Fusarium oxysporum Fo47 confers protection to pepper plants against Verticillium dahliae and Phytophthora capsici,and induces the expression of defence genes

The application of the nonpathogenic isolate Fusarium oxysporum 47 (Fo47) reduced the symptoms of verticillium wilt, phytophthora root rot and phytophthora blight in pepper plants. Botrytis cinerea was also tested on the leaves of plants treated with Fo47, but no protection was observed. Verticillium dahliae colonies cultured in the presence of Fo47 grew slower than control cultures, but Phytophthora capsici growth was unaffected by Fo47. At least part of the protection effect observed against V. dahliae could therefore be due to antagonism or competition. In order to search for induced resistance mechanisms, three defence genes previously related to pepper resistance were monitored over time. These genes encode a basic PR‐1 protein (CABPR1), a class II chitinase (CACHI2) and a sesquiterpene cyclase (CASC1) involved in the synthesis of capsidiol, a phytoalexin. These three genes were transiently up‐regulated in the roots by Fo47 in the absence of inoculation with the pathogen, but in the stem only CABPR1 was up‐regulated. In plants that were inoculated with V. dahliae after the Fo47 treatment, the three genes had a higher relative expression level than the control in both the roots and the stem.     

Verticillium dahliae Modifies the Concentrations of Proline, Soluble Sugars, Starch, Soluble Protein and Abscisic Acid in Pepper Plants

The objective of this research was to study the levels of some organic solutes, such as total protein, total soluble sugars, starch and proline in leaves, as well as abscisic acid concentration in xylem of pepper plants inoculated with Verticillium dahliae Kleb. Healthy and inoculated plants were always kept well watered. Measurements were made at time intervals after inoculation. Leaf water potential rapidly decreased as a consequence of fungal infection. However, relative water content in leaves only changed significantly from day 20 after inoculation, and such decreases coincided with a sharp build up of proline and total soluble sugars in leaves. Starch and protein levels, as well as abscisic acid concentration in xylem, declined in healthy and inoculated peppers as they became older. However, such decreases were more pronounced in infected plants, especially soon after inoculation. Results suggest that proline and total soluble sugars accumulation could be sensors of the damage caused by the fungal infection.     

25%嘧菌酯悬浮剂防治辣椒疫病药效试验

试验结果表明．对辣椒茎基部喷淋施用25％嘧菌酯悬浮剂,随着施药浓度的增大,防病效果逐渐升高,对辣椒疫病发病初期防病效果好、持效期较长,施用本品24-72g／667m^2,加水60kg稀释．对开花座果初期的辣椒具有较高的安全性,并有刺激辣椒生长的作用,具有较高的推广应用价值。推荐本品使用药量以48g／667m^2为宜。     

Survival of strawberry-pathogenic fungi Fusarium oxysporum f. sp. fragariae, Phytophthora cactorum and Verticillium dahliae under anaerobic conditions

The growth and survival of three strawberry pathogens, Fusarium oxysporum f. sp. fragariae (FOF), Phytophthora cactorum, and Verticillium dahliae, were examined in anaerobic (anoxic) conditions at several temperatures (10–40 °C). The growth and survival of these fungi were suppressed by anaerobic conditions in comparison with those in cultured aerobically. Under anaerobic conditions at 22.5 °C, tested isolates of FOF and P. cactorum grew slightly, but V. dahliae did not grow. The three fungi survive for markedly shorter time in the anaerobic conditions compared with the aerobic conditions at all tested temperatures except 40 °C for FOF and P. cactorum. Moreover, survival periods shortened as the cultivation temperature increased. These results demonstrate that anaerobic conditions contribute to eradicating these pathogens during flooding or reductive soil disinfestation.     

Temporal Dynamics of Phytophthora Blight on Bell Pepper in Relation to the Mechanisms of Dispersal of Primary Inoculum of Phytophthora capsici in Soil

ABSTRACT The effect of components of primary inoculum dispersal in soil on the temporal dynamics of Phytophthora blight epidemics in bell pepper was evaluated in field and growth-chamber experiments. Phytophthora capsici may potentially be dispersed by one of several mechanisms in the soil, including inoculum movement to roots, root growth to inoculum, and root-to-root spread. Individual components of primary inoculum dispersal were manipulated in field plots by introducing (i) sporangia and mycelia directly in soil so that all three mechanisms of dispersal were possible, (ii) a plant with sporulating lesions on the soil surface in a plastic polyvinyl chloride (PVC) tube so inoculum movement to roots was possible, (iii) a wax-encased peat pot containing sporangia and mycelia in soil so root growth to inoculum was possible, (iv) a wax-encased peat pot containing infected roots in soil so root-to-root spread was possible, (v) noninfested V8 vermiculite media into soil directly as a control, or (vi) wax-encased noninfested soil as a control. In 1995 and 1996, final incidence of disease was highest in plots where sporangia and mycelia were buried directly in soil and all mechanisms of dispersal were operative (60 and 32%) and where infected plants were placed in PVC tubes on the soil surface and inoculum movement to roots occurred with rainfall (89 and 23%). Disease onset was delayed in 1995 and 1996, and final incidence was lower in plants in plots where wax-encased sporangia (6 and 22%) or wax-encased infected roots (22%) were buried in soil and root growth to inoculum or root-to-root spread occurred. Incidence of root infections was higher over time in plots where inoculum moved to roots or all mechanisms of dispersal were possible. In growth-chamber studies, ultimately all plants became diseased regardless of the dispersal mechanism of primary inoculum, but disease onset was delayed when plant roots had to grow through a wax layer to inoculum or infected roots in tension funnels that contained small volumes of soil. Our data from both field and growth-chamber studies demonstrate that the mechanism of dispersal of the primary inoculum in soil can have large effects on the temporal dynamics of disease.     

Effects of Crop Rotation and Irrigation on Verticillium dahliae Microsclerotia in Soil and Wilt in Cauliflower

ABSTRACT Experiments were conducted in field plots to evaluate the effects of broccoli residue on population dynamics of Verticillium dahliae in soil and on Verticillium wilt development on cauliflower under furrow and subsurface-drip irrigation and three irrigation regimes in 1994 and 1995. Treatments were a factorial combination of three main plots (broccoli crop grown, harvested, and residue incorporated in V.dahliae-infested plots; no broccoli crop or residue in infested plots; and fumigated control plots), two subplots (furrow and subsurface-drip irrigation), and three sub-subplots (deficit, moderate, and excessive irrigation regimes) arranged in a split-split-plot design with three replications. Soil samples collected at various times were assayed for V. dahliae propagules using the modified Anderson sampler technique. Incidence and severity of Verticillium wilt on cauliflower were assessed at 7- to 10-day intervals beginning a month after cauliflower transplanting and continuing until harvest. Number of propagules in all broccoli plots declined significantly (P < 0.05) after residue incorporation and continued to decline throughout the cauliflower season. The overall reduction in the number of propagules after two broccoli crops was approximately 94%, in contrast to the fivefold increase in the number of propagules in infested main plots without broccoli after two cauliflower crops. Disease incidence and severity were both reduced approximately 50% (P < 0.05) in broccoli treatments compared with no broccoli treatments. Differences between furrow and subsurface-drip irrigation were not significant, but incidence and severity were significantly (P < 0.05) lower in the deficit irrigation regime compared with the other two regimes. Abundance of microsclerotia of V. dahliae on cauliflower roots about 8 weeks after cauliflower harvest was significantly (P < 0.05) lower in treatments with broccoli compared with treatments without broccoli. Rotating broccoli with cauliflower and incorporating broccoli residues into the soils is a novel means of managing Verticillium wilt on cauliflower and perhaps on other susceptible crops. This practice would be successful regardless of the irrigation methods or regimes followed on the susceptible crops.     

Genetic variability of tolerance to Verticillium albo-atrum and Verticillium dahliae in Medicago truncatula

Verticillium wilt caused by Verticillium albo-atrum and Verticillium dahliae is responsible for yield losses in many economically important crops. The capacity of pathogenic fungi to adapt to new hosts is a well-known threat to the durability of resistant crop varieties. In the present work, 25 Medicago truncatula genotypes from a core collection and six V. albo-atrum and V. dahliae strains were used to study the potential of non-host Verticillium strains isolated from different plant species to infect this legume plant and the plant’s susceptibility to the pathogens. The experiment was designed as factorial with randomised complete blocks and with three repetitions. The wilt symptoms caused by V. albo-atrum and V. dahliae were scored on a disease index scale from 0 to 4, during 30 days after inoculation of 10-day-old plantlets. Disease severity was quantified by maximum symptom scores (MSS) and areas under the disease progress curves (AUDPC), which integrate the time course of symptom development. Highly significant differences were observed among plant genotypes and fungal strains, and their interaction was also significant. The correlation between MSS and AUDPC was 0.86. The most severe symptoms were caused by the alfalfa strain V. albo-atrum V31-2 and the least severe by V. dahliae JR2, as shown by mean values obtained on the 25 genotypes. M. truncatula genotype TN8.3 was the most susceptible genotype by mean values obtained with the six fungal strains, whereas F11013-3, F83005.9 and DZA45.6 were highly resistant to all strains studied. The results can be used to choose parents for studying the genetics of resistance in Medicago truncatula to Verticillium strains with different levels of aggressiveness.     

Disease Progress Based on Effects of Verticillium dahliae and Pratylenchus penetrans on Gas Exchange in Russet Burbank Potato

ABSTRACT The interactive effects of concomitant infection by the nematode Pratylenchus penetrans and the fungus Verticillium dahliae on symptom expression in Russet Burbank potato was studied in growth chamber experiments. Treatments were P. penetrans at three initial densities, V. dahliae at one inoculum density, the combination of the nematode at these three densities and the fungus, and a noninfested control. Gas exchange was measured nondestructively in leaf cohorts of different ages, one to three times weekly, with a LI-COR portable photosynthesis system. The single-pathogen treatments had no effect on assimilation or transpiration rates, but joint infection had a significant impact. In concomitant infection, photosynthesis was impaired more than transpiration, so estimates of leaf health were based on carbon assimilation rates only. Reductions in assimilation rate were apparent before the onset of visual symptoms. Assimilation rates decreased as much as 44% in the top, and newest, leaves of concomitantly infected plants, compared to rates in control plants. Even so, the health of newly produced leaves did not become progressively worse through time. With light use efficiency less than 0.20 mol of CO(2) fixed per mol of photosynthetically active radiation used as the criterion for disease incidence, disease progressed acropetally from the oldest to the youngest leaves. In plants infected with P. penetrans (0.8 nematodes per cm(3) of soil) in combination with V. dahliae, all leaves in cohorts 1 and 2 were symptomatic by 45 days after planting, and leaves in cohorts 3 to 6 became symptomatic at weekly intervals thereafter. For the control and single-pathogen treatments, the first time that light use efficiency fell below 0.20 in all leaves in cohort 1 was 71 days after planting. Concomitant infection reduced leaf life span by about 3 weeks. Both visual and physiological symptom expression were invariant to differences in initial nematode inoculum densities ranging from 0.8 to 2.5 nematodes per cm(3) of soil in one experiment and from 1.3 to 4.1 nematodes per cm(3) of soil in a second experiment.     

Baseline sensitivity and resistance-risk assessment of Phytophthora capsici to iprovalicarb

Iprovalicarb has been used to control Phytophthora capsici, a devastating pathogen of many economically important crops. To evaluate the risk of fungicide resistance, 158 isolates of P. capsici were examined for sensitivity to iprovalicarb by measuring mycelial growth. Values of effective concentrations for 50% mycelial growth inhibition varied from 0.2042 to 0.5540 μg/ml and averaged 0.3923 (±0.0552) μg/ml, with a unimodal distribution. This is the first report of P. capsici isolates highly resistant to iprovalicarb (resistance factor >100). Resistance of the isolates was stable through 10 transfers on iprovalicarb-free medium, and most resistant isolates had the same level of fitness (mycelial growth, zoospore production, and virulence) as their corresponding parents, indicating that iprovalicarb resistance was independent from other general growth characters. There was cross-resistance among all tested carboxylic acid amide (CAA) fungicides, including iprovalicarb, flumorph, dimethomorph, and mandipropamid, but not with non-CAA fungicides, including azoxystrobin, chlorothalonil, cymoxanil, etridiazole, metalaxyl, and zoxamide. Based on the present results, resistance risk of P. capsici to CAAs could be moderate and resistance management should be considered.     

Molecular detection of Phytophthora capsici in infected plant tissues, soil and water

A species-specific PCR assay was developed for rapid and accurate detection of the pathogenic oomycete Phytophthora capsici in diseased plant tissues, soil and artificially infested irrigation water. Based on differences in internal transcribed spacer (ITS) sequences of Phytophthora spp. and other oomycetes, one pair of species-specific primers, PC-1/PC-2, was synthesized. After screening 15 isolates of P. capsici and 77 isolates from the Ascomycota, Basidiomycota, Deuteromycota and Oomycota, the PC-1/PC-2 primers amplified only a single PCR band of c . 560 bp from P. capsici . The detection sensitivity with primers PC-1/PC-2 was 1 pg genomic DNA (equivalent to half the genomic DNA of a single zoospore) per 25- µ L PCR reaction volume; traditional PCR could detect P. capsici in naturally infected plant tissues, diseased field soil and artificially inoculated irrigation water. Using ITS1/ITS4 as the first-round primers and PC-1/PC-2 in the second round, nested PCR procedures were developed, increasing detection sensitivity to 1 fg per 25- µ L reaction volume. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.     

The detection and estimation of Verticillium dahliae in naturally infested soil

The effects of several factors on the estimation of Verticillium dahliae in soil by the wet-sieving method were studied. The following factors were important for maximising recovery: the removal of soil particles of < 20 μm size from suspensions before plating; the medium used for plating; the amount of sieved soil inoculated to plates; the length of time of incubating plates; and the method of incubating soil on plates. There was no short-term effect of air-drying soil before analysis. Using sodium hexametaphos-phate to aid soil dispersion, treating soil suspensions with sodium hypochlorite, or making suspensions from different amounts of soil had no consistent effect on recovery. Excessive sample-to-sample variability in the results of some experiments was attributed to non-random distribution of fungus propagules in soil, or to the difficulty of standardizing the sieving part of the analysis, or both. Wet-sieving was generally more effective in detecting V. dahliae and gave higher counts than a sucrose-flotation method. Bait methods with eggplants or antirrhinums proved ineffective for estimating V. dahliae in naturally infested soil.     

Biological and genetic characterization of Phytophthora capsici mutants resistant to flumorph

Baseline sensitivity to flumorph, a carboxylic acid amide (CAA) fungicide used to control some oomycetes, was examined using 83 Phytophthora capsici isolates, resulting in a unimodal distribution of effective concentration for 50% inhibition of mycelial growth ranging from 0·716 to 1·363, with a mean of 1·033 ± 0·129 μg mL^?1. To assess the potential risk of developing flumorph resistance, 13 flumorph‐resistant mutants of P. capsici were obtained using ultraviolet irradiation. Most of these mutants and their progeny had high levels of fitness, including mycelial growth, sporulation and virulence. The resistance to flumorph changed slightly, either increasing or decreasing, after 10 transfers on agar media. Cross‐resistance was found between flumorph and other CAA fungicides (dimethomorph and iprovalicarb), but not between flumorph and non‐CAA fungicides (cymoxanil, metalaxyl, azoxystrobin and cyazofamid). To investigate the genetics of the flumorph resistance, 619 progeny were obtained by self‐crossing and sexual hybridization. Segregation of sensitivity to fungicide was measured as a ratio of sensitive (S) to resistant (R) isolates. Segregation of the progeny, from self‐crossed isolate PCAS1 (flumorph resistant), was 1:15 in the first generation; and 0:1 or 1:15 in the second generation. In sexual hybridization, segregation of progeny was 0:1 and 1:7 for R × R hybridization; and 1:3 for R × S hybridization. Therefore, the resistance of P. capsici against flumorph was controlled by two dominant genes.     

Genetic and Morphological Diversity of Temperate and Tropical Isolates of Phytophthora capsici

ABSTRACT Phytophthora capsici is a diverse species causing disease on a broad range of both temperate and tropical plants. In this study, we used cultural characteristics, amplified fragment length polymorphism (AFLP), and DNA sequence analyses of the ribosomal internal transcribed spacer (ITS) region and mitochondrial cytochrome oxidase II (cox II) genes to characterize temperate and tropical isolates from a wide range of host species. All but one temperate isolate grew at 35 degrees C, while all tropical isolates did not. All but two tropical isolates formed chlamydospores, while temperate isolates did not. There was strong bootstrap support for separation of temperate and tropical isolates using AFLP analysis; however, the temperate isolates appeared as a subgroup within the observed variation of the tropical isolates. The majority of temperate isolates clustered within a single clade with low variation regardless of host or geographical origin, while the tropical isolates were more variable and grouped into three distinct clades. Two clades of tropical isolates grouped together and were affiliated closely with the temperate isolates, while the third tropical clade was more distantly related. Phylogenetic analysis of the ITS regions resulted in similar groupings and variation within and between the temperate and tropical isolates as with the AFLP results. Sequence divergence among isolates and clades was low, with more variation within the tropical isolates than within the temperate isolates. Analysis of other species revealed shorter branch lengths separating temperate and tropical isolates than were observed in comparisons among other phylogenetically closely related species in the genus. Analysis of cox II sequence data was less clear. Although the temperate and tropical isolates grouped together apart from other species, there was no bootstrap support for separating these isolates. Restriction fragment length polymorphism (RFLP) analysis of the ITS regions separated the temperate and tropical isolates, as in the AFLP and ITS phylogenetic analyses. However, RFLP analysis of the cox I and II gene cluster did not distinguish between temperate and tropical isolates. The differences in grouping of isolates in these two RFLP studies should be helpful in identifying isolate subgroups. Our data do not fully clarify whether or not temperate and tropical isolates should be separated into different species. The available worldwide data are incomplete and the full range of variation in the species is not yet known. We suggest refraining from using the epithet P. tropicalis until more data are available.     

Phytopathogenic, morphological, genetic and molecular characterization of a Verticillium dahliae population from Crete, Greece

A population of 84?V. dahliae isolates mainly originating from Crete, Greece, was characterized in terms of pathogenicity and virulence on different hosts, in parallel with morphological/physiological characterization, vegetative compatibility grouping and mating type determination. Tomato race 2 was found to have supplanted race 1 and was more virulent on a tomato-susceptible cultivar than race 1. Using a differential host classification system which tests pathogenicity to tomato, eggplant, sweet pepper and turnip, 59 isolates were assigned to tomato, 19 to eggplant, one to sweet pepper and five to tomato-sweet pepper pathogenicity groups. All isolates from Crete fell into VCG subgroups 2A, 2B and 4B, while a remarkably high incidence of bridging isolates (compatible with two or more VCGs) was recorded. The tomato-sweet pepper pathogenicity group was morphologically quite distinct from the others, while conidial length and pigment intensity were discriminatory parameters among VCGs 2A, 2B and 4B. PCR-based molecular marker Tr1/Tr2 was reliable in race prediction among tomato-pathogenic isolates, except for members of VCG 4B, while the application of markers Tm5/Tm7 and 35-1/35-2 was highly successful for tomato-pathogenic isolates. E10 marker was related to VCG 2B, rather than to pathogenicity groups. A single nucleotide polymorphism in the ITS2 region, and two novel molecular markers, M1 and M2, proved useful for the fast and accurate determination of major VCGs 2A, 2B and 4B, and can be used for high-throughput population analyses in future studies. The mating type was unrelated to VCG classification and probably does not control heterokaryon incompatibility in V. dahliae.     

Toxicity of 1,2-dibromoethane and 1,3-dichloropropene to microsclerotia of Verticillium dahliae

The toxicity of two fumigants to microsclerotia (MS) of Verticillium dahliae in air and in soil was determined in sealed containers. 1, 2-Dibromoethane (DBE, ethylene dibromide) at 470 μ ml^?1 of air, or at 12.5 μ g^?1 of soil, killed 97% of the MS, both after incubation for 16 days. 1, 3-Dichloropropene (1, 3-D) at 20 μ ml^?1 of air, however, killed 100% of the MS after incubation for 30 h, and at 100 μ g^?1 of soil after incubation for 3 days. Higher temperatures increased the toxicity of both DBE and 1, 3-D to the MS. The toxicity of DBE increased with increase in soil moisture between 0–80% of field capacity while the toxicity of 1, 3-D increased between 0–20% of field capacity and was constant between 20–80% of field capacity.     

植物内生细菌在辣椒体内的定殖动态及对辣椒疫病的防治效果

为了探讨具生防作用的植物内生细菌在辣椒体内的定殖动态与其防治辣椒疫病的关系,采用对峙培养法和盆栽苗防效法筛选生防菌株,依据菌体形态、生理生化性质和16SrDNA序列鉴定菌种,用抗利福平标记研究菌株在辣椒苗中的定殖动态,在同时接入植物内生细菌和灌根接种辣椒疫霉菌的条件下分析生防菌株的定殖数量与防效的关系。结果表明,菌株G9、R15和J13对辣椒疫病防效最好,经鉴定均为荧光假单胞菌Pseudomonasfluorescens。菌株G9和R15在辣椒根部定殖量高于菌株J13;定殖周期均在30-40d,呈“先增后减”的变化趋势;菌株G9和R15在接种第15d时定殖量最高,菌株J13在根、茎和叶中定殖量达到最高的天数分别为第9、15和15-20d,定殖数量的变化为根〉茎〉叶。菌株G9定殖量达到9．73×10^5cfu·g-1时辣椒疫病的防效达到100％,保持该数量的时间约6d;菌株R15定殖量达到6．30×10^5cfu·g-1以上时对辣椒疫病的防效达到100％,保持该数量的时间约14d。研究结果展现了植物内生细菌在辣椒疫病生物防治上的应用潜力,为制定植物内生细菌防治辣椒疫病的施用技术提供了科学依据。