Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

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.     

Effects of relative humidity on infection,colonization and conidiation of Magnaporthe orzyae on perennial ryegrass

Grey leaf spot, caused by Magnaporthe oryzae, causes severe damage on perennial ryegrass (Lolium perenne) turf. In this study, the effects of relative humidity (RH, 88 to 100% at 28°C) on infection, colonization and conidiation of M. oryzae on perennial ryegrass were investigated in controlled humidity chambers. Results showed that the RH threshold for successful M. oryzae infection was ≥92% at 28°C. The advancement of infection on the leaf tissue was further examined with a green fluorescent protein (GFP)‐tagged M. oryzae strain. No appressorium formation was found when the inoculum was incubated at RH ≤ 88%. Additionally, the GFP‐tagged staining provided a rapid method to quantitatively compare the fungal colonization from leaf tissue at different levels of RH. The fluorescence intensity data indicated that the fungal biomass was highest at 100% RH and there was no fluorescence intensity observed at 88% RH or below. Conidiation was only observed when RH was ≥96%, with the most abundant conidiation occurring 8 days after inoculation. Reduced conidiation was associated with decreasing RH, and no conidiation occurred at RH ≤ 92%. This study indicates that infection and conidiation of M. oryzae on perennial ryegrass required different thresholds: 92% and 96% RH for infection and conidiation, respectively. The quantitative data from this research will assist in prediction of grey leaf spot disease outbreaks and of secondary infection of perennial ryegrass.     

Effects of temperature,inoculum concentration,leaf age,and continuous and interrupted wetness on infection of olive plants by Spilocaea oleagina

Experiments were conducted on olive plants in controlled environments to determine the effect of conidial concentration, leaf age, temperature, continuous and interrupted leaf wetness periods, and relative humidity (RH) during the drier periods that interrupted wet periods, on olive leaf spot (OLS) severity. As inoculum concentration increased from 1·0 × 10² to 2·5 × 10⁵ conidia mL^?1, the severity of OLS increased at all five temperatures (5, 10, 15, 20 and 25°C). A simple polynomial model satisfactorily described the relationship between the inoculum concentration at the upper asymptote (maximum number of lesions) and temperature. The results showed that for the three leaf age groups tested (2–4, 6–8 and 10–12 weeks old) OLS severity decreased significantly (P < 0·001) with increasing leaf age at the time of inoculation. Overall, temperature also affected (P < 0·001) OLS severity, with the lesion numbers increasing gradually from 5°C to a maximum at 15°C, and then declining to a minimum at 25°C. When nine leaf wetness periods (0, 6, 12, 18, 24, 36, 48, 72 and 96 h) were tested at the same temperatures, the numbers of lesions increased with increasing leaf wetness period at all temperatures tested. The minimum leaf wetness periods for infection at 5, 10, 15, 20 and 25°C were 18, 12, 12, 12 and 24 h, respectively. The wet periods during early infection processes were interrupted with drying periods (0, 3, 6, 12, 18 and 24 h) at two levels of RH (70 and 100%). The length of drying period had a significant (P < 0·001) effect on disease severity, the effect depending on the RH during the interruption. High RH (100%) resulted in greater disease severity than low RH (70%). A polynomial equation with linear and quadratic terms of temperature, wetness and leaf age was developed to describe the effects of temperature, wetness and leaf age on OLS infection, which could be incorporated as a forecasting component of an integrated system for the control of OLS.     

Effects of temperature, leaf wetness and cultivar on the latent period of Mycosphaerella graminicola on winter wheat

After inoculation of winter wheat cv. Longbow at a single time, lesions of M. graminicola were produced over a long interval starting 15–35 days after inoculation, dependent on temperature. There was no evidence that a single infection gave rise to more than one lesion. After the initial infection period at 100% relative humidity (r.h.), keeping leaves wet for c. 10 h per day did not shorten latent period on seedlings. Experiments in controlled-environment chambers demonstrated a minimum latent period at approximately 17°C Variation in the latent period of individual lesions was also minimum at this temperature. The latent period varied among the cultivars tested, cv. Longbow having the shortest, cv. Avalon having almost the longest. Field observations broadly confirmed the results of experiments in constant-environment chambers.     

Integrated control of potato late blight: predicting the combined efficacy of host resistance and fungicides

Integrating cultivars that are partially resistant with reduced fungicide doses offers growers an opportunity to decrease fungicide input but still maintain disease control. To use integrated control strategies in practice requires a method to determine the combined effectiveness of particular cultivar and fungicide dose combinations. Simple models, such as additive dose models (ADM) and multiplicative survival models (MSM), have been used previously to determine the joint action of two or more pesticides. This study tests whether a model based on multiplicative survival principles can predict the joint action of fungicide doses combined with cultivars of differing partial host resistance. Data from eight field experiments on potato late blight (Phytophthora infestans) were used to test the model; the severity of foliar blight was assessed and scores used to calculate the area under the disease progress curve (AUDPC). A subset of data, derived from the most susceptible cultivar, King Edward, was used to produce dose–response curves from which parameter values were estimated, quantifying fungicide efficacy. These values, along with the untreated values for the more resistant cultivars, Cara and Sarpo Mira, were used to predict the combined efficacy of the remaining cultivar by fungicide dose combinations. Predicted efficacy was compared against observations from an independent subset of treatments from the field experiments. The analysis demonstrated that multiplicative survival principles can be applied to describe the joint efficacy of host resistance and fungicide dose combinations.     

Effects of temperature and wetness duration on conidial infection, latent period and asexual sporulation of Pyrenopeziza brassicae on leaves of oilseed rape

Experiments in controlled environments were carried out to determine the effects of temperature and leaf wetness duration on infection of oilseed rape leaves by conidia of the light leaf spot pathogen, Pyrenopeziza brassicae . Visible spore pustules developed on leaves of cv. Bristol inoculated with P. brassicae conidia at temperatures from 4 to 20°C, but not at 24°C; spore pustules developed when the leaf wetness duration after inoculation was longer than or equal to approximately 6 h at 12–20°C, 10 h at 8°C, 16 h at 6°C or 24 h at 4°C. On leaves of cvs. Capricorn or Cobra, light leaf spot symptoms developed at 8 and 16°C when the leaf wetness duration after inoculation was greater than 3 or 24 h, respectively. The latent period (the time period from inoculation to first spore pustules) of P. brassicae on cv. Bristol was, on average, approximately 10 days at 16°C when leaf wetness duration was 24 h, and increased to approximately 12 days as temperature increased to 20°C and to 26 days as temperature decreased to 4°C. At 8°C, an increase in leaf wetness duration from 10 to 72 h decreased the latent period from approximately 25 to 16 days; at 6°C, an increase in leaf wetness duration from 16 to 72 h decreased the latent period from approximately 23 to 17 days. The numbers of conidia produced were greatest at 12–16°C, and decreased as temperature decreased to 8°C or increased to 20°C. At temperatures from 8 to 20°C, an increase in leaf wetness duration from 6 to 24 h increased the production of conidia. There were linear relationships between the number of conidia produced on a leaf and the proportion of the leaf area covered by 'lesions' (both log₁₀-transformed) at different temperatures.     

Development of a lateral flow device for in‐field detection and evaluation of PCR‐based diagnostic methods for Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in‐field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross‐reacted with non‐target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 10⁵ cells mL^?1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first‐line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non‐scientists and is cost‐effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.     

Redefining the global populations of Pseudomonas syringae pv. actinidiae based on pathogenic,molecular and phenotypic characteristics

Knowing the population structure of a pathogen is fundamental for developing reliable phytosanitary legislation, detection techniques, and control strategies based on the actual aggressiveness and distribution of the pathogen. Currently, four populations of Pseudomonas syringae pv. actinidiae (Psa) have been described: Psa 1, Psa 2, Psa 3 and Psa 4. However, diagnostic assays specific for Psa populations do not detect Psa 4, the less virulent (LV) strains isolated in New Zealand. Similarly, multilocus sequence typing (MLST) of housekeeping genes, or broad Psa strain genome comparisons, revealed that Psa 4‐LV strains clustered separately from other Psa populations. In order to examine whether the placement of Psa 4 in the pathovar actinidiae was appropriate, various tests were carried out. It was shown that the Psa 4‐LV strains induced leaf and shoot wilting in Prunus cerasus, extensive necrotic lesions in Capsicum annuum fruits, and no significant symptoms in Actinidia deliciosa. Moreover, repetitive‐sequence PCR fingerprinting, type III secretion system effector protein genes detection and colony morphology clearly indicated the distinctiveness of Psa 4‐LV strains from the other three Psa populations. Rep‐PCR molecular typing revealed a high similarity of the Psa 4‐LV strains with members of Pseudomonas avellanae species. The Psa 4‐LV strains, most probably, belong to a new, still unnamed pathovar. It was concluded that the Psa 4‐LV strains isolated in New Zealand do not belong to the pathovar actinidiae, and, consequently, three Psa populations pathogenic to Actinidia spp. should currently include Psa 1, Psa 2 and Psa 3.     

Effects of application rate and interval on the efficacy of sprayable pheromone for mating disruption of the oriental fruit mothGrapholita molesta

The efficacy of microencapsulated sprayable pheromone was evaluated at different application rates and intervals for mating disruption of the oriental fruit moth,Grapholita molesta (Busck), in apple orchards during 2002. The following treatments were arranged in a randomized complete block design with three replications: (i) a low rate of pheromone (6.2 g a.i. ha⁻¹) applied at 14-day intervals, (ii) a medium rate of pheromone (12.4 g a.i. ha⁻¹) applied at 28-day intervals, (iii) a high rate of pheromone (24.7 g a.i. ha⁻¹) applied at 28- day intervals, and (iv) a non-pheromone control (insecticides only). The combination of a single insecticide application against first generationG. molesta at petal fall with one pheromone application each for the second, third and fourth generations at 12.4–24.7 g a.i. ha⁻¹ successfully controlled low populations. Pheromone-treated blocks. had significantly lower trap catches than those in the insecticide-treated control blocks. Among pheromone treatments, significantly more moths were caught in the 6.2 g compared with the 12.4 and 24.7 g rates. Fruit damage was <1% at harvest and there were no significant differences among treatments. Low rate frequent applications of sprayable formulation appeared to be effective under low pest pressure but efficacy declined with increasing populations. Further studies are needed to demonstrate the effectiveness of this approach under higher pest pressure. http://www.phytoparasitica.org posting July 13, 2005.     

The interacting effects of temperature,duration of wetness and inoculum size on the infection of pear blossoms by<Emphasis Type="Italic">Erwinia amylovora</Emphasis>, the causal agent of fire blight

Pear blossoms are the plant tissue that is most vulnerable to infection byErwinia amylovora (Burrill) Winslowet al., the causal agent of fire blight. The interacting effects of temperature, wetness duration and inoculum size on the development of fire blight symptoms in detached pear blossoms were determined in three sets of experiments conducted under controlled conditions. It was expected that this information would facilitate the improvement of a warning system used in fire blight management. Results of the ANOVA tests of the data revealed highly significant interactions among the factors tested. The factors that contributed most to disease incidence were temperature and inoculum size; effects of wetness duration were significant in some cases, but that effect was small. It was further demonstrated that the effects of the interaction of these factors on the incidence of blossom infection may be understood in terms of the general concept of compensation. According to this concept, conditions highly favorable for one of the factors essential for pathogen development may compensate for other factors, for which the conditions are less favorable. As a result of the complex interactions observed between the biotic and abiotic factors, because of compensation relationships and because some of the factors cannot be estimated adequately (for example, inoculum level), it was concluded that it is not yet possible to improve fire blight management by using data on the quantitative relationships between biotic and abiotic factors. Contribution No. 532/02 from the Agricultural Research Organization. http://www.phytoparasitica.org posting Feb. 20, 2003.