Effects of Leaf Wetness Duration, Temperature, and Host Phenological Stage on Infection of Walnut by Xanthomonas arboricola pv. juglandis.

Bacterial blight, caused by Xanthomonas arboricola pv. juglandis, is a significant disease affecting walnut production worldwide. Outbreaks are most severe in spring, and closely tied to host phenology and weather conditions. Pathogen infections are mainly observed in catkins, female flowers, leaves, and fruits. In this study, the effect of wetness duration and temperature on walnut infections by X. arboricola pv. juglandis was determined through two independent experiments conducted under controlled environmental conditions. The combined effect of both climatic parameters on disease severity was quantified using a third-order polynomial equation. The model obtained by linear regression and backward elimination technique fitted well to the data (R2 = 0.94 and R2adj = 0.93). The predictive capacity of the forecasting model was evaluated on pathogen-inoculated walnut plants exposed to different wetness duration-temperature combinations under Mediterranean field conditions. Observed disease severity in all events aligned with predicted infection risk. Additionally, the relationship between leaf and fruit age and the disease severity was quantified and modelled. A prediction model, which has been referred to as the WalBlight-risk model, is proposed for evaluation as an advisory system for timing bactericide sprays to manage bacterial blight in Mediterranean walnut orchards.

First report of Verticillium wilt and mortality of Ailanthus altissima caused by Verticillium dahliae and V. albo-atrum sensu lato in Spain.

Ailanthus altissima (Mill.) swingle is a highly invasive tree that has become established worldwide, especially in the Mediterranean Basin because of its good drought resistance. Ailanthus altissima is included in the list of Invasive Alien Species of the EU, so measures for eradication and management are required. Assessment for potential biological control agents is of great interest to manage this invasive tree in natural ecosystems. Verticillium dahliae Kleb. and Verticillium nonalfalfae Inderb. et al. (formerly V. albo-atrum Reinke & Berthold) have been reported as the causal agents of Verticillium wilt and mortality of ailanthus (Shall and Davis 2009; Rebbeck et al., 2013; Snyder et al., 2013; Brooks et al. 2020). Ailanthus trees with Verticillium wilt symptoms (wilt, premature defoliation, terminal dieback, yellow vascular discoloration, and mortality) were detected for the first time in 2007 in Celrà (42.040466N, 2.864436E) (Catalonia, Northeastern Spain), then spread to neighboring ailanthus populations. In 2018, ailanthus trees in a 570 km2 area in Catalonia were surveyed for disease symptoms. The incidence of wilt disease in ailanthus trees in forest ecosystems ranged from 50 to 90%, and the severity, 60 to 92%. One hundred and fifty branch samples showing wilt symptoms were collected and disinfected by immersion in 1% sodium hypochlorite for 2 min, then cut into 5mm pieces. These were placed onto PDA plates and incubated at 22.5 °C and 12 h light photoperiod for 7-10 days. Eighty-four tentative Verticillium sp. isolates were recovered and subcultured on modified water agar (WA-p) and PDA for identification (Inderbitzin et al. 2011, 2013). The majority of isolates (77 %) were identified as V. dahliae based on morphology; production of brown-pigmented microsclerotia and conidia features and dimensions (5.7 ± 0.9 µm long). Sequencing of mycelial DNA using primer pair ITS1-F and ITS4, resulted in consensus sequences of 503 bp. BLASTn analysis of ITS sequence of native isolate VdGi688 gave 100% identity to the ITS sequences of V. dahliae type strain PD322 (92% coverage) and Vd16_9 (100% coverage). In addition, 23% isolates morphologically corresponded to V. albo-atrum or V. nonalfalfae; melanized resting mycelia and round to oval-shaped conidia (5.2 ± 0.9 µm × 2.2 ± 0.5 µm). The ITS consensus sequence (544 bp) of native isolate VaaGi02 gave 99% identity (90-100 % coverage) to V. albo-atrum isolates CBS 127169, PSU 140, Vaa_TN1 and to V. nonalfalfae type PD592, CBS5451.88 and Vert 18. Sequences from isolates VdGi688 and VaaGi02 were deposited in GenBank as MW624723 and MW624724, respectively. Koch's postulates for seven V. dahliae isolates and eight V. albo-atrum isolates were fulfilled by injection of 1 mL of 1 x 107 conidia/mL suspension into the stem of A. altissima seedlings under greenhouse conditions. Six plants were inoculated per isolate in two independent experiments. Control plants were inoculated with sterile distilled water. All isolates caused leaf chlorosis, defoliation, and apical stem death, as well as internal necrosis and vascular discoloration. Control plants remained asymptomatic. The pathogens were re-isolated from internal symptomatic tissues of inoculated plants. To our knowledge, this is the first report of V. dahliae and V. albo-atrum sensu lato causing Verticillium wilt on A. altissima in Spain. The study suggests the potential of native isolates of Verticillum spp. in the biological control of ailanthus in the Mediterranean Basin. This work was funded by the Diputació de Girona (Spain) (2017/8719, 2019/3091, 2020/7565, and 2021/1468).

Effects of leaf wetness duration and temperature on infection of Prunus by Xanthomonas arboricola pv. pruni.

Xanthomonas arboricola pv. pruni is the causal agent of bacterial spot disease of stone fruits and almond. The bacterium is distributed throughout the major stone-fruit-producing areas of the World and is considered a quarantine organism in the European Union according to the Council Directive 2000/29/EC, and by the European and Mediterranean Plant Protection Organization. The effect of leaf wetness duration and temperature on infection of Prunus by X. arboricola pv. pruni was determined in controlled environment experiments. Potted plants of the peach-almond hybrid GF-677 were inoculated with bacterial suspensions and exposed to combinations of six leaf wetness durations (from 0 to 24 h) and seven fixed temperatures (from 5 to 35°C) during the infection period. Then, plants were transferred to a biosafety greenhouse, removed from bags, and incubated at optimal conditions for disease development. Although leaf wetness was required for infection of Prunus by X. arboricola pv. pruni, temperature had a greater effect than leaf wetness duration on disease severity. The combined effect of wetness duration and temperature on disease severity was quantified using a modification of the Weibull equation proposed by Duthie. The reduced-form of Duthie's model obtained by nonlinear regression analysis fitted well to data (R = 0.87 and R2adj = 0.85), and all parameters were significantly different from 0. The estimated optimal temperature for infection by X. arboricola pv. pruni was 28.9°C. Wetness periods longer than 10 h at temperatures close to 20°C, or 5 h at temperatures between 25 and 35°C were necessary to cause high disease severity. The predictive capacity of the model was evaluated using an additional set of data obtained from new wetness duration-temperature combinations. In 92% of the events the observed severity agreed with the predicted level of infection risk. The risk chart derived from the reduced form of Duthie's model can be used to estimate the potential risk for infection of Prunus by X. arboricola pv. pruni based on observed or forecasted temperature and wetness duration.

Epidemiological Features and Trends of Brown Spot of Pear Disease Based on the Diversity of Pathogen Populations and Climate Change Effects.

Brown spot of pear, caused by the fungus Stemphylium vesicarium, is an emerging disease of economic importance in several pear-growing areas in Europe. In recent years, new control strategies combining sanitation practices and fungicide applications according to developed forecasting models have been introduced to manage the disease. However, the pathogenic and saprophytic behavior of this pathogen makes it difficult to manage the disease. In addition, climate change can also result in variations in the severity and geographical distribution of the disease. In this study, ecological and epidemiological aspects of brown spot of pear disease related to inoculum characterization and climate change impact were elucidated. The pathogenic variation in S. vesicarium populations from pear orchards and its relationship to inoculum sources (air samples, leaf debris, and infected host and nonhost tissues) was determined using multivariate analysis. In total, six variables related to infection and disease development on cultivar Conference pear detached leaves of 110 S. vesicarium isolates were analyzed. A high proportion of isolates (42%) were nonpathogenic to pear; 85% of these nonpathogenic isolates were recovered from air samples. Most isolates recovered from lesions (93%) and pseudothecia (83%) were pathogenic to pear. A group of pathogenic isolates rapidly infected cultivar Conference pear leaves resulted in disease increase that followed a monomolecular model, whereas some S. vesicarium isolates required a period of time after inoculation to initiate infection and resulted in disease increase that followed a logistic model. The latter group was mainly composed of isolates recovered from pseudothecia on leaf debris, whereas the former group was mainly composed of isolates recovered from lesions on pear fruit and leaves. The relationship between the source of inoculum and pathogenic/aggressiveness profile was confirmed by principal component analysis. The effect of climate change on disease risk was analyzed in two pear-growing areas of Spain under two scenarios (A2 and B1) and for three periods (2005 to 2009, 2041 to 2060, and 2081 to 2100). Simulations showed that the level of risk predicted by BSPcast model increased to high or very high under the two scenarios and was differentially distributed in the two regions. This study is an example of how epidemiological models can be used to predict not only the onset of infections but also how climate change could affect brown spot of pear. [Formula: see text] Copyright © 2018 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

A model for predicting Xanthomonas arboricola pv. pruni growth as a function of temperature.

A two-step modeling approach was used for predicting the effect of temperature on the growth of Xanthomonas arboricola pv. pruni, causal agent of bacterial spot disease of stone fruit. The in vitro growth of seven strains was monitored at temperatures from 5 to 35°C with a Bioscreen C system, and a calibrating equation was generated for converting optical densities to viable counts. In primary modeling, Baranyi, Buchanan, and modified Gompertz equations were fitted to viable count growth curves over the entire temperature range. The modified Gompertz model showed the best fit to the data, and it was selected to estimate the bacterial growth parameters at each temperature. Secondary modeling of maximum specific growth rate as a function of temperature was performed by using the Ratkowsky model and its variations. The modified Ratkowsky model showed the best goodness of fit to maximum specific growth rate estimates, and it was validated successfully for the seven strains at four additional temperatures. The model generated in this work will be used for predicting temperature-based Xanthomonas arboricola pv. pruni growth rate and derived potential daily doublings, and included as the inoculum potential component of a bacterial spot of stone fruit disease forecaster.

Interaction of antifungal peptide BP15 with Stemphylium vesicarium, the causal agent of brown spot of pear.

Peptide BP15 has shown antifungal activity against several plant pathogenic fungi, including Stemphylium vesicarium, the causal agent of brown spot of pear. BP15 inhibits the germination, growth and sporulation of S. vesicarium and displays post-infection activity by stopping fungal infection in pear leaves. In this work, live-cell imaging was undertaken to understand the antifungal mechanism of BP15. A double-staining method based on the combination of calcofluor white and SYTOX green coupled with epifluorescence microscopy was used to investigate fungal cell permeabilization and alterations in fungal growth induced by BP15. GFP-transformants of S. vesicarium were obtained and exposed to rhodamine-labelled BP15. Confocal laser microscopy provided evidence of peptide internalization by hyphae, resulting in fungal cell disorganization and death. S. vesicarium membrane permeabilization by BP15 was found to be peptide-concentration dependent. BP15 at MIC and sub-MIC concentrations (10 and 5 µM, respectively) inhibited S. vesicarium growth and produced morphological alterations to germ tubes, with slow and discontinuous compromise of fungal cell membranes. Fungal cell membrane disruption was immediately induced by BP15 at 100 µM, and this was accompanied by rapid peptide internalization by S. vesicarium hyphae. Peptide BP15 interacted with germ tubes and hyphae of S. vesicarium but not with conidial cells.

Combined morphological and molecular approach for identification of Stemphylium vesicarium inoculum in pear orchards.

Stemphylium vesicarium is the causal agent of brown spot of pear, an important disease reported in pear-growing areas of Europe. The pathogen is able to colonize pear leaf debris and dead tissues of herbaceous plants and produce abundant ascospores and conidia that are capable of infecting pear trees. Inoculum monitoring in pear orchards is mainly achieved through spore traps and species identification is based on conidial morphology, but the similarities on conidial traits among species of Stemphylium make correct identification difficult. In this work a total of thirty-seven Stemphylium isolates from pear orchards were characterized at the morphological, pathogenic, and molecular level. Correspondence among ITS and gpd sequences and morphological traits were evaluated. Species identification based exclusively on morphological data was not feasible. Combined morphological and molecular data were necessary for unambiguous identification of isolates in the S. vesicarium species group. Only isolates identified as S. vesicarium were pathogenic on pear. The study revealed that several species of Stemphylium coexist in pear orchards with S. vesicarium, the causal agent of BSP, and that combined morphological and molecular data are needed to differentiate them. Consequently, direct measurements of the airborne inoculum using volumetric spore traps may overestimate the actual pathogen population.

Controlling Brown Spot of Pear by a Synthetic Antimicrobial Peptide Under Field Conditions.

Brown spot of pear, caused by Stemphylium vesicarium, is a fungal disease of increasing importance in several pear-growing areas of Europe. Disease control measures include the application of fungicides and sanitation methods. Antimicrobial peptides may be a complement or alternative to conventional fungicides used to manage brown spot disease. In a previous study, the synthetic peptide BP15 showed postinfection fungicidal activity against S. vesicarium in in vitro and detached-leaf assays. In the present study, the efficacy of BP15 (KKLFKKILKVL-NH2) in controlling brown spot of pear was evaluated under field conditions using potted plants and pear trees in orchards. In field trials, the treatments with BP15 or with the fungicide thiram were scheduled according to the infection risk predicted by the BSPcast model. Potted pear plants treated with BP15 showed a disease reduction of about 42 to 60% in five of seven trials. In three of four tree trials, the disease severity on shoots treated with BP15 was significantly lower than in the nontreated controls, with a mean efficacy of 38.2%. It was concluded that BP15 is a good candidate to be further developed as a fungicide for controlling brown spot of pear.

An update on control of brown spot of pear.

Brown spot of pear is a fungal disease producing high economical losses in several pear-growing areas in Europe. Fungicide applications during the growing period either at fixed schedule or delivered according to the BSPcast forecasting system are not enough to control the disease under favorable conditions. New strategies have been introduced to control the inoculum production using sanitation methods. These methods are based on combinations of leaf litter removal during winter and biological control agent applications during late winter, spring and summer. These practices reduce both the inoculum pressure and disease levels. Therefore, the resulting optimized disease management consists of a combination of sanitation methods applied during the whole year with chemical fungicides scheduled according to the BSPcast forecasting model during the vegetative period. It is expected that the control of brown spot could be further refined upon availability of rapid methods for inoculum potential analysis. However, this analysis is difficult due to the variability in pathogenicity within the pathogen population.

Apical Necrosis and Premature Drop of Persian (English) Walnut Fruit Caused by Xanthomonas arboricola pv. juglandis.

Etiological and epidemiological aspects of apical necrosis of walnut fruit were studied on cultivars Chandler, Franquette, and Hartley in a Spanish walnut orchard during 2007 and 2008. Affected fruit showed brown necrosis beginning at the blossom end of nuts; these symptoms differed from lesions of common blight of walnut (Xanthomonas arboricola pv. juglandis). X. arboricola pv. juglandis was consistently isolated from apical lesions throughout the growing season. Field isolates reproduced symptoms observed in the orchard when inoculated on immature detached walnut fruit in the laboratory. Sporadic occurrence of Fusarium spp. and Alternaria spp., mainly in dropped fruit, was attributed to secondary colonization of apical lesions that were originally caused by X. arboricola pv. juglandis. Apical necrosis and common blight were similar in disease epidemiology and cultivar susceptibility; a major increase in epidemics occurred at initial fruit development, and cvs. Chandler and Hartley were more affected than cv. Franquette. Our results suggest that apical necrosis is a new manifestation of walnut blight characterized by distinct symptoms and severe premature fruit drop.

Evaluation of four whole-plant inoculation methods to analyze the pathogenicity of Erwinia amylovora under quarantine conditions.

Four methods were tested to assess the fire-blight disease response on grafted pear plants. The leaves of the plants were inoculated with Erwinia amylovora suspensions by pricking with clamps, cutting with scissors, local infiltration, and painting a bacterial suspension onto the leaves with a paintbrush. The effects of the inoculation methods were studied in dose-time-response experiments carried out in climate chambers under quarantine conditions. A modified Gompertz model was used to analyze the disease-time relatiobbnships and provided information on the rate of infection progression (rg) and time delay to the start of symptoms (t0). The disease-pathogen-dose relationships were analyzed according to a hyperbolic saturation model in which the median effective dose (ED50) of the pathogen and maximum disease level (ymax) were determined. Localized infiltration into the leaf mesophile resulted in the early (short t0) but slow (low rg) development of infection whereas in leaves pricked with clamps disease symptoms developed late (long t0) but rapidly (high rg). Paintbrush inoculation of the plants resulted in an incubation period of medium length, a moderate rate of infection progression, and low ymax values. In leaves inoculated with scissors, fire-blight symptoms developed early (short t0) and rapidly (high rg), and with the lowest ED50 and the highest ymax.

Evaluation of four whole-plant inoculation methods to analyze the pathogenicity of Erwinia amylovora under quarantine conditions

Four methods were tested to assess the fire-blight disease response on grafted pear plants. The leaves of the plants were inoculated with Erwinia amylovora suspensions by pricking with clamps, cutting with scissors, local infiltration, and painting a bacterial suspension onto the leaves with a paintbrush. The effects of the inoculation methods were studied in dose-time-response experiments carried out in climate chambers under quarantine conditions. A modified Gompertz model was used to analyze the disease-time relatiobbnships and provided information on the rate of infection progression (rg) and time delay to the start of symptoms (t0). The disease-pathogen-dose relationships were analyzed according to a hyperbolic saturation model in which the median effective dose (ED50) of the pathogen and maximum disease level (ymax) were determined. Localized infiltration into the leaf mesophile resulted in the early (short t0) but slow (low rg) development of infection whereas in leaves pricked with clamps disease symptoms developed late (long t0) but rapidly (high rg). Paintbrush inoculation of the plants resulted in an incubation period of medium length, a moderate rate of infection progression, and low ymax values. In leaves inoculated with scissors, fire-blight symptoms developed early (short t0) and rapidly (high rg), and with the lowest ED50 and the highest (AU)

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Evaluation of a Reduced Copper Spraying Program to Control Bacterial Blight of Walnut.

Walnut blight, caused by Xanthomonas arboricola pv. juglandis, is currently controlled in western Europe through a standard schedule of seven applications of sprayed copper from bud break until harvest. A reduced spray schedule, with the last four applications omitted, was compared to the standard program in experimental plots for 3 years. Bacterial population levels in the spring were not significantly different between trees subjected to reduced sprays and those subjected to the standard schedule, but in summer they were higher in the trees that received fewer sprays compared with the standard program. However, disease control on nuts was similar or even better with the reduced spraying program than with the standard program, with the additional economic benefit of four fewer copper applications. After 3 years of using the reduced spray program, the amount of copper accumulated in the soil was significantly lower (about half) than that found in the soil where the standard spray program was implemented.