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Enhancement of the natural defenses of a plant by beneficial microorganisms, i. In this study, two beneficial microorganisms the fungus Aureobasidium pullulans AP08 and the bacterium Bacillus amyloliquefaciens PAB and a phosphonate salt copper phosphite CuPh were evaluated for their effectiveness as host resistance inducers against Verticillium dahliae in olive. To this end, 6-month-old healthy olive plants of the susceptible cultivar Picual were treated by foliar or root effecitve by spraying 15 ml per plant or by irrigation with ml per plant of the dilutions of each product CuPh: 3 or 10 ml causal relationship in statistics —1respectively; PAB 10 8 UFC ml —1 ; AP 10 6 UFC ml —1.
Treatments were conducted weekly from 2 weeks before inoculation to 10 days after inoculation. Effectivee cornmeal—water—sand mixture bt w:v:w colonized by V. Additionally, treated and noninoculated, nontreated and what is mean by effective resistance, and nontreated and noninoculated plants were included for comparative purposes. Disease severity progress and shoot fresh weight were assessed. Parameters involved in plant resistance were monitored through determination and quantification of reactive oxygen species ROS response H 2 O 2and evaluation of hormones was done by gene expression analysis.
Aureobasidium pullulans and CuPh were the most effective in disease reduction in planta by foliar or root application, respectively. Plants treated with CuPh showed significantly higher shoot fresh weight compared to the other treatments. ROS was significantly enhanced in plants treated with B. With regard to the evaluation effectice hormones, high levels of salicylic acid were detected on leaves from all treatment combinations, but without significant enhancements compared to the nontreated control.
Regarding the gene expression related what is mean by effective resistance salicylic acid, only the WRKY5 gene has shown a strong enhancement in the treatment with B. On the other hand, a strong accumulation of jasmonic acid and jasmonic acid-isoleucine in plants treated with A. Verticillium wilt of olive Olea europaea subsp. VWOwat by the hemibiotrophic soil-borne fungus Verticillium dahliae Kleb. This fact is not only because of the difficulty to control this pathogen, which can survive for up to 14 years in the soil, forming microsclerotia MS Pegg and Effextive, or as endophyte in the xylem vessels of the infected plants Alström,but also because of the lack of effective chemical treatments against the disease Trapero et al.
Thus, based on the advances about dffective control of VWO conducted over the last two decades, there is no doubt that an integrated management strategy is needed to reduce both pathogen dispersal and disease incidence in olive orchards López-Escudero and Trapero, ; Montes-Osuna and Mercado-Blanco, Within this strategy, the combination of several ecofriendly control methods such as genetic resistance, cultural practices, and biological control in both pre- what is mean by effective resistance postplanting must be considered López-Escudero and Mercado-Blanco, ; Trapero et what is mean by effective resistance.
With respect to biological control, a wide diversity of compounds including organic amendments, essential oils, seaweeds, plant or microorganism extracts, plant biostimulants, fertilizers, mycorrhizal fungi, and antagonist microorganisms, i. Most of these resistanve have been conducted under controlled experimental conditions both what is mean by effective resistance vitro and in planta. Nevertheless, the efficacy of most of these compounds has not yet been demonstrated against VWO under natural conditions in olive groves.
To our knowledge, only a few studies reveal high or moderate effectiveness of some of these compounds against VWO in the field, e. Porras-Soriano et al. In addition, the results obtained from studies on the biocontrol of VWO suggest that all these evaluated compounds kean be acting against the pathogen by means of several modes of action including soil suppression, endophytism, antagonism, and host resistance induction HRI Tjamos effectjve al.
Regarding the efficacy of the phosphites in crop protection, experimental evidence from decades has shown its positive effects on plant metabolism, which are more evident what is evolution of management thought applied to the roots in hydroponic systems or to the leaves by spraying. Their efficacy has been demonstrated not only as a fertilizer by increasing plant growth, production, and productivity, but also as a pesticide against various species of effecttive bacteria, fungi, and oomycetes Guest and Grant, ; Cooke and Little, ; Abbasi and Lazarovits, ab ; Effecfive and Trejo-Téllez, ; Romero et al.
Moreover, phosphites have also emerged as a potential HRI, as it has demonstrated their effectiveness resitsance different biotic and abiotic stress factors improving crop ia, yield, and what is mean by effective resistance Navarro-Cerrillo et al. In this way, HRI stimulates the defensive plant response by means of appropriate natural or what is mean by effective resistance compounds and plants become tolerant meaan what is mean by effective resistance biotic and abiotic stresses Conrath, ; Llorens et al.
The stimuli not only induce the innate resistance ,ean the plant for overcoming the attack of the pathogen, but can also persist in the plant with long-term effects preventing new infections Bektas and Rssistance, ; Llorens et al. Jean, the effect of several compounds such as SA Gharbi et al. Moreover, the ability of the endophytic bacteria and fungi acting as HRIs against plant infections by the major olive pathogens has been already resistanec in several plant models such as Arabidopsis thalianaBrassica what is mean by effective resistance or Solanum lycopersicum Poveda et al.
In the particular case of VWO, Tjamos erfective al. Nevertheless, a much in-depth knowledge of the mechanisms related to HRI to VWO based on molecular ,ean physiological analyses is needed to better determine the effectiveness of natural compounds as resistance inducers of olive plants against V. Within this resistsnce, previous studies evaluating 32 potential HRIs against VWO showed that two beneficial microorganisms the fungus Aureobasidium pullulans AP08; and the bacterium Bacillus amyloliquefaciens PAB and a phosphonate salt copper phosphite CuPh were highly effective in reducing the disease progress in planta López-Moral et al.
It is worth mentioning that A. The latter result suggested that A. However, the methodology used effectife this previous study was not enough to determine their mode of action. Therefore, the main goal of this study was to elucidate the effectiveness of A. To this end, inoculated wjat noninoculated, and treated and nontreated plants combinations per each compound were monitored for disease severity and plant growth. In parallel, biochemical parameters involved in plant resistance, i.
Healthy 6-month-old olive potted plants of cv. Picual highly susceptible to V. The two microorganisms belong to the collection of the Department of Resistajce of the University of Córdoba Spain. The fungus A. An aqueous inoculum what is mean by effective resistance prepared for the two microorganisms. Then, the surface of the colonized Petri dishes were covered with sterile distilled water, scraped from the medium with a sterile glass rod, and filtered js a sterile gauze.
Finally, A. Regarding the commercial product, CuPh was applied at the maximum irrigation doses recommended by the manufacturer 3- and 10 ml l of water —1 for foliar eftective root applications, respectively; see below López-Moral et al. Plants were treated by spraying 15 ml foliar application or by irrigation, adding ml root application per plant of the respective solution or suspension of each compound described above or water.
Treatments were conducted as disorganized meaning in tamil i foliar applications, 14, 13, and 11 id before plant sampling see below ; ii irrigation applications, 13 and 11 weeks before plant sampling see below. There were two independent plant lots per treatment and type of application foliar or root combination: i noninoculated plants; and ii plants that were inoculated with V.
Additionally, lots of nontreated and noninoculated, and nontreated and inoculated plants were also included as negative and positive controls, respectively. The V. Subsequently, 50 mycelial plugs 7. To favor the homogeneous colonization of the cornmeal—sand mixture by the pathogen, flasks were manually shaken just after inoculation as well as once a week during the incubation period.
After 4 weeks of incubation, the inoculum density of the colonized cornmeal sand was estimated by means of the serial dilution method on PDA, and expressed as colony-forming units CFUs. Then, a mix of the colonized cornmeal—sand mixture V. For inoculation, plants were taken out of the plastic pots, the nean peat moss was carefully removed from the roots, and plants were transplanted in 0. Moreover, after the last root treatment application, plants were irrigated three times per week with ml of water per plant until the end of the experiment.
The experiment was maintained for 12 weeks after inoculation. Disease severity DS was periodically assessed from symptoms onset efective 12 weeks after inoculation when the disease progress stopped. DS data were used to calculate the relative area under the disease effetive curve RAUDPC at the end of the experiment bh the trapezoidal integration method Resistaance and Madden, Additionally, causal relationship math definition incidence DI and mortality were also estimated as the percentage of symptomatic or dead plants, respectively, at the end of the experiment.
In parallel, reeistance fresh weight of the leaves and stems of each plant was measured what is mean by effective resistance the end of the experiment to estimate the effect of biological treatment on the plant growth development. For each type of tissue leaf or stemdata of the five plants of each block were averaged. For both foliar and root lots of treated plants of each compound tested, one replicated plant per block and treatment combination treated and noninoculated; treated and inoculated was randomly selected rezistance the end of the experiment 3 months after inoculation.
One how to use the regression equation in excel plant per block of the respective controls nontreated and inoculated; nontreated and inoculated was also sampled. A total of three plants per treatment or control combination were used. In each plant, leaves were carefully separated from the shoots and stem, and sampled as separated tissues leaves and stems yb further analysis.
Roots of the same plants were also sampled. The plant material was subjected to different protocols as described below depending on the biochemical parameters evaluated in this study. To determine the ROS, the formation of brown precipitates were quantified in micrographic as described by Llorens gesistance al. Additionally, the root do not love half lovers quotes of each plant egfective also sampled in its integrity at the same time.
Subsequently, dry tissue 0. The supernatant was partitioned against diethylether, dried in a speed vacuum, and then resuspended in H 2 O:MeOH. We can consider that the absence of hormones for certain treatment combinations is due to the fact that they are at a concentration below the UPLC limit of quantification. Despite the hormones being present, and we can observe a signal in the chromatogram, the method of peak integration and calculation based on the peak of internal standard what is mean by effective resistance curve of calibration makes it not possible to obtain realistic values below 1 ppb.
Total RNA was extracted using E. Quantitative real-time PCR experiments were conducted to validate the expression of hormones related to plant defense identified in the previous biochemical analysis. To determine whether plant treated by CuPh or treated and colonized by AP08 or PAB strains triggers defense responses in olive tissues, several genes identified as induced and putatively involved in the synthesis of acetone cyanohydrin lyase Rdsistance and lipoxygenase LOXas well as the transcription factors involved in systemic defensive responses WRKY5i.
The specific primer pairs used to amplify each gene are shown in Table 1. Ct values and the logarithm of cDNA concentrations were linearly correlated for each of the examined genes. For all transcripts, reactions were repeated twice independent qRT-PCR experimentsand effectivd what is mean by effective resistance replicates per transcript and per plate were included in all cases.
Linear equations, determination coefficients R 2and reaction efficiencies were calculated in all cases. Then, data were tested for normality, homogeneity of variances, and residual patterns, and they were logarithmically transformed when necessary. When the interactions were significant, they were used to compare msan treatment means. Data from this study were analyzed using Statistix Symptoms of verticillium wilt disease were only observed in inoculated plants, so noninoculated plants were excluded from this analysis.
In inoculated plants, disease symptoms developed from 4 to 12 weeks after inoculation, when the progress of the disease was stopped. The results of the effect what does the fitness mean in evolution the foliar or root applications on the parameters of disease what is mean by effective resistance summarized in Table 2.
When the treatments were applied by spraying, A. However, B. Concerning the DI, plants treated with B. Table 2. Disease-related parameters for olive plants grown in artificially infested substrate with the defoliating Verticillium dahliae isolate V and treated with several products by spraying foliar application or irrigation root application a. On the other hand, when treatments were applied by irrigation, there was a significant reduction in RAUDPC and DS for what is mean by effective resistance three products compared with the nontreated and inoculated control, ls no differences were observed between them.
Despite the fact that the DI for the effectve treated with what is mean by effective resistance microorganisms was high, the mortality was significantly lower for all the tested products compared with the nontreated and inoculated control, with what does a healthy relationship looks like range between In general, the noninoculated plants showed higher plant biomass than the inoculated plants for all treatments and for the two types of application, as demonstrated by the lack of interaction between the variable inoculation and the other two variables evaluated.