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First report of Phyllosticta citricarpa and description of two new species, P. paracapitalensis and P. paracitricarpa , from citrus in Europe
Abstract and Figures
The genus Phyllosticta occurs worldwide, and contains numerous plant pathogenic, endophytic and saprobic species. Phyllosticta citricarpa is the causal agent of Citrus Black Spot disease (CBS), affecting fruits and leaves of several citrus hosts (Rutaceae), and can also be isolated from asymptomatic citrus tissues. Citrus Black Spot occurs in citrus-growing regions with warm summer rainfall climates, but is absent in countries of the European Union (EU). Phyllosticta capitalensis is morphologically similar to P. citricarpa, but is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts, and is known to occur in Europe. To determine which Phyllosticta spp. occur within citrus growing regions of EU countries, several surveys were conducted (2015–2017) in the major citrus production areas of Greece, Italy, Malta, Portugal and Spain to collect both living plant material and leaf litter in commercial nurseries, orchards, gardens, backyards and plant collections. A total of 64 Phyllosticta isolates were obtained from citrus in Europe, of which 52 were included in a multi-locus (ITS, actA, tef1, gapdh, LSU and rpb2 genes) DNA dataset. Two isolates from Florida (USA), three isolates from China, and several reference strains from Australia, South Africa and South America were included in the overall 99 isolate dataset. Based on the data obtained, two known species were identified, namely P. capitalensis (from asymptomatic living leaves of Citrus spp.) in Greece, Italy, Malta, Portugal and Spain, and P. citricarpa (from leaf litter of C. sinensis and C. limon) in Italy, Malta and Portugal. Moreover, two new species were described, namely P. paracapitalensis (from asymptomatic living leaves of Citrus spp.) in Italy and Spain, and P. paracitricarpa (from leaf litter of C. limon) in Greece. On a genotypic level, isolates of P. citricarpa populations from Italy and Malta (MAT1-2-1) represented a single clone, and those from Portugal (MAT1-1-1) another. Isolates of P. citricarpa and P. paracitricarpa were able to induce atypical lesions (necrosis) in artificially inoculated mature sweet orange fruit, while P. capitalensis and P. paracapitalensis induced no lesions. The Phyllosticta species recovered were not found to be widespread, and were not associated with disease symptoms, indicating that the fungi persisted over time, but did not cause disease.
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... P. citricarpa causes citrus black spot (CBS) (Kiely, 1948;Van der Aa, 1973), P. citriasiana and P. citrimaxima both cause citrus tan spot of Citrus maxima (Wikee et al., 2013;Wulandari et al., 2009), and P. citrichinaensis causes spots and freckle on several Citrus species (Wang et al., 2012). Recently, Guarnaccia et al. (2017) described a new species, Phyllosticta paracitricarpa, which is genetically closely related to P. citricarpa. P. paracitricarpa was isolated from Citrus limon leaf litter in Greek lemon orchards (Guarnaccia et al., 2017) as well as from lemon fruits in China (Wang et al., 2023) and has been shown to be pathogenic to C. sinensis and C. limon after artificial inoculations on detached leaves and fruits (Guarnaccia et al., 2017;Wang et al., 2023). ...
... Recently, Guarnaccia et al. (2017) described a new species, Phyllosticta paracitricarpa, which is genetically closely related to P. citricarpa. P. paracitricarpa was isolated from Citrus limon leaf litter in Greek lemon orchards (Guarnaccia et al., 2017) as well as from lemon fruits in China (Wang et al., 2023) and has been shown to be pathogenic to C. sinensis and C. limon after artificial inoculations on detached leaves and fruits (Guarnaccia et al., 2017;Wang et al., 2023). It was also retrospectively demonstrated that some Chinese isolates causing black spots on mandarin fruits, tentatively named "P. ...
... Recently, Guarnaccia et al. (2017) described a new species, Phyllosticta paracitricarpa, which is genetically closely related to P. citricarpa. P. paracitricarpa was isolated from Citrus limon leaf litter in Greek lemon orchards (Guarnaccia et al., 2017) as well as from lemon fruits in China (Wang et al., 2023) and has been shown to be pathogenic to C. sinensis and C. limon after artificial inoculations on detached leaves and fruits (Guarnaccia et al., 2017;Wang et al., 2023). It was also retrospectively demonstrated that some Chinese isolates causing black spots on mandarin fruits, tentatively named "P. ...
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. 2023. 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. PeerJ 11:e16354
... Furthermore, the morphological characteristics used to identify the pathogen are confusing within the species of the Phyllosticta genus associated with citrus [9,10,11]. However, five Phyllosticta species have been identified as causal agents of citrus fungal diseases such as Phyllosticta citricarpa, associated with black spot disease; Phyllosticta paracitricarpa, which causes damage to detached sweet orange fruit [12]; Phyllosticta citriasiana [13] and Phyllosticta citrimaxima [14], associated with brown spot of pomelo; and Phyllosticta citrichinaensis which induce symptoms on leaves and fruits of pomelo, sweet orange and mandarin [11]. In addition, Guignardia mangiferae, an endophytic species has been reported. ...
... Black spot is a major constraint to citrus production in Benin. The morphological characteristics of the disease pathogen correspond to those of P. citricarpa [12,25,26,27]. Mycelial growth of the fungus is slow on PDA medium. ...
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.
... However, due to the challenge of similar morphological features for taxonomic identification and homology analysis, the classification of Phyllosticta species has been complex, leading to a clu ered taxonomy. With advancements in molecular biology, the application of molecular data for species phylogeny has become increasingly sophisticated [5,33,43,52]. Wang ...
... However, due to the challenge of similar morphological features for taxonomic identification and homology analysis, the classification of Phyllosticta species has been complex, leading to a cluttered taxonomy. With advancements in molecular biology, the application of molecular data for species phylogeny has become increasingly sophisticated [5,33,43,52]. ...
The genus Phyllosticta has been reported worldwide and contains many pathogenic and endophytic species isolated from a wide range of plant hosts. A multipoint phylogeny based on gene coding combinatorial data sets for the internal transcribed spacer (ITS), large subunit of ribosomal RNA (LSU rDNA), translation elongation factor 1α (TEF1α), actin (ACT), and glycerol-3-phosphate dehydrogenase (GPDH), combined with morphological characteristics, was performed. We describe three new species, P. fujianensis sp. nov., P. saprophytica sp. nov., and P. turpiniae sp. nov., and annotate and discusse their similarities and differences in morphological relationships and phylogenetic phases with closely related species.
... This type of cropping facilitates the development of diseases and the colonization of healthy neighbored citrus orchards by pathogens. Phyllosticta sp. and Colletotrichum sp. are among the fungi that sporulate extensively, with one ascospore capable of releasing over 3,000 lesions (Guarnaccia et al., 2017). In terms of severity, analyses showed that black spot was the most severe (>80%) of all fungal diseases in the agroecological zones. ...
Fungal diseases are a major constraint for the intensification of citrus production in Benin. The aim of this study was to identify the main citrus fungal diseases and to assess their distribution, prevalence, and severity). A total of 315 orchards were surveyed in the four agro-ecological zones (AEZ V, VI, VII, and VIII) where citrus is produced in Benin. During the surveys, samples of diseased fruits were collected for isolation in the laboratory. The results revealed four main fungal diseases including black spot caused by Phyllosticta sp., anthracnose caused by Colletotrichum sp., brown rot disease caused by Curvularia sp., and fruit rot caused by Fusarium sp. Among these diseases, black spot is the most distributed with 76.69% infected plants as compared to anthracnose, brown rot disease and fruit rot which infected 32.4%, 6.3% and 1.9% of plants respectively. The diseases severity was 2.88, 1.46, 1.08 and 0.60 for black spot, anthracnose, brown rot and fruit rot respectively. Of the three cultivated varieties, Pineapple and Valencia were susceptible to the four diseases while the variety Tangelo was less susceptible. The highest severity (3.51) was recorded in the AEZ VI and the lowest (0.81) in the AEZ VII. This study showed that citrus tree was susceptible to several diseases in Benin with black spot disease caused by Phyllosticta sp., as the most important. Any effort to increase qualitatively and quantitatively citrus productivity should be based on sustainable management of diseases
... The infected leaf litter provides suitable conditions for pseudothecia to establish, grow and reproduce. These pseudothecia then release ascospores as primary inoculum, starting new infections (Dummel et al., 2015;Fourie et al., 2013;Guarnaccia et al., 2017;Huang and Chang, 1972;Kotzé, 1963). The release of mature ascospores from the asci of pseudothecia is triggered by rainfall, irrigation, or heavy dew (Baldassari et al., 2006), excessive rainfall, however, will disrupt ascospore discharge and cause the dead leaves to decompose, destroying the P. citricarpa substrate (Kotzé, 1963(Kotzé, , 1981Lee and Huang, 1973). ...
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.
Yerba mate (Ilex paraguariensis St. Hil.), with its health benefits and socioeconomic significance, plays a crucial role in Argentina and other South American countries like Brazil and Paraguay. Its cultivation in the Province of Misiones (Argentina) supports various sectors, contributes to regional development, and provides employment opportunities. However, the transition from extractive practices to monoculture, accompanied with increased demand, has led to phytosanitary challenges. Imbalanced native microbiota, disease development, and pathogen dispersion have become prevalent issues. Understanding the known pathogens associated with yerba mate plants is crucial for developing effective agricultural strategies. The primary objective of this study is to synthesise current knowledge on prevalent fungal diseases in yerba mate cultivation, as well as to provide agricultural management recommendations for effective disease control. Fungal diseases can cause significant damage to different parts of the plant, resulting in economic losses. The proximity of neighbouring plantations to yerba mate crops may contribute to the cross-contamination of pathogens, emphasizing the need for comprehensive epidemiology and accurate diagnosis. Multiple fungal genera have been reported to cause pathologies in yerba mate. Among the fungi causing foliar diseases are Ceratobasidium niltonsouzanum, Cylindrocladium spathulatum, Pseudocercospora mate, Asterina sphaerelloides, Colletotrichum gloeosporioides aff var. yerbae, and Phyllosticta sp. Caulinary diseases are caused by Alternaria sp., Phoma sp., Colletotrichum sp., and Ceratocystis fimbriata. Regarding root rot, the genera Rhizoctonia sp., Pythium sp., Fusarium sp., and Rosellinia sp. have been reported. Proper crop management practices and monitoring are essential for effective disease control. To reduce reliance on chemical compounds, the use of biocontrol agents like Trichoderma sp. has shown promise in regulating phytopathogenic fungi populations. Continued research is vital to preserve the yerba mate industry and ensure its long-term viability while minimizing environmental impact.
This review article communicates the integration of metabolomics and genomics approaches to unravel the citrus host–pathogen interaction.
This review deals with major diseases caused by fungi and oomycetes in the citrus supply chain, including post-harvest fruit diseases, and summarizes the strategies and techniques that may be adopted to prevent the damages and losses they cause. Its scope is to highlight the contribute that smart technologies provide towards new solutions for sustainable and safe management strategies of these diseases. Particular attention is given to the application of biopesticides, natural substances, resistance inducers and biostimulants to prevent fruit rots. The review focuses also on mycotoxins and mycotoxigenic fungi that contaminate fresh fruit and food products derived from citrus fruit, an aspect that has been little investigated and regulated so far. An additional relevant aspect addressed by the review is the early detection and routine diagnosis of fungal and oomycete pathogens that threat the international trade and long-distance shipment of citrus fruit, with a particular emphasis on quarantine pathogens. In this respect, the opportunities offered by new practical, rapid, sensitive and robust molecular diagnostic methods are briefly discussed.
World trade has greatly increased in recent decades, together with a higher risk of introducing non-indigenous pests. Introduction trends show no sign of saturation, and it seems likely that many more species will enter and establish in new territories in the future. A key challenge in analysing pest invasion patterns is the paucity of historical data on pest introductions. A comprehensive dataset of pests’ introductions in the EU, including their spatial occurrences, is not currently available and information is scattered across different sources or buried in the scientific literature. Therefore, we collected pests’ introduction information (e.g., year, host) from online scientific databases and literature; we then gathered primary spatial data related to the site of first introductions. Finally, we identified the potential pathways of entry for each pest. The dataset contains expert-revised data on 278 pests introduced in the EU between 1999 and 2019, alongside their spatial occurrence and potential pathways of entry, providing a basis to better understand the factors associated with the likelihood of pest introduction. It is important to note that this dataset does not contain the current distribution of the introduced pests, but only records of their first introduction in the EU.
Species of Colletotrichum are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on citrus, either as agents of pre- or post-harvest infections, such as anthracnose, postbloom fruit drop, tear stain and stem-end rot on fruit, or as wither-tip of twigs. In this study we explored the occurrence, diversity and pathogenicity of Colletotrichum spp. associated with Citrus and allied genera in European orchards, nurseries and gardens. Surveys were carried out during 2015 and 2016 in Greece, Italy, Malta, Portugal and Spain. A total of 174 Colletotrichum strains were isolated from symptomatic leaves, fruits, petals and twigs. A multi-locus phylogeny was established based on seven genomic loci (ITS, GAPDH, ACT, CAL, CHS1, HIS3 and TUB2), and the morphological characters of the isolates determined. Preliminary pathogenicity tests were performed on orange fruits with representative isolates. Colletotrichum strains were identified as members of three major species complexes. Colletotrichum gloeosporioides s.str. and two novel species (C. helleniense and C. hystricis) were identified in the C. gloeosporioides species complex. Colletotrichum karstii, C. novaezelandiae and two novel species (C. catinaense and C. limonicola) in the C. boninense species complex, and C. acutatum s.str. was also isolated as member of C. acutatum species complex. Colletotrichum gloeosporioides and C. karstii were the predominant species of Colletotrichum isolated. This study represents the first report of C. acutatum on citrus in Europe, and the first detection of C. novaezelandiae from outside New Zealand. Pathogenicity tests revealed C. gloeosporioides s.str. to be the most virulent species on fruits. The present study improves our understanding of species associated with several disease symptoms on citrus fruits and plants, and provides useful information for effective disease management.
The diversity of fusaria in symptomatic Citrus trees in Greece, Italy and Spain was evaluated using
morphological and molecular multi-locus analyses based on fragments of the calmodulin (CAM), intergenic spacer region of the rDNA (IGS), internal transcribed spacer region of the rDNA (ITS), large subunit of the rDNA (LSU), RNA polymerase largest subunit (RPB1), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-alpha (EF-1α) and beta-tubulin (TUB) genes. A total of 11 species (six Fusarium spp., and five Neocosmospora spp.) were isolated from dry root rot, crown, trunk or twig canker or twig dieback of citrus trees. The most commonly isolated species were Fusarium sarcochroum, F. oxysporum and Neocosmospora solani. Three new Fusarium species are described, i.e., F. citricola and F. salinense belonging to the newly described F. citricola species complex; and F. siculi belonging to the F. fujikuroi species complex. Results of pathogenicity tests showed this new complex to include prominent canker causing agents affecting several Citrus spp. In addition, two new species are described in Neocosmospora, named N. croci and N. macrospora, the latter species being clearly differentiated from most members of this genus by producing large, up to nine-septate sporodochial conidia.
Citrus black spot (Phyllosticta citricarpa) causes fruit blemishes and premature fruit drop, resulting in significant economic losses in citrus growing areas with summer rainfall across the globe. The mating type locus of P. citricarpa has recently been characterized, revealing the heterothallic nature of this pathogen. However, insight into the occurrence of mating and the impact of completing the sexual cycle of P. citricarpa was lacking. To investigate the occurrence and impact of sexual reproduction, we developed a method to reliably, and for the first time, produce ascospores of P. citricarpa on culture media. To demonstrate meiosis during the mating process, we identified recombinant genotypes through multilocus genotyping of single ascospores. Because the process of fertilization was not well understood, we experimentally determined that fertilization of P. citricarpa occurs via spermatization. Our results demonstrate that P. citricarpa is heterothallic and requires isolates of different MAT idiomorphs to be in direct physical contact, or for spermatia to fulfill their role as male elements to fertilize the receptive organs, in order to initiate the mating process. The impact of mating on the epidemiology of citrus black spot in the field is discussed.
The citrus pathogen Phyllosticta citricarpa was first described 117 years ago in Australia, and subsequently from the summer rainfall citrus-growing regions in China, Africa, South America and recently the United States of America (USA). Limited information is available on the pathogen's population structure, mode of reproduction and introduction pathways, which were investigated by genotyping 383 isolates representing 12 populations from South Africa, USA, Australia, China and Brazil. Populations were genotyped using seven published and eight newly developed polymorphic simple sequence repeat (SSR) markers. The Chinese and Australian populations had the highest genetic diversities, whereas populations from Brazil, USA and South Africa exhibited characteristics of founder populations. The USA population was clonal. Based on principal coordinate and minimum spanning network analyses the Chinese populations were distinct from the other populations. Population differentiation and clustering analyses revealed high connectivity and possibly linked introduction pathways between South Africa, Australia and Brazil. With the exception of the clonal USA populations that only contained one mating type, all the other populations contained both mating types in a ratio that did not deviate significantly from 1:1. Whilst most populations exhibited sexual reproduction, linkage disequilibrium analyses indicated that asexual reproduction is important in the pathogen's life cycle.
The aim of this study was to investigate in vitro and model the effect of temperature (T) and water activity (aw) conditions on growth and toxin production by some toxigenic fungi signaled in cheese. Aspergillus versicolor, Penicillium camemberti, P. citrinum, P. crustosum, P. nalgiovense, P. nordicum, P. roqueforti, P. verrucosum were considered they were grown under different T (0–40 °C) and aw (0.78–0.99) regimes. The highest relative growth occurred around 25 °C; all the fungi were very susceptible to aw and 0.99 was optimal for almost all species (except for A. versicolor, awopt = 0.96). The highest toxin production occurred between 15 and 25 °C and 0.96–0.99 aw. Therefore, during grana cheese ripening, managed between 15 and 22 °C, ochratoxin A (OTA), penitrem A (PA), roquefortine-C (ROQ-C) and mycophenolic acid (MPA) are apparently at the highest production risk. Bete and logistic function described fungal growth under different T and aw regimes well, respectively. Bete function described also STC, PA, ROQ-C and OTA production as well as function of T. These models would be very useful as starting point to develop a mechanistic model to predict fungal growth and toxin production during cheese ripening and to help advising the most proper setting of environmental factors to minimize the contamination risk.
This book is intended to provide consolidated information on citrus breeding in the era of biotechnology, which is likely to hasten the pace of variety development aimed at resolving the problems faced by grove owners growing currently available cultivars. The subjects covered are focused on citrus while providing information equally useful to the breeders of other tree crops. It will also help students of genetic and breeding identify appropriate applications of biotechnology in citrus breeding. While providing information on future avenues, it also reviews the past progress and achievements ensuring continuity of the subject. Several chapters include protocols for novel techniques that should facilitate their broader application by citrus breeders.
A diverse collection of isolates identified as Colletotrichum acutatum, including a range of fruit-rot and foliar pathogens, was examined for mtDNA RFLPs and RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase (GS) gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene. RFLPs of mtDNA, RFLPs of the 900-bp GS intron and sequence analysis of each intron identified the same seven distinct molecular groups, or clades, within C. acutatum sensu lato. Sequence analysis produced highly concordant tree topologies with definitive phylogenetic relationships within and between the clades. The clades might represent phylogenetically distinct species within C. acutatum sensu lato. Mating tests also were conducted to assess sexual compatibility with tester isolates known to outcross to form the teleomorph Glomerella acutata. Mating compatibility was identified within one clade, C, and between two phylogenetically distinct clades, C and J4. The C clade represented isolates from a wide range of hosts and geographic origins. J4 clade contained isolates from Australia or New Zealand recovered from fruit rot and pine seedlings with terminal crook disease. That isolates in two phylogenetically distinct clades were capable of mating suggests that genetic isolation occurred before reproductive isolation. No other isolates were sexually compatible with the mating testers, which also were in groups C and J4. Certain clades identified by mtDNA and intron analysis (D1, J3 and J6) appeared to represent relatively host-limited populations. Other clades (C1, F1 and J4) contained isolates from a wide range of hosts. Isolates described as C. acutatum f. sp. pineum were clearly polyphyletic.
Sexual reproduction in fungi is controlled by mating type genes, which are located at the MAT locus. In this study, we investigated the structure of this locus in the phytopathogenic fungus Phyllosticta citricarpa, the causal agent of citrus black spot disease. Despite intensive study, its sexual state has never been observed in single-spore culture. Through analysis of the genome sequences of two individual P. citricarpa isolates, the sequence of the DNA lyase gene was identified and, as previously reported in the literature, the mating type genes were located in the 3′ flanking region of this gene. The results suggested that P. citricarpa is heterothallic, owing to the exclusive presence of the MAT1–1 or MAT1–2 gene in individual strains. In order to characterize the MAT locus, we designed primers to amplify this region. P. citricarpa was found to have complete and apparently functional copies of MAT genes, containing α-1 and HMG domains, present in different isolates. In addition to MAT1–2-1 and MAT1–1-1 genes, the MAT1–1-4 gene was located in the 5′ flanking region of the MAT1–1-1 gene and the MAT1–2-5 gene was located in 5′ flanking region of the MAT1–2-1 gene. A multiplex PCR protocol was also developed to differentiate P. citricarpa idiomorphs, which can be used in distribution and incidence studies of mating type strains, in order to determine the occurrence of sexual reproduction and to facility crossing studies. Furthermore, in Brazil, the two idiomorphs occur in a 1:1 ratio, which is expected in sexually reproducing populations.