Monitoring benzimidazole resistance in Phyllosticta citricarpa using a molecular assay targeting mutations in codons 198 and 200 of the beta-tubulin gene

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is an economically important disease, which is effectively controlled by repeated fungicide applications to protect fruit from infection. Systemic fungicides such as benzimidazoles are widely used for controlling CBS in South Africa, but the molecular mechanisms of benzimidazole resistance in P. citricarpa had not been investigated. Analysis of the nucleotide sequence of the beta-tubulin gene in P. citricarpa revealed mutations inducing three amino acid replacements in benzimidazole-resistant isolates when compared to that of sensitive strains. Amino acid replacements in benzimidazole-resistant isolates included the change of glutamic acid to either alanine or lysine at codon 198 of the beta-tubulin gene and the change from phenylalanine to tyrosine at codon 200. All three mutations were previously implicated in benzimidazole resistance in several fungal pathogens. A polymerase chain reaction (PCR) assay was designed to amplify a portion of the beta-tubulin gene, which is subsequently sequenced to identify benzimidazole resistance in P. citricarpa. This PCR and sequence assay was found to be a more rapid and reliable method for detecting resistance compared to the fungicide-amended plate tests and is valuable for monitoring the occurrence of benzimidazole-resistant P. citricarpa and for assessment of the need for alternative CBS management practices.

Phytosanitary risk associated with illegal importation of pest-infested commodities to the South African agricultural sector

We evaluated the phytosanitary risk associated with illegal importation of pest-infested plant commodities into South Africa. Samples were collected from different South African ports of entry over 8 years (2011 to 2019) and data were analysed descriptively using Statistical Software Package. Pests were frequently detected on commodity species such as Citrus (18.31%), Zea mays (13.22%), Phaseolus vulgaris (12.88%), Musa spp. (9.15%) and Fragaria ananassa (5.08%). The highest number of pests intercepted occurred on fresh fruits (44.06%), followed by grains (26.44%) and vegetables (14.23%). The most intercepted organisms were Callosobruchus rhodesianus (7.79%), Dysmicoccus brevipes (7.11%), Callosobruchus maculates (6.10%) and Phyllosticta citricarpa (4.74%). The majority of intercepted organisms were non-quarantine organisms (70.50%), followed by pests of unknown status (17.28%), quarantine pests (10.84%) and potential quarantine pests (1.35%). Phyllosticta citricarpa, Bactrocera dorsalis, Spodoptera frugiperda and Prostephanus truncatus were the only quarantine pests intercepted in terms of South African regulatory status. The interception was mainly from southern African countries, particularly Mozambique, Zimbabwe and Eswatini. The findings present the level of phytosanitary risk associated with illegal importation and/or non-compliance in regard to plants and plant commodities from different countries through South African ports of entry. Crop production, biodiversity, food security, existing export markets, and access to new export markets could be threatened as importing countries may impose stringent phytosanitary measures to limit the chances of introduction and establishment of quarantine pests into their territories.

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.

Models for predicting pseudothecium maturity and ascospore release of Phyllosticta spp. in South African citrus orchards

Ascosporic infection plays a major role in the epidemiology of citrus black spot (CBS) in South Africa, a disease caused by Phyllosticta citricarpa. Phyllosticta pseudothecium maturation and ascospore release models have been integrated in infection models to predict the availability of the primary inoculum source. However, these models have not been validated on a broader data set and this study aimed to validate and improve these epidemiological models. New pseudothecium maturation and ascospore release models for P. citricarpa were developed, based on weather and ascospore trap data from 13 locations and up to five seasons. From the 29 data sets analysed, 3775 3-hourly periods with ascospore events were recorded on 1798 days; 90% of these events occurred between 16.0 °C and 32.1 °C (daily Tmin and Tmax of 15.4 °C and 33.5 °C, respectively) and 75% occurred above a relative humidity (RH) of 55.9% (daily RH > 47.9%). Rain was recorded during 13.8% of these ascospore events and 20.0% of ascospore days. Using logistic regression, a Gompertz model that best predicted pseudothecium maturation, or the probability of onset of ascospore release, was developed and was markedly more accurate than the previously described models. The model consisted of DDtemp [cumulative degree-days from midwinter (1 July) calculated as (minimum + maximum daily temperature) / 2 – 10 °C] and DDwet (DDtemp accumulated only on days with >0.1 mm rain or vapour pressure deficit <5 hPa) as variables in the formula: probability of first ascospore event = exp(-exp(-(-3.131 + 0.007 × DDtemp - 0.007 × DDwet))). A Gompertz model [PAT = exp(-2.452 × exp(-0.004 × DDwet2))] was also developed for ascospore release; DDwet2 = DDtemp accumulated, from first seasonal ascospore trap day, only on days with >0.1 mm rain or vapour pressure deficit <5 hPa. Similar to the DDwet2 model described in a previous study, this model adequately predicted the general trend in ascospore release but poorly predicted periods of daily, 3-day and 7-day ascospore peaks.

Spatial and temporal genetic analyses of Phyllosticta citricarpa in two lemon orchards in South Africa reveal a role of asexual reproduction within sexually reproducing populations

Citrus Black Spot (CBS), caused by Phyllosticta citricarpa, is a disease that affects citrus worldwide. In different regions of the world where both mating types occur, reports differ as to whether asexually produced pycnidiospores play an important role in the epidemiology of CBS and fruit infections. We, therefore, investigated the potential role of pycnidiospores in two lemon orchards in South Africa using microsatellite-based analysis of fruit populations over time (two seasons) and space (distance). The two orchards were situated in the semi-arid North West province (NW) and subtropical Mpumalanga province (MP). Each population contained both mating types in 1:1 ratios, and linkage disequilibrium analysis indicated a random mating population. A total of 109 and 94 multilocus genotypes (MLGs) were detected across the two seasons in the NW and MP orchards respectively. Psex analyses indicated that most MLGs probably resulted from sexual reproduction, but there were six predominant MLGs in each orchard that were probably replicated via asexual reproduction. Each of the predominant MLGs was monomorphic for mating type. In the NW, five predominant and widespread MLGs caused 46% and 44% of the fruit infections in the two seasons, whereas in MP, three MLGs caused 34% and 48% of the infections. Asexual reproduction in both orchards was supported by relatively low MLG evenness values in all populations. In both orchards, distance was not a reliable predictor of population genetic substructuring, nor season. Populations of P. citricarpa in the MP and NW orchards were significantly genetically differentiated from each other.