Modes of action of the protective strain Fo47 in controlling verticillium wilt of pepper

The protective fungus Fusarium oxysporum Fo47 reduces the severity of wilt caused by the soilborne pathogen Verticillium dahliae in pepper. Modes of action responsible for the biocontrol activity were studied. Microscopic observations of fluorescent protein‐transformed strains colonizing the root surface show that the colonization patterns of Fo47 and V. dahliae were similar. Pixel counting of the images obtained by confocal microscopy showed that Fo47 reduces colonization of the root surface by V. dahliae, suggesting a possible role of competition for nutrients at the root surface. Besides these effects on surface colonization, the hormonal pathways activated during priming of plant defence responses were identified by measuring the amount of some phytohormones and their derivatives in roots and stems of pepper. Results showed an early, slight increase of jasmonyl isoleucine, followed by a transient increase of salicylic acid during the pre‐challenged phase of priming and an increase of 12‐oxo‐phytodienoic acid during the challenge phase of priming. The caffeic, ferulic and chlorogenic acids, known to play a role in plant defence reactions, showed a strong antimicrobial activity against V. dahliae in vitro. In pepper roots, Fo47 stimulated the biosynthesis of caffeic acid and primed that of chlorogenic acid. These results demonstrated that the effective control of V. dahliae provided by Fo47 is based on different but complementary mechanisms.     

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.     

Interactions between Trichoderma harzianum and defoliating Verticillium dahliae in resistant and susceptible wild olive clones

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.     

The β‐amylase genes: negative regulators of disease resistance for Verticillium dahliae

Little is known about the role of plant primary metabolism in defence against pathogens. The present study is the first investigation published that examines the role of β‐amylase (BAM) genes upon fungal, Verticillium dahliae, infection. The responses of Arabidopsis thaliana plants impaired in BAM1, BAM2, BAM3, BAM4 genes, along with double, triple and quadruple mutants of those genes, were used to explore the involvement of BAM in the host plant–V. dahliae interaction. Less severe symptoms were recorded in bam mutants compared to wild type. Real‐time quantitative PCR (qPCR) revealed that the decrease in symptom severity shown in bam plants was correlated with reductions in the growth of the pathogen in the plants. Confocal microscopy of the most and least susceptible bam mutants and the wildtype plants showed that there were no differences between them in the number of attached conidia and penetration sites on the roots. BAM1, BAM2 and BAM3 expression was altered upon V. dahliae infection in the aerial tissues of the wild type. Analysis by qPCR of the PR1 and PDF1.2 expression in the bam3, bam1234, bam14 and wildtype plants showed that PR1 was up‐regulated in the roots of bam plants upon V. dahliae infection.     

Increased resistance to Verticillium dahliae in Arabidopsis plants defective in auxin signalling

Auxin signalling and transport participate in plant–microbe interactions as positive or negative regulators of disease resistance. The present study investigated the responses of Arabidopsis thaliana plants impaired in the auxin receptors TIR1, AFB1 and AFB3 and the auxin transporter AXR4, upon infection by the soilborne root pathogen Verticillium dahliae. Fewer symptoms were recorded in afb1, afb3 and axr4 plants compared to the wild type (wt). qPCR analysis revealed that the decrease in symptom severity in afb1, afb3 and axr4 was correlated with reduction in the growth of the pathogen in the plants. Therefore, afb1, afb3 and axr4 are partially resistant to V. dahliae. Upon V. dahliae infection, the expression of TIR1, AFB1, AFB3 and AXR4 was up‐regulated in roots, while indole‐3‐acetic acid levels were similar to mocks. The partial resistance of afb1, afb3 and axr4 against V. dahliae can be attributed in part to the up‐regulation of defence‐related genes, as it was observed that afb1 and axr4 had higher PR1 levels than wt, while afb3 had higher PDF1.2 levels than wt. The findings of the present study suggest that the auxin signalling defective mutants afb1, afb3 and axr4 show increased resistance against V. dahliae.     

Assessment of real-time PCR as a method for determining the presence of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in different Solanaceae cultivars

Real-time PCR was used to detect and quantify Verticillium dahliae and to assess the susceptibility of four Capsicum annuum cultivars (Luesia, Padrón, SCM331 and PI201234) and the Capsicum chinense cv. C118 to this pathogen. The symptoms which developed after infection included stunting and yellowing, and were more acute in the cv. SCM331, which also suffered defoliation in later stages of the disease and in C118, which suffered severe stunting. Quantification of the pathogen DNA in roots 23 and 34 days post-inoculation (dpi) revealed that there were significantly higher amounts of Verticillium dahliae DNA in C118 than in the other cultivars, followed by SCM331, Padrón and PI201234. The lowest amounts of fungal DNA in roots were found in Luesia. In hypocotyls, the highest amounts of fungal DNA were found in SCM331, while Luesia, Padrón and PI201234 had much lower amounts, and C118 had intermediate levels. When a compatible versus an incompatible system was studied, using the near-isogenic tomato lines LA3030 (susceptible) and LA3038 (resistant to V. dahliae), we were able to detect fungal DNA in both lines. As expected, the fungus/plant DNA ratio was lower in LA3038 than in LA3030 and it decreased with time in LA3038. The amount of Verticillium dahliae DNA in the roots of LA3030 remained constant between days 23 and 34 post-inoculation, but increased 10-fold in collars. Finally, when real-time PCR was applied as a diagnostic method to samples from pepper plants, soil and water collected from farms in northwest Spain, we were able to detect V. dahliae DNA in these samples even when symptoms of the disease were not evident.     

Colonization in cotton plants by a green fluorescent protein labelled strain of Verticillium dahliae

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease caused by the soil-borne fungal pathogen Verticillium dahliae. In this study, a green fluorescent protein (GFP) labelled V. dahliae strain (TV7) was obtained by transforming gfp into defoliating strain V991. Strain TV7 was used to study infection and colonization of wilt resistant cotton cultivar Zhongzhimian KV1 and susceptible cultivar 861 with the aid of confocal laser scanning microscopy. The results showed that initial infection and colonization of V. dahliae in Zhongzhimian KV1 and 861 were similar. Conidia and hyphal colonies formed and penetrated in the root meristematic and elongation zones and in the conjunction of the lateral and main roots. The invaded conidia started to germinate by 2 hpi (hours post-inoculation), penetrated into the root cortex and vascular bundles, eventually colonized in the stem xylem vessels and grew restrictedly in the individual tracheae of both resistant and susceptible cultivars. Moreover, pathogen DNA could be detected by qPCR in roots and stems of both cultivars, but its content in the wilt susceptible cultivar 861 was much higher than that in the wilt resistant cultivar Zhongzhimian KV1. The results indicated that the resistant cultivar has ability to suppress V. dahliae reproduction.     

Induced resistance to Verticillium longisporum in Brassica napus by β‐aminobutyric acid

A preinoculative soil drench application of 0·5 mm β‐aminobutyric acid (BABA) significantly inhibited colonization of oilseed rape (Brassica napus, susceptible cultivar Falcon) by Verticillium longisporum and also prevented plant stunting caused by the pathogen. To better understand the defence responses induced by BABA, the presence of occlusions in the plant hypocotyl, levels of salicylic acid (SA) and hydrogen peroxide (H₂O₂), phenylalanine ammonia lyase (PAL) activity and expression of PR‐1 and PDF1.2 genes were examined. Transverse sections through the hypocotyl region of BABA‐treated plants showed clear vessels surrounded by phenol‐storing cells, in contrast to numerous obstructed vessels in water‐treated plants, in response to the pathogen. A significant increase in SA levels was observed in the hypocotyls of both water‐ and BABA‐treated plants in response to the pathogen; however, SA levels were unrelated to disease resistance. The level of H₂O₂ decreased in both treatments in response to the pathogen. A significant increase in PAL activity was observed in hypocotyl tissues of BABA‐treated plants. The expression patterns of PR‐1 and PDF1.2 were similar in the two treatments in response to the pathogen, indicating no involvement of these genes in resistance. The results indicate a similar organ specificity of the plant hypocotyl for chemically induced internal resistance as for genotype‐related resistance, two phenomena which, however, are based on contrasting cytological responses in the vascular tissues. Nonetheless, evidence is provided that the activity of the phenylpropanoid pathway plays a crucial role in both types of resistance.     

Up‐regulation of PR1 and less disruption of hormone and sucrose metabolism in roots is associated with lower susceptibility to ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las), is a devastating disease of citrus trees in Florida. Previous work showed that the rootstock cultivar Cleopatra mandarin (Citrus reticulata) has a higher population of Las in roots than Swingle citrumelo (C. paradisi × Poncirus trifoliata). Las reduced fibrous root biomass and sucrose content in Cleopatra mandarin more than in Swingle citrumelo. To understand the mechanisms for susceptibility to Las infection, sucrose and hormone metabolism status were evaluated in Cleopatra mandarin and Swingle citrumelo. In fibrous roots of Cleopatra mandarin, higher expression of genes related to sucrose cleavage was consistent with lower sucrose content compared to noninoculated seedlings at 5 weeks post‐root trimming (wpt). In fibrous roots of Swingle citrumelo, both sucrose content and gene expression related to sucrose cleavage were less disrupted by Las infection compared to Cleopatra mandarin at 5 wpt. Genes associated with salicylic acid (SA), ethylene (ET) and abscisic acid (ABA) synthesis, and ABA signalling, phospholipases D (PLD), and phospholipase A2 (PLA2) were activated by Las infection at 5 wpt in Cleopatra mandarin. Expression of downstream effectors of SA, i.e. NPR1, WRKY70 and PR1, did not change in Cleopatra mandarin, suggesting inhibition of the response to SA by the elevation of ABA, ET and PLD. In contrast, the up‐regulation of PR1, lower response of sucrose metabolism genes and down‐regulation of biosynthesis of phytohormones indicates that Swingle citrumelo activates a more effective defence against this biotrophic pathogen than Cleopatra mandarin.     

New insights into radiata pine seedling root infection by Fusarium circinatum

Pine root infection by Fusarium circinatum has been reported in the literature, but the underlying pathogenic interaction is poorly understood. A green fluorescent protein (GFP)‐tagged F. circinatum isolate, together with confocal microscopy, was used in order to monitor the events associated with root infection of Pinus radiata seedlings. It was found that in order to reach and successfully infect pine roots, F. circinatum employed features that are similar to those previously described for other root‐infecting pathogens, such as mycelial strands, single runner hyphae and simple hyphopodia as well as other features that are reminiscent of those that are known to be involved in biotrophic invasion, such as bulbous invasive hyphae and filamentous invasive hyphae. Abundant sporulation was observed at the root surface as well as inside tracheids both in roots and in the root collar region. The fungus can spread from the roots to the aerial parts of the plant, and once there, colonization appears to be similar to the process that occurs when the pathogen is inoculated in the stem. Wilting symptoms and plant demise may be the result of a reduction in water uptake by roots and of the blockage of the vascular system by fungal hyphae and resin.     

Sulphur supply impairs spread of Verticillium dahliae in tomato

Vascular wilt caused by the soil-borne fungus Verticillium dahliae is a major yield and quality-limiting disease across a broad spectrum of crop plants worldwide. Sulphur-enhanced plant defence mechanisms provide an opportunity to effectively and environmentally safely constrain the wilt disease levels in planta. To evaluate the influence of sulphur nutrition on the protective potential of these mechanisms, two near-isogenic tomato genotypes differing in fungal susceptibility, were treated with low or supra-optimal sulphur supply. Microscopic analysis revealed a significant sulphur-induced decrease in the amount of infected vascular cells in both genotypes. However, plant shoot and severely pathogen-affected root growth did not display this distinct alleviating influence of sulphur nutrition. Rates of leaf photosynthesis were impeded by Verticillium dahliae infection in both genotypes especially under low sulphur nutrition. However, assimilate transport rates in the phloem sap were enhanced by fungal infection more in the resistant genotype and under high sulphur nutrition suggesting a stronger sink for assimilates in infected plant tissues possibly involved in sugar-induced defence. A SYBR Green-based absolute quantitative Real-Time assay using a species-specific primer was developed which sensitively reflected sulphur nutrition-dependent changes in fungal colonization patterns. High sulphur nutrition significantly reduced fungal spread in the stem in both tomato genotypes. Concentrations of selected sulphur-containing metabolites revealed an increase of the major anti-oxidative redox buffer glutathione under high sulphur nutrition in response to fungal colonization. Our study demonstrates the existence of sulphur nutrition-enhanced resistance of tomato against Verticillium dahliae mediated by sulphur-containing defence compounds.     

Effect of plant roots on the germination of microsclerotia ofVerticillium dahliae

Induction of germination of microsclerotia by exudates from plant roots may be important for the control ofV. dahliae. Laboratory experiments with root observation boxes were carried out to assess the influence of root tips of seven crop species and cultivars on the germination of microsclerotia ofVerticillium dahliae in soil under controlled conditions. The root density of crops was measured in a field experiment. The results of the laboratory experiments and the field experiment were combined to estimate the total effect of crops on the population of microsclerotia in the field. Germination of microsclerotia was stimulated by all crops compared to a control without a crop. Among crops, roots of potato cvs Element and Astarte had a larger stimulation effect on microsclerotia than that of potato Ostara, pea, flax, sugar beet or onion. The number of hyphae per microsclerotium decreased with distance from the root surface regardless of the crop species or cultivar. Differences in root densities, in the affected root zones and in the stimulation effect on germination of microsclerotia caused large differences among crops in the effect on the population of microsclerotia in the soil. However, growing a rop with the special purpose to reduce the level ofV. dahliae inoculum in the soil is an inefficient control measure, because only a small part of the total soil volume is affected by roots and the number of hyphae per microscleroium affected is too low.Abbreviations MS microsclerotia, microsclerotium     

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.     

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

Histopathology of infection and colonization of Quercus ilex fine roots by Phytophthora cinnamomi

Quercus ilex is one of the European forest species most susceptible to root rot caused by the oomycete Phytophthora cinnamomi. This disease contributes to holm oak decline, a particularly serious problem in the ‘dehesas’ ecosystem of the southwestern Iberian Peninsula. This work describes the host–pathogen interaction of Q. ilex and P. cinnamomi, using new infection indices at the tissue level. Fine roots of 6‐month‐old saplings inoculated with P. cinnamomi were examined by light microscopy and a random pool of images was analysed in order to calculate different indices based on the measured area of pathogen structures. In the early stages of invasion, P. cinnamomi colonizes the apoplast and penetrates cortical cells with somatic structures. On reaching the parenchymatous tissues of the central cylinder, the pathogen develops different reproductive and survival structures inside the cells and then expands through the vascular system of the root. Some host responses were identified, such as cell wall thickening, accumulation of phenolic compounds in the middle lamella of sclerenchyma tissues, and mucilage secretion blocking vascular cells. New insights into the behaviour of P. cinnamomi inside fine roots are described. Host responses fail due to rapid expansion of the pathogen and a change in its behaviour from biotrophic to necrotrophic.     

Tolerance to a non-host isolate of Verticillium dahliae in tomato

Tolerance to Verticillium spp. is a condition in which a host plant develops few symptoms despite substantial colonization by the pathogen. In the present paper we have shown that Craigella tomatoes are tolerant to a non-host isolate of V. dahliae, Dvd E6. Symptom expression was used to quantify disease and quantitative PCR to assess the amount of fungus in the stems. The classical incompatible and compatible interactions between Craigella resistant or Craigella susceptible near isolines and V. dahliae, race 1 were used for comparative purposes. Additional experiments using cytological assessment and quantitative PCR showed that in the tolerant interactions one plant defence response, vascular coating, was deployed as effectively as in resistant plants, limiting pathogen distribution. However, a second defence response, which causes the cyclical elimination of fungus from the stem in the classical interactions either does not occur or is substantially delayed in tolerant plants. Thus, the Verticillium population remains stable and substantial throughout the studied time course.     

Reliable detection of unevenly distributed Verticillium dahliae in diseased olive trees

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.     

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.