Phyllosticta citricarpa is a fungal pathogen causing citrus black spot (CBS). As a regulated pest in some countries, the presence of the pathogen limits the export of fruit and is therefore of agricultural and economic importance. In this study, we used high throughput sequencing data to infer the global phylogeographic distribution of this pathogen, including 71 isolates from eight countries, Argentina, Australia, Brazil, China, Cuba, Eswatini, South Africa and the United States of America. We assembled draft genomes and used a pairwise read mapping approach for the detection and enumeration of variants between isolates. We performed SSR marker discovery based on the assembled genome with the best assembly statistics, and generated genotype profiles for all isolates with 1987 SSR markers in silico. Furthermore, we identified 32,560 SNPs relative to a reference sequence followed by population genetic analyses based on the three datasets; pairwise variant counts, SSR genotypes and SNP genotypes. All three analysis approaches gave similar overall results. Possible pathways of dissemination among the populations from China, Australia, southern Africa and the Americas are postulated. The Chinese population is the most diverse, and is genetically the furthest removed from all other populations, and is therefore considered the closest to the origin of the pathogen. Isolates from Australia, Eswatini and the South African province Mpumalanga are closely associated and clustered together with those from Argentina and Brazil. The Eastern Cape, North West, and KwaZulu-Natal populations in South Africa grouped in another cluster, while isolates from Limpopo are distributed between the two aforementioned clusters. Southern African populations showed a close relationship to populations in North America, and could be a possible source of P. citricarpa populations that are now found in North America. This study represents the largest whole genome sequencing survey of P. citricarpa to date and provides a more comprehensive assessment of the population genetic diversity and connectivity of P. citricarpa from different geographic origins. This information could further assist in a better understanding of the epidemiology of the CBS pathogen, its long-distance dispersal and dissemination pathways, and can be used to refine phytosanitary regulations and management programmes for the disease.

Citrus production is a significant component of the Australian horticulture portfolio. [...]

Citrus black spot, caused by Phyllosticta citricarpa, is characterized by fruit blemishes and premature fruit drop, resulting in significant economic losses in summer rainfall areas. The pathogen forms both conidia and ascospores during its life cycle. However, the occurrence of these spores and their contributions to infection of fruit in field conditions are not well understood. Our research using direct leaf litter monitoring and volumetric spore trapping in Queensland orchards revealed that pseudothecia and ascospores in leaf litter as well as trapped ascospores had low abundance, while pycnidia and conidia were highly abundant. Both P. citricarpa and endophytic Phyllosticta spp. were identified, with P. citricarpa being dominant. In replicated field trials, we determined that infection of Imperial mandarin fruit by P. citricarpa occurred from fruit set until week 20 of fruit development, with the key infection events taking place between weeks 4 and 16 in Queensland subtropical conditions. These results demonstrate that protecting fruit during weeks 4 to 16 significantly reduced P. citricarpa infection. We found no significant correlation between the disease incidence in fruit and P. citricarpa conidial abundance in leaf litter or ascospore abundance measured by volumetric spore trapping. Therefore, it is suggested that inoculum sources in the tree canopy other than those detected by spore trapping and direct leaf litter monitoring may play a major role in the epidemiology of citrus black spot. Improved knowledge regarding epidemiology of P. citricarpa and an understanding of propagules causing infection may aid in development of more effective disease management strategies.

Citrus black spot is an important fungal disease of citrus resulting in fruit drop and rind blemish in tropical and subtropical production areas. The disease is incited by the fungus Phyllosticta citricarpa (McAlpine) van der Aa (synonym: Guignardia citricarpa Kiely), with control currently relying on the application of fungicides. Because the presence and expression of resistance is poorly understood, we sought to develop a method for inoculating fruit in the field that gives reproducible symptoms of citrus black spot consistent with natural field infection. We subsequently validated this method by screening 49 citrus accessions and characterized their qualitative expression of citrus black spot symptoms. Challenge inoculations were undertaken with a known isolate of P. citricarpa, and control fruit were inoculated with water or the endophyte P. paracapitalensis Guarnaccia & Crous. Our results showed that all mandarin, sweet orange, lemon and papeda types were susceptible; pummelo, lime, and sour orange types expressed immunity; while various hybrids were susceptible, resistant and immune. Hybrid progeny from crosses using pummelo [Citrus maxima (Burm.) Merr.] as a parent showed preliminary evidence of segregation for citrus black spot immunity. The implications of these results to achieve genetic improvement for citrus black spot resistance in citrus breeding programs are discussed.

Citrus black spot ( Phyllosticta citricarpa ) is an economically important disease of citrus in Australia. A closely related endophyte, P. capitalensis , also occurs on citrus in Australia, and the two fungi are known to co-exist in orchards. The diversity of other species of Phyllosticta on Citrus in Australia is unknown. Citrus black spot is managed by fungicide treatment and options such as biological control may be better economic alternatives. We studied the diversity of Phyllosticta on Citrus in Australia with a phylogenetic species hypothesis. We report P. paracapitalensis for the first time and confirm the presence of P. capitalensis in Queensland. Many examined isolates of Phyllosticta previously identified as P. capitalensis were reidentified as P. paracapitalensis . We also provide evidence through Koch’s postulates that the two endophytic species are non-pathogenic on citrus fruit. In addition, disease incidence and severity of citrus black spot was significantly reduced when fruit were pre-inoculated with one of the endophytes 14 days prior to pathogen inoculation on the same fruit. Our results indicate these endophytes may have potential antagonistic effects against P. citricarpa under field conditions.

Citrus black spot, caused by Phyllosticta citricarpa, is one of the most important fungal diseases in many citrus-growing regions with hot and humid summers. Ascospores and conidia are known to contribute to epidemic development of the disease. However, pathogenicity testing has never been done for pure ascospores produced from fully characterized P. citricarpa isolates, due to the inability to induce the sexual state in vitro. Recently, an in vitro mating technique was developed to readily produce pure P. citricarpa ascospores for use in host inoculation studies. To test the pathogenicity of P. citricarpa ascospores, we inoculated Troyer citrange leaves and Murcott tangor fruit with ascospores produced in vitro from characterized P. citricarpa isolates. Typical symptoms of citrus black spot occurred. Recovery of P. citricarpa isolates from symptomatic lesions and their characterization using genetic markers enabled us to identify recombinant genotypes among the isolates recovered from ascospore inoculations and, as such, fulfill Koch's postulates for ascospores. We have also identified Troyer citrange seedlings as a potential model system for citrus black spot inoculation studies, because it allows typical symptoms of citrus black spot to be expressed with a much shorter latent period than on fruit. This will facilitate future studies of epidemiological aspects of P. citricarpa ascospores relative to conidia and improve our understanding of the citrus black spot pathosystem. The susceptibility of Troyer citrange seedlings will also facilitate experimenting with disease management methods, aimed at reducing the impact of citrus black spot.

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; subsequently, from the summer rainfall citrus-growing regions in China, Africa, and South America; and, recently, the United States. 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, the United States, Australia, China, and Brazil. Populations were genotyped using seven published and eight newly developed polymorphic simple-sequence repeat markers. The Chinese and Australian populations had the highest genetic diversities, whereas populations from Brazil, the United States, and South Africa exhibited characteristics of founder populations. The U.S. 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 U.S. 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. Although most populations exhibited sexual reproduction, linkage disequilibrium analyses indicated that asexual reproduction is important in the pathogen's life cycle.

Leaf litter is a major inoculum source for citrus diseases such citrus black spot caused by Phyllosticta citricarpa, and greasy spot caused by Mycosphaerella citri. In order to reduce this inoculum source, the efficacy of urea, dolomitic lime, a commercial compost accelerator, and an organic mulch, was assessed for enhanced leaf decomposition and reduction in sporocarps. However, due to the potential for run-off from high volume fungicide applications to disrupt leaf decomposition and microbial antagonism, the amendments were compared with and without simulated fungicide run-off. Mature green leaves of Citrus sinensis were removed from trees and placed inside mesh bags before being pinned to the orchard floor. The amendments were applied, and then simulated run-off from a typical citrus black spot fungicide program (copper, mancozeb, azoxystrobin) was applied. Leaf degradation was assessed every 2-3 weeks by visual ratings and dry weight. No direct effects on sporocarps could be observed due to insufficient infection. The results showed that the organic mulch was the most effective at enhancing decomposition, while there was significantly (P < 0.05) less decomposition in the presence of fungicide run-off.

A simple and inexpensive technique has been developed to allow the simultaneous screening of young hybrid seedlings for susceptibility to Alternaria Brown Spot (ABS) caused by Alternaria alternata and Citrus Scab (CS) caused by Elsinoë fawcettii. It is employed within six months of seed sowing, prior to hybrids being field planted, and has significantly improved breeding efficiency. By using the same culture techniques to multiply each pathogen, greater flexibility and ease of inoculation has been achieved. Plates of each pathogen are scraped/macerated to remove spores and other colony-forming units, strained through a coarse filter, combined and immediately sprayed onto vigorously growing seedlings. These seedlings are then incubated at ~25°C and high humidity for 4-7 days before being returned to the greenhouse. Hybrids susceptible to ABS develop symptoms within two weeks and are immediately discarded. A second round of culling is performed after four weeks when CS symptoms have expressed. The process is repeated three times to minimise 'disease escapes' and has virtually eliminated these diseases from our field plantings of new hybrids. Nucellar seedlings of genotypes known to be resistant and/or susceptible to each pathogen were used to confirm that mixing the two pathogens did not cause a change in symptom development for either disease.

Macadamia is indigenous to Australia. Only two species; M. integrifolia and M. tetraphylla and their interspecific hybrids are considered edible and they constitute the commercial macadamia industry. A fungal pathogen, Pseudocercospora macadamiae, causes husk spot in Australia, resulting in premature fruit abscission giving rise to nuts with low oil content. P. macadamiae is believed to have co-evolved with macadamia and the fungus infects the fruit pericarp (husk) via open stomata and grows intercellularly throughout the parenchymatous tissue where it causes accelerated abscission of immature and mature fruit. Depending on the time of infection, environmental conditions, and cultivar, the time to fruit abscission at the onset of husk spot symptoms may take up to 18 weeks. Factors that break the latency and influence fruit abscission due to P. macadamiae infection are unknown and may be involved in the production of ethylene by the husk tissue and/or abscisic acid by the fungus. Comparison of 18 macadamia genotypes revealed variations to accelerated fruit abscission from husk spot lesions on abscised fruit. This may be related to variation in stomatal abundance in the genotypes. The relationship between kernel maturation, in terms of oil content, fruit abscission in diseased and healthy husk is discussed.

An hourly infection model was used for a risk assessment of citrus black spot (CBS) caused by Phyllosticta citricarpa. The infection model contained a temperature-moisture response function and also included functions to simulate ascospore release and dispersal of pycnidiospores. A validation data set of 18 locations from South Africa and Australia was developed based on locations with known citrus black spot prevalence. An additional 67 sites from Europe and the United States with unknown prevalence were also identified. The model was run for each location with 9 years of hourly weather data from the National Centers for Environmental Prediction (NCEP) Climate Forecast System Reanalysis (CFSR) database. The infection scores for the sites with known prevalence where ranked and a threshold for suitability in a given year was derived from the average score of the lowest ranked moderate prevalence site. The results of the simulation confirm that locations in Florida were high risk while most locations in California and Europe were not at risk. The European location with the highest risk score was Andravida, Greece which had 67% of years suitable for ascosporic infection but only 11% of years were suitable for pycnidiosporic infection. There were six other sites in Europe that had frequency of years suitable for ascosporic infection greater than 22% including Pontecagnano, Italy; Kekrya, Greece; Reggio Calabria, Italy; Cozzo Spadaro, Italy; Messina, Italy; and Siracusa, Italy. Of these six sites only Reggio Calabria had a frequency of years suitable for pycnidiosporic infection greater than 0%. These six sites are predicted to have prevalence similar or less than Messina, South Africa, i.e. low and occasional. Other sites in Europe would best be described as likely to have no prevalence based on very low simulated scores for both spore types. Although Andravida had a similar risk of infection to moderate locations in South Africa there was a difference in the seasonality of infection periods. The ascosporic infection period score was similar between the two sites, but Andravida had a much lower pycnidiosporic infection score in the middle of the period of fruit susceptibility than Addo, South Africa. In Europe favorable climatic conditions are discontinuous, i.e., there is a low frequency of suitable seasons. This raises doubts about the ability of the pathogen to persist at a location and cause disease loss when favorable seasons reoccur. These results suggest that Europe is less suitable for CBS than suggested by an earlier study produced by the European Food Safety Authority using a similar model. The findings from our model simulations suggest that only a few isolated locations in the extreme south of Europe are likely to have a low to marginal risk of P. citricarpa establishment.

Molecular phylogenetic analysis, morphology and pathogenicity to citrus fruit were used to study two isolates of Elsinoë australis associated with scab-like symptoms on a fruit of Citrus australasica (finger lime) and Simmondsia chinensis (jojoba) in Australia. In addition to being associated with finger lime, the isolate from finger lime could cause scab symptoms on C. × aurantium cv. Murcott tangor in pathogenicity tests, but could not cause scab symptoms on the other orange, mandarin, lemon or grapefruit tested. Pathogenicity tests also support previous studies showing the isolate from jojoba could not produce symptoms on fruit of C. natsudaidai. Based on the findings of this study, two novel pathotypes of E. australis are designated from Australia; namely the Finger Lime (FL) pathotype associated with finger lime, and the Jojoba Black Scab (JBS) pathotype associated with black scab of jojoba. The significance of these novel E. australis pathotypes on market access and biosecurity issues for citrus are briefly discussed.

Rapid screening tests and an appreciation of the simple genetic control of Alternaria brown spot (ABS) susceptibility have existed for many years, and yet the application of this knowledge to commercial-scale breeding programs has been limited. Detached leaf assays were first demonstrated more than 40 years ago and reliable data suggesting a single gene determining susceptibility has been emerging for at least 20 years. However it is only recently that the requirement for genetic resistance in new hybrids has become a priority, following increased disease prevalence in Australian mandarin production areas previously considered too dry for the pathogen. Almost all of the high-fruit-quality parents developed so far by the Queensland-based breeding program are susceptible to ABS necessitating the screening of their progeny to avoid commercialisation of susceptible hybrids. This is done effectively and efficiently by spraying 3-6 month old hybrid seedlings with a spore suspension derived from a toxin-producing field isolate of Alternaria alternate, then incubating these seedlings in a cool room at 25°C and high humidity for 5 days. Susceptible seedlings show clear disease symptoms and are discarded. Analysis of observed and expected segregation ratios loosely support the hypothesis for a single dominant gene for susceptibility, but do not rule out the possibility of alternative genetic models. After implementing the routine screening for ABS resistance for three seasons we now have more than 20,000 hybrids growing in field progeny blocks that have been screened for resistance to the ABS disease.

The occurrence of pathogenic and endophytic species of Phyllosticta on cultivated Citrus in Australia was investigated by DNA sequence analysis of specimens held in plant pathology herbaria and culture collections. Sequences of the internal transcribed spacer region (ITS1, 5.8S, ITS2), and partial translation elongation factor 1-alpha (TEF) gene of 41 Phyllosticta -like isolates from Citrus were compared to those sequences from the type specimens of Phyllosticta recorded from around the world. Phylogenetic analysis resolved all the sequences of Australian accessions into two major clades. One clade corresponded to P. citricarpa , which causes citrus black spot disease. The other clade contained P. capitalensis , which is a known endophyte of Citrus and many other plant species. All included herbarium accessions previously designated as Guignardia mangiferae are now designated P. capitalensis . No Australian isolates were identified as the newly described pathogens of citrus P. citriasiana or P. citrichinaensis , or the endophytes Guignarida mangiferae , P. brazilianiae , or P. citribraziliensis .

Pseudocercospora macadamiae causes husk spot of macadamia. Husk spot control would be improved by verifying the stages in fruit development susceptible to infection, and determine some of the climatic conditions likely to lead to high disease pressure periods in the field. Our results showed that the percent conidia germination and growth of germ tubes and mycelia of P. macadamiae were greatest at 26°C, with better conidia germination associated with high relative humidity and free water. The exposure of match-head-sized and pea-sized fruit stages to natural P. macadamiae inoculum in the field led to 2–5-fold increases in husk spot incidence, and up to 8.5-fold increases in premature abscission, compared with unexposed fruit. Exposure of fruit stages later than match-head-sized and pea-sized fruit generally caused no further increases in disease incidence or premature abscission. Climatic conditions were found to have a strong influence on the behaviour of P. macadamiae, the host, oil accumulation, and the subsequent impact of husk spot on premature abscission. Our findings suggest that fungicide application should target fruit at the match-head-sized stage of development in order to best reduce yield losses, particularly in seasons where oil accumulation in fruit is prolonged and climatic conditions are optimal for P. macadamiae.

Pseudocercospora macadamiae causes husk spot of macadamia. Husk spot control would be improved by knowledge of the persistence of the pathogen in orchards between seasons, and by characterising macadamia cultivars for susceptibility to infection, premature fruit abscission, and spatial escape from inoculum sources. Field observation of seven macadamia cultivars found a significant exponential relationship between husk spot incidence and the prevalence of fruit pericarp that failed to abscise (sticktights) in the canopy. Viable conidia of P. macadamiae were readily (>100 conidia per fruit) produced on sticktights for 30 months. The incidence of husk spot was up to four times greater in trees with sticktights than trees without sticktights. Assessment of the propensity to form sticktights, husk spot incidence and fruit abscission after the insertion/removal of sticktights, demonstrated differences in macadamia cultivars; cv. A16 was susceptible to infection and formation of sticktights, but tolerant of premature abscission; cv. 246 was susceptible to infection, tolerant of premature abscission, and spatially escapes P. macadamiae inoculum due to a lack of sticktights; cv. A38 was susceptible to infection, premature abscission, and sticktight formation. Our findings suggest that disease could be reduced by removing sticktights from trees, and that fungicide inputs could be reduced in trees with naturally low numbers of sticktights. In addition we show that sticktights can be used as an inoculum source for field assays to better characterise macadamia cultivars for better informed cultivar selection, and as a tool in breeding programs to screen for germplasm with high levels of resistance.