Nondefoliating Research Articles

Molecular characterizations of Verticillium dahliae isolates from grafted tomatoes grown in Antalya province of Türkiye

AbstractVerticillium wilt caused by soil‐borne fungus Verticillium dahliae results in serious quality, yield and economic losses in tomato fruit production in tomato‐growing areas around the world. In this study, V. dahliae isolates obtained from six different major grafted tomato production areas in Antalya province of Türkiye were characterized for races, pathotypes and vegetative compatibility groups using the specific PCR primers. Out of 66 isolates, 60 were determined to be race 2. Six isolates were not identified as race 1 or race 2. Fifty‐six isolates were determined as defoliating pathotypes (D) while 10 isolates were identified as non‐defoliating (ND) pathotypes. The isolates were clustered into four vegetative compatibility groups (VCGs): VCG1A (61), VCG2A or VCG4B (3), VCG2B (1) and VCG6 (1). According to the results of this study, the race 2, D pathotype and VCG1A isolates were dominant and widely distributed in the region. Additionally, the V. dahliae isolates fall into two major groups based on the dendrogram constructed from molecular data by Dice similarity index. The dendrogram showed significant diversity among the V. dahliae isolates particularly in the Elmalı region compared to other regions. The study is the first comprehensive research with the current disease and race 2 was identified for the first time in grafted tomatoes. These findings will lead to the development of V. dahliae‐resistant grafted tomatoes and breeding programs in Türkiye.

Genetic Diversity and Pathogenicity of Verticillium dahliae Isolates and Their Co-occurrence with Fusarium oxysporum f. sp. vasinfectum Causing Cotton Wilt in Xinjiang, China.

Cotton production in Xinjiang, the largest cotton-producing area in China, has an increasingly serious disease threat from Verticillium dahliae. Eighty-five V. dahliae isolates were obtained from wilted cotton plants collected from eight counties in Xinjiang. The isolates were assessed for genotypic diversity by DNA sequence analysis and PCR molecular genotyping with specific markers for race 1, race 2, defoliating (D) pathotype, nondefoliating (ND) pathotype, and mating type idiomorph Mat1-2. Isolates belonged to lineages 1A or 2B, with three subgenotypes found in each lineage. All isolates tested positive for race 2 and Mat1-2 markers. All isolates in lineage 2B tested positive for the ND pathotype marker but only isolates in the major subgenotype in lineage 1A tested positive for the D pathotype marker. Pathogenicity assays on Gossypium hirsutum 'Acala 44' demonstrated no significant difference among subgenotypes within each lineage. Isolates in lineage 1A caused greater shoot weight reductions, percent leaf drop, and percent diseased leaves than isolates in lineage 2B. One isolate in each lineage for 1A and 2B was avirulent. Isolates in lineage 1A caused greater than 50% leaf drop and a 17-g shoot weight reduction compared with a 9% leaf drop and a 6-g shoot weight reduction by isolates in lineage 2B. Overall, 42% of the V. dahliae isolates from Xinjiang were D pathotype but the percentage varied widely among locations. Two plants had both pathotypes. Nineteen isolates of Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) also were recovered from wilted plants in Xinjiang. Two plants had both Verticillium wilt and Fusarium wilt pathogens. Both pathogens should be considered when using or developing wilt resistant or tolerant materials for Xinjiang.

Co-Occurrence of Defoliating and Non-Defoliating Pathotypes of Verticillium Dahliae in Field-Grown Cotton Plants in New South Wales, Australia.

Verticillium wilt (VW) is a major constraint to cotton production in Australia and worldwide. The disease is caused by a soilborne fungus, Verticillium dahliae, a highly virulent pathogen on cotton. Commonly, V. dahliae is designated into two pathotypes: defoliating (D) and non-defoliating (ND), based on induced symptoms. In the previous two survey seasons between 2017 and 2019, stems with suspected VW were sampled for the confirmation of presence and distribution of D and ND pathotypes across New South Wales (NSW), Australia. A total of 151 and 84 VW-suspected stems sampled from the 2017/18 and 2018/19 seasons, respectively, were subjected to pathogen isolation. Of these, 94 and 57 stems were positive for V. dahliae; and 18 and 20 stems sampled respectively from the two seasons yielded the D pathotype isolates. Two stems from the 2017/18 season and one stem from 2018/19 season yielded both D and ND pathotype isolates. We also successfully demonstrated the co-infection of both pathotypes in pot trials, which was driven predominantly by either of the pathotypes, and appeared independent on vegetative growth, fecundity and spore germination traits. Our study is the first report of the natural co-occurrence of both D and ND pathotypes in same field-grown cotton plants in NSW, to which a challenge to the disease management will be discussed.

Heterologous Expression of the AtNPR1 Gene in Olive and Its Effects on Fungal Tolerance.

The NPR1 gene encodes a key component of systemic acquired resistance (SAR) signaling mediated by salicylic acid (SA). Overexpression of NPR1 confers resistance to biotrophic and hemibiotrophic fungi in several plant species. The NPR1 gene has also been shown to be involved in the crosstalk between SAR signaling and the jasmonic acid-ethylene (JA/Et) pathway, which is involved in the response to necrotrophic fungi. The aim of this research was to generate transgenic olive plants expressing the NPR1 gene from Arabidopsis thaliana to evaluate their differential response to the hemibiotrophic fungus Verticillium dahliae and the necrotroph Rosellinia necatrix. Three transgenic lines expressing the AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained using an embryogenic line derived from a seed of cv. Picual. After maturation and germination of the transgenic somatic embryos, the plants were micropropagated and acclimated to ex vitro conditions. The level of AtNPR1 expression in the transgenic materials varied greatly among the different lines and was higher in the NPR1-780 line. The expression of AtNPR1 did not alter the growth of transgenic plants either in vitro or in the greenhouse. Different levels of transgene expression also did not affect basal endochitinase activity in the leaves, which was similar to that of control plants. Response to the hemibiotrophic pathogen V. dahliae varied with pathotype. All plants died by 50 days after inoculation with defoliating (D) pathotype V-138, but the response to non-defoliating (ND) strains differed by race: following inoculation with the V-1242 strain (ND, race 2), symptoms appeared after 44–55 days, with line NPR1-780 showing the lowest disease severity index. This line also showed good performance when inoculated with the V-1558 strain (ND, race 1), although the differences from the control were not statistically significant. In response to the necrotroph R. necatrix, all the transgenic lines showed a slight delay in disease development, with mean area under the disease progress curve (AUDPC) values 7–15% lower than that of the control.

Genetics, Host Range, and Molecular and Pathogenic Characterization of Verticillium dahliae From Sunflower Reveal Two Differentiated Groups in Europe.

Verticillium wilt and leaf mottle of sunflower, caused by the fungus Verticillium dahliae (Vd) has become a major constraint to sunflower oil production in temperate European countries. Information about Vd from sunflower is very scarce despite genetics, molecular traits and pathogenic abilities of fungal strains affecting many other crops being widely known. Understanding and characterizing the diversity of Vd populations in those countries where sunflowers are frequent and severely affected by the fungus are essential for efficient breeding for resistance. In this study, we have analyzed genetic, molecular and pathogenic traits of Vd isolates affecting sunflower in European countries. When their genetics was investigated, almost all the isolates from France, Italy, Spain, Argentina, and Ukraine were assigned to vegetative compatibility group (VCG) 2B. In Bulgaria, Turkey, Romania, and Ukraine, some isolates were assigned to VCG6, but some others could not be assigned to any VCG. Genotyping markers used for Vd affecting crops other than sunflower showed that all the isolates were molecularly identified as race 2 and that markers of defoliating (D) and non-defoliating (ND) pathotypes distinguished two well-differentiated clusters, one (E) grouping those isolates from Eastern Europe and the other (W) all those from the Western Europe and Argentina. All the isolates in cluster W were VCG2B, while the isolates in cluster E belonged to an unknown VCG or to VCG6. When the host range was investigated in the greenhouse, the fungus was highly pathogenic to artichoke, showing the importance of farming alternatives in the management of Verticillium attacks. Sunflower genotypes were inoculated with a selection of isolates in two experiments. Two groups were identified, one including the isolates from Western Europe, Argentina, and Ukraine, and the other including isolates from Bulgaria, Romania, and Turkey. Three pathogenic races were differentiated: V1, V2-EE (Eastern Europe) and V2-WE (Western Europe). Similarly, three differentials are proposed for race identification: HA 458 (universal susceptible), HA 89 (resistant to V2-EE, susceptible to V2-WE) and INRA2603 (susceptible to V2-EE, resistant to V2-WE). The diversity found in Vd affecting sunflower must be taken into account in the search for resistance to the pathogen for European environments of sunflower production.

Searching for resistance sources to Verticillium wilt of cotton in seedlings from Gossypium spp.

Verticillium dahliae is a major wilt pathogen impacting cotton production in Turkey. Isolates of V. dahliae can be characterized as defoliating (D) or nondefoliating (ND) pathotypes based on symptom expression in cotton. Currently there are no fungicides identified that can manage this pathogen. Host resistance is the most economical and environmentally friendly means to manage this disease. Cotton genotypes, comprising the main cultivars and some breeding lines available in Turkey, were evaluated under controlled conditions for resistance to verticillium wilt. To establish adequate conditions for resistance expression, experiments examining isolate pathogenicity and the most appropriate inoculum concentration were carried out first. Based on the obtained data, 10 cultivars were then inoculated with D and ND pathotypes by the “pot immersion” method at 106 conidia/mL inoculum concentration and the plants were evaluated considering both internal and external disease symptoms. Plants were scored on a 0 to 4 scale for above-ground symptoms (0 = healthy; 4 = dead plant) ca. two weeks after inoculation. Significant differences in resistance level were observed among the genotypes (p < 0.05). The cultivar Maydos Yerlisi had the lowest level of wilt, with a mean rating of 1.0. The cultivar Nazilli NDT-15 had the strongest wilt, with a mean rating of 3.3. These results suggest that ‘Maydos Yerlisi’ can be used in cotton breeding programs to develop resistant varieties to V. dahliae.

Trichoderma asperellum is effective for biocontrol of Verticillium wilt in olive caused by the defoliating pathotype of Verticillium dahliae

Verticillium wilt caused by a highly virulent, defoliating (D) pathotype of Verticillium dahliae is threatening olive production in Spain and other Mediterranean countries. This disease must be managed by an integrated strategy, in which biocontrol agents can play an important role. We have investigated the potential of Trichoderma asperellum strains for antagonism against V. dahliae and suppression of Verticillium wilt of olive caused by the D pathotype. First, we tested the antagonistic potential of T. asperellum strains Bt2, Bt3 and T25 against six V. dahliae isolates, four of the D and two of the nondefoliating (ND) pathotypes, in different in vitro assays. All T. asperellum strains overgrew the colonies of all V. dahliae isolates to a similar extent. However, extracellular compounds from strains Bt3 and T25 showed higher anti-V. dahliae activities than those of Bt2 in membrane assays. Also, growth of Bt2 was reduced by ND V. dahliae whereas that of Bt3 and T25 was not affected by V. dahliae-secreted compounds. In planta assays using strains Bt3 and T25, and ’Picual’ olive plants, showed that the two T. asperellum strains significantly reduced the severity of symptoms and the standardized area under the disease progress curve caused by highly virulent D V. dahliae, but not the final disease incidence. Strain T25 significantly increased growth of ‘Picual’ plants and displayed higher ability for colonizing the olive rhizosphere and establishing endophytic infection in olive roots than Bt3.

Genetic structure of Verticillium dahliae isolates infecting olive trees in Tunisia using AFLP, pathogenicity and PCR markers

Since 2006, verticillium wilt of olive induced by Verticillium dahliae has caused considerable economic losses in olive orchards in Tunisia. The genetic structure of V. dahliae isolates collected from different olive growing regions was investigated using virulence tests, vegetative compatibility grouping (VCG) and amplified fragment length polymorphism (AFLP) analyses. In total, 42 isolates of V. dahliae from diseased olive trees were tested. Cluster analysis and principal coordinate analysis revealed that geographic origin was the main factor determining the genetic structure of V. dahliae populations and both methods indicated a genetic separation between the central and coastal isolates. Isolates were divided into two major groups: the AFLP‐I group included all isolates from Sidi Bouzid, Kairouan, Kasserine and Sfax (centre of the country) and the AFLP‐II group included isolates from Monastir, Zaghouane, Sousse, Mahdia (coastal region), and two isolates from Sfax. Analysis of the molecular variance (amova) indicated a significant level of genetic differentiation among (76%) and within (23%) the two populations. Analyses of both the defoliating (D) and non‐defoliating (ND) pathotypes and VCG markers indicated that most of the isolates belong to VCG 2A and 4B/ND pathotype. The disease severity was highly variable among the isolates tested (P &lt; 0·05) with no evidence of association between aggressiveness and geographical origin of the isolates. Overall, results of this study revealed a clear association between the genetic diversity of the isolates and their geographic origin, but not between genetic diversity and virulence patterns.

A Comparison of Real-Time PCR Protocols for the Quantitative Monitoring of Asymptomatic Olive Infections by Verticillium dahliae Pathotypes

Early, specific, and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity, and efficiency of eight real-time quantitative polymerase chain reaction protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems, and leaves. Results showed that some of those protocols were not specific for V. dahliae or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocols TAQ (based on intergenic spacer ribosomal DNA target gene) and SYBR-4 (based on the β-tubulin 2 target gene) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and stem) tested. Use of TAQ and SYBR-4 protocols allowed accurate quantification of V. dahliae DNA regardless of the background DNA, with a detection limit being fixed at a cycle threshold of 36 (≈18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae. The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant 'Frantoio' olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D V. dahliae DNA were measured in olive stems, whereas the average amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amounts in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days after inoculation). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing certification schemes of pathogen-free planting material as well as helpful tools in breeding resistance to V. dahliae in olive.

Effect of temperature on conidial germination, mycelial growth and aggressiveness of the defoliating and nondefoliating pathotypes of Verticillium dahliae from cotton in China

Three defoliating (D) pathotype isolates and five nondefoliating (ND) pathotype isolates of Verticillium dahliae from cotton in central China were tested for adaptation to various temperatures in conidial germination on water agar (10–33°C), mycelial growth on potato dextrose agar (10–33°C) and infection of cotton seedlings (Gossypium hirsutum cvs. ‘E Mian 24’ and ‘Yin Rui 361’) (25–33°C). Results showed that the D-pathotype isolates adapted better than the ND-pathotype isolates to 30°C for conidial germination and mycelial growth, although the isolates of the two pathotypes had the same optimum temperature of 25°C. Under day/night temperatures of 25/25, 27/27 and 30/25°C for 20 and 25 days, the D-pathotype isolates induced the defoliation syndrome on seedlings of the two upland cotton cultivars, whereas the ND-pathotype isolates did not induce defoliation syndrome. The values for the areas under the disease progress curve (AUDPC) and the vascular discoloration index (VDI) were higher for the D-pathotype isolates than those for the ND-pathotype isolates in each temperature treatment. Under 30/30 or 33/27°C, at least two of the three D-pathotype isolates still had higher AUDPC values and/or VDI values than all the ND-pathotype isolates on E Mian 24. Therefore, the D-pathotype isolates appear more aggressive than the ND-pathotype isolates in infection of cotton. Results also showed that the cotton cultivar Yin Rui 361 was more tolerant than the cotton cultivar E Mian 24 to infection by both pathotypes of V. dahliae. This study suggests that the D-pathotype isolates can well adapt to high temperature and heavily infect cotton under 25–30°C, and these features might be responsible for the rapid spread of this pathotype in central China.

Correlation Between Virulence and Morphological Characteristics of Microsclerotia of Verticillium dahliae Isolates Infecting Olive

AbstractThe spreading of highly virulent isolates of Verticillium dahliae, causing Verticillium wilt of olive, is one of the most threatening concerns for olive cultivation. Using an isolate collection from infected olive trees in southern Spain, the morphology of microsclerotia (MS) produced on water agar was correlated with their molecular characteristics by a PCR‐based pathotyping. Defoliating isolates (D) produced MS with a significantly higher length/width ratio than non‐defoliating (ND) ones. These parameters were correlated using the logistic model log (y/1 − y) = 3.73L/W − 6.95, when the pathotype was regressed on length/width ratio of the propagules. Inflection point of the logistic curve corresponded to length/width = 1.86. This morphological differentiation of virulence groups could be a simple and useful tool at commercial laboratories for the assignation of the pathotype of V. dahliae isolates during routine microbiological‐based diagnosis.

Region-Wide Analysis of Genetic Diversity in Verticillium dahliae Populations Infecting Olive in Southern Spain and Agricultural Factors Influencing the Distribution and Prevalence of Vegetative Compatibility Groups and Pathotypes

Severity of Verticillium wilt in olive trees in Andalusia, southern Spain is associated with the spread of a highly virulent, defoliating (D) Verticillium dahliae pathotype of vegetative compatibility group 1A (VCG1A) but the extent of this spread and the diversity of the pathogen population have never been documented. VCG typing of 637 V. dahliae isolates from 433 trees in 65 orchards from five olive-growing provinces in Andalusia indicated that 78.1% were of VCG1A, 19.8% of VCG2A, 0.6% of VCG2B, 1.4% of VCG4B, and one isolate was heterokaryon self-incompatible. A single VCG prevailed among isolates within most orchards but two and three VCGs were identified in 12 and 3 orchards, respectively, with VCG1A+VCG2A occurring in 10 orchards. VCG1A was the predominant VCG in the three most important olive-growing provinces, and was almost as prevalent as VCG2A in another one. Molecular pathotyping of the 637 isolates using specific polymerase chain reaction assays indicated that VCG1A isolates were of the D pathotype whereas isolates of VCG2A, -2B, and -4B were of the less virulent nondefoliating (ND) pathotype. The pathotype of isolates correlated with the disease syndrome affecting sampled trees. Only three (seq1, seq2, and seq4) of the seven known sequences of the V. dahliae-specific 539- or 523-bp amplicon were identified among the 637 isolates. Distribution and prevalence of VCGs and seq sequences among orchards indicated that genetic diversity within olive V. dahliae in Andalusia is higher in provinces where VCG1A is not prevalent. Log-linear analysis revealed that irrigation management, source of irrigation water, source of planting stock, and cropping history of soil were significantly associated with the prevalence of VCG1A compared with that of VCG2A. Multivariate analyses using a selected set of agricultural factors as variables allowed development of a discriminant model for predicting the occurrence of D and ND pathotypes in the area of the study. Blind tests using this model correctly indentified the V. dahliae pathotype occurring in an orchard. The widespread occurrence and high prevalence of VCG1A/D pathotype in Andalusia have strong implications for the management of the disease.

Suppression of Verticillium Wilt of Olive by Pseudomonas fluorescens

Protection of pathogen-free olive planting material from infection by Verticillium dahliae during plant propagation and/or at planting would help in the management of Verticillium wilt of olive. Despite the importance for rhizosphere functioning, rhizobacterial Pseudomonas spp. have been mainly studied in a cultivation-based manner. In this study, 8 isolates of Pseudomonas fluorescens obtained from roots of olive plants were tested for suppression of Verticillium wilt in nursery-produced olive planting stocks under controlled conditions. The antagonistic activity of P. fluorescens isolates from olive against defoliating (D) and nondefoliating (ND) V. dahliae pathotypes. The isolates of P. fluorescens isolates from olive varied in the ability to inhibit hyphal growth of D and ND V. dahliae on PDA. In planta bioassays were conducted under greenhouse conditions, by inoculating bacterial-treated and -nontreated 3- to 4-month-old, own-rooted plants of susceptible olive cv. Zard with the highly and less virulent V. dahliae . Results indicated that root treatment with some of P. fluorescens isolates significantly reduced the final disease incidence and severity, compared with the nontreated controls. Our results indicate that root treatment of olive plants with selected P. fluorescens isolates during nursery propagation can help in the biocontrol of V. dahliae in olive especially for ND pathotypes. No correlation was found between efficacy of tested bacterial isolates for in vitro antagonism of the pathogen and in planta suppression of Verticillium wilt.

Phenolic Responses of Resistant and Susceptible Olive Cultivars Induced by Defoliating and Nondefoliating Verticillium dahliae Pathotypes.

Verticillium wilt is the most serious olive disease worldwide. The olive-infecting Verticillium dahliae pathotypes have been classified as defoliating (D) and nondefoliating (ND), and the disease is mainly controlled in olive orchards by using resistant or tolerant cultivars. Limited information is available about the nature of resistance in most of the olive cultivars. In the present study, the phenolic responses of the susceptible to V. dahliae olive cv. Amfissis and the resistant cv. Koroneiki upon D and ND V. dahliae infection were monitored in relation to the fungal DNA levels in the vascular tissues with the purpose to explore the defense mechanisms of olive trees against V. dahliae. Quantitative polymerase chain reaction revealed that the decrease in symptom severity shown in Koroneiki trees was associated with significant reduction in the growth of both V. dahliae pathotypes in the vascular tissues compared with Amfissis. In Koroneiki trees, the levels of o-diphenols and verbascoside were positively associated with the DNA levels of the D and ND pathotypes. In addition, a positive association was observed between the levels of verbascoside and the fungal DNA level in Amfissis trees, whereas a negative association was revealed between the fungal DNA level and the total phenols and oleuropein content in both cultivars. The levels of verbascoside were clearly higher in Koroneiki trees compared with Amfissis trees, indicating for the first time in the literature the involvement of verbascoside in the defense mechanism of olive trees against V. dahliae.

Verticillium wilt of olive in Turkey: a survey on disease importance, pathogen diversity and susceptibility of relevant olive cultivars

A comprehensive survey on the prevalence and incidence of Verticillium wilt of olive in Turkey has been conducted over 6 years (2003–2008). Vegetative compatibility group (VCG) assessment and PCR-based molecular pathotyping were used to evaluate the distribution of the defoliating (D) and nondefoliating (ND) pathotypes of Verticillium dahliae in surveyed areas. Pathogen prevalence was 35% of all olive orchards inspected and incidence of the disease reached 3.1%. VCG1A was predominant (29.3%) and infected all major cultivars grown in Turkey. The other two VCGs detected (2A and 4B) were of minor relevance (4.9% and 0.9%, respectively). Disease incidence caused by VCG1A infections was higher (ranging from 1.1% to 6.9%) than that caused by VCG2A and VCG4B in 10 provinces (Manisa, Aydin, Kahramanmaras, Izmir, Mugla, Kilis, Denizli, Gaziantep, Mardin and Balikesir). However, VCG2A and 4B were more prevalent (and responsible for higher disease incidence) than VCG1A in three provinces (Hatay, Osmaniye and Bursa). Finally, VCG1A isolates were found in all provinces except Canakkale, and simultaneous presence of the three VCGs was only verified in Hatay province. An artificial inoculation bioassay (19 representative V. dahliae isolates included) revealed that VCG1A (13) isolates as a group were more aggressive and caused defoliation, whereas VCG2A (5) and VCG4B (1) isolates induced milder symptoms. Within a VCG group, virulence varied among isolates infecting the same olive cultivar and this virulence was also related to the differential susceptibility of the cultivars (‘Manzanilla’, ‘Ayvalik’ and ‘Gemlik’) tested. Molecular pathotyping allowed the identification of D (VCG1A) and ND (VCG2A/4B) pathotypes, which correlated with results from pathogenicity tests. Remarkably, the V. dahliae VCG1A/D pathotype population infecting olive in Turkey was molecularly different from that one previously identified in Spain.

Verticillium wilt of olive in the Guadalquivir Valley (southern Spain): relations with some agronomical factors and spread of Verticillium dahliae

Verticillium wilt of olive (VWO) is now the most destructive olive disease in the Guadalquivir valley in Andalucia (southern Spain). Disease surveys, conducted to assess the association of agronomical and geographical factors with the current spread of the disease, have shown that VWO is widespread in the valley, with a mean disease incidence (DI) in infested plots reaching 20.4% (9000 inspected trees), but with significant differences among the provinces surveyed (25.7, 23.7 and 12%, for Jaen, Cordoba and Seville, respectively). The DI was significantly higher in irrigated (20.7%) than in dry-farming (18.3%) orchards, and also higher in non-tilled orchards (25.6%) than in regularly-tilled orchards (16.3%). The DI was likewise significantly lower for tree densities above 200 trees ha -1 ; and it was higher (21.5%) when the orchards were located near areas where other V. dahliae host plants were cultivated, than if the orchards were surrounded by non-host plants (11.9%). Lastly, the DI was significantly higher in plots where the tree were less than 25 year old and in plantations close to the Guadalquivir River (less than 10 km). ‘Picual’ was the cultivar most often affected with the disease, reaching a DI of 41.9% in orchards where this cultivar was grown. Highly virulent defoliating (D) isolates in the plantation surveyed were significantly more common (67.7%) than non-defoliating (ND) isolates (32.3%). These factors could explain the substantial increase in incidence and severity of VWO seen in the valley during the last decade.

Symptom development, pathogen isolation and Real-Time QPCR quantification as factors for evaluating the resistance of olive cultivars to Verticillium pathotypes

Verticillium wilt is the most serious olive disease in the Mediterranean countries and worldwide. The most effective control strategy is the use of resistant cultivars. However, limited information is available about the level and source of resistance in most of the olive cultivars and there are no published data using microsclerotia, the resting structures of Verticillium dahliae, as the infective inoculum. In the present study, we correlated symptomatology and the presence of the fungus along with the DNA relative amount (molecules μl−1) of a defoliating (D) and a non-defoliating (ND) V. dahliae strain in the susceptible cv. Amfissis and the tolerant cvs Kalamon and Koroneiki, as quantified by the Real-Time QPCR technology. The viability of the pathogen in the plant tissues was confirmed by isolating the fungus on PDA plates, while symptom assessment proved the correlation between the DNA relative amount of V. dahliae in plant tissues and cultivar susceptibility. It was further demonstrated that the D and ND strains were present at a significantly higher level in cv. Amfissis than in cvs Kalamon and Koroneiki. It was finally observed that the relative amount of the pathogen in roots was lower than in stems and shoots and declined in plant tissues over time. These data constitute a valuable contribution in evaluating resistance of olive cultivars or olive root-stocks to V. dahliae pathotypes.

Evaluation of cotton cultivars for resistance to pathotypes of Verticillium dahliae

After the recent detection of serious losses caused by Verticillium wilt of cotton, incited by the defoliating pathotype of Verticillium dahliae in the Aegean Region of Turkey, 28 of the most commonly grown cotton cultivars ( Gossypium hirsutum L.) of Turkey, were evaluated for the presence of field resistance to wilt. Six-week-old plants were inoculated with a cotton nondefoliating (ND) or a cotton defoliating (D) pathotype of V. dahliae under controlled conditions. Resistance was evaluated on the basis of external symptoms by calculating areas under disease progress curves. The percentage of plants killed and of those which recovered from the disease was used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both pathotypes of V. dahliae. All cultivars were more susceptible to the D than to the ND pathotype. The most promising cultivars in the experiments appeared to be Carmen and ST-373. Carmen showed differential resistance: it was susceptible to the D but resistant to the ND pathotype. ST-373 was moderately susceptible to both pathotypes of V. dahliae. A resistance related phenotypic reaction to the disease was quantified by using six growth parameters (plant height, number of nodes, leaf weight, stem weight, leaf to stem ratio, and total shoot weight) measured 13 d after inoculation. The percentage decrease in leaf–stem ratio and leaf weight were found to be the best indicators of resistance. Results obtained in this study will be useful to quantify resistance to V. dahliae and identify the best parameters to phenotype in genetic studies.

Large-scale epidemiological study and spatial patterns of Verticillium wilt in olive orchards in southern Spain

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 = 3.01) and there was a close association between the ND pathotype and irrigated orchards (relative risk = 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.

Vegetative compatibility of cotton-defoliating Verticillium dahliae in Israel and its pathogenicity to various crop plants

Verticillium dahliae isolates recovered from a new focus of severe Verticillium wilt of cotton in the northeast of Israel were tested for vegetative compatibility using nitrate non-utilizing (nit) mutants and identified as VCG1, which is a new record in Israel. Other cotton isolates of V. dahliae from the northern and southern parts of the country were assigned to VCG2B and VCG4B, respectively. VCG1 isolates induced severe leaf symptoms, stunting and defoliation of cotton cv. Acala SJ-2, and thus were characterized as the cotton-defoliating (D) pathotype, whereas isolates of VCG2B and VCG4B were confirmed as the earlier described defoliating-like (DL) and non-defoliating (ND) pathotypes, respectively. This is the first record of the D-pathotype in Israel. The host range of representative isolates of each VCG-associated pathotype was investigated using a number of cultivated plants. Overall, the D isolates were more virulent than DL isolates on all tested host plants, but the order of hosts (from highly susceptible to resistant) was the same: okra (Hibiscus esculentus local cultivar), cotton (Gossypium hirsutum cv. Acala SJ2), watermelon (Citrullus lanatus cv. Crimson Sweet), safflower (Carthamus tinctorius cv. PI 251264), sunflower (Helianthus annuum cv. 2053), eggplant (Solanum melongena cv. Black Beauty), and tomato (Lycopersicon esculentum cv. Rehovot 13). The pattern of virulence of ND isolates differed from that of D and DL isolates, so that the former were highly virulent on eggplant but mildly virulent on cotton. Tomato was resistant to all cotton V. dahliae isolates tested. RAPD and specific PCR assays confirmed that the D isolates from Israel were similar to those originating from other countries.