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1.
Spread of Verticillium wilt into newly established olive orchards in Andalucía, southern Spain, has caused concern in the olive industry in the region. This spread may result from use of Verticillium dahliae-infected planting material, which can extend distribution of the highly virulent, defoliating (D) pathotype of V. dahliae to new areas. In this study, a molecular diagnostic method for the early in planta detection of D V. dahliae was developed, aimed especially at nursery-produced olive plants. For this purpose, new primers for nested PCR were designed by sequencing a 992-bp RAPD marker of the D pathotype. The use of the specific primers and different nested-PCR protocols allowed the detection of V. dahliae pathotype D DNA in infected root and stem tissues of young olive plants. Detection of the pathogen was effective from the very earliest moments following inoculation of olive plants with a V. dahliae pathotype D conidia suspension as well as in inoculated, though symptomless, plants.  相似文献   

2.
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.  相似文献   

3.
Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were 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 isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.  相似文献   

4.
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.  相似文献   

5.
Verticillium wilt caused by Verticillium dahliae is a disease highly prevalent in newly established olive orchards in Andalucía, southern Spain. Two syndromes of the disease occur in Andalucia, namely apoplexy and slow decline. Apoplexy is characterized by quick dieback of twigs and branches while slow decline consists of rapid drying out of inflorescences together with leaf chlorosis and necrosis. Systematic disease observations carried out in two experimental orchards planted with susceptible cv. Picual indicated that natural recovery of diseased trees occurred over time. Infection and vascular colonization of olive plants by V. dahliae were studied in susceptible (Picual) and resistant (Oblonga) cultivars inoculated with a mildly virulent or a highly virulent cotton-defoliating isolate of V. dahliae. Disease symptoms developed 24–32 days after inoculation in cv. Picual, but at that time plants of cv. Oblonga remained free from symptoms. However anatomical observations and isolations indicated that systemic infections by the two isolates had occurred to a large extent in both cultivars.  相似文献   

6.
Verticillium wilt (VW) in olive is best managed by an integrated disease management strategy, of which use of host resistance is a key element. The widespread occurrence of a highly virulent defoliating (D) Verticillium dahliae pathotype has jeopardized the use of commercial olive cultivars lacking sufficient resistance to this pathogen. However, the combined use of resistant wild olive rootstocks and Trichoderma spp. effective in the biocontrol of VW can improve the management of VW in olive. In vivo interactions between D V. dahliae and Trichoderma harzianum were studied in olive and wild olive plants displaying different degrees of resistance against this pathogen using confocal microscopy. This multitrophic system included wild olive clones Ac‐4 and Ac‐15, olive cv. Picual, and the fungal fluorescent transformants T. harzianum GFP22 and V. dahliae V138I‐YFP, the latter being obtained in this study. In planta observations indicated that V138I‐YFP colonizes the roots and stems of the olive and wild olive genotypes, and that GFP22 grows endophytically within the roots of them all. YFP fluorescence signal quantifications showed that: (i) the degree of root and stem colonization by the pathogen varied depending upon the susceptibility of the tested wild olive genotype, being higher in Ac‐15 than in Ac‐4 plants; and (ii) treatment with T. harzianum GFP22 reduced the extent of pathogen growth in both clones. Moreover, root colonization by strain GFP22 reduced the percentage of pathogen colonies recovered from stems of olive and wild olive plants.  相似文献   

7.
Verticillium wilt caused by Verticillium dahliae is one of the most threatening diseases of olive worldwide. For pre‐planting and post‐planting control of verticillium wilt in olive trees, availability of a rapid, reliable and non‐destructive method for detection of V. dahliae is essential. For such a method, suitable and easily performed sampling and efficient processing of samples for extraction of DNA are necessary. In this study, the suitability of young twig and leaf samples of olive trees, which are easy to collect and extract DNA from, were assessed for the detection of V. dahliae in routine procedures. The lower (about 50 cm from the tip) and top parts (about 5 cm from the tip) of twigs, as well as leaves from infected olive trees were screened for V. dahliae infection and distribution using real‐time PCR. The biomass of V. dahliae detected in individual twigs was highly variable, but there was no significant difference between mean quantities of V. dahliae DNA detected in top and lower parts of twigs. Furthermore, it was demonstrated that analysis of combined samples containing DNA extracted from five twigs of an infected tree accurately detected the presence of the pathogen. Similarly, testing combined samples of 5–10 leaves enabled reliable detection of the pathogen in an infected tree. The development of this assay enables reliable detection of V. dahliae in infected olive trees that can aid in management decisions for the implementation of integrated disease management.  相似文献   

8.
An innovative inoculation process, involving the drilling of a trunk hole in 3 year-old olive trees and injecting a dense conidial suspension of Verticillium dahliae, was developed to study differentiation in foliar symptom expression between olive cultivars tolerant or susceptible to the pathogen. It was demonstrated that V. dahliae conidia could be translocated and colonize the xylem at the same distance above and below the point of trunk injection in both cultivars. However, the pathogen could be subsequently isolated at statistically significant percentages in susceptible cv. Amphissis compared to the tolerant cv. Kalamon, indicating operation of resistance mechanisms in the vascular phase of the disease. Consequently symptom development in the susceptible cultivar was at least sixfold more intensive compared to the tolerant cultivar, 6–11 months after trunk inoculation. Perennial olive orchard experiments, aimed at selecting Verticillium-resistant root-stocks, were conducted by applying the novel method in 2–3 year-old root-stock suckers of Amphissis olive trees and in the tolerant cvs Lianolia of Corfu and Koroneiki. It was indicated that potentially resistant root-stocks could be obtained following the trunk drilling technique. Resistance differentiation between cvs Amphissis and Kalamon was further verified through root inoculation by various V. dahliae microsclerotial concentrations and demonstrated that the trunk drilling inoculation procedure is equally efficient in resistance evaluation of olives to Verticillium wilt. The trunk inoculation procedure could be useful in selecting and screening root-stocks for resistance to V. dahliae and other vascular pathogens and could elucidate resistance mechanisms in woody plants against vascular wilt diseases.  相似文献   

9.
Genetic diversity and phenotypic diversity in Verticillium dahliae populations on cotton were studied among 62 isolates from Spain and 49 isolates from Israel, using vegetative compatibility grouping (VCG), virulence and molecular assays. In Spain, defoliating V. dahliae isolates (D pathotype) belong to VCG1, and non-defoliating isolates (ND) belong to VCG2A (often associated with tomato) and VCG4B (often associated with potato). The D pathotype was not identified in Israel. The ND pathotype in Israel is comprised of VCG2B and VCG4B. Isolates in VCG2B and VCG4B ranged in virulence from weakly virulent to highly virulent. The highly virulent isolates induced either partial defoliation or no defoliation. Virulence characteristics varied with inoculation method and cotton cultivar. Highly virulent isolates from Israel were as virulent as D isolates from Spain under conditions conducive to severe disease. The D pathotype is pathologically and genetically homogeneous, whereas the ND pathotype is heterogeneous with respect to virulence, VCG, and molecular markers based on single-primer RAPD and on PCR primer pairs.  相似文献   

10.
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.  相似文献   

11.
Verticillium dahliae is a soilborne fungus that causes Verticillium wilt disease in a plethora of crops. Based on symptoms that develop on cotton, olive and okra, V. dahliae isolates are categorized into two pathotypes, namely defoliating and nondefoliating, with the former showing increased virulence and causing severe defoliation. Reliable differentiation between V. dahliae pathotypes is crucial for the management of Verticillium wilt in cotton and olive. In the present study, a polymorphism was detected among isolates of defoliating and nondefoliating pathotypes in Southern blots using the VdNEP gene as a probe. The regions flanking this gene were isolated by inverse PCR and sequence differences in the 3′ untranslated region (3′-UTR) of the VdNEP gene were detected between the two pathotypes. Based on these sequences, primers were designed and assessed to develop a multiplex PCR detection assay. Using this assay, a collection of cotton and olive V. dahliae isolates from Greece and Cyprus was screened, revealing that the defoliating pathotype is present in several regional units of Greece. Thus, this work presents a new, sensitive molecular marker for the differentiation between V. dahliae pathotypes based on the VdNEP gene. Because the 3′-UTR is involved in the phenotypes displayed by the pathotypes, an expression experiment was conducted under conditions simulating the xylem of a host plant. Expression of the VdNEP gene was elevated at all time points in the defoliating compared to the nondefoliating strain, suggesting a possible involvement of VdNEP expression in the defoliation process.  相似文献   

12.
Verticillium wilt of olive (VWO) is probably the most devastating fungal disease for olive trees worldwide, and currently the main cultivars are susceptible or moderately susceptible to this disease. The evaluation of resistant cultivars as rootstocks to control the disease has scarcely been explored, and mainly in short-term studies under controlled conditions, which usually do not correspond with field evaluations. The main objective of this study was to assess the responses to VWO of different scion × rootstock combinations of the olive cultivars Picual, Arbequina, Changlot Real, and Frantoio in a long-term field experiment with a soil highly infested with the defoliating pathotype of Verticillium dahliae. The results showed that grafting the susceptible cultivar Picual onto resistant rootstocks delayed the onset of the disease symptoms; however, after 4 years, it was observed that all combinations that contain Picual (a) were extensively colonized by V. dahliae; (b) developed severe symptoms of the disease; and (c) had plant mortality similar to Picual growing on its own roots. This result highlights the importance of long-term field experiments to evaluate VWO and shows that grafting susceptible olive cultivars onto resistant ones does not provide a durable control of VWO under high inoculum potential, as V. dahliae is able to progress through the resistant rootstock and then extensively colonize and kill the susceptible scion. However, the high inoculum potential observed in this study does not allow us to consider the evaluated resistant cultivars as completely ineffective under lower inoculum densities.  相似文献   

13.
Verticillium wilt of olive is best managed by resistant cultivars, but those currently available show incomplete resistance to the defoliating (D) Verticillium dahliae pathotype. Moreover, these cultivars do not satisfy consumers' demand for high yields and oil quality. Highly resistant rootstocks would be of paramount importance for production of agronomically adapted and commercially desirable olive cultivars in D V. dahliae‐infested soils. In this work, resistance to D V. dahliae in wild olive clones Ac‐13, Ac‐18, OutVert and StopVert was assessed by quantifying the fungal DNA along the stem using a highly sensitive real‐time quantitative polymerase chain reaction (qPCR) protocol and a stem colonization index (SCI) based on isolation of V. dahliae following artificial inoculations under conditions highly conducive for verticillium wilt. Ac‐13, Ac‐18, OutVert and StopVert showed a symptomless reaction to D V. dahliae. The mean amount of D V. dahliaeDNA quantified in stems of the four clones ranged from 3.64 to 28.89 pg/100 ng olive DNA, which was 249 to 1537 times lower than that in susceptible Picual olive. The reduction in the quantitative stem colonization of wild olive clones by D V. dahliae was also indicated by a sharp decrease in the SCI. Overall, there was a pattern of decreasing SCI in acropetal progression along the plant axis, as well as correlation between positive reisolation and quantification of pathogen DNA. The results of this research show that wild olive clones Ac‐13, Ac‐18, OutVert and StopVert have a valuable potential as rootstocks for the management of verticillium wilt in olive.  相似文献   

14.
Verticillium dahliae Klebahn is the causal agent of tomato wilt disease. Isolates of V. dahliae can be classified based on pathogenicity to tomato, but the pathotypes are indistinguishable in morphology. We designed PCR primers for specific detection of isolates pathogenic to tomato (tomato pathotype) from the sequences of a pathotype-specific gene, vdt1. With the primer pair Tg5/Tc3, a PCR product (approximately 3.2 kb) specific to tomato pathotype was amplified from the genomic DNA of isolates. Using the primer pair, a tomato pathotype isolate was specifically detected from hypocotyls of inoculated tomato and eggplant. On the other hand, no amplification was observed from non-tomato pathotype isolates of V. dahliae, some other wilt pathogens of tomato and a healthy host plant. Therefore, the primer pair can be useful for pathotype-specific detection of V. dahliae as well as for diagnosis of wilt disease of tomato plant. Received 7 September 2001/ Accepted in revised form 3 December 2001  相似文献   

15.
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 < 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.  相似文献   

16.
A study was conducted during 2 years to determine the effects of three different control measures on the development of Verticillium wilt on olive trees cv. ‘Nabali’ at Al-Hallabat, Jordan. The causal agent of the wilt wasVerticillium dahliae Kleb. Treating diseased trees with Cryptonol (8-hydroxyquinoline sulfate) in a soil drench, or covering trees with a solar chamber for 15 days, was effective in suppressing disease development. The fertilizer treatment (NPK, 15:15:30) decreased disease severity and percent infection. All decreases are in comparison with the untreated control, and as recorded during the active phase of the pathogen. The treatments did not differ significantly from each other, and disease incidence in treated trees remained lower than in the control throughout the examined period.  相似文献   

17.
Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.  相似文献   

18.
Understanding pathogenic variation in plant pathogen populations is key for the development and use of host resistance for managing verticillium wilt diseases. A highly virulent defoliating (D) pathotype in Verticillium dahliae has previously been shown to occur only in one clonal lineage (lineage 1A). By contrast, no clear association has yet been shown for race 1 with clonal lineages. Race 1 carries the effector gene Ave1 and is avirulent on hosts that carry resistance gene Ve1 or its homologues. The hypothesis tested was that race 1 arose once in a single clonal lineage, which might be expected if V. dahliae acquired Ave1 by horizontal gene transfer from plants, as hypothesized previously. In a diverse sample of 195 V. dahliae isolates from nine clonal lineages, all race 1 isolates were present only in lineage 2A. Conversely, all lineage 2A isolates displayed the race 1 phenotype. Moreover, 900‐bp nucleotide sequences from Ave1 were identical among 27 lineage 2A isolates and identical to sequences from other V. dahliae race 1 isolates in GenBank. The finding of race 1 in a single clonal lineage, with identical Ave1 sequences, is consistent with the hypothesis that race 1 arose once in V. dahliae. Molecular markers and virulence assays also confirmed the well‐established finding that the D pathotype is found only in lineage 1A. Pathogenicity assays indicated that cotton and olive isolates of the D pathotype (lineage 1A) were highly virulent on cotton and olive, but had low virulence on tomato.  相似文献   

19.
Surveys of 94 artichoke fields throughout the artichoke production areas of Comunidad Valenciana (eastern Spain) were conducted from 1999 to 2002 to determine the incidence and distribution of Verticillium wilt.Verticillium dahliae was isolated from 80.9% of the sampled fields, and detected in all artichoke-growing areas, with a mean disease incidence of 53.8% infected plants. The disease was found to cause severe damage to cv. ‘Blanca de Tudela’, which is the most important artichoke cultivar grown in Spain, and was also observed on the seed-propagated cv. ‘Imperial Star’. In field trials to study the role of infected planting material and soil inoculum on infection of artichoke plants during the cropping season,V. dahliae was transmitted from infected stumps to the plants, confirming that the use of infected stumps could have greatly contributed to the dissemination of the pathogen. Inoculum density ofV. dahliae in soil had an effect on crop infection, in that a higher number of microsclerotia per gram of soil resulted in a higher percentage of infected plants. In addition, yield of cv. Blanca de Tudela was significantly affected byV. dahliae infection, showing that a higher percentage of infection corresponded with lower yield. http://www.phytoparasitica.org posting July 21, 2005.  相似文献   

20.
Verticillium wilt of olive was first recorded in Syria in mid-1978 and confirmed as due to Verticillium dahliae. This article reports observations made in a comprehensive survey of the disease in nine provinces over 7 years. Percent infection varied from 0.85 to 4.5 in different provinces. Newly planted groves in lowland areas showed more infection than older groves in hilly areas. Isolation of V. dahliae was possible at all times of year. Young trees were more susceptible to wilt than older ones. The performance of 13 local cultivars against wilt was studied under natural field conditions and found to vary greatly from susceptible to resistant. Agricultural practices greatly affect spread of the disease. High disease incidence was observed in irrigated groves compared with non-irrigated, and correlated positively with number of ploughings. Verticillium wilt causes a loss between 1 and 2.3% of total olive production annually.  相似文献   

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