Search

Citrus black spot (CBS), caused by Phyllosticta citricarpa , is an important fungal disease of citrus. Higher CBS severity has been associated with P. citricarpa infections at the young and green stages of fruit. The length of the fruit susceptibility period may be influenced by the amount of inoculum and the climate of the citrus growing region. This study was conducted in orchards across two South African provinces to assess the intensity of CBS under two conditions: (i) unsprayed fruit of Valencia sweet orange, Nova, and Empress mandarins inoculated with P. citricarpa at 10 1 , 10 3 , and 10 5 pycnidiospores/mL at various intervals after fruit set, from October (petal fall stage) to March (for mandarins) or June (for sweet orange), and (ii) non-inoculated Valencia fruit from orchards subjected to spray programs ranging from one to nine applications between October and June. Higher CBS intensity was observed in Valencia fruit inoculated between November and January, with an average disease severity index (DSI) of 0.94, compared to a significantly lower (0.07) for fruit inoculated from February to June. Inoculations in November or December for Nova mandarins and in December for Empress with 10 5 pycnidiospores/mL resulted in higher CBS intensity compared to those with lower pycnidiospore concentrations. The absence of fungicide applications from October to December during the 2017-2018 season led to increased CBS intensity, with a DSI of ~0.47, a value which was not significantly different from that of non-treated control (NTC) trees. Leaving sweet orange trees unprotected from January onwards did not significantly increase the CBS intensity. These results confirm that citrus growers should focus on protecting citrus fruit, against P. citricarpa infection, in their dark green and young stages (c.15-30 mm diameter) to control CBS., Competing Interests: The authors declare no conflicts of interest., (© 2025 The Author(s). Plant‐Environment Interactions published by New Phytologist Foundation and John Wiley & Sons Ltd.)

The COVID-19 pandemic highlighted the need for rapid and sensitive diagnostic tools. In this work, the Magnetophoretic Slider Assay (MeSA) was integrated with electrochemical detection (eMeSA) using screen-printed carbon electrodes for the first time for the detection of SARS-CoV-2 nucleocapsid protein (NP). A sandwich enzyme-linked immunosorbent assay (ELISA) was performed on streptavidin-labeled magnetic beads (MBs). The streptavidin MB/biotinylated antibody/NP complexes were added into the sample inlet, where the beads were trapped using an external magnet while the solution rehydrated the HRP-labeled antibody (HRP-Ab) and 3,3',5,5'-tetramethylbenzidine (TMB) pads. By sliding the external magnet along the channel, the bead complexes were moved to the reservoir under the HRP-Ab pad, forming sandwich complexes. These complexes were subsequently moved back across the device to reach the electrochemical detection zone, where they reacted with released TMB, which underwent oxidation upon reacting with HRP attached to the detection antibody, followed by reduction due to the voltage applied to the working electrode (0.0 V vs. Ag reference electrode). The assay showed promising results in detecting SARS-CoV-2 in 10 min, with a limit of detection of 8.89 ng/mL NP and 78.02 PFU/mL inactivated virus. The results from 15 human samples demonstrated 100% clinical specificity and 100% clinical sensitivity for samples with RT-PCR cycle threshold (C t ) values from 19 to 30, meeting WHO criteria for COVID-19 diagnostics. The eMeSA offers an alternative to traditional ELISA for a wide range of point-of-care and point-of-need diagnostic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this work, the electrochemical behavior of the glycosylated flavonoid kaempferitrin was studied, and an electroanalytical methodology was developed for its determination in infusions of Bauhinia forficata using a boron-doped diamond electrode (BDD). The electrochemical behavior of the flavonoid was studied by cyclic voltammetry, and two irreversible oxidation peaks at 0.80 and 1.0 V vs Ag/AgCl were observed. The influence of the pH on the voltammograms was examined, and higher sensitivity was found at pH 7.0. The electrochemical process corresponding to peak 1 at 0.80 V is predominantly diffusion-controlled, as the study shows at varying scan rates. An analytical plot was obtained by square wave voltammetry at optimized experimental conditions (frequency = 100 s -1 , amplitude = 90 mV, and step potential = 8 mV) in the concentration range from 3.4 μmol L -1 to 58 μmol L -1 , with a linearity of 0.99. The limit of detection and limit of quantification values were 1.0 μmol L -1 and 3.4 μmol L -1 , respectively. Three samples of Bauhinia forficata infusions (2 g of sample in 100 mL of water) were analyzed, and the KF values found were 5.0 × 10 -4  mol L -1 , 3.0 × 10 -4  mol L -1 , and 7.0 × 10 -4  mol L -1 , with recovery percentages of 98 %, 106 % and 94 %, respectively. Finally, experiments were performed with two other flavonoids (chrysin and apeginin) to compare and propose an electrochemical oxidation mechanism for kaempferitrin, which was supported by quantum chemical calculations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H 2 O 2 ), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at -0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 μg kg -1 and 10 μg kg -1 , and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Classical approaches for controlling plant pathogens may be impaired by the development of pathogen resistance to chemical pesticides and by limited availability of effective antimicrobial agents. Recent increases in consumer awareness of and/or legislation regarding environmental and human health, and the urgent need to improve food security, are driving increased demand for safer antimicrobial strategies. Therefore, there is a need for a step change in the approaches used for controlling pre- and post-harvest diseases and foodborne human pathogens. The use of light-activated antimicrobial substances for the so-called antimicrobial photodynamic treatment is known to be effective not only in a clinical context, but also for use in agriculture to control plant-pathogenic fungi and bacteria, and to eliminate foodborne human pathogens from seeds, sprouted seeds, fruits, and vegetables. Here, we take a holistic approach to review and re-evaluate recent findings on: (i) the ecology of naturally-occurring photoantimicrobials, (ii) photodynamic processes including the light-activated antimicrobial activities of some plant metabolites, and (iii) fungus-induced photosensitization of plants. The inhibitory mechanisms of both natural and synthetic light-activated substances, known as photosensitizers, are discussed in the contexts of microbial stress biology and agricultural biotechnology. Their modes-of-antimicrobial action make them neither stressors nor toxins/toxicants (with specific modes of poisonous activity), but a hybrid/combination of both. We highlight the use of photoantimicrobials for the control of plant-pathogenic fungi and quantify their potential contribution to global food security., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Citrus black spot (CBS) is a disease caused by the fungus Phyllosticta citricarpa that affects citrus plants, causing fruit blemish and premature drop that result in severe economic losses in commercial citrus orchards. However, CBS symptoms and effects may vary depending on the citrus species: Citrus limon (lemon) is susceptible and highly affected by the disease, while no CBS-related damage has ever been observed for Citrus latifolia (Tahiti lime), implying that it must be resistant to the disease. The difference in the response to this disease provided the opportunity to gain insight into the metabolites responsible for the resistance by comparison of the metabolomic profiles of these two citrus species. Metabolic variations of C. limon and C. latifolia inoculated with P. citricarpa were analyzed using various metabolomic-based platforms including 1 H NMR for overall metabolic profiling, and LC-MS and HPTLC for targeted analysis. The 1 H NMR spectra of the samples demonstrated that certain phenolics were strongly induced after pathogenic inoculation, especially in the resistant species. The induced phenolics were identified from C. latifolia by further 1 H NMR, LCMS and HPTLC analysis yielding six prenylated and methoxy coumarins, i.e., 5,7-dimethoxycoumarin, 5-geranyloxy-7-methoxycoumarin, 7-geranyloxycoumarin, 8-methoxypsoralen, 5,8-dimethoxypsoralen and 5-geranyloxypsoralen. These isolated coumarins and a coumarin-rich fraction were tested against the fungal pathogen, P. citricarpa , to evaluate their activity. None of the individual coumarins exhibited a significant inhibition, while the coumarin fraction exhibited a strong antifungal activity suggesting a synergistic interaction of its components. To obtain further insight into the roles of these compounds in the plant defense, the possible mechanisms of the individual coumarins were tested using an in-silico model, the PASS Online Tool; the analysis showed that each coumarin appeared to have a unique defense mechanism, even with very slight variations in the chemical structures. The results could provide evidence of the existence of a complex plant defense mechanism consisting in a multitude of synergistic interactions between compounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fernandes, Salomé-Abarca, Gonçalves Pereira, Brandão Seibert, Silva-Junior, Das Graças Fernandes da Silva and Choi.)

Background: Despite technical improvements in the citrus chain and leadership in orange production achieved in the past decades, premature fruit drop remains a major component of crop loss in São Paulo state citrus belt, the largest sweet orange production area in the world. The present study aimed to determine, during five consecutive seasons, the impact of the diseases and pests on premature fruit drop in the orange belt., Results: Fruit drop due to the main diseases and pests averaged approximately 11.0%, which corresponded to approximately 63% of the annual fruit drop. The average fruit drop rate due to fruit borer and fruit flies combined was 4.0%, Huanglongbing (HLB) 3.3%, black spot 2.6%, leprosis 1.0% and citrus canker 0.3%. The average amount of fruit drop (million 40.8 kg boxes) and value of crop losses (million US$ dollars), in five seasons, were 12.7 and 66.2 for fruit borer/fruit flies, 11.0 and 57.9 for HLB, 8.1 and 42.2 for black spot, 3.1 and 15.6 for leprosis, and 0.9 and 4.9 for citrus canker, respectively., Conclusion: Fruit borer and fruit flies (combined), HLB, black spot, leprosis and citrus canker are, in this order, the main diseases and pests in the orange belt of São Paulo state. All of these causes significantly increased the overall fruit drop rate in the evaluated seasons. The results will contribute to the development of the Brazilian citrus industry, while showing to other citrus-growing regions the potential that diseases and pests have to jeopardize production. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Background: Mineral oil added to fungicide spray mixtures has been a frequently used strategy to control citrus black spot (CBS) worldwide. Although mineral oil may increase the efficacy of control, its use represents around 15% of the costs of a CBS spray program. This study aimed to assess the performance of different proportions of mineral oil added to a fungicide tank mixture for CBS control in young (less than 10 years old) and old (more than 12 years old) sweet orange orchards of early ('Hamlin'), mid-season ('Pera') and late-maturing ('Valencia') cultivars in São Paulo state, Brazil. The efficacy of 0.15%, 0.20% or 0.25% mineral oil added to a fungicide spray mixture was determined by assessing CBS incidence, severity and fruit drop in six orchards over two seasons., Results: Fungicide programs with or without oil were effective in reducing 100% CBS symptom expression in both young and old 'Hamlin' orchards and in the young 'Pera' orchard. The lowest mineral oil rate tested (0.15%) showed a reduction in CBS intensity of around 90%, similar to the highest rate tested in the old 'Pera' orchard. The highest cost-benefit program to control CBS in the old 'Valencia' orchard was obtained with the mineral oil rate of 0.25%, commonly used in the São Paulo citrus belt, which reduced CBS severity by up to 97%., Conclusion: This study demonstrated that mineral oil rates for CBS control can be adjusted according to tree age and cultivar. These findings contribute to the establishment of more sustainable citrus production by reducing spray costs while maintaining the efficacy of CBS control. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

The widespread use of conventional chemical antifungal agents has led to worldwide concern regarding the selection of resistant isolates. In this scenario, antimicrobial photodynamic treatment (APDT) has emerged as a promising alternative to overcome this issue. The technique is based on the use of a photosensitizer (PS) and light in the presence of molecular oxygen. Under these conditions, the PS generates reactive oxygen species which damage the biomolecules of the target organism leading to cell death. The great potential of APDT against plant-pathogenic fungi has already been reported both in vitro and in planta, indicating this control measure has the potential to be widely used in crop plants. However, there is a lack of studies on environmental risk with ecotoxicological assessment of PSs used in APDT. Therefore, this study aimed to evaluate the environmental toxicity of four phenothiazinium PSs: i) methylene blue (MB), ii) new methylene blue N (NMBN), iii) toluidine blue O (TBO), and iv) dimethylmethylene blue (DMMB) and also of the commercial antifungal NATIVO®, a mixture of trifloxystrobin and tebuconazole. The experiments were performed with Daphnia similis neonates and zebrafish embryos. Our results showed that the PSs tested had different levels of toxicity, with MB being the less toxic and DMMB being the most. Nonetheless, the environmental toxicity of these PSs were lower when compared to that of NATIVO®. Furthermore, estimates of bioconcentration and of biotransformation half-life indicated that the PSs are environmentally safer than NATIVO®. Taken together, our results show that the toxicity associated with phenothiazinium PSs would not constitute an impediment to their use in APDT. Therefore, APDT is a promising approach to control plant-pathogenic fungi with reduced risk for selecting resistant isolates and lower environmental impacts when compared to commonly used antifungal agents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this work, nanoporous gold (NPG) was prepared according to three different approaches, such as (i) anodization-electrochemical reduction (A-ECR, NPG A ), (ii) dynamic hydrogen bubble template (DHBT, NPG B ), and (iii) the combination of both methods (NPG A+B ). Field-emission scanning electron microscopy (FE-SEM) and cyclic voltammetry (CV) were used to investigate the structural morphology and the electrochemical behavior of the fabricated materials. The NPG A+B electrode showed a large amount of surface defects and/or edges, greater electrochemical surface area (2.5 cm 2 ), and increased roughness factor (35.4). Such outstanding features of the NPG A+B platform were demonstrated by the sensitive detection of methyl parathion (MP) in river water samples. CV results indicated nearly 25-fold, 6-fold, and 2.5-fold higher sensitivity for NPG A+B compared to that of bare Au, NPG A , and NPG B , respectively. Differential pulse voltammetry (DPV) results show a linear behavior in the MP concentration range of 5-50 ng mL -1 with a limit of detection (LOD) of 0.6 ng mL -1 and limit of quantification (LOQ) of 2.0 ng mL -1 . Besides, the NPG A+B sensor also revealed excellent selectivity towards MP detection in the presence of other interfering molecules or ions, reproducibility, and repeatability., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Copper is the most efficient pesticide for the control of citrus canker ( Xanthomonas citri subsp. citri ). To mitigate the environmental impacts and costs, the copper sprays in citrus orchards are being optimized based on the tree row volume (TRV). A previous investigation allowed for significant reductions of the spray volume and copper rates. Nevertheless, the results also indicated the need for additional studies. The aim of this work was to assess whether both the spray volume and the metallic copper rate based on the TRV may be further reduced. A field trial was carried out during two seasons in a 3-year-old commercial orchard of Pera sweet orange located in the municipality of Paranavaí, Paraná, Brazil. The volumes of 20 and 40 ml of spray mixture per m 3 of the tree canopy were assessed in combination with the metallic copper rates of 10.5, 21.0, 36.8, or 52.5 mg/m 3 . Disease was measured as the temporal progress of canker incidence on leaves, cumulative dropped fruit with canker, and incidence of diseased fruit at harvest. The quality of sprays was assessed by measuring the copper deposition and leaf coverage. The treatment with the highest citrus canker control for the lowest use of water and copper was the combination of 40 ml and 36.8 mg/m 3 . Regression analyses indicated that the minimum threshold deposition of copper was ∼1.5 µg Cu 2+ /cm 2 leaf area. In addition, the lowest spray volume and copper rate necessary to achieve this deposition are 35 ml/m 3 and 30 mg/m 3 . The use of 20 ml/m 3 did not efficiently control the disease due to the deficient coverage of treated surfaces. This study demonstrated that it is possible to use even lower amounts of copper and water without interfering with the efficiency of control of citrus canker.

Postbloom fruit drop (PFD) of citrus is caused by the Colletotrichum acutatum and C. gloeosporioides species complexes. The disease is important when frequent rainfall occurs during the flowering period of citrus trees. In Brazil, until 2012, PFD was mainly controlled by preventive applications of the methyl-benzimidazole carbamate (MBC) carbendazim and demethylation-inhibitor (DMI) fungicides such as difenoconazole. Since then, mixtures containing the DMI tebuconazole and the quinone-outside inhibitor (QoI) trifloxystrobin have been commonly used. Fungicides are often applied preventively, sometimes even when conditions are not conducive for PFD development. Excessive fungicide applications may favor the selection of resistant populations of Colletotrichum spp. In this study, we assessed the fungicide sensitivity of C. acutatum isolates collected during the two distinct periods of PFD management in Brazil: before and after the trifloxystrobin and tebuconazole mixture became widely employed. The sensitivity of 254 C. acutatum isolates to carbendazim and difenoconazole and of 164 isolates to tebuconazole and trifloxystrobin was assessed. Mycelial growth inhibition of these isolates was evaluated for all the fungicides using either serial dilution of fungicide rates or the spiral gradient dilution method. In addition, inhibition of conidial germination was also assessed for trifloxystrobin. Analysis of partial β-tub , cytb , and cyp51b gene sequences did not reveal any mutations related to resistance to MBCs, QoIs, and DMIs, respectively. In mycelial growth assays, mean EC 50 values were 0.14, 0.11, and 0.21 μg/ml for difenoconazole, tebuconazole, and trifloxystrobin, respectively. The conidial germination inhibition by trifloxystrobin was similar among the tested isolates, and the mean EC 50 value was 0.002 μg/ml. All isolates had similar mean mycelial growth inhibition for carbendazim, regardless of the fungicide concentrations. Therefore, based on similar EC 50 values and molecular analyses, no shift in the sensitivity of isolates has been observed to the fungicides commonly used in different citrus-producing areas in Brazil.

Citrus postbloom fruit drop, caused by Colletotrichum spp., is an important disease in the Americas. The pathogen infects citrus flowers, produces orange-brown lesions on petals, and may cause the abscission of young fruit. In diseased flowers, the calyxes remain attached to the peduncle after the young fruit drop. No anatomical and microanalysis studies have been conducted to determine whether calyx tissues can be infected by Colletotrichum spp. and why calyxes remain attached to the peduncle. Based on light microscopy, we demonstrate that the ovary abscission zone exhibits a separation region composed of layers of thickened lignified walled cells, indicating that abscission involves the disruption of cell walls. The first layers of the protective zone (PZ) are composed of densely packed cells with suberized walls produced by the wound meristem. Beneath the PZ, there is a compact mass of small cells that accumulate starch grains. X-ray fluorescence microanalysis (µ-XRF) confirmed the increased accumulation of calcium in the receptacle of the persistent calyxes compared to non-inoculated citrus flowers. Moreover, the peduncle pith and the receptacle exhibit hypertrophied cells with thick walls that may be related to calyx retention. Fungal structures are not observed inside the persistent calyx tissues.

Several Phyllosticta species are known as pathogens of Citrus spp., and are responsible for various disease symptoms including leaf and fruit spots. One of the most important species is P. citricarpa, which causes a foliar and fruit disease called citrus black spot. The Phyllosticta species occurring on citrus can most effectively be distinguished from P. citricarpa by means of multilocus DNA sequence data. Recent studies also demonstrated P. citricarpa to be heterothallic, and reported successful mating in the laboratory. Since the domestication of citrus, different clones of P. citricarpa have escaped Asia to other continents via trade routes, with obvious disease management consequences. This pathogen profile represents a comprehensive literature review of this pathogen and allied taxa associated with citrus, focusing on identification, distribution, genomics, epidemiology and disease management. This review also considers the knowledge emerging from seven genomes of Phyllosticta spp., demonstrating unknown aspects of these species, including their mating behaviour., Taxonomy: Phyllosticta citricarpa (McAlpine) Aa, 1973. Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Botryosphaeriales, Family Phyllostictaceae, Genus Phyllosticta, Species citricarpa., Host Range: Confirmed on more than 12 Citrus species, Phyllosticta citricarpa has only been found on plant species in the Rutaceae., Disease Symptoms: P. citricarpa causes diverse symptoms such as hard spot, virulent spot, false melanose and freckle spot on fruit, and necrotic lesions on leaves and twigs., Useful Websites: DOE Joint Genome Institute MycoCosm portals for the Phyllosticta capitalensis (https://genome.jgi.doe.gov/Phycap1), P. citriasiana (https://genome.jgi.doe.gov/Phycit1), P. citribraziliensis (https://genome.jgi.doe.gov/Phcit1), P. citrichinaensis (https://genome.jgi.doe.gov/Phcitr1), P. citricarpa (https://genome.jgi.doe.gov/Phycitr1, https://genome.jgi.doe.gov/Phycpc1), P. paracitricarpa (https://genome.jgi.doe.gov/Phy27169) genomes. All available Phyllosticta genomes on MycoCosm can be viewed at https://genome.jgi.doe.gov/Phyllosticta., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Postbloom fruit drop (PFD) of citrus, caused by Colletotrichum acutatum sensu lato and C. gloeosporioides sensu lato, is an important disease in the humid tropics of the American continent. PFD mainly affects flowers, on which typical symptoms are characterized by orange-brown lesions with presence of acervuli. The disease has a sporadic occurrence, but preventative fungicide sprays are applied every season. The objective of this study was to evaluate the effectiveness of a fungicide spray strategy for PFD based on a predictive model of C. acutatum conidium germination linked to weather conditions. Fungicide sprays were performed when the model predicted pre-established thresholds of 10, 15, 20, and 25% of germinated spores (T10, T15, T20, and T25, respectively). Five experiments were conducted in two different seasons in the state of São Paulo, Brazil. PFD control efficacy of the threshold-based treatments was compared with a nontreated control and to a calendar-based spray system. Additionally, an economic analysis was performed to assess the gross income revenues of the fungicide spraying strategies. Disease control in plots treated at T10, T15, and T20 was as effective as the calendar-based strategy. The number of fungicide applications was reduced by 33 to 71% when sprays were applied at T15 and T20, and gross income increased or was comparable to that of the other treatments. Therefore, using a conidium germination model with a threshold of 15 or 20% is recommended as a spraying strategy for PFD management in Brazil.

Citrus black spot (CBS), caused by Phyllosticta citricarpa , affects different citrus species worldwide. CBS is mainly expressed as false melanose and hard spot symptoms. There is no consensus in the literature about the period when fruit are susceptible to P. citricarpa infection and the length of the CBS incubation period. Therefore, this study aimed to assess the influence of sweet orange variety, fruit age, and inoculum concentration on the incubation period and the expression of different CBS symptoms. Attached fruit of Hamlin, Pera, and Valencia sweet orange at 1.5, 3.0, 5.0, and 7.0 cm diameter were inoculated with suspensions containing 10 3 and 10 5 conidia/ml of P. citricarpa . The percent conidial germination was quantified using scanning electron microscopy. The CBS symptoms on fruit were assessed monthly. The four diameters did not significantly affect conidial germination on the inoculated fruit, although CBS incidences were lower when larger fruit were inoculated. Hard spot symptoms on sweet orange fruit did not develop from the false melanose symptoms and vice versa. The incubation periods for false melanose were shorter than those observed for hard spot. False melanose began to appear 44 days after inoculation, but hard spot only formed at 113 days or later. Incubation periods were shorter and incidences of false melanose were higher following inoculation with higher inoculum concentration and smaller fruit diameter. The incubation period of hard spot varied among varieties and fruit diameters. However, there was no relationship between hard spot incidence and variety. This study provides a better understanding of the factors affecting the variation in the CBS incubation period and disease incidence on fruit.

The period of citrus black spot (CBS) control used in South Africa (SA) and Australia, from October to January or February, has not been as effective in São Paulo (SP), Brazil. This study aimed to evaluate different periods of protection and determine the critical period for CBS control in SP. A field trial was carried out for two seasons in a mature Valencia sweet orange orchard located in Mogi Guaçu, SP. Spray programs with a total of 60, 100, 140, 180, and 220 days of fruit protection (DFP) were evaluated. CBS symptoms and fruit drop decreased exponentially as the length of the period of protection increased. The reductions in CBS intensity and crop loss with these programs ranged from 34 to 96 and 50 to 77%, respectively. The programs with 180 and 220 DFP, which protected the fruit from September to March and May, showed the highest cost benefit. The critical period needed for CBS control in SP is longer than that in SA and Australia. The results obtained with the present study are helpful for scheduling a more efficient and rational program for CBS control not only in SP but also in other tropical and subtropical regions with similar weather conditions.

Antimicrobial photodynamic treatment (APDT) is a promising light based approach to control diseases caused by plant-pathogenic fungi. In the present study, we evaluated the effects of APDT with the phenothiazinium photosensitizer methylene blue (MB) under solar radiation on the germination and viability of conidia of the pathogenic fungus Colletotricum abscissum (former Colletotrichum acutatum sensu lato). Experiments were performed both on petals and leaves of sweet orange (Citrus sinensis) in different seasons and weather conditions. Conidial suspensions were deposited on the leaves and petals surface, treated with the PS (25 or 50μM) and exposed to solar radiation for only 30min. The effects of APDT on conidia were evaluated by counting the colony forming units recovered from leaves and petals and by direct evaluating conidial germination on the surface of these plant organs after the treatment. To better understand the mechanistic of conidial photodynamic inactivation, the effect of APDT on the permeability of the conidial plasma membrane was assessed using the fluorescent probe propidium iodide (PI) together with flow cytometry and fluorescence microscopy. APDT with MB and solar exposure killed C. abscissum conidia and prevented their germination on both leaves and petals of citrus. Reduction of conidial viability was up to three orders of magnitude and a complete photodynamic inactivation was achieved in some of the treatments. APDT damaged the conidial plasma membrane and increased its permeability to PI. No damage to sweet orange flowers or leaves was observed after APDT. The demonstration of the efficacy of APDT on the plant host represents a further step towards the use of the method for control phytopathogens in the field., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The increasing tolerance to currently used fungicides and the need for environmentally friendly antimicrobial approaches have stimulated the development of novel strategies to control plant-pathogenic fungi such as antimicrobial phototreatment (APT). We investigated the in vitro APT of the plant-pathogenic fungus Colletotrichum acutatum with furocoumarins and coumarins and solar radiation. The compounds used were: furocoumarins 8-methoxypsoralen (8-MOP) and 5,8-dimethoxypsoralen (isopimpinellin), coumarins 2H-chromen-2-one (coumarin), 7-hydroxycoumarin, 5,7-dimethoxycoumarin (citropten) and a mixture (3:1) of 7-methoxycoumarin and 5,7-dimethoxycoumarin. APT of conidia with crude extracts from 'Tahiti' acid lime, red and white grapefruit were also performed. Pure compounds were tested at 50μM concentration and mixtures and extracts at 12.5mgL(-1). The C. acutatum conidia suspension with or without the compounds was exposed to solar radiation for 1h. In addition, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP was the most effective treatment, killing 100% of the conidia followed by the mixture of two coumarins and isopimpinellin that killed 99% and 64% of the conidia, respectively. APT with the extracts killed from 20% to 70% of the conidia, and the extract from 'Tahiti' lime was the most effective. No damage to sweet orange leaves was observed after APT with any of the compounds or extracts., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The constitutive characters of plants can be structural or biochemical and play an important role in their defence against pathogens. Citrus postbloom fruit drop (PFD) caused by Colletotrichum spp. is one of the most important fungal diseases of citrus. The pathogen infects the flowers, leading to premature fruit drop and reducing citrus production. However, flower buds smaller than 8 mm long are usually not infected by Colletotrichum spp. Thus, this study investigated whether there are constitutive mechanisms in flower buds related to Colletotrichum spp. infection. We studied flower buds that were 2, 3, 4, 8, 12 and 15 mm long and petals, after anthesis, of sweet orange 'Valência' using light and scanning electron microscopy and histochemistry. We evaluated the effect of volatile organic compounds (VOCs) in flowers (R-limonene and linalool) on the in vitro growth of Colletotrichum acutatum. We found that the arrangement of the epidermal papillae in the petal primordia, the occurrence of prismatic crystals and the distribution of oil glands are the main differences between buds smaller than 8 mm and buds 8-15 mm long. Osmophores at the tips of petals produced and accumulated phenols, terpenes and lipophilic compounds. Flower buds smaller than 8 mm long have constitutive structural and biochemical barriers to Colletotrichum spp. infection. In addition, this is the first time that osmophores have been reported in citrus. Our study shows that natural terpenes of Citrus flowers inhibit the fungal growth in vitro, highlighting the potential use of terpenes for the chemical control of PFD in citrus., (Published by Oxford University Press on behalf of the Annals of Botany Company.)

Citrus crops in São Paulo State, Brazil, have been severely affected by postbloom fruit drop disease (PFD), which is caused by Colletotrichum acutatum. This disease leads to the drop of up to 100% of young fruits. Previous studies have assumed that this pathogen exhibits a clonal reproductive mode, although no population genetic studies have been conducted so far. Thus, the genetic structure of six C. acutatum populations from sweet orange orchards showing PFD symptoms was determined using nine microsatellite markers, enabling inference on predominant mode of reproduction. C. acutatum populations exhibit a nearly panmictic genetic structure and a high degree of admixture, indicating either ongoing contemporary gene flow at a regional scale or a recent introduction from a common source, since this pathogen was introduced in Brazil only very recently. Sharing haplotypes among orchards separated by 400 km suggests the natural dispersal of fungal propagules, with the possible involvement of pollinators. A significant population expansion was detected, which was consistent with an increase in host density associated with crop expansion toward new areas across the state. Findings of moderate to high levels of haplotypic diversity and gametic equilibrium suggest that recombination might play an important role in these pathogen populations, possibly via parasexual reproduction or a cryptic sexual cycle. This study provides additional tools for epidemiological studies of C. acutatum to improve prevention and management strategies for this disease.