0
Exterior Interior”
14.8&2-3 18.8k l-6
2
0
Exterior Interior
1:3
Exterior Interior
1:8
Exterior Interior
in soil abraded them to
in plants
of necrosis
(cm)
TMV
t values
4.3 + 2.0 6.9 f 2.3
3.35** 4*19**
7.4kO.6 6.2kO.7
2.6+0-7 1*5+0*5
5*03** 5.19;’
5.3 f o-9 4*4* 1.0
0.7kO.4 0*4+0-3
4.69** 3.94**
6.6k2.0 5*7+ 1.7
3.12** 4.33**
14*2* 14.5*
1.6 1.1
W The adaxial surface of the youngest fully expanded leaf amcl one leaf below it of plants at the six leaf stage was inoculated by rubbing with 25 pg ml-’ TMV. Plants were transplanted 7 days later to soil naturally infested with Pp. e Soil was diluted as indicated with Pro Mix. svalues significant at P
on the physiological and biochemical changes that result in induced protection. The root and stem protection suggests that by further understanding and then exploiting this system, control of black shank in field situations may be achieved by induced resistance. Two observations indicated that a host response was required for protection to occur. A time period was necessary between TMV inoculation and the onset of protection, and a direct interaction between viru.s and fungus did not occur 22
J. L. McIntyre and J. A. Dodds
328
since protection
was systemic and virus particles are localized in the TMV
lesions
[201The level of protection in induced plants was routinely high, and often was absolute. The reduction in lesion numbers on leaves of protected as compared to control plants was 25 + ‘I-fold (5 f s.e.r.). This equalled or exceeded protection levels for other host-parasite systems where lesion numbers were used to assay protection [5, 6, 8, 17, 19, 22, 25, 34, 46, 47, 541. Local and systemic leaf protection occurred 3 days after TMV inoculation, coincident with the appearance of TMV lesions, whereas opposite half-leaf protection occurred 7 days after TMV inoculation. If phloem transport of the inducing principle or protectant is assumed, materials would translocate throughout the induced leaf half and to the remainder of the plant prior to their appearance in the opposite leaf half [52]. Ross [46, 471 demonstrated that tobacco plants hypersensitive to TMV developed protection against several different viruses following inoculation with TMV. Results of the present study and those of Ross show similar kinetics for local, opposite half leaf, and systemic protection. Protection of tobacco against bacteria and other fungi has also been induced by TMV [17, 301. In those studies only local protection was reported, but methods of assay may have precluded the observation of systemic protection. Resistance of tobacco to fungi, bacteria, and viruses can also be induced in ways other than by the use of TMV [6, 8, 14, 17, 23, 27-29, 31, 34, 35, 42-44, 50, 531. Sequeira & Hill [49] have noted that it seems obvious that all of these protection phenomena cannot be due to the presence of a single inhibitor or the result of a single protective response. However, our results in combination with the numerous reports cited above of methods to induce protection of tobacco against a wide array of micro-organisms, suggests that it is unlikely that tobacco plants are capable of responding specifically to the varied stresses that induce protection, We therefore propose a generalized stress response to these many different stimuli. This stress response may in turn initiate several different protection mechanisms. However, a specific stress or challenge inoculation may favour the activity of a certain protection mechanism. The technical assistance of M. Reisner and M. Tiffany
is gratefully
acknowledged.
REFERENCES 1. BATEMAN, D. F. (1961). Synergism between cucumber mosaic virus and Rhizoctoaia in relation to Rhizoctonia damping-off of cucumber. Phytopathology 51, 574-575 (Abstr.). 2. BEUTE, M. K. (1970). Effect of virus infection on susceptibility to certain fungus diseases and yield of Gladiolus. Phytopathology 60, 1809-1813. 3. BEUTE, M. K. & LOCKWOOD, J. L. (1967). Mechanism of increased susceptibility to root rots in virus-infected pea. Phytofiathologv 57, 804 (Abstr.). 4. CARROLL, R. B. & LUKEZIC, F. L. (1972). Induced resistance in alfalfa to Copmebacterium insidiosum by prior treatment with avirulent cells. Phytopathology 62, 555-564. 5. CARUSO, F. L. & Kuc, J. (1977). Protection of watermelon and muskmelon against Collctotrichum lagenarium by Colletotrichum lagenarium. Phytopathology 67, 1285-1289. 6. COHEN, Y., REUVENI, M. & KENNETH, R. G; (1975). Resistance to powdery mildew in tobacco induced by Peronospora tabacina. Phytopathology 65, 1313-1315. 7. CRANE, G. L. & CALPOUZOS, L. (1969). Synergism of Cercospora beticola and beet yellows virus in Idling sugar beet leaves. Phytopatholog)r 59, 1338-1339.
lnductlon
8.
of locallsed
CRUKXSHANK, Peronos$ora
820
and systemic protection
I. A. M. & MANDRYK, M. (1960). The tabacina on foliage reaction to Blue Mould.
effect of stem infection Journal of ths Australian
of tobacco with Institute of Agri-
cultural.%ience 26, 369-372. 9. DIAZ-POLAN~~, C., Surrrr, S. H. & H~~cocx, J. G. (1969). Effect of virus infection on stem rot of squash caused by Fusarium solani f. sp. -bitae. Phytopathology 59, 18-22. 10. DODDS, J. A. & ELTON, R. I. (1974). Masking of the RNA genome of tobacco mosaic virus virology 59, 418-427. by the protein of barley stripe mosaic virus in doubly infected barley. 11, ELLISTON, J., KIJC, J. & WILLLUS, E. (197 1). Induced resistance to bean anthracnose at a distance from the site of the inducing interaction. Phyto~atholo~ 61, I1 10-l 112. 12. ERCOLANI, G. L. (1970). Bacterial canker of tomato. IV. The interaction between virulent and avirulent strains of Coqwbacterium michiganense (E. F. Sm.) Jens. in vivo. Phytopathologia MediterfUfleU 9, 151-159. 13. FARLEY, J. D. & LOCKWOOD, J. L. (1964). Increased susceptibility to root rots in virus-infected peas. Phytojathology 54, 1279-1280. 14. GLWNAZZI, S. & -19, B. (1974). Virus resistance induced in plants by._ polyacrylic acid. Jownal of General Virology 23,‘1-9.. 15. GOHEEN. A. C. & SCHNATHORST W. C. 11961). Resistance to uowderv mildew in leaf roll-affected ’ grap&Gnes. Plant Disease Re#orter 45; 641L643. [ T\ A 16. ~R~CHMIDT, R., ACRES, S. & Kuc J. (1976). Protection of cucumber against Col[ctotrihum lagenarium and Claa@nium cucumerinum. Phytopathology 790-794. 17. Hxcnrr, E. I. & BATEMAN, D. F. (1964). Nonspecific acquire% resistance to pathogens resulting from localized infections by Thhiclaoiopsis basicola or viruses in; tobacco leaves. Phytobathology
54, 523530. 18. HOI-IL, H. R. (1975). Levels of nutritional complexity in PhytoOp?iwra: lipids, nitrogen sources and growth factors. Phytofiathologische
330
J. L. McIntyre and J. A. Dodds
36. MCINTYRE, 37. 38. 39. 46. 41. 42. 43. 44. 45. 46.
47. 48. 49.
50. 51. 52.
53. 54.
J. L. & TAYLOR, G. S. (1978). Race 3 of Phytophthora parasitica var. nicotianae. Phytopathology 68, 35-38. MCINTYRE, J. L., KUC, J. & WILLIAMS, E. B. (1973). Protection of pear against tire blight by bacteria and bacterial sonicates. Phytopathalogy 63, 872-877. MCINTYRE, J. L., KUC, J. & WILLIAMS, E. B. (1975). Protection of Bartlett pear against 6re blight with deoxyribonucleic acid from virulent and avirulent Erwinia amylovora. Physiological Plant Pathology 7, 153-170. MILLER, P. M. & MCINTYRE, J. L. (1976). Oxamyl-treated soil protects tobacco against black shank. Phytopathology 66, 221-224. MWANZA, W. P. & WILLIAMS, L. E. (1966). Viruses as predisposing factors in the susceptibility of corn and wheat plants to other pathogens. Phytopathology 56, 892 (Abstr.). NITZANY, F. E. (1966). Synergism between Pythium uitimum and cucumber mosaic virus. Phytopatholoo 56, 1386-1389. NOVACKY, A. (1972). Suppression of the bacterially induced hypersensitive reaction by cytokinins. Physiological Plant Pathology 2, 101-104. NOVACKY, A. & HANCHEY, P. (1976). Effect of internal leaf injury on bacterial hypersensitivity. Acta Phytnpatlwlogica Acaakmiae Scientiarum Hungaricae 11, 217-222. NOVACKY, A., ACEDO, G. & GOODMAN, R. N. (1973). Prevention of bacterially induced hypersensitive reaction by living bacteria. Physiological Plant Pathology 3, 133-136. RAJU, D. G., SILL, W. H., JR & BRO~XR, L. E. (1969). The combined effects of two viral diseases and leaf rust on wheat. Phytopathology 59, 1488-1492. Ross, A. F. (1961). Localized acquired resistance to plant virus infection in hypersensitive hosts. Virology 14, 329-339. Ross, A. F. (1961). Systemic acquired resistance induced by localized virus infections in plants. Virology 14, 340558. RUSSELL, G. E. (1966). Some effects of inoculation with yellowing viruses on the susceptibility of sugar beet to fungal pathogens. Transactions of ti Britih Mycological So&& 49, 611619. SEQ~~IRA, L. & HILL, L. M. (1974). Induced resistance in tobacco leaves: the growth of Pseudomonas solanacearum in protected tissues. Physiologkal Plant Pathology 4, 447-455. SEQUUEIRA, L., Arsr, S. & AINSLEE, V. (1972). Prevention of the hypersensitive reaction in tobacco by proteinaceous constituents of Pseudomonas solanacearum. Phytopathology 62, 536-541. SMITH, H. C. (1962). Is barley yellow dwarf virus a predisposing factor in the common root rot disease of wheat in Canada? Canadian Plant Disease Survey 42, 143-148. THAINE, R., OVENDEN, S. L., & TURNER, J. S. (1959). Translocation of labelled assimilates in the soybean. Australian 3oumai of Biological S&rue 12, 349-372. WACEK, T. J. & SEQUEIRA, L. (1973). The peptidoglycan of Pseudomonas solanaceanun: chemical composition and biological activity in relation to the hypersensitive reaction in tobacco. Physiological Plant Pathologv 3, 363-369. WILSON, E. M. (1958). Rust-TMV cross-protection and necrotic-ring reaction in bean. Phytepathology 48, 228-231.