First report of Phyllosticta citricarpa and description of two new species, P. paracapitalensis and P. paracitricarpa, from citrus in Europe.

The genus Phyllosticta occurs worldwide, and contains numerous plant pathogenic, endophytic and saprobic species. Phyllosticta citricarpa is the causal agent of Citrus Black Spot disease (CBS), affecting fruits and leaves of several citrus hosts (Rutaceae), and can also be isolated from asymptomatic citrus tissues. Citrus Black Spot occurs in citrus-growing regions with warm summer rainfall climates, but is absent in countries of the European Union (EU). Phyllosticta capitalensis is morphologically similar to P. citricarpa, but is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts, and is known to occur in Europe. To determine which Phyllosticta spp. occur within citrus growing regions of EU countries, several surveys were conducted (2015-2017) in the major citrus production areas of Greece, Italy, Malta, Portugal and Spain to collect both living plant material and leaf litter in commercial nurseries, orchards, gardens, backyards and plant collections. A total of 64 Phyllosticta isolates were obtained from citrus in Europe, of which 52 were included in a multi-locus (ITS, actA, tef1, gapdh, LSU and rpb2 genes) DNA dataset. Two isolates from Florida (USA), three isolates from China, and several reference strains from Australia, South Africa and South America were included in the overall 99 isolate dataset. Based on the data obtained, two known species were identified, namely P. capitalensis (from asymptomatic living leaves of Citrus spp.) in Greece, Italy, Malta, Portugal and Spain, and P. citricarpa (from leaf litter of C. sinensis and C. limon) in Italy, Malta and Portugal. Moreover, two new species were described, namely P. paracapitalensis (from asymptomatic living leaves of Citrus spp.) in Italy and Spain, and P. paracitricarpa (from leaf litter of C. limon) in Greece. On a genotypic level, isolates of P. citricarpa populations from Italy and Malta (MAT1-2-1) represented a single clone, and those from Portugal (MAT1-1-1) another. Isolates of P. citricarpa and P. paracitricarpa were able to induce atypical lesions (necrosis) in artificially inoculated mature sweet orange fruit, while P. capitalensis and P. paracapitalensis induced no lesions. The Phyllosticta species recovered were not found to be widespread, and were not associated with disease symptoms, indicating that the fungi persisted over time, but did not cause disease.

Partial Cold Treatment of Citrus Fruit for Export Risk Mitigation for Thaumatotibia leucotreta (Lepidoptera: Tortricidae) as Part of a Systems Approach.

Some of South Africa's citrus export markets require mandatory postharvest cold treatment of citrus fruit as a phytosanitary risk mitigation treatment for Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). An alternative to this may be partial cold treatment as one of the final steps in a systems approach to mitigate phytosanitary risk. Consequently, the efficacy of such partial cold treatments was evaluated. It was first determined that a 2°C cold treatment was significantly more effective against fourth and fifth instars (the most cold-tolerant instars) than treatments at 3°C and 4°C for a duration of 18 d. Secondly, it was determined that 2°C for 18 d and 1°C for 16 d were similarly effective, but both treatments were significantly more effective than 1°C for 14 d. Mean mortality of fourth and fifth instars treated with 2°C for 18 d in seven replicates from four trials was 99.94%. Finally, it was determined that the inability of the majority of surviving larvae to develop to adulthood would further increase the efficacy of a 2°C for 18 d treatment to 99.96%. Inclusion of reproductive nonviability of survivors increased mortality to 99.99%.

Verification of Inspection Standards and Efficacy of a Systems Approach for Thaumatotibia leucotreta (Lepidoptera: Tortricidae) for Export Citrus From South Africa.

A systems approach has been developed for mitigation of risk associated with Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), in citrus fruit exported from South Africa, as an alternative to a stand-alone cold treatment. This study was undertaken to assess compliance with inspection standards applicable to various steps within the systems approach and to determine its overall efficacy. Larval infestation of fruit was monitored weekly in fruit from 33 orchards, until the time of harvest, postpicking, and postpacking into export cartons. Significant positive regressions were recorded between infestation of fruit during the full monitoring period in the orchard and the last 4 wk before harvest, between the last 4 wk before harvest and on delivery to the packinghouse, and on delivery to the packinghouse and in the packed carton. There was an improvement in the level of compliance with each of these successive steps in the system, thus verifying that the grading and inspection thresholds were appropriately sensitive and confirmed the effectiveness of the system. The overall risk mitigation efficacy of the systems approach was calculated. The calculation included several known compounding under estimations of efficacy. Nonetheless, the proportion of fruit that could be infested with T. leucotreta after application of the systems approach was between P ≤ 5.328 × 10(-6) and P ≤ 8.380 × 10(-7), 6-38 times less than the proportion associated with the probit 9 (P ≤ 3.2 × 10(-5)) standard for a stand-alone cold treatment, being three survivors in 100,000 at the 95% confidence level.

Comparing the Use of Laboratory-Reared and Field-Collected Thaumatotibia leucotreta (Lepidoptera: Tortricidae) Larvae for Demonstrating Efficacy of Postharvest Cold Treatments in Citrus Fruit.

Some of South Africa's export markets require postharvest cold treatment of citrus fruit for phytosanitary risk mitigation for Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). An alternative to a standalone cold treatment may be a reduced intensity cold treatment as a step in a systems approach. For cold treatment trials, large numbers of larvae are required. Due to recent dramatic improvement of T. leucotreta control in the field, sufficient naturally infested citrus fruit are no longer available. Artificial infestation of fruit is not viable due to rapid decay of the fruit. Consequently, it is necessary to use laboratory-reared T. leucotreta larvae in artificial diet. In trials, field-collected larvae from the Eastern Cape were at least as cold-tolerant as those from other regions. Larvae in Navel oranges showed the median level of susceptibility in a range of citrus types evaluated at 6°C, and their use in trials was considered acceptable due to their greater natural susceptibility to T. leucotreta infestation. We demonstrated that larvae at high density in artificial diet were at least as cold-tolerant as larvae at lower densities. When exposed to 2°C for 18 d or longer, larvae in artificial diet as used in the trials were at least as cold-tolerant as larvae in fruit. Very few surviving larvae from fruit completed development, with no subsequent generation. Consequently, it is considered justifiable to conduct cold-treatment trials with laboratory-reared T. leucotreta larvae in artificial diet without risk of overestimating the effect of cold on feral larvae in citrus fruit. [corrected]

The potential distribution of Bactrocera dorsalis: considering phenology and irrigation patterns.

A species in the Bactrocera dorsalis (Hendel) complex was detected in Kenya during 2003 and classified as Bactrocera invadens Drew, Tsuruta & White. Having spread rapidly throughout Africa, it threatens agriculture due to crop damage and loss of market access. In a recent revision of the B. dorsalis complex, B. invadens was incorporated into the species B. dorsalis. The potential distribution of B. dorsalis has been previously modelled. However, previous models were based on presence data and did not incorporate information on the seasonal phenology of B. dorsalis, nor on the possible influence that irrigation may have on its distribution. Methyl eugenol-baited traps were used to collect B. dorsalis in Africa. Seasonal phenology data, measured as fly abundance throughout the year, was related to each location's climate to infer climatic growth response parameters. These functions were used along with African distribution records and development studies to fit the niche model for B. dorsalis, using independent global distribution records outside Africa for model validation. Areas at greatest risk of invasion by B. dorsalis are South and Central America, Mexico, southernmost USA, parts of the Mediterranean coast, parts of Southern and Eastern Australia and New Zealand's North Island. Under irrigation, most of Africa and Australia appear climatically suitable.

Combining field phenological observations with distribution data to model the potential distribution of the fruit fly Ceratitis rosa Karsch (Diptera: Tephritidae).

Despite the potential for phenological and abundance data to improve the reliability of species niche models, they are seldom used. The aim of this study was to combine information on the distribution, relative abundance and seasonal phenology of Natal fruit fly, Ceratitis rosa Karsch (Diptera: Tephritidae), in South Africa to model its potential global distribution. Bucket traps, baited with Biolure, were used to trap C. rosa in different climatic regions of South Africa over a two-year period. A CLIMEX niche model of the potential global distribution of C. rosa was fitted using the collected trapping data and other distribution records from South Africa. Independent distribution records for elsewhere in Africa were reserved for model validation. The CLIMEX model results conformed well to the South African trapping data, including information on relative abundance and seasonal phenology, as well as to the pattern of presence records of the species elsewhere in Africa. The model suggests that under recent historical conditions a large part of South America, Central America, Mexico and southern USA may be climatically suitable for establishment of C. rosa. In Europe, climatically suitable habitat is restricted to coastal regions of the Mediterranean, in Asia, mostly to the southern and south eastern countries, and in Australia mostly to the wetter south and east. The independent cross-validation provided by South African relative abundance and seasonal phenology data, central African distribution data and relevant species specific biological information provides greater confidence in the modelled potential distribution of C. rosa.

Cold susceptibility and disinfestation of Bactrocera invadens (Diptera: Tephritidae) in oranges.

To develop a cold disinfestation treatment for the fruit fly Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) that is rapidly spreading across Africa, research was conducted in Nairobi, Kenya, using flies from a laboratory culture and 'Valencia' orange (Citrus sinensis L. Osbeck) as the host. The developmental rate of B. invadens in Valencia oranges was determined at 28 degrees C, and the third instar was found to be the least susceptible of the egg and larval life stages to cold treatment at 1.1 degrees C in oranges. When 22,449 B. invadens third instars were exposed in oranges to a cold treatment with an approximate midpoint of 1.1 +/- 0.5 degrees C, the results suggested that a period of 16 d would be worthwhile verifying on a larger scale in oranges. Results from the first replicate of 16,617 larvae showed no survivors, but the second replicate of 23,536 larvae had three survivors. Because a longer cold treatment based on a mean temperature of 1.1 degrees C would create logistical difficulties for some export markets, further replicates were conducted at an approximate midpoint of 0.5 degrees C and at mean hourly maximum of 0.9 +/- 0.5 degrees C, for 16 d. After three replicates, in which 65,752 B. invadens third instars in total were treated with no survivors, the Japanese requirement of 99.99% mortality at the 95% confidence level was surpassed. The following treatment protocol for B. invadens larvae in oranges can therefore be recommended: fruit pulp to be maintained at temperatures of 0.9 degrees C or lower for 16 consecutive days.