leaf injury to three commercially-grown mosaic virus, tobacco ringspot virus, tomato

OZONE RESPONSE OF TOMATO PLANTS INFECTED WITH CUCUMBER MOSAIC VIRUS AND/OR TOBACCO MOSAIC VIRUS D. P. ORMROD1 and W. c. KEMP, Departme-nt of Horticultural science, (Jniversity of Guetph, Guelph, ontario NIG 2wI and zresearch station, Agriculture canadi, vinelaid statioi, ont. L1R 2E. Received 24 Apr. 1979, accepted 2 July 1979. Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 46.3.21.212 on 3/13/18 onvrnbo, D. P. ano Ksnap, w. G. r9jg. ozone response of tomato plants infected with cucumber mosaic virus and/or tobacco mosiic virus. Can. J. plant Sci.59: 177-183. The sensitivity of three tomato cultivars to several concentrations of ozone was evaluated after prior sequential inoculations with tobacco mosaic virus (TMV) and/or cucumber mosaic virus (CMV). Ozone injury in inoculated and uninoculated tomatoes varied from slight to severe depending on the virus, cultivar,.ozone concentration and virus incubation period. The frequency of increased ozone injury was about twice as great as that of suppressed injury on infected plants. Ozone injury occurred more frequently in TMV-inoculated plants than in thbse inoculated with CMV. There were more increases than decreasei in ozone injury after 7 or 14 days of virus infection, but mainly decreases in injury after 2l days infection. Growttrwas significantly reduced in plants exposed to ozone after a 2l-day virus incubation period, particularly when they were inoculated with both viruses. On a 6valu6 la sensibilit6 de trois cultivars de tomate b plusieurs concentrations d'ozone aprbs des inoculations successives avec le virus de la mosaique du tabac et (ou) celui de la mosaique du concombre. Les d6gats caus6s par I'ozone chez les tomates inocul6es et non inocul6es ont vari6 de l6gers d graves, selon le type de virus, de cultivar, la concentration d'ozone et la p6riode d'incubation. Li fr6quence d'apparition des d6gats par I'ozorle fut environ le double de celle de leur suppr-ession sur les plants infect6s_. Les d6gats caus6s par I'ozone sont apparus plus fr6qu^emment sur les plants inocul6s avec la mosaique du tabac que chez ceui inocul6s avec la mosaique du concombre. On a observ6 plus d'accroissement que de r6duction des d6gits aprbs 7 ou l4jours d'infection viraie, mais principalemenfdes r6ductions aprds 2l jours. La croissance a 6t6 significativement r6duiie chez les plants expos3s ir I'ozone aprbs une p6riode d'incubation de 2l jours, en particulieriorsqu'ils 6taient inocul6s avec les deux types de virus. The suppression of ozone injury in plant tobacco (Nicotiana tabacum L.) cultivars species infected with various viruses has infectedforgdays(moyerandsmith 1975). been well documented. In general, both Pinto bean (Phiseolus- vulgaris L,) leaves systemic and localized virus infections from plants inoculated with bean common reportedly reduce ozone injury. Leaves of mosaic virus 4-5 days before exposure to Nicotiana sylvestris exposed to ozone 12 ozone were also less sensitive to thi oxidant days after inoculation with tobacco mosaic (Davis and Smith lg74). A reduction in virus (TMV) were generally less sensitive to severity of ozone injury was noted on two oxidant injury than were comparable leaves field-giown tobacco cuitivars infected with on-uninoculated plants (Brennan and Leone TMV (Bisessar and Temple 1977). Local 1969). Tobacco etch virus decreased ozone lesions induced in Pinto beans by alfalfa leaf injury to three commercially-grown mosaic virus, tobacco ringspot virus, tomato ringspot virus and TMV also reduced ozone Can. J. Plant Sci. 59: 177-1E3 (October 1979) sensitivity in the areas immediately surto17

178 CANADIAN JOURNAL OF PI.ANT SCIENCE Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 46.3.21.212 on 3/13/18 rounding the restricted infections (Davis and Smith 1976). More recently, Reinert and Gooding (1978) have reported that a 3- to 4-wk streak virus infection of tobacco increases rather than decreases ozone sensitivity. Leafinjury was increased and plant weight decreased by the interaction of virus and ozone compared to ozone alone. Experiments described herein were designed to examine the response of three commercially grown tomato (Lycopersicon esculentum Mill.) cultivars exposed to several concentrations of ozone after inoculation with cucumber mosaic virus (CMV), TMV or both. MATERIALS AND METHODS Seeds of the tomato cultivars Vendor, Fireball and Heinz 135 were treated with 17o tri-sodium phosphate solution for 15 min to inactivate any seed-borne TMV, washed thoroughly in tap water, air-dried, sown in 5-cm peat pots containing steamed sandy loam soil and placed in a growth chamber at about 2"C, 16lx,TOVoRH and l6-h photoperiod. Approximately 3 wk later, these potted seedlings at the second leaf stage were replanted in larger lo-cm clay pots and maintained under the same conditions subsequent to sequential virus inoculations. About 6 days from transplanting, at about the seventh-leaf stage, they were exposed to ozone under similar environmental conditions using four plexiglass chambers. Plants ofeach cultivar were inoculated with CMV or TMV, alone or in combination, 2l, 14 or 7 days before a 3-h exposure in mid-photoperiod to, 15, 3 or pphm (vol/vol) (first experiment with Vendor and Fireball);, 3, 6 or 9 pphm (second experiment with Vendor); or, 3, or 6 pphm (experiment with Heinz 135) ozone. Ozone exposures took place over a 3-day period because each chamber could accommodate only 2 plants. Inoculation dates were adjusted accordingly. Comparable uninoculated controls were also exposed to ozone. After ozone exposure, the plants were transferred to a greenhouse compartment for 7 days, after which injury on each plant was rated by three independent observers. The rating system used initially was: : no ozone injury; to 5 : severe necrosis on all leaves. In later experiments, leaf injury was rated by a modified Horsfall-Barratt system (Horsfall and Barraft 19) which included two additional injury classes. Injury on individual leaves was assessed at the same time using indices from to 12 representing, -l, l-3, 3-6, 6-12, 12-25' 25-5, 5-75, 75-87, 87-94, 94-97, 97-99, 99-lOOVo of the leaf area injured, respectively. Only the injury ratings for one or two of the middle-aged leaves are reported. Dry weight of the top growth of each plant was also determined in the second experiment with Vendor and in the experiment with Heinz 135. Isolatds of TMV and CMV were obtained from naturally infected tomatoes in Ontario. One isolate of each virus was used in this study. They were maintained by serial transfer in Harrow Velvet tobacco and N. glutinosa, respectively' The TMV isolate was the tomato strain (No. 156). Crude juice inocula were prepared from systemically infected leaves of tobacco (TMV) and N. glutinosa (CMV) 1-14 days after inoculation. Inocula consisted of infected leaf discs (ca. 3 g fresh weight) ground in l- 15 ml of.25vo NazSOg. Inoculum was rubbed on a carborundum-dusted single cotyledonary leaf of each tomato seedling and plants were then rinsed with tap water. Where both viruses were applied to a plant, CMV and TMV were rubbed on sep:uate cotyledonary leaves. It was assumed that the only statistically comparable means in these experiments were those of plants treated together throughout the entire inoculation and ozone exposure procedure, that is, of five replicate plants for each of four virus combinations, within each inoculation time and each ozone'concentration. Only 2 plants were exposed to ozone at the same time in the same chamber. Analyses of variance were performed on these data and means were separated by Duncan's multiple range test at the 57o probability level. RESULTS Effect of Ozone-Virus Interaction on Tomato Leaf Injury Injury on Vendor and Fireball plants exposed to 15 pphm ozone was enhanced by double virus infection for 7 days (Table l). Injury was also increased on Fireball inoculated with TMV alone 7 days before exposure to 15 pphm ozone. With plants inoculated 14 days before ozone exposure' the most striking effects were on Vendor

ORMROD AND KEMP- OZONE RESPONSE OF INFECTED TOMATO PLANTS ro79 Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 46.3.21.212 on 3/13/18 treated with 15 or 3 pphm ozone where CMV induced significantly less injury than TMV or double infection. Twenty-one days after inoculation, TMV reduced ozone injury on Vendor exposed to 3 pphm ozone compared to all other treatments and on Fireball exposed to l5 pphm compared with CMV. The sixth leaves of Vendor tomatoes inoculated 7 days before exposure to 9 pphm ozone w,ere much more severely injured than wer,e the corresponding leaves on the control plants (Table 2). Fourteen days after inoculation, injury on sixth leaves of plants treated with 6 pphm ozone was significantly greater than on controls when the plants had prr:viously been infected with CMV or TMV but not with both viruses. Plants treated with 6 pphm ozone 2l days after inoculation with both viruses exhibited much less injury than those either inoculated with TMV or left uninoculated. Less injury was also noted on plants exposed to 9 pphm ozone than on non-exposed ones provided they were inoculated with CMV and TMV ot TMV alone 3 wk in advance of ozone treatment. Injury ratings on the third leaf of Heinz 135. which was somewhat more sensitive than others, indicated that plants inoculated 7 days before exposure to 6 pphm ozone with CMV or both CMV and TMV were less injured (Table 3). Leaves on plants inoculated 14 days earlier with TMV and Table L Average ozone injury rating of Vendor and Fireball tomato plants inoculated with cucumber mosaic virus (CMV) and/ortobac,:omosaic virus (flvl$ Z, 14 or2l days prior to ozone exposure at, 15,3 or pphm(vol/vol) : inoculation 2r Virus Ozone conc CMV TMV CMV+TMV l5 3 l5 3 15 3 15 3 l5 3 15 3 Cultivar Vendor O.Mf a O.33 ab l.2oa 1.93a 2.87 a 3.6Oa O.2ab.6a l.nab O.'73 a 2.13 a 2.2O a O.67 a O.4a r.33b 1.8, 3.2Oa 2.87 a Cultivar Fireball O.l3 ab O.7 a 3.8 a 3.@a 3.53 a 3.73 a.2oa O.2a O.93 a LI3 a 2.86a 2.27 a.53 ab r.t3 b 2.47 a 2.13 a 2.8Oa 3.66a.4O ab l.2oa 3.73 a.86 c 2.6b 2.67 a O.l3 a O.33 a 2.8a.87 c 3.47 a 3.2Oa.6o 1.4 a 2.87 a O.4Oa 2.2Oa 3.6Oa.67 b 1.8 a 3.33 a.53 bc t.67 b 2.67 a.4a r.47 b 3. a.8 bc 3.33 a 3.41a.73 a l.6o a 2.'73a.4'7 ab t-t5a 3.53 a faverage of five replicate plants by three observers; injury rating scale, = no injury, 5 : severe necrosis on all leaves. c-c Virus means within cultivar, days after inoculation and ozone concentration followed by the same letter are not significantly differerrt (P :.5).

18 CANADIAN JOI,JRNAL OF PLANT SCIENCE Table2. AverageinjuryratingofthesixthleafofVendortomatoesinoculatedwithCMVand/orTMV7,14or2l days prior to ozone exposure at, 3, 6 or 9 pphm (vol/vol) Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 46.3.21.212 on 3/13/18 Ozone Virus inoculation None cmv CMV+TMV 21 3 6 9 3 6 9 3 6 9.3a 2.4a 1.5 a 2.1a 4.8a l.l a 4.bc 6.2c 1.3 a 2.7 a 4.8 b 1.7 a 5.5 b 7.7 a l.6a 2.5 ab 4.7 bc.5a 5.2b 2.4a 5.3 b 7.1a l.l a 4.1 bc 3.3 b O.l a 5.1a 4.7 b O.6a 3.1ab.a.7 a.5 a faverage.of five replicate ilants by three observers; Horsfall-Barratt rating scale, : no injury, 12 : entire leaf surface injured. a-c-virus means within days after inoculation and ozone concentration followed by the same letter are not significantly different (P :.5). both viruses had more injury than leaves on plants inoculated with CMV after exposure to 3 pphm, while TMV-inoculated ones exposed to pphm ozone had more injury than controls. Twenty-one days after inoculation with both viruses, plants were injured more by pphm ozone than were the controls while at 6 pphm both TMV and double virus inoculations induced significantly more injury than was found on CMV-inoculated plants. Statistical analyses of the injury ratings of the fifth leaf of Heinz 135 revealed few significant differences among virus treatments. Plants inoculated for 7 days with each virus alone or in combination had less injury due to 6 pphm ozone than did control plants. A 3 pphm ozone exposure 2l days after inoculation Table3. Averageinjuryratingof the thirdleaf of Heinz 135tomatoesinoculatedwithCMVand/orTMVT days prior to ozone exposure at, 3, or 6 pphm (vol/vol) inoculation 2l Ozone 3 6 3 6 3 6 Virus.14or2l None TMV CMV+TMV l.7i a 6.a 7.9c 1.4 ab 5.1a.2a 1.3 a 2.7 ab 2.4a 5.Oa b.5 a 5.3 ab 1.3 a 2.8 ab 1.4 a 3.a 6.O bc 2.8 b 7.1b 6.1a.2a 2.6ab 4.6 b l.l a 2.9a 3.1a 2.8b 5.8 ab 6.Oa.a 4.4 b 4.7 b taverage of five replicate plants by three observers, Horsfall-Barratt rating scale, = no injury, 12 : entire leaf injured. c-c_virus means within days after inoculation and ozone concentration followed by the same letter are not significantly different (P =.5).

ORMROD AND KEMP - OZONE RESPONSE OF INFECTED TOMATO PLANTS r8l Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 46.3.21.212 on 3/13/18 with CMV caused more injury than ozone exposure without virus inoculation. A direct comparison of leaf 5 injury and leaf 3 injury (Table 3) revealed that the corresponding injury ratings were similar on both leaves but the significance of differences varied between leaves. Effects of Ozone-Virus Interaction on Tomato Growth The growth of Heinz 135 was not retarded by either virus alone after 2l-day incubation periods (Table 4), but growth was significantly decreased in plants inoculated with CMV or both CMV and TMV and exposed 2l days later to 3 pphm ozone. Growth was also reduced in plants inoculated with both viruses and2l days later exposed to either or 6 pphm ozone. Growth of Vendor plants was significantly decreased when they were inoculated 14 days previously with any virus and exposed to 9 pphm ozone, inoculated 2l days previously with CMV plus TMV and exposed to 3 pphm ozone, or inoculated with both viruses or TMV alone 21 days previously and treated with 9 pphm ozone (Table 5). DISCUSSION The responses to ozone of Vendor, Fireball and Heinz 135 tomato plants inoculated with either CMV or TMV or a combination of the two viruses were easily differentiated visually and often could be separated by weight differences. Infected tomatoes may be either more sensitive or more insensitive to ozone than virus-free plants of the same cultivars grown under the same conditions. Although increased sensitivity of tobacco to ozone after tobacco streak virus inoculation has been reported (Reinert and Gooding 1978) this tendency towards more injury to virus-infected plants after ozone exposure is contrary to the results of many other investigations (Brennan and Leone 197; Moyer and Smith 1975; and' others) using different virus/ozone combinations. Our experiments demonstrate that both effects may occur. Of the 17 significant differences compared with virus-free plants (Tables 1-3), l were of increases in ozone injury as a result of virus infection while 7 were of decreases. TMV was involved with significant effects most frequently, with 7 cases, whereas the double virus infection was involved in 6, and Table 4. Average shoot dry wt (g/plant) of Heinz 135 tomato plants inoculated with CMV andlottmy 7, 14 or 2l days prior to ozone exposure at, 3, or 6 pphm (vol/vol) inoculation Virus Ozone conc None CMV CMV+TMV 3 6 3 6 3 6 5.21 a 6.6a 5.9 a ).) a 4.9 a 5.7 a 3.6a 5.2 c 4.9 b 5. D +- I A 6.Oa 5.1a 4.4 a 5.3 a 4.6a 4.4 a 4.9 a 3.9 ab 4.9 b 4.6 b 5.8 a 6.5 a 6.1a 5.Oa 5.4 a 4.4 a 4.8 a 4.7 a 4.7 bc 4.6ab 4.2ab 6.2a 7.5 a 6.'7 a 5.8 a 5.4a 5.4 a 5.6a 3.5 a 3.7 a 3.5 a tmean of five replicate plants. a-c Virus means within days after inoculation and ozone concentration followed by the same letter are not significantly different (P :.5).

182 CANADIAN JOURNAL OF PLANT SCIENCE Table 5. Average shoot dry wt (g/plant) of Vendor tomato plants inoculated with CMV and/or TMV 7, 14 or 2 I days prior to ozone exposure at, 3, 6 or 9 pphm (vol/vol) Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 46.3.21.212 on 3/13/18 Vinrs inoculation conc None CMV TMV CMV+TMV 3 6 9 3 6 9 3 6 9 4.31 a 3.9 a 5.2 a 4.6 a 5.Oa 4.6a 5.9 a 5.7 b 3.7 a 4.8 b 5. a 4.6 b 4.6a 3.6 a 3.7 a 4.1 a 3.8 a 3.6ab 3.8 a 3.5 ab 3.9 a 3.5 a 4.O a 4.4a 4.8 a 4.2a 3.1ab 2.6a 2.8a 5.O a 4.6 a 3.9 a 4.1 a 4.9 a 3- t a 4.6a z.ta 3.7 a 2.5 a tmean of five replicate plants. a-c Virus means within incubation period and ozone concentration followed by the same letter are not significantly different (P =.5). CMV in 4. In general, the virus combination was about as effective as TMV alone in affecting ozone response whereas CMV alone demonstrated less interaction with ozone. The number of casbs of significant virus-ozone interactions was about the same after 7 -, 14-, and 2l-day incubation periods. There were more significant increases than decreases in ozone injury to inoculated leaves incubated for 7 and 14 days. Almost all significant effects after infection for 2l days were decreases in injury. Length of the virus incubation period appears not to have been associated previously with such a change in ozone response. Decreases in ozone sensitivity have been reported for brief incubation times (Davis and Smith 1974) and increases with longer ones (Reinert and Gooding I 978). Clearly, length of the incubation period of each virus in its host could be of some importance but its mode of action is unclear in these experiments since age of plants and concentration of inocula differed at each inoculation date. Both of these factors do have an effect on the rate of systemic infection of a host. Infected tomato cultivars afpeared to differ in theirresponse to ozone although this may have been associated with the use of somewhat different ozone concentrations and assessment methods for different cultivars. The frequency of significant increases and decreases in ozone injury in Heinz 135 after virus inoculation was about the same. However, increases in ozone injury were twice as frequent in Vendor as were decreases. Ozone concentrationhad no interacting effect on enhanced severity of injury noted in virus-inoculated plants in these experiments while decreases in ozone injury were largely confined to 6 and 9 pphm ozone exposures. Growth was decreased significantly in plants exposed to ozone after long virus inoculation periods. A combination of both viruses and ozone caused top growth reduction on six occasions and TMV and CMV alone with the oxidant each decreased the weight twice. The only previous study of growth responses clearly demonstrated an additive effect of ozone and virus in decreasing the weight of plants (Reinert and Gooding 1978). BISESSAR, S. and TEMPLE, P. J. 1977. Reduced ozone injury on virus-infected tobacco in the field. Plant Dis. Rep. 61: 961-963. BRENNAN, E. and LEONE, I. A. 1969. Suppression of ozone toxicity symptoms in

Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 46.3.21.212 on 3/13/18 ORMROD AND KEMP _ OZONE RESPONSE OF INFECTED TOMATO PLANTS virus-infected tobacco. Phytopathology 59: 263-264. DAVIS, D. D. and SMITH, S. H. 1974. Reduction of ozone-sensitivity of pinto bean by bean common mosaic virus. Phytopathology 64: 383-385. DAVIS, D. D. and SMITH, S. H. 19'76. Reduction of ozone sensitivity of pinto bean by virus-induced local lesions. Plant Dis. Rep. 6: 3l-34. HORSFALL, J. G. andbarratt, R. W. 19. r83 An improved grading system for measuring plant diseases. Phytopathology 35: 655. MOYER, J. W. and SMITH, S. H. 1975. Oxidant injury reduction on tobacco induced by tobacco etch virus infection. Environ. Pollut. 9: 1 3-1 6. REINERT, R. A. and GOODING, G. V' Jr. 1978. Effect of ozone and tobacco streak virus alone and in combination onnicotiana tabacum. Phytopathology 68: 15-17.

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