Citrus Black Spot Research Articles

Monitoring quantities of Phyllosticta citricarpa inoculum in sweet orange orchards using qPCR and young citrus plants as spore traps

AbstractMonitoring of Phyllosticta citricarpa (causal agent of citrus black spot [CBS]) inoculum in orchards has previously been performed using volumetric spore traps. However, volumetric traps are nonspecific, and only capture ascospores (not pycnidiospores) of different Phyllosticta species. This research aimed to monitor and quantify the DNA of P. citricarpa using young citrus plants as a spore trap combined with quantitative PCR (qPCR), as well as quantify the number of ascospores and pycnidiospores, and to correlate results with weather variables. Citrus nursery plants were placed as traps under and outside tree canopies during fruit developmental stages (from October to July) in two seasons in commercial ‘Valencia’ sweet orange orchards. DNA was extracted from trap leaves to quantify P. citricarpa inoculum by qPCR of the ribosomal internal transcribed spacer 1 (ITS1 rRNA) region of P. citricarpa (Pc‐ITS). Correlations of Pc‐ITS to rainy days, leaf wetness and temperature were performed. Overall, the highest numbers of 400 Pc‐ITS/cm2 of leaf tissue, which represented up to 12 ascospores or pycnidiospores per cm2, were detected on leaves sampled from October to March, regardless of the trap position, season and orchard. Trap plants placed under canopies had up to 20‐fold more Pc‐ITS than those placed outside. Rainy days and leaf wetness were the variables most positively correlated with Pc‐ITS. Both results in the fluctuation of P. citricarpa inoculum in orchards and the most favourable weather variables associated with inoculum production contribute to better understanding of the critical periods for CBS management in citrus‐growing areas.

Identification of Biomarkers Associated with Phyllosticta citricarpa Tolerance.

Citrus black spot (CBS) is a fungal disease caused by Phyllosticta citricarpa Kiely, (McAlpine Van der Aa), with most cultivars being susceptible to infection. Currently, disease control is based on the application of protective fungicides, which is restricted due to resistance, health and environmental concerns. Although using natural products for disease management is gaining momentum, more advances are required. This study obtained the metabolic profiles of the essential oil and cuticular waxes of two citrus cultivars with a varying susceptibility to CBS infection using gas chromatography-mass spectrometry. A multivariate data analysis identified possible biomarker compounds that contributed to the difference in susceptibility between the two cultivars. Several identified biomarkers were tested in vitro for their antifungal properties against P. citricarpa. Two biomarkers, propanoic acid and linalool, were able to completely inhibit pathogen growth at 750 mg/L and 2000 mg/L, respectively.

Breeding for citrus black spot resistance

Breeding for citrus black spot resistance

Isolation, Identification and Characterization of Phyllosticta citricarpa Causing Citrus Black Spot Disease in Benin

Citrus black spot is a major constraint to citrus production in Benin. Identification of the pathogen causing citrus black spot disease was carried out in the laboratory on symptomatic fruits. Fruit samples were collected from orchards in 4 citrus-growing agro-ecological zones. A total of 66 representative isolates of Phyllosticta sp. were obtained from isolation. Molecular analysis using PCR of the nucleotide sequences of the ITS regions with universal primers ITS1/ITS4 and specific primers GcF1/GcR and the phylogenetic tree showed that the sequences of all isolates obtained in the different agroecological zones were identical to those of Phyllosticta citricarpa. The pathogenicity test satisfied Koch's postulates by re-isolation of Phyllosticta citricarpa from inoculated fruits. A thorough study of genetic diversity and a full understanding of the behavior of P. citricarpa will pave the way for more targeted approaches to the prevention, control and sustainable management of citrus black spot disease in Benin.

Mating type and microsatellite genotyping indicate that the Tunisian population of Phyllosticta citricarpa is clonal and thrives only asexually

Citrus black spot (CBS) caused by Phyllosticta citricarpa was reported for the first time in Tunisia in 2019. This was also the first reported occurrence of the disease in a Mediterranean climate. In Tunisia, CBS is mainly found in lemon (Citrus limon) orchards, and is seldom observed on sweet orange (Citrus × sinensis). This recent finding in North Africa raises questions about how the disease has been able to spread under Mediterranean climatic conditions. In this work, 216 Phyllosticta strains collected from lemon orchards in 2021, 2022 and 2023 throughout the country's main citrus-growing provinces were characterised by species morphological and molecular identification, mating type and Simple Sequence Repeats (SSR) microsatellite genotyping (MLG). P. citricarpa was the only species found to be associated with CBS in Tunisia. Although P. citricarpa is a heterothallic fungal species, potentially able to reproduce both sexually and asexually, a single mating type (MAT 1-1-1) idiomorph was found in the population. In addition, three MLGs were observed, across ten microsatellite loci, one of which was massively represented (93 %), indicating a clonal population. The clonality observed suggests a single recent introduction of the pathogen into the country. These findings support the idea that in Tunisia, P. citricarpa only reproduces asexually by pycniospores, with a relatively limited dispersal potential. This is consistent with the absence of pseudothecia on the leaf litter. These results show that CBS is able to thrive under Mediterranean conditions, even in the absence of sexual reproduction. This should be taken into consideration for CBS risk assessment and management.

Characterization of volatile compounds from healthy and citrus black spot-infected Valencia orange juice and essential oil by using gas chromatography–mass spectrometry

Citrus black spot (Phyllosticta citricarpa, CBS) is an important fungal disease that causes rind blemishes and affects quality of citrus fruits. The response of citrus to CBS in terms of volatiles was evaluated using molecular sensory science approaches. Fifty and twenty-one volatiles were identified in the orange juice and essential oil samples, respectively, via gas chromatography–mass spectrometry (GC–MS). The total volatile content in the samples increased after CBS infection, especially in the severe-infection group (SEG) juice and moderate-infection group (MOG) essential oil, which reached the highest levels. CBS enhanced floral, fruity, and off-flavor aromas and decreased the green aroma in citrus juice. Citrusy, floral, and green aromas increased in the CBS-infected essential oil. Six/five potential markers were screened in citrus juice/essential oil, respectively using the orthogonal partial least-square discriminant analysis (OPLS-DA) model. The changes in aroma profile and the difference in infection levels in citrus were attributed to these odorants.

Survey of Phyllosticta citricarpa mating type in the São Paulo citrus belt and optimization of in vitro ascospore production

AbstractPhyllosticta citricarpa, the citrus black spot causal agent, requires both mating types, designated as MAT1‐1 and MAT1‐2, to produce ascospores. Mating‐type ratio in some citrus‐growing areas has been characterized but remains little known in the São Paulo (SP) citrus belt, the largest sweet orange producer worldwide. A protocol for in vitro ascospore production was developed recently; however, there are few studies regarding the optimal conditions for their production. Thus, this study aimed to determine the mating‐type ratio in SP and quantify ascospores produced under different conditions in vitro. The mating types were identified by duplex PCR, and ascospore production was assessed in five culture media: potato dextrose agar (PDA), ½ PDA, malt extract agar (MEA), oatmeal agar (OMA) and citrus agar (CA), and at temperatures from 10 to 30°C. The ratio of MAT1‐1 and MAT1‐2 was similar to 1:1. Ascospore production reached peaks of 8.0, 7.0 and 3.0 × 103 ascospores/mL in MEA, PDA and ½ PDA, respectively. There were no ascospores observed in OMA and CA. P. citricarpa ascospores were observed only at 15, 20 and 25°C in PDA, with peaks of up to 2.0 × 103 ascospores/mL at 42, 35 and 21 days, respectively. There were no ascospores observed at 10 and 30°C. Both mating types are similarly distributed in the SP citrus belt, and in vitro ascospore production may be optimized by using MEA at 25°C. These data provide a better understanding of P. citricarpa sexual reproduction and a background for further studies with ascospores.

Citrus Black Spot Disease in Ghana: A Review on Impacts, Management Options and Current Status

Citrus is one of the most common seasonal fruit crops cultivated across most semi-deciduous forest zones across the globe. Apart from its refreshing nature when consumed in a fresh state, it is also packed with an abundant supply of vitamins A, B and C. Apart from being served in the raw state as a fruit or a dessert, it also serves as a critical raw material for the beverage industry. In some instances, it also serves as a very important material for the pharmaceutical industry. As important as this fruit can be, its production is challenged by the incidence of pests and diseases. One of the major diseases facing the industry is the Citrus Black Spot (CBS), caused by Phyllosticta citricarpa. In Ghana, CBS is known to affect almost all citrus species and varieties in commercial groves, except for sour orange (C. aurantium) and its hybrids and Tahiti lime (C. latifolia). It is estimated to be responsible for about 22% of fruit/yield loss annually. This review highlighted the impact on the citrus production industry in Ghana, the management options available and future perspectives.

Geographic distribution, prevalence, and incidence of citrus black spot caused by Phyllosticta citricarpa in Tunisia

AbstractCitrus black spot (CBS), caused by Phyllosticta citricarpa, is an important disease of citrus, an economically important fruit crop worldwide and particularly in Tunisia. CBS was recently detected in Tunisia and this study revealed that it is widespread in the governorates of Nabeul and Sousse. The highest incidence of CBS was found in the Bouargoub region (73.7%), while the lowest was observed in Soliman (25.0%) in the Nabeul governorate. The surveys revealed that CBS affected mainly lemon (Citrus limon). The symptoms of CBS observed were hard spots on fruits, twigs, and leaves, as well as false melanose on fruits. Symptoms were observed in both mature and immature citrus fruits, but disease incidence was higher in mature (97.0%) than that in immature fruits (2.95%). In this study, morphological and molecular identifications confirmed the presence of P. citricarpa. This is the first time that the extent of CBS has been documented in a Mediterranean citrus area.

Investigation into the role of phenolic compounds in the protection of citrus against Phyllosticta citricarpa

IntroductionThe use of chemical fungicides to combat disease has made a substantial contribution to food quality and security. Nonetheless, their applications have been limited due to environmental and health concerns, unaffordability, and the fact that pathogens have acquired resistance to some of these fungicides. Alternative eco-friendly and safe control methods should be explored. The current study investigated the influence of citrus rind phenolic compounds against Phyllosticta citricarpa infection by metabolic profiling of two citrus cultivars with varying degrees of susceptibility to infection.MethodsChromatographic data obtained by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC) was subjected to multivariate data analysis to identify biomarkers associated with the tolerant cultivar. The identified biomarkers were tested in vitro against P. citricarpa.ResultsSeville oranges, a tolerant cultivar, displayed higher levels of phenolic content and lower total sugar content, that are both associated with lower susceptibility to citrus black spot infection. The generated Principal Component Analysis (PCA) and Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA) models gave an overview of the data set and identified components that may be responsible for the differences in susceptibility between the two cultivars. Candidate biomarkers associated with tolerance were identified as naringin, neoeriocitrin, bruteiridin, melitidin, and lucenin-2.ConclusionNaringin, a major candidate biomarker was able to inhibit the growth of the pathogen at 10 000 ppm.

Citrus black spot caused by Phyllosticta citricarpa found on Valencia oranges in Botswana

Citrus black spot caused by <i>Phyllosticta citricarpa</i> found on Valencia oranges in Botswana

Neem Essential Oil as an Antifungal Agent against Phyllosticta citricarpa.

The fungus Phyllosticta citricarpa is a quarantine phytopathogen responsible for causing citrus black spot (CBS) disease. To export fruits to CBS-free countries, they must undergo a sanitation process to ensure disease control. In this study, neem essential oil (NEO) was tested against P. citricarpa for the first time as an alternative sanitizer. In vitro experiments were conducted to determine the inhibition concentration of NEO for P. citricarpa, and the mode of action of the essential oil was evaluated. In vivo assays were performed to simulate the sanitization process used in packinghouses. NEO was characterized by GC-MS/MS. The results revealed that NEO at 100 μL·mL-1 exhibited a similar inhibitory effect as copper oxychloride, suppressing 89.68 ± 1.14% of fungal mycelium growth. Fluorescence microscopy experiments demonstrated that NEO functions by disrupting the cytoplasmic membrane of fungal hyphae, leading to their death within 30 minutes of contact with NEO. GC-MS/MS characterization revealed a high presence of phenolic compounds, which serve as the primary antifungal agents responsible for the action against fungal hyphae. In vivo assays showed that NEO at 100 μL·mL-1 also reduced microorganisms (CFU mL-1) by 93.00 ± 3.88% compared to the negative control. Overall, the results demonstrate that NEO can effectively serve as an alternative sanitizer against P. citricarpa in citrus packinghouses. Our findings allow future studies to explore the use of NEO for sanitizing other fruits and combating different phytopathogens to broaden its potential application in fruit sanitation for export.

Metabolomics approach to understand molecular mechanisms involved in fungal pathogen-citrus pathosystems.

Citrus is a crucial crop with a significant economic impact globally. However, postharvest decay caused by fungal pathogens poses a considerable threat, leading to substantial financial losses. Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii and Phyllosticta citricarpa are the main fungal pathogens, causing green mold, blue mold, sour rot and citrus black spot diseases, respectively. The use of chemical fungicides as a control strategy in citrus raises concerns about food and environmental safety. Therefore, understanding the molecular basis of host-pathogen interactions is essential to find safer alternatives. This review highlights the potential of the metabolomics approach in the search for bioactive compounds involved in the pathogen-citrus interaction, and how the integration of metabolomics and genomics contributes to the understanding of secondary metabolites associated with fungal virulence and the fungal infection mechanisms. Our goal is to provide a pipeline combining metabolomics and genomics that can effectively guide researchers to perform studies aiming to contribute to the understanding of the fundamental chemical and biochemical aspects of pathogen-host interactions, in order to effectively develop new alternatives for fungal diseases in citrus cultivation. We intend to inspire the scientific community to question unexplored biological systems, and to employ diverse analytical approaches and metabolomics techniques to address outstanding questions about the non-studied pathosystems from a chemical biology perspective.

Selecting hyperspectral bands and extracting features with a custom shallow convolutional neural network to classify citrus peel defects

Citrus Black Spot (CBS) causes considerable damage to the Florida citrus industry. Early detection of CBS, especially in the presence of other peel blemishes, would enable better mapping and control of CBS spread, reduce wasted fruit, and permit early removal of culls from the packing stream. Oranges whose peels bore the symptoms of four defects/disease (CBS, greasy spot, melanose, and wind scar), as well as a normal control group, were imaged with a hyperspectral imaging system. Principal Component Analysis- (PCA) and Linear Discriminant Analysis (LDA) -based methods were employed to select bands from these images, and a custom convolutional neural network (CNN) for feature extraction was trained with these bands. The extracted features permitted classification of the peel conditions with four classifiers: SoftMax, Support Vector Machines (SVM), Random Forest Classifier (RFC), and K-Nearest Neighbors (KNN). A mean overall accuracy of 94.9 % was achieved using an SVM classifier on five bands selected with PCA, and 90.2 % with LDA-selected bands. These results show the potential of CNNs to extract features for automated postharvest citrus inspection.

Harnessing the power of comparative genomics to support the distinction of sister species within Phyllosticta and development of highly specific detection of Phyllosticta citricarpa causing citrus black spot by real-time PCR.

Citrus crops are affected by many fungal diseases. Among them, Citrus Black Spot caused by the ascomycete Phyllosticta citricarpa is particularly economically damaging wherever it occurs. Many other species of Phyllosticta are described on Citrus, but only P. citricarpa is considered a quarantine pest on the European continent. In order to prevent the introduction of this species into Europe, it is essential to have a detection test which can reliably identify it, and not confuse it with other species present on citrus, notably P. paracitricarpa. The latter taxon has recently been described as very close to P. citricarpa, and most detection tests do not allow to distinguish the two species. In this work, we exploited the genomic data of 37 isolates of Phyllosticta spp. from citrus, firstly to assess their phylogenetic relationships, and secondly to search for genomic regions that allowed the definition of species-specific markers of P. citricarpa. Analysis of 51 concatenated genes separated P. citricarpa and P. paracitricarpa in two phylogenetic clades. A locus was selected to define a hydrolysis probe and primers combination that could be used in real-time PCR for the specific detection of the quarantine species, to the exclusion of all others present on Citrus. This test was then thoroughly validated on a set of strains covering a wide geographical diversity, and on numerous biological samples to demonstrate its reliability for regulatory control. The validation data highlighted the need to check the reliability of the test in advance, when a change of reagents was being considered.

Paecilins Q and R: Antifungal Chromanones Produced by the Endophytic Fungus Pseudofusicoccum stromaticum CMRP4328.

Chemical investigation of the endophyte Pseudofusicoccum stromaticum CMRP4328 isolated from the medicinal plant Stryphnodendron adstringens yielded ten compounds, including two new dihydrochromones, paecilins Q (1: ) and R (2: ). The antifungal activity of the isolated metabolites was assessed against an important citrus pathogen, Phyllosticta citricarpa. Cytochalasin H (6: ) (78.3%), phomoxanthone A (3: ) (70.2%), phomoxanthone B (4: ) (63.1%), and paecilin Q (1: ) (50.5%) decreased in vitro the number of pycnidia produced by P. citricarpa, which are responsible for the disease dissemination in orchards. In addition, compounds 3: and 6: inhibited the development of citrus black spot symptoms in citrus fruits. Cytochalasin H (6: ) and one of the new compounds, paecilin Q (1: ), appear particularly promising, as they showed strong activity against this citrus pathogen, and low or no cytotoxic activity. The strain CMRP4328 of P. stromaticum and its metabolites deserve further investigation for the control of citrus black spot disease.

Duration of fungicide spray program for the control of citrus black spot in sweet orange based on cultivar maturation and orchard age

Citrus black spot (CBS) damage and Phyllosticta citricarpa inoculum tend to increase in old orchards and in late-maturing cultivars of sweet orange. However, CBS control in São Paulo citrus belt is based on standard fungicide program of 180–220 days of fruit protection, regardless of cultivar and orchard age. This study aimed to investigate whether the protection period may be adjusted based on the cultivar maturation and age of orchards intended for juice processing. The efficacy of protection periods of 60, 100, 140, 180, 220 and 260 days was determined by assessing CBS incidence and severity and CBS-related fruit drop in 12 orchards over two seasons. The standard protection period, with 180 days targeted for mid-age and 220 days for old orchards, was the most cost effective for mid-season and late-maturing cultivars. These protection periods resulted in a significant reduction in CBS severity and fruit drop by up to ∼95% and ∼50%, respectively. For early-maturing cultivars, the duration of CBS spray program may be drastically reduced to 60 days for mid-age orchards and to 100 days for old orchards. These protection periods significantly reduced CBS severity and fruit drop by up to ∼92% and ∼50%, respectively. These adjustments in CBS fungicide protection period based on cultivar and orchard age may save up to US$14.3 million per season if adopted throughout the main Brazilian citrus belt. The results of this study allow citrus growers to save inputs, reduce costs, improve the operational efficiency within the farm and contribute to a more sustainable citrus industry.

Clonal Expansion in Multiple Phyllosticta Species Causing Citrus Black Spot or Similar Symptoms in China.

Phyllosticta spp. are important pathogens of citrus plants. Several Phyllosticta species associated with Citrus species grown in China have been reported; however, the relative prevalences of individual species and the distributions of their genotypes among host Citrus species remain largely unknown. In this study, we conducted an extensive survey of Phyllosticta species across 11 citrus-producing provinces in southern China. From fruits and leaves with black spots or black-spot-like symptoms, a total of 461 Phyllosticta strains were isolated. Based on molecular (ITS, actA, tef1, gapdh, LSU, and rpb2 sequences) and morphological data, the strains were systematically identified as belonging to five species: P. capitalensis, P. citrichinaensis, P. citriasiana, P. citricarpa, and P. paracitricarpa. To further understand intraspecific genetic diversity and relationships, strains of five species from different geographic and host sources were analyzed based on the multilocus sequence data. Our population genetic analyses revealed that all five Phyllosticta species on citrus showed evidence for clonal dispersals within and among geographic regions. In addition, pathogenicity tests using representative strains showed that all five species can cause disease on the tested Citrus spp. We discuss the implications of our results for the control and management of Citrus Black Spot and related diseases.

CITRUS DISEASE CLASSIFICATION WITH TRANSFER LEARNING AND CNN BASED MODELS

In recent years, image processing and deep learning have been widely used in the detection and classification of plant diseases. These uses offer great opportunities for the early detection of plant diseases in agriculture. Early detection of the disease is essential to prevent disease symptoms from spreading to intact leaves and to reduce crop damage. For the stated reasons, a deep learning model with three different approaches has been proposed and used for the classification of diseases that are most common in citrus leaves and affect citrus export to a great extent. Training and test data used in the proposed model are separated according to the K-fold 5 value. For this reason, the average of the performance values obtained according to the K-fold 5 value is presented in the study. As a result of the experimental studies, with the fine-tuned DenseNet201 model, which is the first model, an accuracy rate of 0.95 was achieved. In the second model, with the proposed 21-layer CNN model, an accuracy rate of 0.99 was achieved. The third model is defined to show the progress of the proposed DenseNet201 model over the basic DenseNet201 model. With the CNN method recommended for the classification of citrus grades, Blackspot (citrus black spot (CBS), canker (citrus bacterial cancer (CBC)), greening (huanglongbing (HLB)), and (healthy) Healthy) 100%, 100%, 98% and 100% rates have been reached.

Antimicrobial, antifungal and antioxidant activity from the mucus cocoon of the Parrotfish (Genus Scarus : Laboridei: Scaridae)

The ocean harbors one of the largest sources of important bioactive molecules due to the complex, dynamic nature of the various ecosystems and biodiversity interactions. The complexity and inaccessibility of certain species or component of a species make it difficult to collect and preserve samples for testing. In this study, the cocoon produced by Parrotfishes (genus Scarus) at night was collected using scuba diving techniques and tested for antimicrobial, antifungal, and antioxidant properties. Five extracts (crude, acidic, aqueous 1, aqueous 2, and organic) were obtained from the mucus and antimicrobial tests were carried out using well diffusion and minimal inhibitory concentration (MIC) against the following bacteria: Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Klebsiella pneumoniae ATCC 700603, Salmonella enterica ATCC 13076, Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 and four fungi: Fusarium equiseti ATCC 200472, Alternaria spp. (environmental isolate), Guignardia citricarpa (environmental isolate) and Candida albicans ATCC 10231. The 1-diphenyl-2-picrylhydrazyl (DPPH) assay was used to measure the scavenging activity of the extracts. The acidic extract showed the highest (11.56 mm) zone of inhibition (ZOI) against E. coli and the MIC (0.406 mg/ml). In terms of ZOI, the least effective extracts were the crude (2.67 mm) followed by the aqueous 1 (3.33 mm) while for the MIC the least effective extract was the aqueous 2 (13 mg/ml). Significant inhibition was observed on the gram-negative bacteria compared to gram-positive bacteria in both methods. Regarding the antifungal assay, the organic extract was most effective against C. albicans (9.03 mm), and only the acidic extract showed inhibition zone (14.0 mm) against Guignardia citricarpa. The aqueous 2 extract exhibited the highest inhibition (71.96 %), while the crude extract showed the lowest inhibition (34.11 %). The present findings indicate that Parrotfish mucus can further be explored in terms of its pharmacological properties.