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 the ascomycete, Phyllosticta citricarpa, is a fruit, foliar, and twig spotting fungal disease affecting the majority of commercial cultivars of citrus. The disease causes cosmetic lesions, may cause fruit drop and P. citricarpa is considered a quarantine pathogen by some countries, impacting domestic and international trade of citrus fruit. Regulatory requirements affecting fruit trade exist even though there is no documented case of disease spread via infected fruit into previously disease-free areas. To clarify the risk of fruit as a potential pathway for the spread of CBS, we developed a quantitative, probabilistic risk assessment model. The model provides an assessment of all steps in the fruit pathway, including production, packinghouse handling, transportation, export-import distribution channels, and consumer endpoints. The model is stochastic and uses Monte Carlo simulation to assess the risk of P. citricarpa moving through all steps in the pathway. We attempted to use all available literature and information to quantitate risk at each point in the potential pathway and by sequentially linking all steps to determine the overall quantitative risk. In addition, we assessed climatological effects on incidence of diseased fruit at production sites and on fungal reproduction and infection, as well as criteria for establishment at endpoints. We examined ten case studies between exporting and importing locations/countries. Model results indicated fruit to be an epidemiologically insignificant means for CBS spread, even between producing countries where CBS occurs and CBS-free importing countries with disease-conducive climates. We created a second model to examine the introduction of infected plant material from countries where CBS occurs. This model demonstrated significant probability of introduction via such infected material. However, pathogen establishment and disease development was still restricted only to areas with conducive climatological conditions. We created a tool to quantitatively explore the viability of various potential pathways via combinations of CBS-present production sites and corresponding pathway endpoints, including environments conducive and non-conducive to CBS. The tool is provided to aid decision makers on phytosanitary risk relative to international trade of citrus fruit.

The alternation of spray programmes consisting of tank mixtures of a benzimidazole, mancozeb and oil in November followed by a strobilurin, copper and spray oil (A+B; C+D sequence) in January show great potential for the control of citrus black spot even in orchards where the benzimidazole resistant citrus black spot population comprises 87% (2003/2004 season) and 63-66% (2004/2005 season). The future use of the benzimidazoles will depend on the outcome of a carbendazim residue study (study number 051 1119) that is currently underway on citrus in RSA as conducted by CRI and the SABS for the support of an EU import tolerance. If the study shows that a single benzimidazole application will enable compliance with the revised EU import tolerance, a spray programme that includes an early benzimidazole application in November (A+B; C+D sequence), could be considered.

The global distribution of citrus black spot (CBS) disease, caused by Phyllosticta citricarpa , is climatically constrained, which is evident from its occurrence in citrus growing areas with warm, summer rainfall and its absence from areas with cooler, Mediterranean-type winter rainfall. Various epidemiological and modelling studies have supported this observation, predominantly estimating unsuitability for P. citricarpa in Mediterranean type climates, with no more than marginal suitability estimated at a few localities within some regions with Mediterranean type climates. The study by Martínez-Minaya et al. (European Journal of Plant Pathology, 143, 69–83, 2015 ), describes an historic sequence of recorded CBS occurrence in parts of South Africa, conducts an autocorrelation analysis and a correlative analysis with Köppen-Geiger climate zones and makes observations about the occurrence of certain Köppen-Geiger climate zones in the European Union. The study suggests that significant portions of the European Union and the broader Mediterranean basin are climatically similar to warm, summer rainfall areas in South Africa where P. citricarpa persists and causes CBS disease and concludes that the potential distribution of P. citricarpa is less constrained by climatic factors than spatial contagion. However, in this critique we expose methodological shortcomings in the Martínez-Minaya et al. (European Journal of Plant Pathology, 143, 69–83, 2015 ) study and conclude that the study grossly overestimated the extent of the geographical area that could support P. citricarpa , thereby rendering the findings scientifically unreliable.

'Navel' sweet orange [Citrus sinensis (L.) Osbeck] fruit are characterized by the presence of a secondary fruitlet (navel) located inside the stylar-end of the primary fruit. Fruit with large navel-end openings have increased susceptibility to develop navel-end fruit splitting and navel-end rot. Furthermore, large navel-end openings can provide refuge for insect pests including mealybugs. Foliar application of the plant growth regulator and synthetic auxin, 2,4-dichlorophenoxy acetic acid (2,4-D) during flowering time reduces the size of the navel, as well as that of the navel-end opening. The objective of this study was to determine if the closing of the navel-end opening by foliar application of 2,4-D could reduce the susceptibility of 'Navel' sweet orange fruit to mealybug [Planococcus citri (Risso)] infestation, Alternaria [Alternaria alternata (Fr.) Keissl] infection and the subsequent development of Alternaria black core rot (ABCR). Foliar application of 2,4-D at full bloom (FB) increased the percentage of fruit with fully closed navel-ends significantly. These results concur with previous research and were consistent across different production areas, cultivars and seasons, irrespective of the concentration and formulation of 2,4-D used. Apart from two anomalies, all 2,4-D treatments at FB resulted in a lower percentage of mealybug infested fruit at time of harvest and significantly reduced the percentage of fruit infected with ABCR. A combination treatment of 10 mg L-1 2,4-D and 0.20 mL L-1 tebuconazole applied at FB was most successful in reducing ABCR infection and therefore provides a novel control method for black core rot in 'Navel' sweet orange fruit. (C) 2017 Elsevier Ltd. All rights reserved.

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.

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.

1 Citrus Research International, PO Box 28, Nelspruit 1200, South Africa 2 Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Stellenbosch 7602, South Africa 3 Fundecitrus, Araraquara, Brazil 4 University of Sao Paulo, Piracicaba, Brazil 5 Department of Conservation Ecology and Entomology, Stellenbosch University, Private Bag X1, Stellenbosch 7602, South Africa 6 U.S. Horticultural Research Laboratory, Agricultural Research Services, United States Department of Agriculture, Fort Pierce, FL, USA 7 ExperiCo (Agri-Research Solutions), A Division of Agri R&D (Pty) Ltd, PO Box 4022, Ida’s Valley, Stellenbosch, 7609, South Africa 8 Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA

Spray application forms the backbone of pre-harvest pest and disease management strategies in South African citrus production. Due to zero tolerance status of Citrus black spot (caused by Phyllosticta citricarpa) (McAlpine) for export to the European Union, growers tend to use high application fungicide volumes ranging from 8000 to 12000 l ha-1. These high volumes realise good disease control whilst growers are hesitant to risk compromising disease control by applying at reduced volumes. Moreover, high spray volumes act as a “safety buffer” for poor sprayer maintenance, calibration and application techniques.

Inadequate disease control on citrus foliage and fruit is often attributed to insufficient fungicide spray deposition on target surfaces. This study describes a novel spray deposition assessment protocol and determines deposition benchmarks indicative of the biological effectiveness for better interpretation of spray deposition results. Suitability of a yellow fluorescent pigment as tracer for copper oxychloride deposition was demonstrated through its similar particle concentration and size. Spray deposition assessment of spray targets, which were sprayed with a mixture that included the fluorescent pigment, involved photomacrography of whole leaf or fruit surfaces, followed by digital image analyses. This protocol proved to be very accurate in determining the quantity and quality of deposition. To determine deposition benchmarks, detached young 'Nova' mandarin leaves were sprayed with copper oxychloride and fluorescent pigment at different concentrations (0.1-2 times the recommended concentration) and spray deposition assessed. Subsequently, leaves were spray inoculated with a spore suspension of Altenaria alternata [causal agent of Alternaria brown spot (ABS) of mandarins], moist-incubated for c. 48 h and symptom expression rated. A very good linear relationship was found between fungicide concentration, leaf area covered by fluorescent pigment particles (%FPC) (r = 0.879) and Cu residue analysis (r = 0.992). A von Bertalanffy growth curve best fitted the relation between ABS control and deposition quantity (%FPC) data (91% of the percentage variance accounted for) with a good correlation between observed and predicted values (r = 0.825). Benchmarks for 50% and 75% disease control were calculated as 2.07 %FPC and 4.14 %FPC, respectively. These corresponded with Cu residue levels of 59.4 and 91.0 mg kg(-1), respectively. These FPC benchmarks can be used to evaluate spray technology research, specifically for control of ABS and similar citrus fruit and foliar diseases. (C) 2012 Elsevier Ltd. All rights reserved.

The South African citrus industry is strongly focused on exports and South Africa is a signatory member of both the World Trade Organisation Agreement on the application of Sanitary and Phytosanitary Measures and the International Plant Protection Convention. Citrus black spot, caused by Guignardia citricarpa, does not occur in all the South African citrus production areas and, therefore, South Africa has a responsibility to provide those trading partners that have identified G. citricarpa as a regulated pest with reliable information about the distribution of citrus black spot within South Africa. Detection surveys were conducted in citrus production areas in the Western Cape, Northern Cape and Free State Provinces and appropriate diagnostic protocols were used to ensure reliable detection of G. citricarpa. Trees in commercial orchards and home gardens on farms and in towns of 17, 9 and 5 magisterial districts in the Western Cape, Northern Cape and Free State Provinces, respectively, were sampled between 1995 and 2010. Fruit samples were taken during June and July, and leaf samples from November to January. None of the 3060 fruit and leaf samples collected during these surveys tested positive for G. citricarpa. Phyllosticta capitalensis, a non-pathogenic, ubiquitous, endophytic species was, however, detected during these surveys. In compliance with relevant International Standards for Phytosanitary Measures and based on the outcome of these official surveys, these three provinces in South Africa can be recognised as citrus black spot pest free areas.

High volumes of copper hydroxide, cuprous oxide and copper oxychloride were sprayed under natural conditions onto mature orange trees to compare the retention on citrus leaves and fruit over a period of 56 days by means of copper residue analyses and a spray deposition assessment protocol using fluorometry, photomacrography and digital image analyses. Rainfall and increase in fruit size were also recorded to determine if it had an influence on weathering of copper residues. Initial retention following application of the different fungicides differed on Valencia leaves and fruit: applications with cuprous oxide retained significantly more copper residue and fluorescent pigment, while copper hydroxide retained higher copper and pigment levels on Navel leaves and fruit. Nonetheless, persistence of copper residues deposited by the three copper fungicide formulations was similar and decreased at the same tempo during both seasons; initially a fast reduction (48 and 60% for year one and two respectively) in residue during the first 14 days followed by a more gradual decline (41 and 24% for year one and two respectively) from 14 to 56 days. The loss of copper residues was attributed to weathering (days after treatment), fruit growth and cumulative rainfall as these factors were inversely correlated with copper residue levels (Pearson's r = -0.840, -0.722 and -0.733 respectively). A 76% and 90% correlation was observed between the copper residue analyzed and the quantitative fluorescent pigment measurements on mature leaves and fruit, respectively; showing that fruit is more reliable for fluorometry analyses and that this technique proved to be an effective tool for spray deposition and persistence assessment of copper fungicides. All copper formulations tested at these registered rates at 35-day spray intervals were effective in controlling Guignardia citricarpa. (C) 2012 Elsevier Ltd. All rights reserved.

Alternaria alternata pv. citri is the causal agent of Alternaria brown spot on tangerines and their hybrids and infects young leaves and fruit of all ages. In South Africa, repeated high volume (∼9000 L/ha) fungicide sprays on susceptible cultivars are the only effective control measure of this disease. The effect of runoff on spray deposition and biological efficacy was largely unknown. The aims of this study were first to characterise spray deposition and runoff on fruit and leaves, and second to determine the effect of runoff on biological efficacy. Mature Nova mandarin leaves (upper and lower leaf surfaces), Valencia Late oranges and Eureka lemons were sprayed with different volumes of, or dipped in, a mixture of water and a yellow fluorescent pigment. Sprayed parts were illuminated under black light, visualised under a stereomicroscope and digitally photographed at 10magnification. Quantitative and qualitative deposition assessment of the spray deposition was performed by means of digital image analyses. Hoerl regression curves were fitted to quantitative and qualitative deposition values on upper and lower leaf surfaces over spray volume (R 2 -values >0.95) and trends clearly indicated that deposition on young or mature leaves and fruit improved as spray volume increased, but only until the point of runoff was reached, thereafter deposition quantity and quality decreased. Deposition values following dip treatments were in all cases significantly subordinate to those of the best spray volumes. Mature upper leaf surfaces and Eureka lemon fruit generally retained less spray deposits than lower leaf surfaces and Valencia Late orange fruit, respectively. In order to determine the effect of runoff on biological efficacy of copper hydroxide against Alternaria brown spot, young Nova leaves were treated in a similar fashion and subsequently drop-inoculated with a virulent strain of A. alternata pv. citri and incubated for 3.5 days in moist chambers at 25°C. Biological efficacy of sprays followed a quadratic trend over spray volume and clearly demonstrated the detrimental effect of runoff on biological efficacy of fungicide sprays. Sigmoidal regression analyses of mean infection percentages against quantitative and qualitative deposition on upper and lower surfaces of young Nova leaves yielded very good fits indicating the correlation between biological and deposition data.

Azoxystrobin was evaluated in replicated small-plot trials from 1995 to 1999 for control of citrus black spot (CBS) on ‘Valencia’ oranges caused by Guignardia citricarpa. Applications of different rates of tank mixes of azoxystrobin and mancozeb during the susceptible period from October to January were compared with an untreated control as well as the standard four applications of mancozeb with or without mineral oil (1.20 g a.i./liter + 0.5% [vol/vol]/liter and 1.60 g a.i./liter of water, respectively). Two applications of azoxystrobin in tank mixtures with mancozeb and mineral oil (0.5% [vol/vol]/liter) in mid-November and mid-January at rates of 0.10, 0.15, and 0.20 g a.i./liter controlled CBS by more than 98 to 99%, 99 to 100% and 95 to 98%, respectively. Concomitantly, where mineral oil was not added to the fungicide mixture, azoxystrobin and mancozeb resulted only in 73 to 95%, 74 to 93% and 92.2 to 92.3% CBS control, respectively. Tank mixtures of benomyl, mancozeb, and mineral oil reduced CBS by only 29%, which could be attributed to the presence of benomyl-resistant pathogen isolates in the experimental orchard. Azoxystrobin applied at rates of 0.05, 0.075, and 0.10 g a.i./liter in tank mixtures with mancozeb (1.2 g a.i./liter) and mineral oil (0.5% [vol/vol]/liter of water) or Agral 90 (0.5% [vol/vol]/liter of water) were equally effective, reducing CBS by more than 99%. When mineral oil was compared to different adjuvants in tank mixtures with azoxystrobin and mancozeb, only mineral oil resulted in 100% clean exportable fruit. There was no difference between Sunspray 6E and Bac oil when mixed with azoxystrobin and mancozeb on the degree of disease control. Furthermore, the concentration of mineral oil in water can be lowered from 0.5% (vol/vol)/liter of water to 0.3% (vol/vol)/liter of water without a loss in efficacy against CBS. It is therefore, recommended that azoxystrobin (0.075 g a.i./liter) must be applied in tank mixtures with mancozeb (1.2 g a.i./liter) and mineral oil, which can be applied at either 0.5% (vol/vol)/liter of water or 0.3% (vol/vol)/liter of water.

The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa. This distinction was supported by amplified fragment length polymorphism analysis that also supported the exclusion of two isolates that had apparently been misclassified as G. citricarpa. On cherry decoction agar, but not on other media, growth rates of group I isolates were lower than those of group II isolates. Conidial dimensions were similar, but group I isolates formed conidia with barely visible mucoid sheaths, whereas those of group II formed conidia with thick sheaths. Cultures of isolates belonging to group I produced rare infertile perithecia, whereas fertile perithecia were formed by most isolates of group II. Colonies of isolates belonging to group I were less dark than those of group II, with a wider translucent outer zone and a lobate rather than entire margin. On oatmeal agar, exclusively group I isolates formed a yellow pigment, Group I harbored strains from citrus fruits with classical black spot lesions (1 to 10 mm in diameter) usually containing pycnidia. Group II harbored endophytic strains from a wide range of host species, as well as strains from symptomless citrus fruits or fruits with minute spots (< 2-mm diameter) without pycnidia. These observations support the historic distinction between slowly growing pathogenic isolates and morphologically similar fast-growing, nonpathogenic isolates of G. citricarpa. The latter proved to belong to G. mangiferae (P. capitalensis), a ubiquitous endophyte of woody plants with numerous probable synonyms including G. endophyllicola, G. psidii, P. anacardiacearum, and P. theacearum. G. mangiferae occurs in the European Union and the United States on many host species including citrus, and does not cause symptoms of citrus black spot, justifying its exclusion from quarantine measures.

Conidia of Phyllosticta citricarpa (Pc) did not infect unwounded packhouse-treated citrus fruit maintained at -0.5 and 25 °C in artificial and natural inoculation studies, but infrequently produced black spot lesions from which the pathogen could be isolated on artificially inoculated wounded fruit kept at 25 °C. Conidial germination and appressorium formation by seven Pc isolates from various countries were completely inhibited in vitro by the postharvest fungicides guazatine and o-phenylphenol (sodium salt) at recommended rates, whereas thiabendazole, imazalil sulphate, two emulsion formulations of imazalil, and particularly prochloraz, provided significant inhibition. Chlorine dioxide suppressed conidial germination more effectively and at lower concentrations than calcium hypochlorite. Packhouse treatments which consistently reduced the viability of Pc in black spot lesions on fruit included warm water (43-47 °C for three minutes), chemical tank (guazatine, imazalil sulphate, 2,4-D), and a combination of chlorine, high-pressure spraying, warm water, chemical tank, and polyethylene waxing. Conidial viability was reduced to zero on black spot-infected fruit stored for three weeks at 25 °C, as well as on fruit exposed to chlorine, warm water, the above chemicals, or all treatments combined.