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Large-scale epidemiological study and spatial patterns of Verticillium wilt in olive orchards in southern Spain
Crop Protection 28 (2009) 46–52
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Crop Protection journal homepage: www.elsevier.com/locate/cropro
Large-scale epidemiological study and spatial patterns of Verticillium wilt in olive orchards in southern Spain Estefanı´a Rodrı´guez a, *, Jose M. Garcı´a-Garrido a, Pedro A. Garcı´a b, Mercedes Campos a a b
´n Experimental del Zaidı´n (CSIC), Profesor Albareda 1, 18008 Granada, Spain Departments of Agroecology-Plant Protection and Soil Microbiology and Symbiotic Systems, Estacio Department of Statistics and O.R. University of Granada, Campus de Fuente Nueva s/n, 18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 April 2008 Received in revised form 15 August 2008 Accepted 18 August 2008
Verticillium wilt (VW) is caused by the soilborne pathogen, Verticillium dahliae. The fact that available control measures are not completely effective, together with an increasing spread of the highly virulent defoliating pathotype (D) in the last two decades, makes VW one of the most threatening olive tree disease. For better management, epidemiology and spatial patterns of VW were investigated in an important olive-growing area in southern Spain. A sampling survey was conducted from 2002 to 2005 in 873 olive orchards, which were assessed for the presence of V. dahliae. PCR assays were used to identify the highly virulent defoliating (D) and nondefoliating (ND) pathotypes of V. dahliae. Ordinary kriging, a geostatistical tool that uses regression techniques for interpolation of spatial data, was applied to obtain predictive V. dahliae incidence maps and to evaluate the correlation between distance from the river and VW. Prevalence in olive orchards was 14.1%, with a disease incidence of 1.9% and mean incidence per olive orchard of 9.5%. Prevalence of the ND pathotype was higher (10.7%) than for the D pathotype (3.4%). VW was three times more prevalent in irrigated than in non-irrigated orchards (relative risk ¼ 3.01) and there was a close association between the ND pathotype and irrigated orchards (relative risk ¼ 2.03). Spatial pattern analysis showed several factors involved in the spread of the pathogen. VW was more prevalent in the north-west, suggesting the spreading of the disease from other bordering areas known to be contaminated, in southwest Spain. The D pathotype had a very restricted and aggregated distribution, suggesting the introduction of this pathotype in new olive orchards areas. The incidence map generated by kriging displayed an increase in north, northeast and south areas with numerous subareas with probabilities equalling or exceeding 53%, showing the effect of intensive agriculture (high plant density and irrigation) on incidence of the disease. Assessment of rivers-pathogen relationship revealed an association of VW with the distance to the nearest river, indicating that soils through the rivers’ axis and previously cropped with horticultural crops susceptible to V. dahliae, have implications for the establishment of new olive orchards and the occurrence of the disease. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Epidemiology Disease incidence GIS Olea europaea Pathotypes Pathogen prevalence
1. Introduction The control of Verticillium wilt (VW) in olive, caused by the soilborne, fungal pathogen, Verticillium dahliae Kleb., is of great concern in Andalusia (southern Spain), where olives are the most widespread traditional crop. About 30% of European olive oil production comes from this area (COI, 2005). VW is one the most serious olive tree diseases facing farmers worldwide because it has become increasingly widespread in the last two decades. Available control measures are not completely effective. Because systemic fungicides are unable to prevent the disease, control of VW currently depends on preventive measures * Corresponding author. Tel.: þ34 958 181 600; fax: þ34 958 189 600. E-mail address: [email protected] (E. Rodrı´guez). 0261-2194/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2008.08.009
such us using pathogen-free soil and propagation material. The most practical management after planting at present is a combination of cultural methods (Jime´nez-Dı´az et al., 1998; Tjamos, 2008). The disease has become even more important with the spread of the highly virulent defoliating pathotype (D) (Bejarano-Alca´zar et al., 2001; Lo´pez-Escudero and Blanco-Lo´pez, 2001; Navas-Corte´s et al., 2001; Mercado-Blanco et al., 2003; Rodrı´guez Navarro, 2006). V. dahliae isolates infecting olive (Olea europaea) and cotton (Gossypium hirsutum) show cross-virulence (Schnathorst and Mathre, 1966; Schnathorst and Sibbet, 1971) and they can be classified into defoliating (D) or nondefoliating (ND) pathotypes, based on their ability to cause defoliation of green leaves from shoots and twigs (Schnathorst, 1973). The D pathotype has been reported in cotton-growing countries since 1966 (Mathre et al., 1966;
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Schnathorst, 1973; Korolev et al., 1999; Elena and Paplomatas, 2001), but was not known to occur in Spain until 1983, when it was found in an area of intensive cotton cultivation in southwest Spain (Bejarano-Alca´zar et al., 1996) with recent evidence of longdistance spread of this pathotype to olive-growing regions. D isolates of V. dahliae from China, Spain, and the United States appear molecularly similar (Pe´rez-Arte´s et al., 2000), suggesting that this pathotype may have been introduced into Spain, and that the worldwide spread of the D pathotype may have occurred with contaminated cotton seed. To date, a PCR-based procedure has been developed for consistent detection of D and ND pathotypes of V. dahliae as key components of successful management of VW of olive (Mercado-Blanco et al., 2003). An important strategy of disease management is to analyze spatial patterns of VW, because they can provide important clues to the ecology of disease (e.g., direction and distance of spread or importance and proximity of the sources). Spatial patterns of most variables can be described, analyzed, and displayed at any scale with geographical information systems (GIS) and geostatistics. One of the more popular geostatistical technique is Kriging, which refers to a group of linear regression techniques that use models of the spatial autocorrelation to estimate values at unsampled locations (Beagle Ristaino and Gumpertz, 2000; Jaime-Garcı´a and Cotty, 2006). Although previous studies have provided information about the importance and distribution of VW in the most important areas for olive production (Thanassoulopoulos et al., 1979; Cirulli, 1981; Blanco-Lo´pez et al., 1984; Al-Ahmad and Mosli, 1993; Serrihini and Zeroual, 1995; Levin et al., 2003; Nigro et al., 2005), more epidemiological studies, particularly in case of the D pathotype, are required to obtain detailed information on pathogen prevalence or disease incidence. Few studies of the spatial pattern and pathogen dispersal have been conducted in olive crops (Navas-Corte´s et al., 2008). Epidemiology is concerned with the patterns of disease occurrence and the factors that influence these patterns, and provide the basis for the development and evaluation of preventive crop protection measures. Therefore, the aim of this study was to investigate the epidemiology of the fungus for better management. The objectives were to (i) assess the prevalence, incidence and map the V. dahliae pathotypes, (ii) analyze the spatial patterns of spread of the disease and the association of the pathogen with the environment. Using a double-stratified sampling technique, a sampling survey was conducted in an important olive-growing area in southern Spain. PCR assays were used to identify V. dahliae pathotypes. Kriging was applied to reconstruct the spatial patterns of VW. 2. Materials and methods 2.1. Field surveys The sampling survey was conducted in olive orchards in the province of Granada in Andalusia (southern Spain). Andalusia, which accounts for 75% of Spanish production, has the biggest olive crop under cultivation in the world. Granada province is the third largest producer in the Andalusia region with 175,000 ha distributed among five olive-growing areas: Norte, Vega, Alhama, Sur and Levante (Fig. 1) (MAPA, 1975). Each production region differs greatly from other regions in terms of the physical characteristics of the plantations, management practices, socioeconomic situation and environmental effects (MAPA, 1975). The sampling survey was designed in collaboration with the Local Government (Modelo de Explotaciones Olivareras, Unidad de Prospectiva, Consejerı´a de Agricultura y Pesca. Junta de Andalucı´a) which supported the information about the olive orchards in Granada province including area (ha), number of olive trees and type of cultivars.
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Because of heterogeneity between the different olive regions and use of irrigation, we designed a stratified double-sampling technique. A stratified sampling technique is generally used when the population is heterogeneous and the sample size is proportional to the relative size of the strata (Kish, 1995). The proportional location in the first sampling level is based on the percentage of orchards in each region. The second level consists of olive orchards with probability proportional to size (PPS). PPS gives a probability of selecting a sampling unit (e.g., olive orchards) proportional to the size of its population (Kish, 1995). The sample size for estimating proportions was 998 olive orchards out of a total population of 129,962 in Granada province, assuming that a priori probability of an olive orchard infected by V. dahliae (p) is equal to the probability of an olive orchard not infected (q) such that p ¼ q ¼ 0.5 (Kish, 1995). Standard error was less than 3.1% (95% confidence interval). Finally, the total number of olive orchards surveyed was actually 873 covering an area of 4087.2 ha and 527,903 olive trees, with a standard error 3.3% (95% confidence interval). The olive orchards needed for the sample survey in each area were: 380 in Norte, 231 in Vega, 107 in Alhama, 68 in Sur, and 87 in Levante region. The centroid for each olive orchard surveyed was identified using information from the Ministry of Agriculture and Fisheries’ (online). From 2002 to 2005 a questionnaire was sent to randomly chosen farmers who answered details about agronomic features (e.g., irrigation, age of plantation, crop management), origin of plant material, previous cropping history and symptomatology for each olive orchard surveyed (Rodrı´guez Navarro, 2006; Rodrı´guez et al., 2008). To reduce bias from farmer’s subjectivity, all olive orchards with VW symptoms were visited directly by the authors in order to validate survey results. 2.2. Pathogen prevalence, disease incidence and V. dahliae isolation from olive trees For disease assessment we used the terms pathogen prevalence and disease incidence (Nutter et al., 2006). Pathogen prevalence is a measure of the proportion (or percentage) of geographical sampling units (olive orchards) where the pathogen has been detected relative to the total number of geographical sampling units (olive orchards) that were inspected. Disease incidence measures the proportion of olive trees where disease symptoms were observed relative to the total number. Mean incidence per olive orchard or ‘‘MI’’ is defined as the mean of number of olive trees affected per olive orchard. The samples were taken from March to June and from September to November when evaluation of the presence of V. dahliae in plants is most appropriate (Levin et al., 2003). Once in an orchard, olive trees showing wilt symptoms were inspected, counted and sampled. Branches and stems from at least 10 of the affected olive trees were collected. Samples were assessed for the presence of V. dahliae in the laboratory. Twenty-four small internal fragments of vascular tissue from different affected branches per tree were surface-sterilized with NaClO (10%) for 1 min and subsequently rinsed with sterile water for 1 min and segments were put on a petri plate in water agar with chlortetracycline (30 mg l1), and colonies were periodically examined for V. dahliae identification. 2.3. Pathotype identification using PCR Isolates obtained from the plant material were assayed using specific PCR assays for identifying V. dahliae pathotypes. Fungal isolates were grown on potato dextrose broth (PDB, Sigma) for 7 d at 24 C in the dark on an orbital shaker (125 rpm). Mycelia were harvested, lyophilized and ground as previously described by Pe´rez-Artes et al. (2000). DNA was extracted from ground mycelia
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Fig. 1. Geographic distribution of olive crop in Granada province (Andalusia, southern Spain). Each dot represents the central point of the olive orchards randomly selected to survey Verticillium wilt; ‘‘n’’ indicates the number of olive orchards surveyed in each olive-growing area depending on olive surface.
using the DNeasy Plant Kit (Qiagen, Hilden, Germany). DNA concentration was assessed using Nanodrop and by agarose gel electrophoresis according to standard procedures. DNA solutions were stored at 20 C for further use. Isolates were characterized by the PCR assays using a 3-primer set as previously described by Mercado-Blanco et al. (2003). Primer pair DB19/DB22 yields V. dahliae-specific polymorphic DNA bands of 539 or 523 bp (Carder et al., 1994). The combined use of primers DB19, DB22 and espdef01 yields one of the V. dahliae-specific markers together with an amplicon of 334 bp, in case of isolates of the D pathotype, which allow differentiation between D and ND V. dahliae pathotypes in a single PCR assay (Mercado-Blanco et al., 2003). Amplification reactions (25 ml) consisted of micromolar ratio of 0.7:0.1:0.2 (DB19/ DB22/espedef01) for primers, 200 mM each dNTP, 2.5 ml of 10 reaction buffer, 0.75 U of EcoTaq (Ecogen S.R.L., Barcelona, Spain), 1.5 mM MgCl2, and 10 ng of template DNA. Reaction conditions included denaturation at 94 C for 4 min followed by 30 cycles of 1 min of annealing at 60 C, extension for 30 s at 72 C, denaturation for 1 min at 94 C, and a final extension step of 6 min at 72 C. 2.4. Statistical analyses Mean values of pathogen prevalence were compared using the
c2 test and Relative Risk (RR) values. In statistics and mathematical epidemiology, RR is the risk of an event (or of developing a disease) relative to exposure (Kish, 1995). Relative risk is calculated by dividing the death or disease risk in a specific population group (group A) by the risk of population from all other groups. For example, if the relative risk were 3.0, population in group A would be three times as likely as populations from other groups to become diseased. Classical ANOVA and post hoc comparisons (by means of Tamhane’s T2 method because equal variance was not assumed) were performed to show significant differences for incidence between areas. Distance of olive orchards surveyed to the nearest river was assayed by Welch’s t-test (Kish, 1995). 2.5. ArcGIS A GIS database was developed for Granada province olive orchards. Data included information questionnaires, information
from the Ministry of Agriculture and Fisheries’ and results of isolation and PRC assays. ArcGIS (version 9.1) was used to data display, to interpolate with kriging and to calculate the distance from each olive orchard surveyed (point) to the nearest river (polyline). Ordinary kriging was applied to provide a surface map of V. dahliae incidence in Granada province and to reproduce the main characteristics of the spatial pattern of VW, including correlation with the nearest rivers. An olive surface layer (1995–1996 landcrop/land-use maps from Consejerı´a de Agricultura y Pesca, Junta de Andalucı´a, Granada province) was used to mask the surface to remove non-olive areas and to reduce the proportions based on olive surface because kriging is a technique that assumes that spatial homogeneity is fundamental for the interpolation.
3. Results 3.1. Pathogen prevalence and disease incidence From 2002 to 2005, 873 olive orchards in five olive-growing areas were surveyed (Fig. 1). Of the 873 olive orchards, 14.1% were positive for V. dahliae, 11.5% were negative for V. dahliae and observed symptoms were induced by other (a)biotic factors, and 74.4% of the total olive orchards surveyed did not show any wilt symptoms. The geographical distribution of V. dahliae is shown in Fig. 2. The distribution of VW appeared to be more clustered in the north-west of the province, as evidenced by differences in pathogen prevalence (c2 ¼ 27.49; p < 0,005) (Table 1; Fig. 2). Norte and Vega had the highest percentage of olive orchards infected by V. dahliae, whereas Levante and Sur had similar levels of pathogen prevalence, and Alhama had the lowest percentage (Table 1). 76% of the total isolates recovered belonged to the ND pathotype and 24% belonged to the D pathotype, indicating a pathogen prevalence of 10.7 and 3.4% for ND and D pathotypes, respectively (Table 1). In all areas surveyed, prevalence of the ND pathotype was greater than the D pathotype. Overall pathotypes were associated with specific geographical regions (c2 ¼ 76.21; p < 0.0001) (Table 1; Fig. 2). The ND pathotype was observed in all the areas surveyed, with the highest prevalence in Levante and the lowest in Alhama. The D pathotype was found in the north-west of the region,
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Fig. 2. Geographic distribution of defoliating and nondefoliating pathotypes of Verticillium dahliae in olive orchards in Granada province.
especially in Norte area. The D pathotype was rare in Levante and absent in Sur (Table 1; Fig. 2). Incidence of VW was 1.9% (Table 1), corresponding to 103,073 olive trees that were infected or dead in Granada province. Mean incidence (MI) was 9.5% in a range from 0.1 to 97%. Incidence variations between areas were significant (p < 0.024). The incidence was higher in Levante, Sur and Norte than in Vega and Alhama (Table 1). Data were subjected to kriging to construct a probability map of incidence of VW (Fig. 3). Mapping showed increased disease incidence in north, northeast and south areas of Granada province with numerous subareas with disease probabilities equalling or exceeding 53% in Norte area. However, the disease probability throughout the central and southern Granada province was less than 20%. ND pathotype isolates were associated with higher levels of disease incidence (Table 1), with twice the number of olive trees
being affected by ND isolates (incidence of 2.2 and 1.2%, respectively). The incidence of the ND pathotype was greater in the northeast and south of the region, particularly in Levante and Sur (Table 1) whereas D pathotype incidence was higher the west of the region, especially in Norte area. However, differences between areas were not significant (Table 1). 3.2. Irrigation–V. dahliae relationship Survey results indicated that 33% of the total orchards were under irrigation management. Pathogen prevalence was three times higher in irrigated orchards (c2 ¼ 46.4, p < 0.0001, RR ¼ 3.01). VW was present in 25.6% of irrigated olive orchards and in 8.5% of non-irrigated orchards, but there was no significant difference in disease incidence between the two water treatments (1.95% and 2% for irrigated and non-irrigated olive orchards, respectively).
Table 1 Pathogen prevalencea, disease incidenceb, and mean incidence (MI)c of Verticillium wilt in olive orchards in the five areas surveyed in Granada province (Andalusia, southern Spain).
Province Norte Vega Alhama Sur Levante
a b c d
Olive orchards surveyed
Mean Incidence (%)
Prevalence (%)
Incidence (%)
V. dahliae
pathotype ND
pathotype D
V. dahliae
pathotype ND
pathotype D
873 380 231 107 68 87
9.5 11.6 (a) 3.4 (b) 4.6 (b) 12.1 (a,b) 18.9 (a,b) F4,119 ¼ 2.918 p < 0.024
14.1 15.5 15.2 9.3 10.3 13.8 c2 ¼ 27.49 p < 0.005
10.7 11.6 10.4 6.5 10.3 12.6 c2 ¼ 76.21 p < 0.0001
3.4 3.7 4.8 2.8 0.0 1.1 c2 ¼ 76.21 p < 0.0001
1.9 2.6 (a)d 1.3 (b) 0.8 (b) 6.2 (a,b) 8.2 (a,b) F4,119 ¼ 2.918 p < 0.024
2.2 2.5 1.7 0.9 6.2 11.9 F4,90 ¼ 2.076 p ¼ 0.091
1.2 3.7 0.5 0.6 0.3 F3,26 ¼ 1.164 P ¼ 0.343
Pathogen prevalence measures the percentage of olive orchards in which pathogen was detected. Chi-square value is significant between areas. Disease incidence measures the pertentage of trees diseased. Classical ANOVA was performed. MI is the mean incidence of the total olive orchards surveyed. Classical ANOVA was performed. a,b denote homogeneous subgroups after post-hoc Tamhane’s T2 comparisons (p < 0.05).
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Fig. 3. Kriging map of the distribution of VW incidence in Granada province. Kriging estimation included the proportion of olive trees affected in orchards where V. dahliae was detected. Olive surface layer was used to generate kriging estimation.
Both D and ND pathotypes were more prevalent in irrigated olive orchards (Table 2). Nevertheless, we found statistical differences in the disease levels caused by both pathotypes depending on the type of water treatment. D isolates were significantly more common in non-irrigated orchards. Conversely, ND isolates were twice as common in irrigated orchards (c2 ¼ 5.392; p < 0.02; RR ¼ 2.03). Thus, contribution to prevalence was 83.5% and 16.5% for ND and D, respectively, in irrigated, and 35.0% and 65.0%, respectively, for ND and D in non-irrigated orchards. Incidence of the D pathotype was also higher in non-irrigated orchards, but incidence of the ND pathotype was similar in both irrigated and non-irrigated orchards (Table 2). 3.3. Rivers–V. dahliae relationship Statistical comparisons using the Welch t-test showed that the mean distance from the river in olive orchards affected by VW (1.48 km) was significantly less than in olive orchards free from symptoms of VW (1.94 km) (p < 0,001; t ¼ 3.378; d.f. ¼ 179.97). Most of the orchards affected (70%) by V. dahliae were located less
than 1000 m from the river. Correlation analysis indicated that the negative associations between distance and incidence for both pathotypes were not significant (rDpathotype ¼ 0.298, p ¼ 0.11; rNDpathotype ¼ 0.114, p ¼ 0.28). Kriging interpolation of distance of each olive orchard to the nearest river exhibited a strong aggregative spatial pattern to neighbouring rivers. Fig. 4 shows this clustered distribution pattern and dots indicate orchards located less than 1000 m from the river. Kriging gave a pattern of disease closely related to the Welch t-test results. Infection was not homogenous. The observed disease gradient along the rivers axis was characterized by a higher disease in the orchards nearest to the rivers and ranged from 70 to 100%, decreasing towards 50–15% with increasing distance from the rivers axis. The disease gradient was clearly marked in the Norte area.
4. Discussion This study provides data on epidemiology and the spatial patterns of Verticillium wilt in olive, allowing a spatial perspective
Table 2 Pathogen prevalencea and disease incidenceb of V. dahliae and pathotypes nondefoliating (ND) and defoliating (D) in irrigated and non-irrigated olive orchards. Pathogen prevalence (%)
Non-irrigated Irrigated c2 value a b
Disease incidence (%)
V. dahliae
Pathotype ND
Pathotype D
V. dahliae
Pathotype ND
Pathotype D
8.5 25.6 46.4 (p < 0.0001)
5.4 21.4 5.392 (p < 0.02)
2.9 4.2 5.392 (p < 0.02)
1.95 2.0 (p > 0.05)
2.0 2.3
1.7 0.8
Pathogen prevalence measures the percentage of olive orchards in which V. dahliae were detected. Disease incidence is the number of olive trees infected with V. dahliae.
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Fig. 4. Kriging map of spatial distribution pattern of Verticillium dahliae incidence associated with rivers in the province of Granada (Andalusia, southern Spain). Olive surface layer as a mask, and the distance of each olive orchard surveyed to the nearest river were used to generate kriging estimation.
on disease and ultimately a better knowledge for disease management. 4.1. Pathogen prevalence, disease incidence and irrigation– V. dahliae relationship To date, the highly virulent, defoliating pathotype of V. dahliae had not been reported in the area surveyed and no studies had previously quantified and mapped the disease. In our study, VW was found in 14.1% of the olive orchards surveyed and the D pathotype had a prevalence of 3.4%. Although VW was widespread throughout the olive-growing areas, the geographic distribution of the disease was clustered in the north-west province, and was more prevalent in Norte and Vega areas (north-west), indicating a quick spread of the disease throughout these orchards. Norte and Vega border on the infected olive orchards in southwest Spain where the disease has been well established since the 1980s (Sa´nchez-Herna´ndez et al., 1998; Blanco-Lo´pez et al., 1984). Leaves from the affected olive trees contribute, through formation of microsclerotia, to increasing the inoculum level in the soil and by disseminating the pathogen with wind-blown leaves of diseased host plants (Navas-Corte´s et al., 2001). The D pathotype showed a very restricted distribution within the north-west areas. Navas-Corte´s et al. (2008) studied the development of Verticillium wilt epidemics in olive cv. Arbequina and they indicated that infections by the D pathotype were aggregated around initial infections. It has been suggested that the
worldwide spread of the D pathotype may have occurred with contaminated cotton seed (Pe´rez-Artes et al., 2000; Collins et al., 2005) and frequently, serious outbreaks of VW in olive orchards, not previously infested with V. dahliae, have been attributed to the cultivation of cotton in neighbouring fields (Wilhelm and Taylor, 1965; Navas-Corte´s et al., 2008). The D pathotype was first reported in cotton crops in southwest Spain in the mid-1980s (BejaranoAlca´zar et al., 1996), and subsequently it was located next to bordering olive orchards. However, cotton is not cultivated in the area surveyed or in neighbouring fields, indicating that introduction of the D pathotype could occur through infected planting stock. The distribution of V. dahliae via greenhouse and nursery stock has been reported in countries such as Greece (Thanassoulopoulos, 1993) and Italy (Nigro et al., 2005). Analysis of disease incidence and the probability map generated by kriging reveal the heterogeneity among the different olivegrowing areas studied. Incidence is affected by multiple factors including pathogen prevalence in the soil, virulence of the pathotype, management practices and agronomic features such us irrigation, plant density, age of trees and cultivars (Jime´nez-Dı´az et al., 1998; Rodrı´guez et al., 2008). Certainly, the high plant density and irrigated management applied in olive orchards from Levante and Sur have produced favourable conditions for disease development, specially for the ND pathotype. Geographic distribution of ND isolates and the significant association of this pathotype with irrigation management support this idea. Also, many studies (Cirulli, 1981; Blanco-Lo´pez et al.,1984; Al-Ahmad and Mosli,1993; Serrihini
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and Zeroual, 1995), have related the spread of VW and irrigated olive orchards and Rodrı´guez et al. (2008) reported that irrigation combined with high plant density caused outbreaks of disease. Conversely, D isolates were more important in non-irrigated orchards, typical of traditional cultivation systems located in Norte area, resulting in higher disease incidence in this area. In fact, only 6–8 cfu g1 of soil of the D pathotype can produce 100% incidence of infected plants in cotton fields, while the ND pathotype needs between 35 and 40 cfu g1 to cause the same level of disease incidence (Bejarano-Alca´zar et al., 1995). 4.2. Rivers–V. dahliae relationship The probability of VW occurrence was dependent on the distance of the orchard from the nearest river. Orchards, which were at a distance of 1 km or less, were heavily infected by V. dahliae. The kriging map showed that disease incidence was higher in the olive orchards sited nearest to the rivers and decreased with increasing distance from this border. However, there was no correlation between distance and incidence. Thus, the distance from a river determined the presence/absence of the pathogen in an orchard, but not the number of trees diseased. Landuse/land-cover maps of Andalusia (1995–1996) together with data from the surveys, showed that the principal land use of soils through the rivers axis was horticultural crops susceptible to V. dahliae, especially irrigated crops. The use of these contaminates soils for the establishment of new olive orchards explaining the map generated by kriging and predicts the risk of disease occurrence in orchards located near to neighbouring rivers. Finally, the use of plants infected by V. dahliae appears to be the main factor influencing the spread of the highly defoliating pathotype in the area surveyed, whereas infections by the nondefoliating pathotype are related to the use of contaminated soils previously cropped with horticultural crops. Incidence of the disease depends on multiple factors, but the presence of the pathogen in a soil combined with irrigation causes disease to peak, especially for the ND pathotype. Acknowledgements We thank the Caja Rural de Granada Foundation for funding this work. We also thank M. Jaramillo and G. Ferna´ndez for assistance in the orchard and H. Barroso for technical assistance. References Al-Ahmad, M.A., Mosli, M.N., 1993. Verticillium wilt of olive in Syria. Bull. OEPP/ EPPO 23, 521–529. Beagle Ristaino, J., Gumpertz, M.L., 2000. New frontiers in the study of dispersal and spatial analysis of epidemics caused by species in the genus Phytophthora. Annu. Rev. Phytopathol. 38, 541–576. Bejarano-Alca´zar, J., Melero-Vara, J.M., Blanco-Lo´pez, M.A., Jime´nez-Dı´az, R.M., 1995. Influence of inoculum density of defoliating and nondefoliating pathotypes of Verticillium dahliae on epidemics of Verticillium wilt of cotton in southern Spain. Pthytopathology 85, 1474–1481. Bejarano-Alca´zar, J., Blanco-Lo´pez, M.A., Melero-Vara, J.M., Jime´nez-Dı´az, R.M., 1996. Etiology, importance and distribution of Verticillium wilt of cotton in southern Spain. Plant Dis. 80, 1233–1238. Bejarano-Alca´zar, J., Pe´rez-Arte´s, E., Jime´nez-Dı´az, R.M., 2001. Spread of the defoliating pathotype of Veticillium dahliae to new cotton-and olive-growing areas in Southern Spain. In: Proceedings of the Eighth International Verticillium Symposium, Co´rdoba, Spain (Abstr.) 57. Blanco-Lo´pez, M.A., Jime´nez-Dı´az, R.M., Caballero, J.M., 1984. Symptomatology, incidence and distribution of Verticillium wilt of olive tree in Andalucı´a. Phytopathol. Mediterr. 23, 1–8. Carder, J.H., Morton, A., Tabrett, A.M., Barbara, D.J., 1994. Detection and differentiation by PCR of sub-specific groups within two Verticillium species causing vascular wilts in herbaceous hosts. In: Schots, A., Dewey, F.M., Oliver, R. (Eds.),
Modern Assays for Plant Pathogenic Fungi: Identification, Detection and Quantification. CAB International, Oxford, pp. 91–97. Cirulli, M., 1981. Attualli cognizioni sulla Verticilliosi dell´olivo. Informatore Fitopatologico 1-2, 1001–1005. COI, 2005. El mercado mundial del aceite de oliva. El mercado mundial de las aceitunas de mesa. Olivae 104, 31–35. Collins, A., Mercado-Blanco, J., Jime´nez-Dı´az, R.M., Olivares, C., Clewes, E., Barbara, D.J., 2005. Correlation of molecular markers and biological properties in Verticillium dahliae and the possible origins of some isolates. Plant Pathol. 54, 549–557. Elena, K., Paplomatas, E.J., 2001. The defoliating strain of Verticillium dahliae on cotton: first report for Greece (Abstr.). Phytopathol. Mediterr. 40, 70. Jaime-Garcı´a, R., Cotty, P.J., 2006. Spatial relationships of soil texture and crop rotation to Aspergillus flavus community structure in south texas. Phytopathology 96 (6), 599–607. Jime´nez-Dı´az, R.M., Tjamos, E.C., Cirulli, M., 1998. Verticillium wilt of major tree hosts. Olive. In: Hiemstra, J.A., Harris, D.C. (Eds.), A Compedium of Verticillium Wilts in Tree Species. Ponsen & Looijen, Wageningen, the Netherlands, pp. 13–16. Kish, L., 1995. Survey Sampling. John Wiley & Sons, New York. Korolev, N., Katan, J., Katan, T., 1999. New Verticillium dahliae pathotype highly aggressive to cotton in Israel. Phytoparasitica 27 (Abstr.) 144. Levin, A.G., Lavee, S., Tsror, (Lahkim) L., 2003. Epidemiology of Verticillium dahliae on olive (cv. Picual) and its effect on yield under saline conditions. Plant Pathol. 52, 212–218. Lo´pez-Escudero, F.J., Blanco-Lo´pez, M.A., 2001. Effect of a single or double soil solarization to control Verticillium wilt in established olive orchards in Spain. Plant Dis. 85, 489–496. MAPA, 1975. Inventario Agrono´mico del olivar V: Provincia de Granada. Publicaciones del Ministerio de Agricultura, Madrid, Spain. Mathre, D.E., Erwin, D.C., Paulus, A.O., Ravenscroft, A.V., 1966. Comparison of the virulence of isolates of Verticillium albo-atrum from several of the cotton growing regions of the United States, Mexico and Peru. Plant Dis. Rep. 50, 930–933. Mercado-Blanco, J., Rodrı´guez-Jurado, D., Parrilla-Arau´jo, S., Jime´nez-Dı´az, R.M., 2003. Simultaneous detection of the defoliating and nondefoliating Verticillium dahliae pathotypes in infective olive plants by duplex, nested polymerase chain reaction. Plant Dis. 87, 1487–1494. Navas-Corte´s, J., Rodrı´guez-Jurado, D., Trapero-Casas, J.L., Landa, B.B., MercadoBlanco, J., Pe´rez-Artes, E. Jime´nez.Dı´az, R.M., 2001. Spatio-temporal dynamics of Verticillium wilt epidemics in an olive orchard. In: Proceedings of the Eighth International Verticillium Symposium, Co´rdoba, Spain (Abstr.) 59. Navas-Corte´s, J.A., Landa, B.B., Mercado-Blanco, J., Trapero-Casas, J.L., Rodrı´guezJurado, D., Jime´nez-Dı´az, R.M., 2008. Spatiotemporal analysis of spread of infections by Verticillium dahliae pathotypes within a high tree density olive orchard in southern Spain. Phytopathology 98, 167–180. Nigro, F., Gallote, P., Romanazzi, G., Schena, L., Ippolito, A., Salerno, M.G., 2005. Incidence of Verticillium wilt on olive in Apulia and genetic diversity of Verticillium dahliae isolates from infected trees. J. Plant Pathol. 87, 13–23. Nutter, F.W., Esker, P.D., Coelho-Netto, R.A., 2006. Disease assessment concepts and the advancements made in improving the accuracy and precision of plant disease data. Eur. J. Plant Pathol. 115, 95–113. Pe´rez-Artes, E., Garcı´a-Pedrajas, M.D., Bejarano-Alca´zar, J., Jime´nez-Dı´az, R.M., 2000. Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses. Eur. J. Plant Pathol. 106, 507–517. Rodrı´guez, E., Garcı´a, P.A., Garcı´a Garrido, J.M., Campos, M., 2008. Agricultural factors affecting Verticillium wilt in olive orchards in Spain. Eur. J. Plant Pathol. 122, 287–295. doi:10.1007/s10658-008-9287-0. Rodrı´guez Navarro, E., 2006. Estudio epidemiolo´gico de la Verticilosis del olivo (Verticillium dahliae, [Klebahn 1913]) en la provincia de Granada. Ph.D. thesis, University of Granada, Spain. Sa´nchez-Herna´ndez, M.E., Ruiz-Da´vila, A., Pe´rez de Algaba, A., Blanco-Lo´pez, M.A., Trapero-Casas, A., 1998. Occurrence and etiology of death of young olive trees in southern Spain. Eur. J. Plant Pathol. 104, 347–357. Schnathorst, W.C., 1973. Additional strains of Verticillium dahliae from cotton in California. In: Proceedings of Beltwide Cotton Production Research Conference, National Cotton Council of America, Memphis, TN, pp. 22–23. Schnathorst, W.C., Mathre, D.E., 1966. Host range and differentiation of a severe form of Verticillium albo-atrum in cotton. Phytopathology 56, 1155–1161. Schnathorst, W.C., Sibbet, G.S., 1971. The relation of strains of Verticillium albo-atrum to severity of Verticillium wilt in Gossypium hirsutum and Olea europaea in California. Plant Dis. Rep. 55, 780–782. Serrihini, M.N., Zeroual, A., 1995. La Verticilosis del olivo en Marruecos. Olivae 58, 58–61. Thanassoulopoulos, C.C., 1993. Spread of Verticillium wilt by nursery plants in olive groves in the Chalkidiki area (Greece). Bull. OEPP/EPPO 23, 517–520. Thanassoulopoulos, C.C., Biris, D.A., Tjamos, E.C., 1979. Survey of Verticillium wilt of olive trees in Greece. Plant Dis. Rep. 63, 936–940. Tjamos, E.C., 2008. Prospects and strategies in controlling Verticillium wilt of olive. EPPO Bull. 23, 505–512. Wilhelm, S., Taylor, J.B., 1965. Control of Verticillium wilt of olive through natural recovery and resistance. Phytopathology 55, 310–316.
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Crop Protection journal homepage: www.elsevier.com/locate/cropro
Large-scale epidemiological study and spatial patterns of Verticillium wilt in olive orchards in southern Spain Estefanı´a Rodrı´guez a, *, Jose M. Garcı´a-Garrido a, Pedro A. Garcı´a b, Mercedes Campos a a b
´n Experimental del Zaidı´n (CSIC), Profesor Albareda 1, 18008 Granada, Spain Departments of Agroecology-Plant Protection and Soil Microbiology and Symbiotic Systems, Estacio Department of Statistics and O.R. University of Granada, Campus de Fuente Nueva s/n, 18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 April 2008 Received in revised form 15 August 2008 Accepted 18 August 2008
Verticillium wilt (VW) is caused by the soilborne pathogen, Verticillium dahliae. The fact that available control measures are not completely effective, together with an increasing spread of the highly virulent defoliating pathotype (D) in the last two decades, makes VW one of the most threatening olive tree disease. For better management, epidemiology and spatial patterns of VW were investigated in an important olive-growing area in southern Spain. A sampling survey was conducted from 2002 to 2005 in 873 olive orchards, which were assessed for the presence of V. dahliae. PCR assays were used to identify the highly virulent defoliating (D) and nondefoliating (ND) pathotypes of V. dahliae. Ordinary kriging, a geostatistical tool that uses regression techniques for interpolation of spatial data, was applied to obtain predictive V. dahliae incidence maps and to evaluate the correlation between distance from the river and VW. Prevalence in olive orchards was 14.1%, with a disease incidence of 1.9% and mean incidence per olive orchard of 9.5%. Prevalence of the ND pathotype was higher (10.7%) than for the D pathotype (3.4%). VW was three times more prevalent in irrigated than in non-irrigated orchards (relative risk ¼ 3.01) and there was a close association between the ND pathotype and irrigated orchards (relative risk ¼ 2.03). Spatial pattern analysis showed several factors involved in the spread of the pathogen. VW was more prevalent in the north-west, suggesting the spreading of the disease from other bordering areas known to be contaminated, in southwest Spain. The D pathotype had a very restricted and aggregated distribution, suggesting the introduction of this pathotype in new olive orchards areas. The incidence map generated by kriging displayed an increase in north, northeast and south areas with numerous subareas with probabilities equalling or exceeding 53%, showing the effect of intensive agriculture (high plant density and irrigation) on incidence of the disease. Assessment of rivers-pathogen relationship revealed an association of VW with the distance to the nearest river, indicating that soils through the rivers’ axis and previously cropped with horticultural crops susceptible to V. dahliae, have implications for the establishment of new olive orchards and the occurrence of the disease. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Epidemiology Disease incidence GIS Olea europaea Pathotypes Pathogen prevalence
1. Introduction The control of Verticillium wilt (VW) in olive, caused by the soilborne, fungal pathogen, Verticillium dahliae Kleb., is of great concern in Andalusia (southern Spain), where olives are the most widespread traditional crop. About 30% of European olive oil production comes from this area (COI, 2005). VW is one the most serious olive tree diseases facing farmers worldwide because it has become increasingly widespread in the last two decades. Available control measures are not completely effective. Because systemic fungicides are unable to prevent the disease, control of VW currently depends on preventive measures * Corresponding author. Tel.: þ34 958 181 600; fax: þ34 958 189 600. E-mail address: [email protected] (E. Rodrı´guez). 0261-2194/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2008.08.009
such us using pathogen-free soil and propagation material. The most practical management after planting at present is a combination of cultural methods (Jime´nez-Dı´az et al., 1998; Tjamos, 2008). The disease has become even more important with the spread of the highly virulent defoliating pathotype (D) (Bejarano-Alca´zar et al., 2001; Lo´pez-Escudero and Blanco-Lo´pez, 2001; Navas-Corte´s et al., 2001; Mercado-Blanco et al., 2003; Rodrı´guez Navarro, 2006). V. dahliae isolates infecting olive (Olea europaea) and cotton (Gossypium hirsutum) show cross-virulence (Schnathorst and Mathre, 1966; Schnathorst and Sibbet, 1971) and they can be classified into defoliating (D) or nondefoliating (ND) pathotypes, based on their ability to cause defoliation of green leaves from shoots and twigs (Schnathorst, 1973). The D pathotype has been reported in cotton-growing countries since 1966 (Mathre et al., 1966;
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Schnathorst, 1973; Korolev et al., 1999; Elena and Paplomatas, 2001), but was not known to occur in Spain until 1983, when it was found in an area of intensive cotton cultivation in southwest Spain (Bejarano-Alca´zar et al., 1996) with recent evidence of longdistance spread of this pathotype to olive-growing regions. D isolates of V. dahliae from China, Spain, and the United States appear molecularly similar (Pe´rez-Arte´s et al., 2000), suggesting that this pathotype may have been introduced into Spain, and that the worldwide spread of the D pathotype may have occurred with contaminated cotton seed. To date, a PCR-based procedure has been developed for consistent detection of D and ND pathotypes of V. dahliae as key components of successful management of VW of olive (Mercado-Blanco et al., 2003). An important strategy of disease management is to analyze spatial patterns of VW, because they can provide important clues to the ecology of disease (e.g., direction and distance of spread or importance and proximity of the sources). Spatial patterns of most variables can be described, analyzed, and displayed at any scale with geographical information systems (GIS) and geostatistics. One of the more popular geostatistical technique is Kriging, which refers to a group of linear regression techniques that use models of the spatial autocorrelation to estimate values at unsampled locations (Beagle Ristaino and Gumpertz, 2000; Jaime-Garcı´a and Cotty, 2006). Although previous studies have provided information about the importance and distribution of VW in the most important areas for olive production (Thanassoulopoulos et al., 1979; Cirulli, 1981; Blanco-Lo´pez et al., 1984; Al-Ahmad and Mosli, 1993; Serrihini and Zeroual, 1995; Levin et al., 2003; Nigro et al., 2005), more epidemiological studies, particularly in case of the D pathotype, are required to obtain detailed information on pathogen prevalence or disease incidence. Few studies of the spatial pattern and pathogen dispersal have been conducted in olive crops (Navas-Corte´s et al., 2008). Epidemiology is concerned with the patterns of disease occurrence and the factors that influence these patterns, and provide the basis for the development and evaluation of preventive crop protection measures. Therefore, the aim of this study was to investigate the epidemiology of the fungus for better management. The objectives were to (i) assess the prevalence, incidence and map the V. dahliae pathotypes, (ii) analyze the spatial patterns of spread of the disease and the association of the pathogen with the environment. Using a double-stratified sampling technique, a sampling survey was conducted in an important olive-growing area in southern Spain. PCR assays were used to identify V. dahliae pathotypes. Kriging was applied to reconstruct the spatial patterns of VW. 2. Materials and methods 2.1. Field surveys The sampling survey was conducted in olive orchards in the province of Granada in Andalusia (southern Spain). Andalusia, which accounts for 75% of Spanish production, has the biggest olive crop under cultivation in the world. Granada province is the third largest producer in the Andalusia region with 175,000 ha distributed among five olive-growing areas: Norte, Vega, Alhama, Sur and Levante (Fig. 1) (MAPA, 1975). Each production region differs greatly from other regions in terms of the physical characteristics of the plantations, management practices, socioeconomic situation and environmental effects (MAPA, 1975). The sampling survey was designed in collaboration with the Local Government (Modelo de Explotaciones Olivareras, Unidad de Prospectiva, Consejerı´a de Agricultura y Pesca. Junta de Andalucı´a) which supported the information about the olive orchards in Granada province including area (ha), number of olive trees and type of cultivars.
47
Because of heterogeneity between the different olive regions and use of irrigation, we designed a stratified double-sampling technique. A stratified sampling technique is generally used when the population is heterogeneous and the sample size is proportional to the relative size of the strata (Kish, 1995). The proportional location in the first sampling level is based on the percentage of orchards in each region. The second level consists of olive orchards with probability proportional to size (PPS). PPS gives a probability of selecting a sampling unit (e.g., olive orchards) proportional to the size of its population (Kish, 1995). The sample size for estimating proportions was 998 olive orchards out of a total population of 129,962 in Granada province, assuming that a priori probability of an olive orchard infected by V. dahliae (p) is equal to the probability of an olive orchard not infected (q) such that p ¼ q ¼ 0.5 (Kish, 1995). Standard error was less than 3.1% (95% confidence interval). Finally, the total number of olive orchards surveyed was actually 873 covering an area of 4087.2 ha and 527,903 olive trees, with a standard error 3.3% (95% confidence interval). The olive orchards needed for the sample survey in each area were: 380 in Norte, 231 in Vega, 107 in Alhama, 68 in Sur, and 87 in Levante region. The centroid for each olive orchard surveyed was identified using information from the Ministry of Agriculture and Fisheries’ (online). From 2002 to 2005 a questionnaire was sent to randomly chosen farmers who answered details about agronomic features (e.g., irrigation, age of plantation, crop management), origin of plant material, previous cropping history and symptomatology for each olive orchard surveyed (Rodrı´guez Navarro, 2006; Rodrı´guez et al., 2008). To reduce bias from farmer’s subjectivity, all olive orchards with VW symptoms were visited directly by the authors in order to validate survey results. 2.2. Pathogen prevalence, disease incidence and V. dahliae isolation from olive trees For disease assessment we used the terms pathogen prevalence and disease incidence (Nutter et al., 2006). Pathogen prevalence is a measure of the proportion (or percentage) of geographical sampling units (olive orchards) where the pathogen has been detected relative to the total number of geographical sampling units (olive orchards) that were inspected. Disease incidence measures the proportion of olive trees where disease symptoms were observed relative to the total number. Mean incidence per olive orchard or ‘‘MI’’ is defined as the mean of number of olive trees affected per olive orchard. The samples were taken from March to June and from September to November when evaluation of the presence of V. dahliae in plants is most appropriate (Levin et al., 2003). Once in an orchard, olive trees showing wilt symptoms were inspected, counted and sampled. Branches and stems from at least 10 of the affected olive trees were collected. Samples were assessed for the presence of V. dahliae in the laboratory. Twenty-four small internal fragments of vascular tissue from different affected branches per tree were surface-sterilized with NaClO (10%) for 1 min and subsequently rinsed with sterile water for 1 min and segments were put on a petri plate in water agar with chlortetracycline (30 mg l1), and colonies were periodically examined for V. dahliae identification. 2.3. Pathotype identification using PCR Isolates obtained from the plant material were assayed using specific PCR assays for identifying V. dahliae pathotypes. Fungal isolates were grown on potato dextrose broth (PDB, Sigma) for 7 d at 24 C in the dark on an orbital shaker (125 rpm). Mycelia were harvested, lyophilized and ground as previously described by Pe´rez-Artes et al. (2000). DNA was extracted from ground mycelia
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48
Fig. 1. Geographic distribution of olive crop in Granada province (Andalusia, southern Spain). Each dot represents the central point of the olive orchards randomly selected to survey Verticillium wilt; ‘‘n’’ indicates the number of olive orchards surveyed in each olive-growing area depending on olive surface.
using the DNeasy Plant Kit (Qiagen, Hilden, Germany). DNA concentration was assessed using Nanodrop and by agarose gel electrophoresis according to standard procedures. DNA solutions were stored at 20 C for further use. Isolates were characterized by the PCR assays using a 3-primer set as previously described by Mercado-Blanco et al. (2003). Primer pair DB19/DB22 yields V. dahliae-specific polymorphic DNA bands of 539 or 523 bp (Carder et al., 1994). The combined use of primers DB19, DB22 and espdef01 yields one of the V. dahliae-specific markers together with an amplicon of 334 bp, in case of isolates of the D pathotype, which allow differentiation between D and ND V. dahliae pathotypes in a single PCR assay (Mercado-Blanco et al., 2003). Amplification reactions (25 ml) consisted of micromolar ratio of 0.7:0.1:0.2 (DB19/ DB22/espedef01) for primers, 200 mM each dNTP, 2.5 ml of 10 reaction buffer, 0.75 U of EcoTaq (Ecogen S.R.L., Barcelona, Spain), 1.5 mM MgCl2, and 10 ng of template DNA. Reaction conditions included denaturation at 94 C for 4 min followed by 30 cycles of 1 min of annealing at 60 C, extension for 30 s at 72 C, denaturation for 1 min at 94 C, and a final extension step of 6 min at 72 C. 2.4. Statistical analyses Mean values of pathogen prevalence were compared using the
c2 test and Relative Risk (RR) values. In statistics and mathematical epidemiology, RR is the risk of an event (or of developing a disease) relative to exposure (Kish, 1995). Relative risk is calculated by dividing the death or disease risk in a specific population group (group A) by the risk of population from all other groups. For example, if the relative risk were 3.0, population in group A would be three times as likely as populations from other groups to become diseased. Classical ANOVA and post hoc comparisons (by means of Tamhane’s T2 method because equal variance was not assumed) were performed to show significant differences for incidence between areas. Distance of olive orchards surveyed to the nearest river was assayed by Welch’s t-test (Kish, 1995). 2.5. ArcGIS A GIS database was developed for Granada province olive orchards. Data included information questionnaires, information
from the Ministry of Agriculture and Fisheries’ and results of isolation and PRC assays. ArcGIS (version 9.1) was used to data display, to interpolate with kriging and to calculate the distance from each olive orchard surveyed (point) to the nearest river (polyline). Ordinary kriging was applied to provide a surface map of V. dahliae incidence in Granada province and to reproduce the main characteristics of the spatial pattern of VW, including correlation with the nearest rivers. An olive surface layer (1995–1996 landcrop/land-use maps from Consejerı´a de Agricultura y Pesca, Junta de Andalucı´a, Granada province) was used to mask the surface to remove non-olive areas and to reduce the proportions based on olive surface because kriging is a technique that assumes that spatial homogeneity is fundamental for the interpolation.
3. Results 3.1. Pathogen prevalence and disease incidence From 2002 to 2005, 873 olive orchards in five olive-growing areas were surveyed (Fig. 1). Of the 873 olive orchards, 14.1% were positive for V. dahliae, 11.5% were negative for V. dahliae and observed symptoms were induced by other (a)biotic factors, and 74.4% of the total olive orchards surveyed did not show any wilt symptoms. The geographical distribution of V. dahliae is shown in Fig. 2. The distribution of VW appeared to be more clustered in the north-west of the province, as evidenced by differences in pathogen prevalence (c2 ¼ 27.49; p < 0,005) (Table 1; Fig. 2). Norte and Vega had the highest percentage of olive orchards infected by V. dahliae, whereas Levante and Sur had similar levels of pathogen prevalence, and Alhama had the lowest percentage (Table 1). 76% of the total isolates recovered belonged to the ND pathotype and 24% belonged to the D pathotype, indicating a pathogen prevalence of 10.7 and 3.4% for ND and D pathotypes, respectively (Table 1). In all areas surveyed, prevalence of the ND pathotype was greater than the D pathotype. Overall pathotypes were associated with specific geographical regions (c2 ¼ 76.21; p < 0.0001) (Table 1; Fig. 2). The ND pathotype was observed in all the areas surveyed, with the highest prevalence in Levante and the lowest in Alhama. The D pathotype was found in the north-west of the region,
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Fig. 2. Geographic distribution of defoliating and nondefoliating pathotypes of Verticillium dahliae in olive orchards in Granada province.
especially in Norte area. The D pathotype was rare in Levante and absent in Sur (Table 1; Fig. 2). Incidence of VW was 1.9% (Table 1), corresponding to 103,073 olive trees that were infected or dead in Granada province. Mean incidence (MI) was 9.5% in a range from 0.1 to 97%. Incidence variations between areas were significant (p < 0.024). The incidence was higher in Levante, Sur and Norte than in Vega and Alhama (Table 1). Data were subjected to kriging to construct a probability map of incidence of VW (Fig. 3). Mapping showed increased disease incidence in north, northeast and south areas of Granada province with numerous subareas with disease probabilities equalling or exceeding 53% in Norte area. However, the disease probability throughout the central and southern Granada province was less than 20%. ND pathotype isolates were associated with higher levels of disease incidence (Table 1), with twice the number of olive trees
being affected by ND isolates (incidence of 2.2 and 1.2%, respectively). The incidence of the ND pathotype was greater in the northeast and south of the region, particularly in Levante and Sur (Table 1) whereas D pathotype incidence was higher the west of the region, especially in Norte area. However, differences between areas were not significant (Table 1). 3.2. Irrigation–V. dahliae relationship Survey results indicated that 33% of the total orchards were under irrigation management. Pathogen prevalence was three times higher in irrigated orchards (c2 ¼ 46.4, p < 0.0001, RR ¼ 3.01). VW was present in 25.6% of irrigated olive orchards and in 8.5% of non-irrigated orchards, but there was no significant difference in disease incidence between the two water treatments (1.95% and 2% for irrigated and non-irrigated olive orchards, respectively).
Table 1 Pathogen prevalencea, disease incidenceb, and mean incidence (MI)c of Verticillium wilt in olive orchards in the five areas surveyed in Granada province (Andalusia, southern Spain).
Province Norte Vega Alhama Sur Levante
a b c d
Olive orchards surveyed
Mean Incidence (%)
Prevalence (%)
Incidence (%)
V. dahliae
pathotype ND
pathotype D
V. dahliae
pathotype ND
pathotype D
873 380 231 107 68 87
9.5 11.6 (a) 3.4 (b) 4.6 (b) 12.1 (a,b) 18.9 (a,b) F4,119 ¼ 2.918 p < 0.024
14.1 15.5 15.2 9.3 10.3 13.8 c2 ¼ 27.49 p < 0.005
10.7 11.6 10.4 6.5 10.3 12.6 c2 ¼ 76.21 p < 0.0001
3.4 3.7 4.8 2.8 0.0 1.1 c2 ¼ 76.21 p < 0.0001
1.9 2.6 (a)d 1.3 (b) 0.8 (b) 6.2 (a,b) 8.2 (a,b) F4,119 ¼ 2.918 p < 0.024
2.2 2.5 1.7 0.9 6.2 11.9 F4,90 ¼ 2.076 p ¼ 0.091
1.2 3.7 0.5 0.6 0.3 F3,26 ¼ 1.164 P ¼ 0.343
Pathogen prevalence measures the percentage of olive orchards in which pathogen was detected. Chi-square value is significant between areas. Disease incidence measures the pertentage of trees diseased. Classical ANOVA was performed. MI is the mean incidence of the total olive orchards surveyed. Classical ANOVA was performed. a,b denote homogeneous subgroups after post-hoc Tamhane’s T2 comparisons (p < 0.05).
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Fig. 3. Kriging map of the distribution of VW incidence in Granada province. Kriging estimation included the proportion of olive trees affected in orchards where V. dahliae was detected. Olive surface layer was used to generate kriging estimation.
Both D and ND pathotypes were more prevalent in irrigated olive orchards (Table 2). Nevertheless, we found statistical differences in the disease levels caused by both pathotypes depending on the type of water treatment. D isolates were significantly more common in non-irrigated orchards. Conversely, ND isolates were twice as common in irrigated orchards (c2 ¼ 5.392; p < 0.02; RR ¼ 2.03). Thus, contribution to prevalence was 83.5% and 16.5% for ND and D, respectively, in irrigated, and 35.0% and 65.0%, respectively, for ND and D in non-irrigated orchards. Incidence of the D pathotype was also higher in non-irrigated orchards, but incidence of the ND pathotype was similar in both irrigated and non-irrigated orchards (Table 2). 3.3. Rivers–V. dahliae relationship Statistical comparisons using the Welch t-test showed that the mean distance from the river in olive orchards affected by VW (1.48 km) was significantly less than in olive orchards free from symptoms of VW (1.94 km) (p < 0,001; t ¼ 3.378; d.f. ¼ 179.97). Most of the orchards affected (70%) by V. dahliae were located less
than 1000 m from the river. Correlation analysis indicated that the negative associations between distance and incidence for both pathotypes were not significant (rDpathotype ¼ 0.298, p ¼ 0.11; rNDpathotype ¼ 0.114, p ¼ 0.28). Kriging interpolation of distance of each olive orchard to the nearest river exhibited a strong aggregative spatial pattern to neighbouring rivers. Fig. 4 shows this clustered distribution pattern and dots indicate orchards located less than 1000 m from the river. Kriging gave a pattern of disease closely related to the Welch t-test results. Infection was not homogenous. The observed disease gradient along the rivers axis was characterized by a higher disease in the orchards nearest to the rivers and ranged from 70 to 100%, decreasing towards 50–15% with increasing distance from the rivers axis. The disease gradient was clearly marked in the Norte area.
4. Discussion This study provides data on epidemiology and the spatial patterns of Verticillium wilt in olive, allowing a spatial perspective
Table 2 Pathogen prevalencea and disease incidenceb of V. dahliae and pathotypes nondefoliating (ND) and defoliating (D) in irrigated and non-irrigated olive orchards. Pathogen prevalence (%)
Non-irrigated Irrigated c2 value a b
Disease incidence (%)
V. dahliae
Pathotype ND
Pathotype D
V. dahliae
Pathotype ND
Pathotype D
8.5 25.6 46.4 (p < 0.0001)
5.4 21.4 5.392 (p < 0.02)
2.9 4.2 5.392 (p < 0.02)
1.95 2.0 (p > 0.05)
2.0 2.3
1.7 0.8
Pathogen prevalence measures the percentage of olive orchards in which V. dahliae were detected. Disease incidence is the number of olive trees infected with V. dahliae.
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Fig. 4. Kriging map of spatial distribution pattern of Verticillium dahliae incidence associated with rivers in the province of Granada (Andalusia, southern Spain). Olive surface layer as a mask, and the distance of each olive orchard surveyed to the nearest river were used to generate kriging estimation.
on disease and ultimately a better knowledge for disease management. 4.1. Pathogen prevalence, disease incidence and irrigation– V. dahliae relationship To date, the highly virulent, defoliating pathotype of V. dahliae had not been reported in the area surveyed and no studies had previously quantified and mapped the disease. In our study, VW was found in 14.1% of the olive orchards surveyed and the D pathotype had a prevalence of 3.4%. Although VW was widespread throughout the olive-growing areas, the geographic distribution of the disease was clustered in the north-west province, and was more prevalent in Norte and Vega areas (north-west), indicating a quick spread of the disease throughout these orchards. Norte and Vega border on the infected olive orchards in southwest Spain where the disease has been well established since the 1980s (Sa´nchez-Herna´ndez et al., 1998; Blanco-Lo´pez et al., 1984). Leaves from the affected olive trees contribute, through formation of microsclerotia, to increasing the inoculum level in the soil and by disseminating the pathogen with wind-blown leaves of diseased host plants (Navas-Corte´s et al., 2001). The D pathotype showed a very restricted distribution within the north-west areas. Navas-Corte´s et al. (2008) studied the development of Verticillium wilt epidemics in olive cv. Arbequina and they indicated that infections by the D pathotype were aggregated around initial infections. It has been suggested that the
worldwide spread of the D pathotype may have occurred with contaminated cotton seed (Pe´rez-Artes et al., 2000; Collins et al., 2005) and frequently, serious outbreaks of VW in olive orchards, not previously infested with V. dahliae, have been attributed to the cultivation of cotton in neighbouring fields (Wilhelm and Taylor, 1965; Navas-Corte´s et al., 2008). The D pathotype was first reported in cotton crops in southwest Spain in the mid-1980s (BejaranoAlca´zar et al., 1996), and subsequently it was located next to bordering olive orchards. However, cotton is not cultivated in the area surveyed or in neighbouring fields, indicating that introduction of the D pathotype could occur through infected planting stock. The distribution of V. dahliae via greenhouse and nursery stock has been reported in countries such as Greece (Thanassoulopoulos, 1993) and Italy (Nigro et al., 2005). Analysis of disease incidence and the probability map generated by kriging reveal the heterogeneity among the different olivegrowing areas studied. Incidence is affected by multiple factors including pathogen prevalence in the soil, virulence of the pathotype, management practices and agronomic features such us irrigation, plant density, age of trees and cultivars (Jime´nez-Dı´az et al., 1998; Rodrı´guez et al., 2008). Certainly, the high plant density and irrigated management applied in olive orchards from Levante and Sur have produced favourable conditions for disease development, specially for the ND pathotype. Geographic distribution of ND isolates and the significant association of this pathotype with irrigation management support this idea. Also, many studies (Cirulli, 1981; Blanco-Lo´pez et al.,1984; Al-Ahmad and Mosli,1993; Serrihini
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and Zeroual, 1995), have related the spread of VW and irrigated olive orchards and Rodrı´guez et al. (2008) reported that irrigation combined with high plant density caused outbreaks of disease. Conversely, D isolates were more important in non-irrigated orchards, typical of traditional cultivation systems located in Norte area, resulting in higher disease incidence in this area. In fact, only 6–8 cfu g1 of soil of the D pathotype can produce 100% incidence of infected plants in cotton fields, while the ND pathotype needs between 35 and 40 cfu g1 to cause the same level of disease incidence (Bejarano-Alca´zar et al., 1995). 4.2. Rivers–V. dahliae relationship The probability of VW occurrence was dependent on the distance of the orchard from the nearest river. Orchards, which were at a distance of 1 km or less, were heavily infected by V. dahliae. The kriging map showed that disease incidence was higher in the olive orchards sited nearest to the rivers and decreased with increasing distance from this border. However, there was no correlation between distance and incidence. Thus, the distance from a river determined the presence/absence of the pathogen in an orchard, but not the number of trees diseased. Landuse/land-cover maps of Andalusia (1995–1996) together with data from the surveys, showed that the principal land use of soils through the rivers axis was horticultural crops susceptible to V. dahliae, especially irrigated crops. The use of these contaminates soils for the establishment of new olive orchards explaining the map generated by kriging and predicts the risk of disease occurrence in orchards located near to neighbouring rivers. Finally, the use of plants infected by V. dahliae appears to be the main factor influencing the spread of the highly defoliating pathotype in the area surveyed, whereas infections by the nondefoliating pathotype are related to the use of contaminated soils previously cropped with horticultural crops. Incidence of the disease depends on multiple factors, but the presence of the pathogen in a soil combined with irrigation causes disease to peak, especially for the ND pathotype. Acknowledgements We thank the Caja Rural de Granada Foundation for funding this work. We also thank M. Jaramillo and G. Ferna´ndez for assistance in the orchard and H. Barroso for technical assistance. References Al-Ahmad, M.A., Mosli, M.N., 1993. Verticillium wilt of olive in Syria. Bull. OEPP/ EPPO 23, 521–529. Beagle Ristaino, J., Gumpertz, M.L., 2000. New frontiers in the study of dispersal and spatial analysis of epidemics caused by species in the genus Phytophthora. Annu. Rev. Phytopathol. 38, 541–576. Bejarano-Alca´zar, J., Melero-Vara, J.M., Blanco-Lo´pez, M.A., Jime´nez-Dı´az, R.M., 1995. Influence of inoculum density of defoliating and nondefoliating pathotypes of Verticillium dahliae on epidemics of Verticillium wilt of cotton in southern Spain. Pthytopathology 85, 1474–1481. Bejarano-Alca´zar, J., Blanco-Lo´pez, M.A., Melero-Vara, J.M., Jime´nez-Dı´az, R.M., 1996. Etiology, importance and distribution of Verticillium wilt of cotton in southern Spain. Plant Dis. 80, 1233–1238. Bejarano-Alca´zar, J., Pe´rez-Arte´s, E., Jime´nez-Dı´az, R.M., 2001. Spread of the defoliating pathotype of Veticillium dahliae to new cotton-and olive-growing areas in Southern Spain. In: Proceedings of the Eighth International Verticillium Symposium, Co´rdoba, Spain (Abstr.) 57. Blanco-Lo´pez, M.A., Jime´nez-Dı´az, R.M., Caballero, J.M., 1984. Symptomatology, incidence and distribution of Verticillium wilt of olive tree in Andalucı´a. Phytopathol. Mediterr. 23, 1–8. Carder, J.H., Morton, A., Tabrett, A.M., Barbara, D.J., 1994. Detection and differentiation by PCR of sub-specific groups within two Verticillium species causing vascular wilts in herbaceous hosts. In: Schots, A., Dewey, F.M., Oliver, R. (Eds.),
Modern Assays for Plant Pathogenic Fungi: Identification, Detection and Quantification. CAB International, Oxford, pp. 91–97. Cirulli, M., 1981. Attualli cognizioni sulla Verticilliosi dell´olivo. Informatore Fitopatologico 1-2, 1001–1005. COI, 2005. El mercado mundial del aceite de oliva. El mercado mundial de las aceitunas de mesa. Olivae 104, 31–35. Collins, A., Mercado-Blanco, J., Jime´nez-Dı´az, R.M., Olivares, C., Clewes, E., Barbara, D.J., 2005. Correlation of molecular markers and biological properties in Verticillium dahliae and the possible origins of some isolates. Plant Pathol. 54, 549–557. Elena, K., Paplomatas, E.J., 2001. The defoliating strain of Verticillium dahliae on cotton: first report for Greece (Abstr.). Phytopathol. Mediterr. 40, 70. Jaime-Garcı´a, R., Cotty, P.J., 2006. Spatial relationships of soil texture and crop rotation to Aspergillus flavus community structure in south texas. Phytopathology 96 (6), 599–607. Jime´nez-Dı´az, R.M., Tjamos, E.C., Cirulli, M., 1998. Verticillium wilt of major tree hosts. Olive. In: Hiemstra, J.A., Harris, D.C. (Eds.), A Compedium of Verticillium Wilts in Tree Species. Ponsen & Looijen, Wageningen, the Netherlands, pp. 13–16. Kish, L., 1995. Survey Sampling. John Wiley & Sons, New York. Korolev, N., Katan, J., Katan, T., 1999. New Verticillium dahliae pathotype highly aggressive to cotton in Israel. Phytoparasitica 27 (Abstr.) 144. Levin, A.G., Lavee, S., Tsror, (Lahkim) L., 2003. Epidemiology of Verticillium dahliae on olive (cv. Picual) and its effect on yield under saline conditions. Plant Pathol. 52, 212–218. Lo´pez-Escudero, F.J., Blanco-Lo´pez, M.A., 2001. Effect of a single or double soil solarization to control Verticillium wilt in established olive orchards in Spain. Plant Dis. 85, 489–496. MAPA, 1975. Inventario Agrono´mico del olivar V: Provincia de Granada. Publicaciones del Ministerio de Agricultura, Madrid, Spain. Mathre, D.E., Erwin, D.C., Paulus, A.O., Ravenscroft, A.V., 1966. Comparison of the virulence of isolates of Verticillium albo-atrum from several of the cotton growing regions of the United States, Mexico and Peru. Plant Dis. Rep. 50, 930–933. Mercado-Blanco, J., Rodrı´guez-Jurado, D., Parrilla-Arau´jo, S., Jime´nez-Dı´az, R.M., 2003. Simultaneous detection of the defoliating and nondefoliating Verticillium dahliae pathotypes in infective olive plants by duplex, nested polymerase chain reaction. Plant Dis. 87, 1487–1494. Navas-Corte´s, J., Rodrı´guez-Jurado, D., Trapero-Casas, J.L., Landa, B.B., MercadoBlanco, J., Pe´rez-Artes, E. Jime´nez.Dı´az, R.M., 2001. Spatio-temporal dynamics of Verticillium wilt epidemics in an olive orchard. In: Proceedings of the Eighth International Verticillium Symposium, Co´rdoba, Spain (Abstr.) 59. Navas-Corte´s, J.A., Landa, B.B., Mercado-Blanco, J., Trapero-Casas, J.L., Rodrı´guezJurado, D., Jime´nez-Dı´az, R.M., 2008. Spatiotemporal analysis of spread of infections by Verticillium dahliae pathotypes within a high tree density olive orchard in southern Spain. Phytopathology 98, 167–180. Nigro, F., Gallote, P., Romanazzi, G., Schena, L., Ippolito, A., Salerno, M.G., 2005. Incidence of Verticillium wilt on olive in Apulia and genetic diversity of Verticillium dahliae isolates from infected trees. J. Plant Pathol. 87, 13–23. Nutter, F.W., Esker, P.D., Coelho-Netto, R.A., 2006. Disease assessment concepts and the advancements made in improving the accuracy and precision of plant disease data. Eur. J. Plant Pathol. 115, 95–113. Pe´rez-Artes, E., Garcı´a-Pedrajas, M.D., Bejarano-Alca´zar, J., Jime´nez-Dı´az, R.M., 2000. Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses. Eur. J. Plant Pathol. 106, 507–517. Rodrı´guez, E., Garcı´a, P.A., Garcı´a Garrido, J.M., Campos, M., 2008. Agricultural factors affecting Verticillium wilt in olive orchards in Spain. Eur. J. Plant Pathol. 122, 287–295. doi:10.1007/s10658-008-9287-0. Rodrı´guez Navarro, E., 2006. Estudio epidemiolo´gico de la Verticilosis del olivo (Verticillium dahliae, [Klebahn 1913]) en la provincia de Granada. Ph.D. thesis, University of Granada, Spain. Sa´nchez-Herna´ndez, M.E., Ruiz-Da´vila, A., Pe´rez de Algaba, A., Blanco-Lo´pez, M.A., Trapero-Casas, A., 1998. Occurrence and etiology of death of young olive trees in southern Spain. Eur. J. Plant Pathol. 104, 347–357. Schnathorst, W.C., 1973. Additional strains of Verticillium dahliae from cotton in California. In: Proceedings of Beltwide Cotton Production Research Conference, National Cotton Council of America, Memphis, TN, pp. 22–23. Schnathorst, W.C., Mathre, D.E., 1966. Host range and differentiation of a severe form of Verticillium albo-atrum in cotton. Phytopathology 56, 1155–1161. Schnathorst, W.C., Sibbet, G.S., 1971. The relation of strains of Verticillium albo-atrum to severity of Verticillium wilt in Gossypium hirsutum and Olea europaea in California. Plant Dis. Rep. 55, 780–782. Serrihini, M.N., Zeroual, A., 1995. La Verticilosis del olivo en Marruecos. Olivae 58, 58–61. Thanassoulopoulos, C.C., 1993. Spread of Verticillium wilt by nursery plants in olive groves in the Chalkidiki area (Greece). Bull. OEPP/EPPO 23, 517–520. Thanassoulopoulos, C.C., Biris, D.A., Tjamos, E.C., 1979. Survey of Verticillium wilt of olive trees in Greece. Plant Dis. Rep. 63, 936–940. Tjamos, E.C., 2008. Prospects and strategies in controlling Verticillium wilt of olive. EPPO Bull. 23, 505–512. Wilhelm, S., Taylor, J.B., 1965. Control of Verticillium wilt of olive through natural recovery and resistance. Phytopathology 55, 310–316.