Epidemiology of Verticillium dahliae on olive (cv. Picual) and its effect on yield under saline conditions

Transcription

1 Plant Pathology (2003) 52, Blackwell Publishing Ltd. Epidemiology of Verticillium dahliae on olive (cv. Picual) and its effect on yield under saline conditions A. G. Levin a,b, S. Lavee b and L. Tsror (Lahkim) c * a Department of Life Sciences, Ben Gurion University, Beer Sheva; b Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot; and c Department of Plant Pathology, Agricultural Research Organization, Gilat Experiment Station, M.P. Negev 85280, Israel The epidemiology of Verticillium dahliae and its effect on yield was studied for 3 years in three plots of olive cv. Picual, planted in soil previously cropped with highly susceptible V. dahliae host plants and irrigated with saline water. Disease incidence increased 2 2-, 2 6- and 1 5-fold in plots 3A, 9A and 9C, respectively, within 39, 25 and 15 months of the first record taken. The highest severities were recorded in spring 2001 (4 0, 5 3 and 5 4 on a scale of 2 10, respectively). Disease incidence and severity increased during winter spring and decreased during summer. Seasonal changes were also observed in the isolation of V. dahliae; the highest isolation rates in diseased trees were in winter (34%) and spring (45%), and the lowest were in autumn and summer (19 20%). Verticillium dahliae was isolated on an average of 27, 28 and 19% from the bottom, middle and top of the tree canopy, respectively, and was isolated from trees with and without symptoms. The fruit yield from diseased trees was reduced by an average of 75% in comparison with symptomless trees in plot 3A each year. A similar yield reduction (89%) was recorded in plot 9A in The severe expression of the disease and its effect on yield in the present study could be due to the fact that the orchard was planted in infested soil, and that saline irrigation probably exacerbates the problem. Keywords: Olea europea, pruning influence, salt-water irrigation, seasonal behaviour, Verticillium wilt Introduction Olive trees are grown extensively and intensively mainly in the Mediterranean region and in California (Wilhelm et al., 1962; Thanassoulopoulos et al., 1979; Cirulli, 1981). Verticillium wilt (Verticillium dahliae) limits production of high-yielding, high-quality cultivars in these areas (Hartmann et al., 1971; Blanco-López et al., 1984). The disease was first recorded in Italy in 1946 (Ruggieri, 1946), and is now present in many Mediterranean countries and in California (Snyder et al., 1950; Zachos, 1963; Saydam & Copcu, 1972; Blanco-López et al., 1984; Al-Ahmad & Mosli, 1993; Serrhini & Zeroual, 1995; Bellahcene et al., 2000). The disease was first recorded in Israel in cv. Manzanillo in the early 1960s (S. Lavee, unpublished data). Growth suppression, defoliation and wilting may occur on part of the branches because this is a vascular disease (Zachos, 1963; Cirulli, 1981; Blanco-López et al., 1984), and the olive is a highly sectored tree with direct vascular connection of specific roots and shoots (Lavee, 1996); in severe attacks, trees die. *To whom correspondence should be addressed. tsror@bgumail.bgu.ac.il Accepted 14 October 2002 Economic damage caused by the disease has increased during the past 20 years because of extensive irrigation and intercropping with V. dahliae-susceptible hosts such as potato and cotton (Tjamos, 1993; Blanco- López & Jiménez-Díaz, 1995). This effect has been reported for intensive (Cirulli, 1981) as well as for extensive olive production. Increase in disease has been shown to be associated with irrigation; disease incidence in nonirrigated orchards was 9, 17 and 4%, while in irrigated orchards it was 21, 40 and 13% in Morocco (Serrhini & Zeroual, 1995), southern Spain (Blanco- López et al., 1984), and Syria (Al-Ahamad & Mosli, 1993), respectively. In Israel olive orchards comprise ha, of which 10 15% are irrigated. Most plantings in the past 10 years have had drip irrigation. Due to a shortage of land, olives are planted in soil where Verticillium-susceptible crops were grown previously, which may increase the risk of disease. A possible interaction between V. dahliae and saline irrigation water was observed on potato: disease expression and colonization levels of the fungus were more severe in V. dahliae-tolerant cultivars (Désirée and Cara) irrigated with saline water (10 ds m 1 ) compared with plots irrigated with fresh water (Nachmias et al., 1993) BSPP

2 Verticillium wilt of olive 213 During the past 10 years, cv. Picual has been introduced as one of the main oil varieties in intensive plantations in Israel. This cultivar tolerates salinity but is very susceptible to Verticillium wilt (Barranco Navero et al., 2000). The objectives of this research were to study the epidemiology and aetiology of V. dahliae in cv. Picual irrigated with saline water, and the effect of the disease on fruit yield. Materials and methods Survey site The study was carried out in a commercial orchard located in southern Israel, where cv. Picual represents 20% of the total planted area. The trees were planted in soil where Verticillium-susceptible field crops had previously been grown for 30 years (including several cycles of potatoes and tomatoes). The soil type is a sandy loam, ph = , with a high concentration of salt (Na +, Meq; Ca 2+ and Mg 2+, Meq; B, mg L 1 ; the sodicity hazard of the soil, expressed as the sodium adsorption ratio (SAR), is and ds m 1, Shainberg & Oster, 1978). The annual rainfall in the region of the orchard is 100 mm, and the trees are drip-irrigated year round (9000 m 3 ha 1 ) with brackish water. The salt concentration of the water is 4 2 ds m 1. Three plots of cv. Picual in the orchard, grown under similar agronomic management, were selected: plot 3A with 474 trees was planted in August 1996, plot 9A with 303 trees and plot 9C with 282 trees were planted in July Disease assessment Disease evaluation was carried out by visual observation, walking between the rows. Disease incidence represents the number of trees with symptoms out of the total number of trees for each plot. Symptoms were evaluated each month over 3 years, based on a scale of 0 5: 0 = healthy tree; 1 = up to 25% of tree with symptoms, including chlorotic leaves and dead twigs; 2 = up to 50% of tree affected, including symptoms on lateral branches; 3 = up to 75% of tree affected including most branches; 4 = up to 95% of tree affected; 5 = tree dead. Diseased branches were removed in summer or winter. After each pruning the trees were classified as symptomless. The severity index (on a scale of 2 10) took into consideration the number of diseased trees with the different levels of symptoms, and was calculated as: SI = [(number of trees in level 1 2) + (number of trees in level 2 4) + (number of trees in level 3 6) + (number of trees in level 4 8) + (number of trees in level 5 10)]/ total diseased trees modified from Tsror et al. (1998). Isolation of V. dahliae from trees with and without symptoms Samples of five diseased or symptomless branches from each of 65 randomly selected trees from plot 3A were collected during different seasons of Thirtyeight trees were diseased at the beginning of the study. Four segments, 4 cm in length, from each branch were surface-sterilized with 0 3% HClO for 7 min and rinsed with sterile water. Three pieces (2 5 mm long) from each were transferred to sorbose agar medium (SA) (0 2% w/v sorbose, 1 5% w/v agar +100 p.p.m. streptomycin), incubated at 25 C in the dark, and examined after 2 weeks for the presence of V. dahliae. Isolation of V. dahliae from different heights of tree Samples of five diseased or symptomless branches from each of 17 randomly selected diseased trees from plot 9A were collected from the bottom, middle and top parts of the trees canopies during autumn 2000 and winter, spring and summer The isolation of V. dahliae was done as described previously. Measurement of fruit yield Fruit yield of each of the 474 and 303 trees in plots 3A and 9A was recorded separately. The number of trees with symptoms in plot 3A was 146, 164 and 190 in 1999, 2000 and 2001, respectively. In plot 9A the number of diseased trees was 56 in 2000 (the only year in which the yield was recorded). The yield in plot 9C was not recorded. Statistical analysis The experimental design in the present epidemiological study considered each tree as a single replicate. Data were analysed using sas (SAS Institute Inc., Cary, NC, USA). anova was followed by mean separation using Tukey Kramer multiple range tests at P = Data recorded as percentages were arcsin-transformed before analysis. Results Disease incidence and severity The first symptoms of Verticillium wilt were observed months after planting. Disease was monitored in plot 3A from January In plot 3A disease incidence was 22% and the severity index was 2 7 (Fig. 1). Disease incidence and severity increased to 41% and 3 4, respectively, by May Immediately after pruning the diseased branches in June 2000, trees were considered symptomless again. One month later, disease incidence increased from 21 to 36% in December 2000, and severity index increased from 2 4 to 2 8. A month after the third pruning of diseased branches (January 2001), disease incidence (23%) was similar to that recorded after the

3 214 A. G. Levin et al. Figure 1 Incidence and severity of Verticillium dahliae in cv. Picual in three plots during Evaluation of disease symptoms was carried out each month based on a scale of 0 5; 0 = healthy tree; 1 = up to 25% of tree with symptoms, including chlorotic leaves and dead twigs; 2 = up to 50% of tree affected, including symptoms on lateral branches; 3 = up to 75% of tree affected including most branches; 4 = up 95% of tree affected; 5 = tree dead. Severity index ( ) took into consideration the number of diseased trees in the different levels of symptoms and was calculated as follows: SI = [(number of trees in level 1 2) + (number of trees in level 2 4) + (number of trees in level 3 6) + (number of trees in level 4 8) + (number of trees in level 5 10)]/total diseased trees. P = pruning of diseased branches. first and second pruning, but the severity index was higher (SI = 3 4) than in December Disease incidence and severity index increased to 50% and 3 8 by March The highest severity indices were recorded during spring. In plot 9A, disease incidence, recorded for the first time in March 2000, was 12% and the severity index 4 7 (Fig. 1). Disease incidence doubled during the first year, while the severity index decreased from 5 3 in May to 3 3 in October A month after pruning the diseased branches (February 2001), disease incidence decreased to 18% and the severity index increased to 4 3. In March 2002 six trees had died because of the disease, and the severity index was 5 3. In plot 9C, disease incidence, recorded for the first time in January 2001, was 24%, and the severity index was 3 9 (Fig. 1). Disease incidence increased by 50% by March The maximum severity index was recorded in April

4 Verticillium wilt of olive 215 Table 1 Frequency (%) of isolations of Verticillium dahliae from diseased and symptomless trees ( ) Winter (Dec Feb) Spring (Mar May) Summer (June Aug) Autumn (Sep Nov) Av. between years Year D a S a D S D S D S D S 1999 nt nt nt nt nt nt 26.3 b (38) c 7 4 (27) (40) y 4 0 (25) 39 0 (41) 0 0 (24) 28 1 (32) 3 0 (33) nt nt (27) 8 3 (38) 51 4 (36) 3 4 (29) 10 8 (38) 3 7 (27) 10 8 (38) 3 7 (27) Average a d 4 2b a D, diseased; S, symptomless. b Samples of five branches with or without symptoms of each 65 trees from plot 3A collected in each season Four segments (4 cm long) from each branch were tested on SA for the presence of V. dahliae. c Number of trees in parentheses. d Means within a column followed by different letters are significantly different (Turkey Kramer test, P = 0 05). nt = not tested (5 4); it decreased (to 3 9) during summer, and increased again (to 5 2) in March Seasonal effects on the isolation of V. dahliae from Picual trees with and without symptoms Verticillium dahliae was isolated during all seasons throughout the study. In 1999 and 2000 an average of 38 out of 65 inspected trees were diseased; in 2001 an average of 34 out of 65 trees were diseased. The highest percentages of isolations (in a sample of 65 trees) were in spring (45%) and winter (34%), in comparison with 20 and 19% in summer and autumn, respectively (Table 1). These differences were not statistically significant. In another sample of 17 diseased trees, with samples from different heights of the trees, isolation of V. dahliae was the highest (P = 0 05) during the winter (49%). The lowest isolation rate was recorded in summer (6%) (Table 2). The percentage of V. dahliae isolations was higher (P = 0 05) in diseased trees (29%) than in symptomless trees (4%) (Table 1). Isolation of V. dahliae from different heights of the trees Verticillium dahliae was isolated from branches at the bottom, middle and top parts of the tree canopy throughout the year. There were no differences in isolation between the different parts of the canopy (Table 2). Table 2 Frequency (%) of isolation of Verticillium dahliae from different heights of tree, according to season Season Bottom Middle Top Average Autumn 29 4 a b b Winter a Spring b Summer b Average 26 5a 27 9a 19 1a a Samples of five branches from each of 17 randomly selected diseased trees were collected from the bottom, middle and high part of the tree s canopy. Samples were taken in autumn 2000 to summer 2001 from plot 9A. b Means followed by the same letter are not significantly different according to Turkey Kramer test (P = 0 05). Effect of V. dahliae on fruit yield The number of trees with symptoms in plot 3A was 146, 164 and 190 in 1999, 2000 and 2001, respectively. In plot 9A the number of diseased trees was 56 in In plot 3A, fruit yields of diseased trees were lower (P = 0 05) in comparison with symptomless trees in all years (Fig. 2). The yield of symptomless trees was normally distributed, with an average of 46 and 48 kg fruit per tree in 1999 and 2001, respectively, while there was an abnormal distribution in the diseased trees, with an average of 6 and 13 kg fruit per tree in the same years. In 2000 (the second harvest), the total yield from plot 3A was low, and the average yield of diseased trees (0 5 kg fruit per tree) was less than that of the symptomless trees (1 5 kg per tree, P = 0 05; data not shown). Differences in yield (P = 0 05) between trees with and without symptoms were observed in plot 9A (1 9 and 18 kg fruit per tree, respectively). Discussion An increase in disease incidence was observed in all three plots. There was an increase in disease incidence from 20 to 50% within 39 months in plot 3A; from 11 to 31% within 25 months in plot 9A; and from 22 to 35% within 15 months in plot 9C. The increase in disease incidence was mainly from the middle of winter to the end of spring. The data on disease severity suggest that the activity of Verticillium wilt was seasonal. The highest severity index (>5 0) was recorded during the spring of During summer and the beginning of autumn disease severity decreased, and a partial and temporary recovery of the trees by the development of new growth was observed. The relatively high temperatures in the summer may be an important factor affecting both fungal activity and growth of the trees. The maximum/minimum average temperatures in summer 1999 and 2000 in the survey site were 35/18 C (National Meteorological Center of Israel). These findings are in agreement with Wilhelm & Taylor (1965), Thanassoulopoulos et al. (1979) and Navas-Cortéz et al. (2001). Seasonal changes were also observed in colonization of the trees by the fungus, where the highest reisolation rates

5 216 A. G. Levin et al. Figure 2 Effect of Verticillium dahliae on yield of fruit in cv. Picual (plot 3A in 1999 and 2001; plot 9A in 2001). The yields of each of 474 trees in plot 3A and of 303 trees in plot 9A were evaluated separately. The number of diseased trees was 146 and 190 (out of 474 trees) in plot 3A in 1999 and 2001, respectively; and was 56 (out of 303 trees) in plot 9A in The 2000 yield in plot 3A was 1 5 and 0 5 kg per tree for symptomless and diseased trees, respectively. The number of diseased trees was 164 (out of 474). Means of yields of diseased and symptomless trees followed by the same letter are not significantly different according to Turkey Kramer test (P = 0 05). in diseased trees were in winter and spring (45 and 34%, respectively). Higher isolation rates were obtained in the winter (P = 0 05) when samples of branches were from different tree heights. These data were recorded from only 17 diseased trees (plot 9A). However, when seasonal effects were studied with a larger sample size (65 trees from plot 3A), no differences between the seasons were observed. The fungus was isolated from branches from all

6 Verticillium wilt of olive 217 parts of the trees in all seasons, but to different extents. This could indicate an infection cycle that starts in autumn with the reinvasion of the fungus, probably by the germination of microsclerotia and the upward spread of conidia in the xylem during winter and spring, when disease expression was at a maximum; and is completed when the fungal population decreases during summer, probably because of high temperatures (Tosi & Zazzerini, 1998). Alternatively, physiological changes may have occurred independently of infection (Wilhelm & Taylor, 1965). Isolation of the fungus from the roots and trunks of trees, after inactivation in branches has occurred, suggests the potential for reinvasion during autumn (Wilhelm & Taylor, 1965). Our data on isolation support the epidemiological data, with the highest disease incidence and severity observed in winter spring and the lowest in summer. The low rates of isolation of V. dahliae during summer were in accordance with some previous reports (Ruggieri, 1948; Bonifacio & Parrini, 1975; Cirulli, 1981), but not with that of Wilhelm & Taylor (1965). Tosi & Zazzerini (1998) consistently related the isolation of the fungus from stem tissues to the occurrence of a mild winter and summer, and sporadic success of isolation to a cold winter (minimum temperature below 0 C) and warm summer (maximum temperature above 30 C). The fungus was isolated from symptomless trees at lower rates than trees with symptoms. This was expected, as parts of the symptomless trees might not be colonized. The isolation of V. dahliae from symptomless trees may indicate that infections were latent, and that symptoms might appear in later stages of growth. Alternatively, weakly virulent isolates of V. dahliae may have been present (Al-Ahmad & Mosli, 1993). Disease severity was apparently affected by the time when trees were pruned. In plot 3A, which was pruned in May 1999 and June 2000, disease severity decreased 1 month later ( in May 1999; in June 2000). Removing the diseased branches in summer may slow the progress of Verticillium wilt. In contrast, when trees were pruned in winter, in both plots 3A ( January 2001) and 9A (February 2001) disease severity 1 month after pruning was higher than in the previous month. This response could be because of a combination of stimulated vegetative growth; enhanced root activity; changes in the hormonal balance within the tree (Lavee, 1996; Pastor Muñoz-Cobo & Humanes Guillén, 1998) that overlap with high activity of the fungus in the tree (Wilhelm & Taylor, 1965), and/or the stimulation of sclerotial germination and infection of roots (Schnathorst, 1981). Natural recovery of trees of cv. Picual was observed in only two plantations in Spain, with a decrease in disease expression over the years, and this hypothesis was confirmed when the observations were made on individual trees (Blanco-López & Jiménez-Díaz, 1995). Yield reductions associated with V. dahliae were observed in all three seasons in plot 3A (1999, 2000 and 2001) and in one season in plot 9A (2000). The average yield reductions were 75 90%. In general, trees that had any disease symptoms before blooming did not set fruit, indicating a possible physiological change in the diseased trees (Bell & Mace, 1981). Thanassoulopoulos et al. (1979) reported that in Greece 2 3% of 14 million trees were affected by Verticillium wilt, of which 1% died, resulting in a yield loss of metric tonnes. In Syria % of 6 5 million trees in nine provinces were affected, and annual yield losses were 1 2 3% (Al- Ahmad & Mosli, 1993). These trees were mostly not irrigated. The severe expression of the disease and its dramatic effect on yield in the present study could be due to the fact that the orchard was irrigated, and the effect may have been enhanced because of the salinity of the water (4 2 ds m 1 ). A possible interaction between V. dahliae and saline irrigation water was investigated in potato (Nachmias et al., 1993), and disease expression and colonization by the fungus was more severe in V. dahliae-tolerant varieties (Désirée and Cara) irrigated with saline water (10 ds m 1 ) in comparison with plots irrigated with fresh water. Phytophthora root rot of citrus was also more severe under saline conditions (Blaker & MacDonald, 1986). In conclusion, this study demonstrates the seasonal behaviour of V. dahliae in the highly susceptible cv. Picual, and its severe effect on yield. The expression of Verticillium wilt in Picual trees irrigated with saline water appears to be independent of tree age in the first 6 years after planting. The seasonal behaviour of the disease was also demonstrated by the different isolation rates of the fungus and its distribution in the different heights of the tree. The findings strongly indicate the potential risk of planting susceptible olive varieties in soil previously cropped with V. dahliae-susceptible crops when high irrigation levels are used, especially with saline water. Acknowledgements We thank Janis Joseph for editing the manuscript. Contribution No. 512/02 from Agricultural Research Organization, Institute of Plant Protection, Bet Dagan, Israel. The research was partially supported by Ramat Negev Desert Agro-Research Center, Israel. References Al-Ahmad MA, Mosli MN, Verticillium wilt in Syria. Bulletin OEPP/EPPO Bulletin 23, Barranco Navero D, Cimato A, Fiorino P, Rallo Romero L, Touzani A, Castaneda C, Serafini F, Trujillo Navas I, World Catalogue of Olive Varieties. Madrid, Spain: International Olive Oil Council. Bell AA, Mace ME, Biochemistry and physiology of resistance. In: Mace ME, Bell AA, Beckman CH, eds. Fungal Wilt Diseases of Plants. New York: Academic Press, Bellahcene M, Fortas Z, Geiger JP, Matallah A, Henni D, Verticillium wilt in olive in Algeria: geographical distribution and extent of the disease. Olivae 82, 41 3.

7 218 A. G. Levin et al. Blaker NS, MacDonald JD, The role of salinity in the development of Phytophthora root rot of citrus. Phytopathology 76, Blanco-López MA, Jiménez-Díaz RM, Una propuesta de lucha integrada contra la verticilosis del olivo. Fruticultura Profesion 70, 52 8 (in Spanish). Blanco-López MA, Jiménez-Díaz RM, Caballero JM, Symptomatology, incidence and distribution of Verticillium wilt of olive trees in Andalucia. Phytopathologia Mediterranea 23, 1 8. Bonifacio A, Parrini C, Presenza di Verticillium dahliae Kleb. in oliveti della Toscana. Informatore Fitopatologico 25, 21 5 (in Italian). Cirulli M, Attuali cognizioni sulla Verticilliosi dell olivo. Informatore Fitopatologico 31, (in Italian). Hartmann H, Schnathorst WC, Whisler WC, Oblonga, a clonal olive rootstock resistant to Verticillium wilts. California Agriculture 25, Lavee S, Biology and physiology of the olive. In: World Olive Encyclopedia. Barcelona, Spain: International Olive Council, Nachmias A, Kaufman Z, Livescu L, Tsror L, Meiri A, Caligari PDS, Effect of salinity and its interaction with disease incidence on potatoes grown in hot climates. Phytoparasitica 21, Navas-Cortéz JA, Rodríguez-Jurado D, Trapero-Casas JL, Landa BB, Mercado Blanco J, Pérez-Artés E, Jiménez-Díaz RM, Spatio-temporal dynamics of verticillium wilt epidemics in an olive orchard, X. Proceedings of the 8th International Verticillium Symposium, Cordoba, Spain: University of Spain. Pastor Muñoz-Cobo M, Humanes Guillén J, Poda del Olivo Moderna Olivicultura, 3 a edn. Madrid, Spain: Agricola Española, Ruggieri G, A new disease of olive. L Italia Agricola 83, Ruggieri G, Ricerche ed esperienze su una tracheoverticilliosi dell olivo. Olivicoltura 3, 6 9. Saydam C, Copcu M, Verticillium wilt of olive in Turkey. Journal of Turkish Phytopathology 1, Schnathorst WC, Life cycle and epidemiology of Verticillium. In: Mace ME, Bell AA, Beckman CH, eds. Fungal Wilt Diseases of Plants. New York: Academic Press, Serrhini MN, Zeroual A, Verticillium wilt in Morocco. Olivae 58, Shainberg I, Oster JD, Properties of irrigation water. In: Shainberg I, Oster JD, eds. Quality of Irrigation Water. Bet Dagan, Israel: International Irrigation Information Center, Snyder WC, Hansen HN, Wilhelm S, New hosts of Verticillium albo-atrum. Plant Disease Reporter 34, Thanassoulopoulos CC, Biris DA, Tjamos EC, Survey of Verticillium wilt of olive trees in Greece. Plant Disease Reporter 63, Tjamos EC, Prospects and strategies in controlling Verticillium wilt of olive. Bulletin OEPP/EPPO Bulletin 23, Tosi L, Zazzerini A, Investigation on the epidemiology of Verticillium wilt in central Italy. Olivae 71, Tsror (Lahkim) L, Erlich O, Amitai S, Hazanovsky M, Verticillium wilt of paprika caused by a highly virulent isolate of Verticillium dahliae. Plant Disease 82, Wilhelm S, Kaiser WJ, Georgopoulus SG, Opitz KW, Verticillium wilt of olives in California. Phytopathology 52, 32 (abstract). Wilhelm S, Taylor JB, Control of Verticillium wilt of olive through natural recovery and resistance. Phytopathology 55, Zachos DG, La verticlliose de l oliver en Gréce. Annales Institut Phytopathologique Benaki (NS) 5,