Phytophthora root-rot of container-grown citrus as affected by foliar sprays and soil drenches of phosphorous and acetyl salicylic acids

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1 Plant and Soil 107, (1988) 9 Kluwer Academic Publishers PLSO 7340 Phytophthora root-rot of container-grown citrus as affected by foliar sprays and soil drenches of phosphorous and acetyl salicylic acids G.E. WALKER South Australian Department of Agriculture, Loxton Research Centre, Box 411, Loxton, South Australia, 5333 Received 14 April Revised November 1987 Key words: acetyl salicylic acid, citrus, metalaxyl, phosphorous acid, Phytophthora citrophthora, P. nicotianae var. parasitica, root-rot Abstract Mycelial growth of Phytophthora nicotianae var. parasitica in vitro was inhibited by phosphorous and acetyl salicylic acids at 10 ppm; mycelial growth of P. citrophthora was inhibited by phosphorous acid at 10ppm and acetyl salicylic acid at 100ppm. Foliar sprays and soil drenches with these chemicals were compared with soil applications of metalaxyl as treatments for Phytophthora root-rot of container-grown citrus (var. Cleopatra mandarin) caused by the aforementioned spp. Foliar sprays with 0.1% phosphorous acid were as effective as the metalaxyl treatment in promoting root growth and phosphorous acid offers considerable potential as an alternative treatment for Phytophthora root-rot of citrus. Introduction Phytophthora citrophthora and P. nicotianae var. parasitica are important causes of root and collar rot in citrus in Australia (Broadbent, 1977) and elsewhere in the world (Chadha et al., 1970; McKenzie, 1985; McMillan, 1970; Rossetti, 1969). Infestations in nurseries may result in heavy losses (Lee and Wicks, 1982). Metalaxyl has proved highly effective for control of Phytophthora infections on citrus, both in the field (Timmer and Castle, 1985) and in nurseries (Lee and Wicks, 1982). However, the demonstration that isolates of Phytophthora spp. may develop resistance to this chemical in the laboratory (Joseph and Coffey, 1984) and the finding of such isolates in the field (Cohen and Reuveni, 1983) have shown the need for alternative fungicides with different modes of action. The fungicide fosetyl-a1 is broken down to phosphorous acid in plant tissues and this chemical has been shown to be active against Phytophthora spp, both in vitro and on avocado seedlings (Coffey and Bower, 1984; Fenn and Coffey, 1984). Trunk injections with 10% and 20% solutions of phosphorous acid partially neutralized to make them more compatible with plant tissue have induced recovery of avocado trees suffering from root-rot caused by P. cinnamomi (Pegg et al., 1985). The mode of action of phosphorous acid is thought to be primarily a direct one (Cohen and Coffey, 1986). Various salicylates are known to exert powerful static effects against fungi, yeast and bacteria (Rainsford, 1984). Salicylic acid was found to completely inhibit the growth and germination of various fungi in vitro (Singh, 1978). Salicylates are commonly produced by plants, often in abundance (Rainsford, 1984). The role they play in plant tissues is not clear but could be related to resistance from attack by micro-organisms, particulaly fungi. Abdelal (1983) found that acetyl salicylic acid inhibited development of urediospore pustules of cereal rusts (Puecinia spp.) and in preliminary studies by the author was found to inhibit growth of Phytophthora spp. in vitro. The purpose of this study was to evaluate the efficacy of foliar sprays and soil drenches of phosphorous and acetyl salicylic acids against Phytophthora root-rot of container-grown citrus. 107

2 108 Walker Materials and methods Growth rate of Phytophthora citrophthora and P. nicotianae var. parasitica in vitro Molten Potato Dextrose Agar (PDA) (Difco Pty. Ltd.) was diluted to 90% of the concentration recommended by the manufacturer (36gl -j) with solutions of phosphorous and acetyl salicylic acids (adjusted to ph 5.7 with potassium hydroxide) (Ajax Chemicals Pty. Ltd.) to obtain final concentrations of the acids of 10 and 100ppm. As a control PDA was similarly diluted with deionized water. Final ph of all media was between 5 and 6. Cultures of P. citrophthora and P. nicotianae vat. parasitica were obtained from diseased roots and soil taken from Riverland citrus trees using the pear-baiting technique. Seven-mm diameter discs were taken from the growing margins of two-week old cultures of PDA, placed in the centre of 9 cm Petri dishes containing test media and incubated in the dark at 24~ Maximum and minimum colony diameters were recorded periodically from each of three colonies per test medium. Greenhouse trials with container-grown citrus Two-year old nursery citrus trees (var. Cleopatra mandarin, Citrus reshni) growing in a potting mix consisting of composted Pinus bark, sand and peat moss in plastic tubes 150 mm in diameter (capacity 4.51) and exhibiting symptoms of poor growth, leaf chlorosis and necrosis were found to be suffering root-rot. P. citrophthora was isolated from potting mix of several specimens using the pear-baiting technique. Trees were divided into two groups according to symptoms on top growth: group 1, those in poor condition with leaf chlorosis and little new growth, and group 2, those in very poor condition without new growth, and with leaf curling and leaf chlorosis and/or necrosis. Trees from group 1 were randomly assigned to the following treatment groups (10 trees per treatment): a) untreated control b) foliar spray to run-off with 10% phosphorous acid adjusted to ph 5.8 with potassium hydroxide c) foliar spray to run-off with 0. 1% acetyl salicylic acid, ph 2.5 d) soil drench with 0.1% phosphorous acid, ph 1.6 e) soil drench with 0.1% acetyl salicylic acid, ph 2.5 f) soil drench with metalaxyl, g per tree Trees from group 2 were assigned to the above treatment groups (8 trees per treatment) plus a foliar spray to run-off with 0.1% phosphorous acid, ph 1.6. Treatments were applied on 3 October, 1985; foliar sprays using a hand-held pressurized sprayer applying approximately 125ml per tree and soil drenches in a volume of 500 ml-- or, in the case of treatment with metalaxyl, a drench of 500 ml of water followed applications of Ridomil 50G granules (Ciba-Geigy Pty. Ltd.) at g a.i. per tree (as recommended by the manufacturer for the size container used). All trees not receiving a fungicide spray or drench were treated with water. Following treatment trees from both groups were arranged in complete randomized block designs in a greenhouse at ambient temperature and watered as required. One week after treatment trees were lopped to 50 cm above the soil level and new growth trained to a single shoot. Shoot height was periodically assessed and fresh shoot and root weights were recorded on 11 April A visual rating of severity of root-rot was also recorded (0 = 0%; 1 = 1-25%;2 = 26-50%; 3 = 51-75%; %; 5 = 100% roots with rotting) and the pear-baiting technique used to determine the presence of Phytophthora spp. in the potting mix. Mean daily maximal (air) temperatures during the course of the trial were as follows: October 24.1 ~ November 26.5 ~ December 28.0 ~ January 29.6 ~ Feburary 29.7 ~ March 30.3 ~ and April (1-11) 27.2~ Analyses of variance were carried out on data and Duncan's new multiple range test applied to those data with significant variance ratios (P = 0.05). Results Growth rates of P. citrophthora and P. nicotianae var. parasitica in vitro

3 Phytophthora root-rot of container-grown citrus 109 Table 1. Per cent inhibition of radial growth of P. citrophthora (P.c.) and P. nicotianae var. parasitica (P.n.p.) on PDA amended with phosphorous acid (PA) and acetyl salicylic acid (ASA) compared with unamended control Amendment Days incubation at 24~ P.c. P.n.p. P.c. P.n.p. P.c. P.n.p. P.c. P.n.p. Control 0.0 a a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 10ppm PA 29.4 b 27.7 b 33.9 b 24.1 b 35.5 b 26.2 b b 100ppm PA 93.5 d 86.7 d 92.2 d 83.9 d 90.5 d 82.3 d 89.6 d 81.7 d 10ppm ASA 7.6 a 55.4 c 7.3 a 52.7 c 6.5 a 51.6 c 3.8 a 53.5 c 100ppm ASA 57.0 c 92.8 d 61.9 c 94.6 e 63.8 c 94.8 e 67.1 c 91.3 d Means within columns followed by the same letter are not significantly different according to Duncan's multiple range test, P = Both phosphorous and acetyl salicylic acids reduced the growth rates in vitro of isolates of P. citrophthora and P. nicotianae var. parasitica; however, acetyl salicylic acid, more effective against P. nicotianae var. parasitica than phosphorous acid at 10 ppm, was not effective against P. citrophthora at this concentration. Acetyl salicylic acid was also less effective against P. citrophthora than phosphorous acid at 100ppm (Table 1). Table 2. Effects of phosphorous acid (PA), acetyl salicylic acid (ASA) and metalaxyl on the growth of group 1 b citrus trees affected by Phytophthora root-rot Treatment Top Root Root-rot weight weight rating (g) (g) Metalaxyl drench 47.5 a a 71.5 a 1.5 a 0.1% PA drench 37.4 ab 47.9 b 2.8 b 0.1% ASA foliar spray 30.8 b 46.1 b 3.2 c 10% PA foliar spray 27.1 b 42.2 b 3.3c 0.1% ASA drench 32.7 b 41.7 b 3.6 cd Control 29.1 b 41.0 b 3.8 d a Means within columns followed by the same letter are not significantly different according to Duncan's multiple range test, P = b For an explanation of the difference between groups 1 and 2 trees see text. Greenhouse trials on container-grown citrus Metalaxyl was the most effective treatment applied to group 1 trees (Tables 2 and 3), consistently yielding the highest values in all indices of plant growth and the lowest root-rot rating. It was the only treatment resulting in significantly higher root weights in group 1 trees. Metalaxyl was less effective when applied to severely affected group 2 trees (Tables 4 and 5) resulting in root weights not significantly different from those trees treated with foliar sprays of 0.1% phosphorous or acetyl salicylic acids, or soil drenches with 0.1% phosphorous acid. Root weights were significantly higher than those of untreated group 2 trees only in those trees treated with metalaxyl or foliar sprays with 0.1% phosphorous acid. Top weights of group 1 trees drenched with 0.1% phosphorous acid were not significantly different from those of trees treated with metalaxyl. No treatment produced significant increases in top weight of group 2 trees. All treatments apart from the drench with acetyl salicylic acid reduced the root-rot rating of group 1 trees (Table 2); however, no treatment was effective in this regard in the case of group 2 trees (Table 4). Table 3. Effects of phosphorous acid (PA), acetyl salicylic acid (ASA) and metalaxyl on shoot height (cm) of group 1 citrus trees affected 'by Phytophthora root-rot Treatment Days post-treatment Metalaxyl drench a a a a a a a a 0.1% PA drench a a a a a ab ab 0.1% ASA drench a a a a a ab abc 0.1% ASA fol.sp a a a a a bc bcd Control a a a a ab bc cd 10% PA fol.sp b 72.3 b b b b c cd a Means within columns followed by the same letter are not significantly different according to Duncan's multiple range test, P = Means within columns not followed by letters not significantly different according to analysis of variance, P = 0.05.

4 110 Walker Table 4. Effects of phosphorous acid (PA), acetyl salicylic acid (ASA) and metalaxyl on the growth of group 2 citrus trees severely affected by Phytophthora root-rot Treatment Top Root Root-rot weight weight rating (g) (g) Metalaxyl drench a a % PA drench ab % PA foliar spray a % ASA foliar spray ab % PA foliar spray b % ASA drench b 2.8 Control b 2.1 a Means within columns followed by the same letter are not significantly different according to Duncan's multiple range test, P = Means within columns not followed by letters not significantly different according to analysis of variance, P = At harvest, shoot heights of group 1 trees treated with metalaxyl and the phosphorous acid drench were significantly higher than those of untreated trees (Table 3); however, no significant increases in shoot heights in group 2 trees were apparent except at 55 days post-treatment in the case of trees treated with metalaxyl or the 0.1% phosphorous acid foliar spray and drench (Table 5). Foliar sprays with partially neutralized 10% phosphorous acid failed to increase plant growth and were observed to cause burning of young leaves on those few trees in group 1 which possessed new growth at the time of treatment. Also shoot growth was significantly reduced in both groups for between 11 and 14 weeks following this treatment (Tables 3 and 5). Foliar sprays or soil drenches with 0.1% acetyl salicylic acid failed to produce any significant increases in plant growth; however, root weights of group 2 trees treated with the foliar spray were not significantly different from those of trees treated with metalaxyl. The foliar spray also reduced the root-rot rating of group 1 trees. No treatment eliminated Phytophthora spp. from the potting mix of container-grown citrus and treatments did not appear to have any consistent effect on incidence of the two spp (Table 6). P. citrophthora was more commonly isolated from the more severely affected group 2 trees, while P. nicotianae var. parasitica was the dominant species isolated from group 1 trees. At harvest, ph of potting mix from trees treated with acid drenches was not different from that of untreated trees (range ). Discussion In vitro tests with phosphorous and acetyl salicylic acids against isolates of P. citrophthora and P. nicotianae var. parasitica demonstrated the potential of these chemicals as fungicides against Phytophthora spp. P. citrophthora was more sensitive to inhibition of mycelial growth than P. nicotianae var. parasitica by phosphorous acid (at 10ppm producing a reduction in radial growth after 8 days of 39 and 24% respectively), a phenomenon also observed for American isolates (Coffey and Bower, 1984). However, inhibition of mycelial growth by phosphorous acid was not as great as found against American isolates--ranging up to 89% for P. citrophthora after 7 days at a concentration of 10 ppm (Coffey and Bower, 1984) on a synthetic medium containing 4.5 g glucose per 1. This may be related to the nutrient-rich medium used in the present study, the greater availability of nutrients perhaps Table 5. Effects of phosphorous acid (PA), acetyl salicylic acid (ASA) and metalaxyl on shoot height (cm) of group 2 citrus trees severely affected by Phytophthora root-rot Treatment Days post-treatment % PA fol.sp a a a a a Metalaxyl drench a a ab a % PA drench 96.3 a ab ab a Control 86.4 b ab ab a % ASA drench 75.8 bc bc ab ab % ASA fol.sp bc be be ab % PA fol.sp c 64.1 c 95.5 c b a Means within columns followed by the same letter are not significantly different according to Duncan's multiple range test, P = 0.05.

5 Phytophthora root-rot of container-grown citrus 111 Table 6. Incidence of P. citrophthora (P.c.) and P. nicotianae var. parasitica (P.n.p.) in (a potting mix containing) citrus trees treated with metalaxyl, phosphorous acid (PA) and acetyl salicylic acid (ASA) as determined by pear-baiting (as % of total trees in groups I and 2 respectively) Treatment Group 1 Group 2 P.c. P.n.p. Total P.c. P.n.p. Total Metalaxyl drench % PA drench % PA foliar spray % ASA foliar spray % PA foliar spray % ASA drench Control t partially overcoming growth inhibition. Acetyl salicylic acid was more effective in vitro against P. nicotianae var. parasitica than phosphorous acid at 10 ppm (producing a reduction in radial growth of 53% after 8 days), but was not effective against P. citrophthora except at a concentration of 100 ppm. In trials with these chemicals on potted citrus infected with P. citrophthora and P. nicotianae var. parasitica, phosphorous acid applied as an 0.1% solution showed greatest promise as a potential treatment. Both the foliar spray with this solution and soil drenches with metalaxyl produced significant increases in root weight of severely affected trees and it appears that foliar sprays with phosphorous acid offer considerable potential as an alternative treatment for Phytophthora root-rot of citrus. However, foliar sprays with partially neutralized 10% phosphorous acid--as used for trunk injection of avocados (Pegg et al., 1985)--caused burning of young foliage and retarded shoot growth for a period of between 11 and 14 weeks after treatment. Such formulations may require considerable dilution before they can be used as foliar sprays on citrus. Drenching with 0.1% phosphorous acid did not increase root weights over untreated trees and repeat applications and/or increases in the concentration of the drench may be required to render this method more effective. However, in the case of severely affected trees this treatment achieved root weights equal to those of trees receiving a foliar spray of this solution. Additionally both this treatment and the foliar spray with 10% phosphorous acid reduced the extent of root rotting evident on group 1 trees. Foliar sprays with 0.1% acetyl salicylic acid reduced the root-rot rating in group 1 trees and produced root weights not significantly different from those of group 2 trees treated with metalaxyl. In vitro tests demonstrated that a concentration of this chemical in excess of 100ppm would be required to substantially reduce mycelial growth of P. citrophthora and higher dosages than used in the present study may be necessary to achieved satisfactory results in vivo. Alternatively trunk injection may be necessary to overcome limitations in uptake or translocation of this chemical; however, this method could not be tested in the present study using young nursery trees. The high in vitro activity of acetyl salicylic acid against P. nicotianae var. parasitica suggests that the potential of this chemical for control of this species at least be investigated further. With severely affected trees, metalaxyl (at the rate used) was unable to rehabilitate plant growth to the extent seen with less severely affected trees producing a 43% increase in root weight over untreated trees compared with a 74% increase in less severely affected trees. Further applications of metalaxyl and the other chemicals tested during the course of these experiments may have increased their efficacy. Both metalaxyl and fosetyl-al are known to remain active in root tissue for at least 2 months (Menge, 1986). Phosphorous acid and metalaxyl signficantly increased the shoot height of severely affected trees at 55 days post-treatment but not subsequently, suggesting a decline in activity beyond this date. However, this trend was not apparent with group 1 trees where significant increases in shoot height were not recorded until 138 and 190 days post-treatment in the case of metalaxyl and phosphorous acid respectively. P. citrophthora was more commonly isolated from severely affected trees than P. nicotianae var.

6 112 Phytophthora root-rot of container-grown citrus parasitica, suggesting a greater pathogenicity. P. citrophthora has been shown to cause significantly greater decay of citrus feeder roots in in vitro trials than P. nicotianae var. parasitica (Klotz et al., 1958), and was found to be the most pathogenic of Phytophthora spp. in field trials on citrus (Rossetti, 1969). Declining temperatures around the time of harvest may have favoured growth and hence isolation of P. citrophthora. Optimal temperatures for growth of P. citrophthora and P. nicotianae var. parasitica are reported to be ~ and ~ respectively (Waterhouse, 1963). Prevailing temperatures attained the higher optimum level for P. nicotianae var. parasitica mainly in the period from January to March. No treatment including metalaxyl eliminated Phytophthora spp. from potting mix. Metalaxyl used at higher rates than in the present study has given greater suppression of P. nicotianae var. parasitica in soil (Lee and Wicks, 1982). It was concluded from these studies that phosphorous acid offered considerable potential for the control of Phytophthora infestations in citrus nurseries. It is readily applied as a foliar spray and is potentially more economical than currently available fungicides. These factors make it suitable for use in the prevention of disease outbreaks in the nursery; however, its routine usage as a preventative would encourage the development of resistance. This risk could be reduced by alternating its use with that of other fungicides such as metalaxyl. Acknowledgement The author thanks Mr M F Wachtel for technical support and Mrs S Wetherall for typing the manuscript. References Abdelal H R Acetyl salicylic acid as a promising fungicide for controlling certain fungi. Egypt. J. Phytopathol. 13, Broadbent P 1977 Phytophthora diseases of citrus: a review. Proc. Int. Soc. Citriculture 3, Chadha K L, Randharwa N S, Bindra O.S. Chohan J S and Knorr L C 1970 Citrus decline in India. Causes and Control. Punjab Agric. Univ., Ludhiana, 97 p. Coffey M D and Bower L A In vitro variability among isolates of eight Phytophthora species in response to phosphorous acid. Phytopathology 74, Cohen Y and Coffey M D 1986 Systemic fungicides and the control of oomycetes. Annu. Rev. Phytopathology 24, Cohen Y and Reuveni M Occurrence of metalaxyl-resistant isolates of Phytophthora infestans in potato fields in Israel. Phytopathology 73, Fenn M E and Coffey M D 1984 Studies on the in vitro antifungal activity of fosetyl-a1 and phosphorous acid. Phytopathology 74, 606~I 1. Joseph M C and Coffey M D Development of laboratory resistance to metalaxyl in Phytophthora citricola. Phytopathology 74, Klotz L J, De Wolfe T A and Wong P-P 1958 Decay of fibrous roots of citrus. Phytopathology 48, Lee T C and Wicks T J 1982 Evaluation of fungicides for the control of Phytophthora nicotianae var. parasitica in container-grown Rough Lemon rootstock. Aust. P1. Pathol. 11, Mclntosh D L 1964 Phytophthora spp. in soil of the Okanagan and Similikameen valleys of British Columbia. Can. J. Bot. 42, McKenzie D The influence of metalaxyl (Ridomil (R)) on Phytophtbora root and collar rot of citrus in South Africa. Citrus Subtropical Fruit J. No. 618, McMillan R T 1983 Control of citrus foot-rot and root-rot. Proc. Fla. State Hort. Soc. 96, Menge J A 1986 Use of new systemic fungicides on citrus. Citrograph 71, Pegg K G, Whiley A W, Saranah J B and Glass R 1985 Control of Phytophthora root rot of avocado with phosphorus acid. Aust. PI. Pathol. 14, Rainsford K D 1984 Aspirin and the Salicylates. Butterworths, London, 335 p. Rossetti V 1969 Studies on Phytophthora species on citrus in Brazil. Proc. 1st. Inst. Citrus Symp. Ed. H D Chapman. Univ. of Calf., Riverside, Vol. 3, Singh L 1978 In vitro screening of some chemicals against phytopathogenic fungi. J. Indian Bot. Soc. 57, Timmer L W and Castle W S 1985 Effectiveness of metalaxyl and fosetyl AI against Phytophthora parasitica on sweet orange. PI. Dis. 69, Waterhouse G M 1963 Key to the species of Phytophthora de Bary. C.M.I. Mycological Papers No. 92.