Enemy or ally: a genomic approach to elucidate the lifestyle of Phyllosticta citrichinaensis

Members of the fungal genus Phyllosticta can colonize a variety of plant hosts, including several Citrus species such as Citrus sinensis (orange), Citrus limon (lemon), and Citrus maxima (pomelo). Some Phyllosticta species have the capacity to cause disease, such as Citrus Black Spot, while others have only been observed as endophytes. Thus far, genomic differences underlying lifestyle adaptations of Phyllosticta species have not yet been studied. Furthermore, the lifestyle of Phyllosticta citrichinaensis is ambiguous, as it has been described as a weak pathogen but Kochs postulates may not have been established and the presence of this species was never reported to cause any crop or economic losses. Here, we examined the genomic differences between pathogenic and endophytic Phyllosticta spp. colonizing Citrus and specifically aimed to elucidate the lifestyle of Phyllosticta citrichinaensis. We found several genomic differences between species of different lifestyles, including groups of genes that were only present in pathogens or endophytes. We also observed that species, based on their carbohydrate active enzymes, group independent of their phylogenetic association, and this clustering correlated with trophy prediction. Phyllosticta citrichinaensis shows an intermediate lifestyle, sharing genomic and phenotypic attributes of both pathogens and endophytes. We thus present the first genomic comparison of multiple citrus-colonizing pathogens and endophytes of the genus Phyllosticta, and therefore provide the basis for further comparative studies into the lifestyle adaptations within this genus.

2021–2022 Florida Citrus Production Guide: Citrus Black Spot

Sections: regulatory considerations; growers, caretakers, harvesters, and haulers; processing and packing facilities and haulers, disposal of citrus debris; Interstate shipment of fruit; export shipment of fruit; regulatory remarks; and recommended chemical controls

Validation of Quantitative and Digital Polymerase Chain Reaction Assays Targeting the Mating Types of Phyllosticta citricarpa, the Causal Agent of Citrus Black Spot

Citrus black spot (CBS) is a disease caused by the ascomycetous fungus Phyllosticta (formerly Guignardia) citricarpa (Botryosphaeriales, Pezizomycotina) currently present in citrus groves in five counties in southwest Florida. Within Florida, P. citricarpa shows limited reproduction via asexual sporulation due to the presence of only one (MAT1-2-1) of the two required mating types for sexual reproduction. Here we present two novel polymerase chain reaction (PCR) assays standardized in quantitative (qPCR) and digital (dPCR) platforms to distinguish both mating types (MAT1-1-1 and MAT1-2-1) of P. citricarpa, to monitor for the potential introduction of the MAT1-1-1 mating type into Florida and a novel protocol for DNA extraction from asymptomatic leaves. During citrus harvesting season 2018−2019, fruit lesions, as well as asymptomatic leaves adjacent to symptomatic fruit and asymptomatic trees in CBS-infected groves were surveyed for P. citricarpa presence and mating types. Results support the presence of only MAT1‑2-1 mating type in Florida, after surveying more than 1,145 citrus fruit lesions. We also confirmed the limited dispersal ability of the asexual state of P. citricarpa in Florida in ten groves using the enhanced capabilities of the dPCR platform in the detection of P. citricarpa directly from asymptomatic leaves with low pathogen inoculum.

The Effects of Postharvest Treatments and Sunlight Exposure on the Reproductive Capability and Viability of Phyllosticta citricarpa in Citrus Black Spot Fruit Lesions

Citrus black spot (CBS) is caused by Phyllosticta citricarpa, which is classified as a quarantine organism in certain countries whose concerns are that CBS-infected fruit may be a pathway for introduction of the pathogen. This study evaluated the reproductive capability and viability of P. citricarpa under simulated conditions in which the whole fruit, peel segments, or citrus pulp with CBS lesions were discarded. Naturally infected ‘Midknight’ Valencia orange and ‘Eureka’ lemon fruit, either treated using standard postharvest sanitation, fungicide, and wax coating treatments or untreated, were placed into cold storage for 5 weeks (oranges at 4 °C and lemons at 7 °C). Thereafter, treated and untreated fruit were incubated for a further 2 weeks at conditions conducive for CBS symptom expression and formation of pycnidia. The ability of pycnidia to secrete viable pycnidiospores after whole fruit and peel segments or peel pieces from citrus pulp were exposed to sunlight at warm temperatures (±28 °C) and ±75% relative humidity levels was then investigated. The combination of postharvest treatments and cold storage effectively controlled CBS latent infections (>83.6% control) and pycnidium formation (<1.4% of lesions formed pycnidia), and the wax coating completely inhibited pycnidiospore release in fruit and peel segments. Pycnidiospores were secreted only from lesions on untreated fruit and peel segments and at low levels (4.3–8.6%) from peel pieces from pulped treated fruit. However, spore release rapidly declined when exposed to sunlight conditions (1.4% and 0% after 2 and 3 days, respectively). The generally poor reproductive ability and viability of CBS fruit lesions on harvested fruit, particularly when exposed to sunlight conditions, supports the conclusion that citrus fruit without leaves is not an epidemiologically significant pathway for the entry, establishment, and spread of P. citricarpa.

Inoculum Dynamics and Infection of Citrus Fruit by Phyllosticta citricarpa

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.

2020–2021 Florida Citrus Production Guide: Citrus Black Spot

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