Epidemiology and effects of verticillium wilt on yield of olive trees (cvs. Barnea and Souri) irrigated with saline water in Israel

Epidemiology ofVerticillium dahliae on olive trees (cvs. ‘Barnea’ and ‘Souri’) irrigated with saline water was studied over a period of 3 years. Disease incidence in cv. Barnea increased from 18.9% to 40.5% in the first year, while disease severity decreased from 4.2 to 2.4 (on a scale of 2–10). In the first month after pruning of diseased branches (June 1999), disease incidence was 26.9% and it increased to 60.6% by May 2000. No changes in disease severity were observed from this time until the end of the study. In cv. Souri, disease incidence doubled during the 3 years of the study (from 22.1% to 44.8%), and disease severity increased from 2.4 to 2.9. The disease caused significant reduction of yield in cv. Barnea in 1999 and 2000 (48.3% and 12.0%, respectively). In cv. Souri yield was measured only in 2001, where a reduction of 66.8% was observed.V. dahliae was isolated from both diseased and symptomless trees. A significant decrease in the isolation rate ofV. dahliae from diseased cv. Barnea trees occurred between the first year (47.9%) and the third year (5.3%); positive isolations were obtained in all seasons, without differences between seasons. In cv. Souri, the fungus was isolated in significantly higher rates during the winter and spring (55% and 50%, respectively). Our findings strongly support the assumption that natural recovery occurs in cv. Barnea 4–5 years after planting, but not in cv. Souri. http://www.phytoparasitica.org posting July 10, 2003.     

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.     

Detection of the Defoliating Pathotype of Verticillium dahliae in Infected Olive Plants by Nested PCR

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.     

Incidence of Verticillium wilt of artichoke in eastern Spain and role of inoculum sources on crop infection

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.     

Effect of temperature,relative humidity,leaf wetness and leaf age on <Emphasis Type="Italic">Spilocaea oleagina</Emphasis> conidium germination on olive leaves

The effects of temperature, relative humidity (RH), leaf wetness and leaf age on conidium germination were investigated for Spilocaea oleagina, the causal organism of olive leaf spot. Detached leaves of five ages (2, 4, 6, 8 and 10 weeks after emergence), six different temperatures (5, 10, 15, 20, 25 and 30°C), eight wetness periods (0, 6, 9, 12, 18, 24, 36 and 48 h), and three RH levels (60, 80 and 100%) were tested. Results showed that percentage germination decreased linearly in proportion to leaf age (P < 0.001), being 58% at 2 weeks and 35% at 10 weeks. A polynomial equation with linear term of leaf age was developed to describe the effect of leaf age on conidium germination. Temperature significantly (P < 0.001) affected frequencies of conidium germination on wet leaves held at 100% RH, with the effective range being 5 to 25°C. The percent germination was 16.1, 23.9, 38.8, 47.8 and 35.5% germination at 5, 10, 15, 20 and 25°C, respectively, after 24 h. Polynomial models adequately described the frequencies of conidium germination at these conditions over the wetness periods. The rate of germ tube elongation followed a similar trend, except that the optimum was 15°C, with final mean lengths of 175, 228, 248, 215 and 135 μm at 5, 10, 15, 20 and 25°C, respectively after 168 h. Polynomial models satisfactorily described the relationships between temperature and germ tube elongation. Formation of appressoria, when found, occurred 6 h after the first signs of germination. The percentage of germlings with appressoria increased with increasing temperature to a maximum of 43% at 15°C, with no appressoria formed at 25°C after 48 h of incubation. Increasing wetness duration caused increasing numbers of conidia to germinate at all temperatures tested (5–25°C). The minimum leaf wetness periods required for germination at 5, 10, 15, 20 and 25°C were 24, 12, 9, 9 and 12 h, respectively. At 20°C, a shorter wetness period (6 h) was sufficient if germinating conidia were then placed in 100% RH, but not at 80 or 60%. However, no conidia germinated without free water even after 48 h of incubation at 20°C and 100% RH. The models developed in this study should be validated under field conditions. They could be developed into a forecasting component of an integrated system for the control of olive leaf spot.     

Possibilities of biological and chemical control of Verticillium wilt in pepper

In pathogen populations in Serbia, the incidence, pathogenic and morphological characters ofVerticillium spp. were studied. Biological and chemical control ofVerticillium was investigated in pepper plants (Capsicum annuum L. cv. ‘Soroksari’) with the biofungicide Polyversum^® (Pythium oligandrum) and the conventional fungicides benomyl and propamocarb-hydrochloride. On the basis of macroscopic and microscopic characters of the isolates originating from eight localities in Serbia, it was established that they apparently belong to the speciesVerticillum dahliae. The isolates differed in their pathogenic characters. However, all of them caused marked wilting symptoms on pepper plants 40 days after inoculation, conducted when there were more than nine fully developed leaves on the primary stem. The fungicides were applied either before or after inoculation. Benomyl was the most efficient fungicide in wilt control (88.2% when applied after inoculation and 94.6% when applied before inoculation). Polyversum proved more efficient (66.6%) when applied before rather than after inoculation. Propamocarb-hydrochloride provided sufficient Verticillium wilt control; its efficacy and that of Polyversum were similar, and less efficient than benomyl, but still significantly different from the disease control.     

Novel methodologies in screening and selecting olive varieties and root-stocks for resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

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.     

Fusarium wilt on sweet basil: Cause and sources in southeastern Spain

This study concerned a new disease detected in 1997 in southeastern Spain — Fusarium wilt in basil (Ocimum basilicum L.), caused byFusarium oxysporum f.sp.basilici. Its importance was evaluated at two locations in the Almería area, where 14% of the plants presented symptoms of the disease after 4 months of cropping. The search for sources of the disease inoculum was centered on the health of the seeds and the polypropylene trays that were reused for plant production. Analysis of four lots of seeds from Germany and Italy showed that two of them harboredF. oxysporum f.sp.basilici. This finding was confirmed by the analysis of seeds collected from diseased plants. Furthermore, analysis of three reused trays revealed the presence of the pathogen on them and it was concluded that the trays acted as the source of dispersion of the mycosis. http://www.phytoparasitica.org posting July 14, 2004.     

Integrated control of verticillium wilt of olive with cryptonol in combination with a solar chamber and fertilizer

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.     

Assessment of the effect of surface drip irrigation on Verticillium dahliae propagules differing in persistence in soil and on verticillium wilt of olive

For efficient integrated management of verticillium wilt in olive (VWO), it is important to establish whether irrigation treatments (with Verticillium dahliae‐free water) that mitigate the disease in V. dahliae‐infested soil, also reduce the levels of more and less persistent propagules of the pathogen in the soil. Effects of irrigation on VWO and V. dahliae propagules were evaluated under natural environmental conditions. Potted plants were irrigated (pathogen‐free water) to two ranges of soil water content (RWC; high and low) at three surface drip‐irrigation frequencies (daily, weekly, and daily during some periods and otherwise weekly). VWO and total inoculum density (ID), density of less persistent micropropagules (MpD) and more persistent sclerotia in wet soil (S_wD), and sclerotia density for air‐dried soil (S_dD) were monitored. A logistic model (multiple sigmoid) of disease incidence revealed the lowest parameter values in treatments irrigated daily. Daily frequency of irrigation showed significantly lower disease incidence (39.2%) and disease intensity index (43.9%) and MpD (88.0%) values as areas compared with other frequencies, regardless of the RWC. High RWC significantly reduced (70.8–84.9%) ID, S_wD and S_dD as areas, but significantly increased (18.0%) the incidence of infected plants (IIP), regardless of the irrigation frequency. The disease incidence was not correlated with temperature. Daily irrigation to low RWC mitigated the VWO and the IIP, kept soil to the lowest MpD and resulted in the lowest S_dD level at the end of the trial. Results suggested that less persistent propagules could have played a part in the disease development.     

Broad bean wilt virus 2 in yams

We identified Broad bean wilt virus 2 (BBWV-2) in yams based on particle morphology, test plant symptoms, protein features, aphid transmission, and molecular classification using nucleotide sequences of coat protein genes.     

A new aggressive strain of Verticillium albo-atrum in Verticillium resistant cultivars of tomato in the Netherlands

In 1991 serious losses caused byVerticillium wilt were found on two holdings in the Westland glasshouse district in the Netherlands in which theVerticillium resistant tomato cultivars Calypso and Criterium were grown in soilless systems. Isolates from diseased plants were identified asVerticillium albo-atrum.In inoculation experimentsVerticillium resistant tomato cultivars were seriously affected by the new isolates but not by a control isolate. Moneydor, a susceptible cultivar without the Ve gene, was the most seriously diseased by all isolates. The isolates from theVerticillium resistant tomato cultivars were less virulent on the susceptible cultivar than the control isolate.     

Carnation Fusarium wilt suppression in four composts

Fusarium wilt is now a major disease of carnation crops worldwide. Methyl bromide, which is used to remedy it, is environmentally unsafe. An alternative approach integrated into biological control is to grow crops in suppressive media. Suppressiveness of seven plant growth media to Fusarium oxysporum f. sp. dianthi was evaluated in bioassays with carnation (Dianthus cariophyllus) cv. Medea. These media were: (1) grape marc compost, (2) cork compost, (3) olive oil husk + cotton gin trash composted and mixed with rice husk, (4) spent mushroom compost mixed with peat, (5) coir fibre, (6) light peat and (7) vermiculite. In order to look for carnation Fusarium wilt suppressiveness indicators, growth medium pH and β-glucosidase activity were evaluated. Furthermore, F. oxysporum populations were measured in plant growth media at the beginning and end of bioassays. The compost media showed a range of suppressiveness in comparison with peat. Grape marc compost was the most effective plant growth medium in suppressing carnation Fusarium wilt. On the other hand coir fibre, peat and vermiculite were conducive for this disease. β-glucosidase activity and pH were positively correlated with disease severity as in other reports for tomato. Therefore, these two parameters are good indicators for carnation Fusarium wilt suppressiveness, and possibly for other F. oxysporum pathosystems. All composts showed similar F. oxysporum populations at the end of the bioassays to peat and vermiculite.     

Suppression of Fusarium wilt of spinach with compost amendments

The effect of different organic composts on the suppression of wilt disease of spinach caused by Fusarium oxysporum f. sp. spinaciae was evaluated in a continuous cropping system in both containers and in microplot field trials. Test soils infested with the pathogen were amended with wheatbran, wheatbran and sawdust, coffee grounds, chicken manure, or mixture of different composts with and without 5% (w/w) crab shell powder either once (5%, w/w) or continuously (2.5%) into the test soils infested with the pathogen. In the container trials, the soil amended with composts became suppressive to disease development on the second and third cropping. The suppressive effect was notable in the soil amended with the mixture of compost with and without crab shell powder. The coffee compost lowered soil pH but became suppressive to the disease after modifying the soil pH. In the field trial using the mixture of the different composts containing 5% crab shell powder, a combination of 5% before the first cropping and 2.5% every second cropping gave stable disease control and promoted plant growth. After compost amendment, populations of fungi, bacteria and actinomycetes as measured by dilution plate counting and the total microbial activity as evaluated by fluorescein diacetate hydrolysis increased and population of the pathogen gradually decreased. These phenomena were especially notable in soils amended with the mixture of different composts. These results indicate that diversity in the organic materials promotes higher microbial activity and population in the soil thereby enhancing disease suppressiveness.     

Comparative Study of Genetic Diversity and Pathogenicity Among Populations of Verticillium Dahliae from Cotton in Spain and Israel

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.     

黄瓜枯萎病拮抗菌的拮抗促生因子检测及生物效应研究

以黄瓜枯萎病病原菌,尖孢镰刀菌黄瓜专化型(FOC,Fusarium oxysporum f.sp.cucumerinum)为靶标筛选拮抗菌。通过初筛和复筛,获得2株对尖孢镰刀菌黄瓜专化型拮抗效果稳定、拮抗能力较强的细菌QHZ-Yb1和QHZ-Yb2(以下简称,Yb1和Yb2),采用平板对峙法测定其拮抗作用,抑菌带宽度达3.37 mm和3.42 mm,抑菌圈直径2.7 cm,抑菌率分别达到61.9%和62.3%;通过形态学结合生理生化及菌株的16S rDNA序列分析,初步确定这两株菌均为萎缩芽孢杆菌(Bacillus atrophaeus);潜在拮抗促生因子的测定结果表明:菌株Yb1和Yb2具有多种拮抗促生因子,其脂肽类粗提取物的抑菌率分别达到57.4%和58.0%,生长素IAA的分泌量可达1.56 mg·L~(-1)和3.69 mg·L~(-1),溶P量为0.77 mg·L~(-1)和1.19 mg·L~(-1),两个菌株均可产嗜铁素、分泌酪蛋白酶;种子萌发试验结果表明:Yb1和Yb2菌液均可促进黄瓜种子的萌发;菌株生物效应的盆栽试验结果表明:Yb1、Yb2以及Yb1+Yb2菌悬液的处理不仅对黄瓜枯萎病有明显的生防效果,而且可显著增加黄瓜植株的生物量,增加黄瓜植株根际土中细菌和放线菌的数量,减少真菌和尖孢镰刀菌的数量,尤其以Yb1+Yb2菌悬液的处理效果最显著,对黄瓜枯萎病的发病率降低11.11个百分点,植株生物量增加56.7%。结论:菌株Yb1和Yb2具有生防潜力,两菌株配合运用效果更好。     

Bacterial wilt of bellflower caused by Ralstonia solanacearum in Japan

A sudden wilt of bellflower (Campanula lactiflora) was observed in Japan in 1997. A bacterium that formed white fluidal and mucoid colonies resembling those of Ralstonia solanacearum was isolated from the infected plants. The bacterium was bacteriologically identified as biovar 3 of R. solanacearum. This is the first report of R. solanacearum affecting a plant species of the Campanulaceae family.     

Efficacy of olive mill waste water and its derivatives in the suppression of crown gall disease of bitter almond

Olive mill waste water (OMW) and some of its indigenous bacterial strains were tested in vitro and in planta for their efficacy against crown gall disease caused by Agrobacterium tumefaciens. OMW and polyphenols displayed a high level of antibacterial activity, however the volatile fraction was less efficient as only a bacteriostatic effect was observed. In pot experiments, the percentage of bitter almond rootstock showing symptoms of crown gall was significantly reduced with the dosage rate of OMW 1% as compared to the control (highly natural infected soil treated with water). Five indigenous bacterial strains isolated from OMW exhibited an antagonistic effect against the bacterium. Based on the gene 16S rRNA sequence analysis, one isolate showed 99.2% similarity to known sequences of Bacillus subtilis, one isolate demonstrated high percentage similarities (99.3%) to the genera Bacillus pumilis, and two isolates were associated with Stenotrophomonas maltophilia and Pseudomonas putida 100% and 99.6% similarities respectively. Among these bacteria, the strain B1 proved efficient against the soil borne pathogen in vitro and pot experiments. Our study in controlled conditions suggested that the addition of OMW to soil exerts significant disease suppressiveness against A. tumefaciens. Thabet Yangui and Ali Rhouma contributed equally to this work and are regarded as joint first authors.     

Plant Host Range of Verticillium longisporum and Microsclerotia Density in Swedish Soils

Verticillium longisporum is a soil-borne fungal pathogen causing vascular wilt of Brassica crops. This study was conducted to enhance our knowledge on the host range of V. longisporum. Seven crop species (barley, oat, oilseed rape, pea, red clover, sugar beet and wheat) and five weed species (barren brome, black-grass, charlock, cleavers and scentless mayweed) all common in southern Sweden were evaluated for infection by response to V. longisporum. Oat, spring wheat, oilseed rape, scentless mayweed and charlock inoculated with V. longisporum in a greenhouse showed stunting to various degrees close to the fully ripe stage. Based on the extent of microsclerotia formation, explants were separated into four groups: for pea and wheat, <5% of the samples had formed microsclerotia; for scentless mayweed, 5–10%; for oat, 10–20%; and for charlock and oilseed rape >80%. The results suggest that plant species outside the Brassicaceae can act as reservoirs of V. longisporum inoculum. Soil inoculum densities in nine fields were monitored over a period of 12 months, which ranged from 1 to 48 cfu g⁻¹ soil. Density of microsclerotia was lowest just after harvest, reaching its maximum six months later. No significant correlation between inoculum density in soil and disease incidence on oilseed rape plants was found. However, the data suggest that a threshold of 1 cfu g⁻¹ soil is needed to cause disease on oilseed rape. Species identification based on microsclerotia morphology and PCR analysis showed that V. longisporum dominated in soil of seven, and V. dahliae in two of the nine fields studied.