Providence Moyo

Fruit protection against infection by Phyllosticta citricarpa , the causal agent of citrus black spot (CBS), relies heavily on fungicides. Quinone-outside inhibitors (QoIs), which have a site-specific mode of action, have been in use since the early 2000s in South African citrus orchards. This study investigated the occurrence of the F129L, G137R or G143A mutations in codons 129, 137 and 143, respectively, in cytochrome b ( cytb ) of P. citricarpa . Molecular analysis detected the F129L mutation in cytb of 73 of 191 isolates tested, but the G143A and G137R mutations were not detected. A non-synonymous nucleotide substitution from TTC to TTA was predominantly detected in 69 isolates, while a codon change from TTC to CTC was detected in only four isolates. Both mutations result in an amino acid substitution from phenylalanine to leucine. To investigate the practical impact of this mutation on the field efficacy of QoI fungicides, trials were performed on a lemon orchard with a history of high CBS pressure and 100% detection rate of the F129L mutation. Results revealed a strong association between the occurrence of the F129L mutation and reduced efficacy in controlling CBS by pyraclostrobin and azoxystrobin, when applied outside the requirements of registration, i.e. not in a mixture with a fungicide from another FRAC group. This study is the first report of the F129L mutation and a shift in sensitivity to QoI fungicides in P. citricarpa populations.

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is an important fungal disease of citrus. Higher CBS severity has been associated with P. citricarpa infections at the young and green stages of fruit. The length of the fruit susceptibility period may be influenced by the amount of inoculum and the climate of the citrus growing region. This study was conducted in orchards across two South African provinces to assess the intensity of CBS under two conditions: (i) unsprayed fruit of Valencia sweet orange, Nova, and Empress mandarins inoculated with P. citricarpa at 10¹, 10³, and 10⁵ pycnidiospores/mL at various intervals after fruit set, from October (petal fall stage) to March (for mandarins) or June (for sweet orange), and (ii) non‐inoculated Valencia fruit from orchards subjected to spray programs ranging from one to nine applications between October and June. Higher CBS intensity was observed in Valencia fruit inoculated between November and January, with an average disease severity index (DSI) of 0.94, compared to a significantly lower (0.07) for fruit inoculated from February to June. Inoculations in November or December for Nova mandarins and in December for Empress with 10⁵ pycnidiospores/mL resulted in higher CBS intensity compared to those with lower pycnidiospore concentrations. The absence of fungicide applications from October to December during the 2017–2018 season led to increased CBS intensity, with a DSI of ~0.47, a value which was not significantly different from that of non‐treated control (NTC) trees. Leaving sweet orange trees unprotected from January onwards did not significantly increase the CBS intensity. These results confirm that citrus growers should focus on protecting citrus fruit, against P. citricarpa infection, in their dark green and young stages (c.15–30 mm diameter) to control CBS.

Monitoring 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/cm ² of leaf tissue, which represented up to 12 ascospores or pycnidiospores per cm ² , 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.

Phyllosticta citricarpa is the causative agent of citrus black spot (CBS), a cosmetic fungal disease that has been reported in most of the citrus-growing regions of the world. The occurrence of CBS in orchards is predominantly controlled by the application of fungicides during the fruit susceptibility period. Benzimidazoles (benomyl and carbendazim) and toluamides (ethaboxam and zoxamide) are two systemic fungicide classes suitable for the control of CBS in South Africa. Both inhibit proper assembly of the fungal β-tubulin protein during mitosis, and resistance to these fungicides is primarily related to alterations in the binding sites on this target protein. In this study, P. citricarpa isolates with known resistance status were subjected to whole genome sequencing, the mutations conferring resistance to benomyl and zoxamide were examined and allele-specific primers targeting these mutations were developed. The allele-specific multiplex PCR assay for the detection of benomyl resistance in P. citricarpa will eliminate the need for laborious and time-consuming sensitivity assays or DNA sequencing.

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 β-tubulin gene in P. citricarpa revealed mutations inducing three amino acid replacements in benzimidazole-resistant isolates when compared with those 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 β-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 PCR assay was designed to amplify a portion of the β-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 with 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.

A recent olive trunk disease survey performed in the Western Cape Province, South Africa, identified several fungi associated with olive trunk disease symptoms, including species of Basidiomycota, Botryosphaeriaceae, Coniochaetaceae, Calosphaeriaceae, Diaporthaceae, Diatrypaceae, Phaeomoniellaceae, Phaeosphaeriaceae, Symbiotaphrinaceae, Togniniaceae, and Valsaceae. Many of the species recovered had not yet been reported from olive trees; therefore, the aim of this study was to determine their pathogenicity toward this host. Pathogenicity tests were first conducted on detached shoots to select virulent isolates, which were then used in field trials. During field trials, 2-year-old olive branches of 15-year-old trees were inoculated by inserting colonized agar plugs into artificially wounded tissue. Measurements were made of the internal lesions after 8 months. In total, 58 isolates were selected for the field trials. Species that formed lesions significantly larger than the control could be considered as olive trunk pathogens. These included Biscogniauxia rosacearum, Celerioriella umnquma, Coniochaeta velutina, Coniothyrium ferrarisianum, isolates of the Cytospora pruinosa complex, Didymocyrtis banksiae, Diaporthe foeniculina, Eutypa lata, Fomitiporella viticola, Neofusicoccum stellenboschiana, Neofusicoccum vitifusiforme, Neophaeomoniella niveniae, Phaeoacremonium africanum, Phaeoacremonium minimum, Phaeoacremonium oleae, Phaeoacremonium parasiticum, Phaeoacremonium prunicola, Phaeoacremonium scolyti, Phaeoacremonium spadicum, Pleurostoma richardsiae, Pseudophaeomoniella globosa, Punctularia atropurpurascens, Vredendaliella oleae, an undescribed Cytospora sp., Geosmithia sp., two undescribed Neofusicoccum spp., and four Xenocylindrosporium spp. Pseudophaeomoniella globosa can be regarded as one of the main olive trunk pathogens in South Africa because of its high incidence from olive trunk disease symptoms in established orchards and its high virulence in pathogenicity trials.

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 (P. citricarpa.

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 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. Significance: We developed a new pseudothecium maturation model from 29 data sets, comprising different climatic regions in South Africa, and validated previously published models. The new model was markedly more accurate in predicting the onset of ascospore release and can be used to improve existing CBS epidemiological models and improve risk assessment and management of CBS in South African citrus orchards.

Loquat (Eriobotrya japonica Lindl., Rosaceae) is a subtropical tree grown for its edible fruit (Tziros 2013). In South Africa, loquat is grown as ornamental tree. Twig and branch dieback symptoms a...