A three-year survey of the ecology of entomopathogenic nematodes (EPN) and entomopathogenic fungi (EPF) was undertaken on soils from citrus orchards of different ages to determine the influence of orchard age on the ecology of entomopathogenic fungi and nematodes. The influence of mulch and irrigation method on the occurrence of EPN and EPF was also determined. Most of the isolates recovered (n = 810) were Beauveria sp. (87.88% of all isolates), followed by Metarhizium sp. (11.87% of all isolates). Only 0.24% of soil samples collected during this study tested positive for EPN. All EPN isolates recovered were Heterorhabditis bacteriophora. No significant differences in EPF occurrence were recorded between orchards under drip and micro-sprinkler irrigation. EPF occurrence was significantly lower (P = 0.016) in orchards covered by mulch (31.85% +/- 2.07% occurrence) than in orchards with no covering (38.57% +/- 1.57% occurrence). EPF occurrence of 40.33 +/- 2.13% was highest in non-bearing orchards, followed by mature orchards (nine years or older) (36.76 +/- 2.05% of samples) with the lowest EPF occurrence of 25.30 +/- 2.02% reported in juvenile orchards (four to eight years old). Juvenile orchards sustain significantly less EPF than mature and non-bearing orchards because of the combined negative impact of less favourable environmental conditions (lower shade density) and fungicide applications.

Several isolates of Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitacae) and Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitacae) have been investigated as possible microbial control agents of key citrus pests in South Africa. Although laboratory results have been promising, field trials against foliar pests have shown limited success. These findings highlighted the need to investigate other biological attributes of these fungal isolates besides virulence in order to select candidates that may be better suited for the foliar environment. Thus, this study investigated the influence of temperature on the in vitro growth of seven indigenous local isolates and the humidity requirements necessary to promote successful infection, in comparison with two commercial isolates (B. bassiana PPRI 5339 and M. anisopliae ICIPE 69). All the fungal isolates grew across a range of temperatures (8-34 degrees C) and optimally between 26 and 28 degrees C. Similarly, fungal infection of Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) fifth instars occurred across a range of humidity levels (12%, 43%, 75%, 98%) regardless of fungal concentration, although external sporulation was restricted to treatments exposed to 98% relative humidity. It was concluded that neither temperature nor humidity, when considered alone, is likely to significantly influence the efficacy of any of the isolates in the field, given that they are active within temperature and humidity ranges experienced in South African citrus orchards.

Fruit-piercing moths are a sporadic pest of citrus, especially in the Eastern Cape Province of South Africa, where the adults can cause significant damage in outbreak years. However, growers confuse fruit-piercing moths with fruit-sucking moths that do not cause primary damage. In this study we trapped these moths during the 2013–2015 growing seasons. A large number of diverse fruit-feeding moths were collected through weekly sampling in citrus orchards in the Eastern Cape and northern Limpopo provinces. Twenty-three species of fruit-feeding moth were trapped. However, only two were fruit-piercing species, capable of causing primary damage, namely Serrodes partita (Fabricius) (Erebidae) and Eudocima divitiosa (Walker) (Erebidae). Surprisingly S. partita, which has been reported as the main fruit-piercing moth pest of citrus in South Africa, comprised only 6.9 % of trap catches. The categorisation of moths as fruit-piercing or fruit-sucking (causing secondary damage) was confirmed by examining the morphological structures (tearing hooks and erectile barbs) of these moths’ proboscides. This study has shown that in non-outbreak seasons, S. partita comprised only a small percentage of fruit-feeding moths in citrus orchards. However, growers may misidentify the harmless fruit-sucking species as fruit-piercing species, and thus overestimate the density of fruit-piercing moths.

Seven indigenous entomopathogenic fungal isolates were identified as promising biocontrol agents of key citrus pests including false codling moth, Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), citrus thrips, Scirtothrips aurantii Faure (Thysanoptera: Thripidae) and citrus mealybug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae) under laboratory conditions. Even though field trials using the two most virulent isolates (Beauveria bassiana G Ar 17 B3 and Metarhizium anisopliae FCM Ar 23 B3) against soil-dwelling life stages of T. leucotreta were positive, foliar application against citrus mealybugs and thrips, has been disappointing. Thus, the UV sensitivity of the seven initial promising isolates (four B. bassiana and three M. anisopliae) in comparison with two commercial isolates (M. anisopliae ICIPE 69 and B. bassiana PPRI 5339) and their formulated products were investigated in this study. All isolates investigated were highly sensitive to UV radiation, and a 2 h exposure to simulated fullspectrum solar radiation at 0.3 W/m(2) killed conidia of all tested isolates. Nonetheless, variability in susceptibility was found amongst isolates after exposure for 1 h. The most virulent M. anisopliae isolate, FCM Ar 23 B3, was the most susceptible to UV radiation with <3 % relative germination, 48-51 h post-exposure. Whilst isolates of the two mycoinsecticides showed similar susceptibility to UV radiation, their formulated products (vegetable oil and emulsifiable concentrate) were tolerant, when tested for 1 h. These findings indicate that a suitable UV protectant formulation of these fungi or a different application strategy will be required for success against P. citri and S. aurantii. (C) 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

False codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is an important indigenous pest of citrus in southern Africa. Successful control is dependent upon integration of area-wide sterile insect releases and other suppression methods. The aim of this work was to test pyrethroid and organophosphate-based insecticides (tau-fluvalinate and chlorpyrifos) for their residual effect on mortality of released irradiated T.leucotreta male moths. Both of these insecticides were effective in killing irradiated T.leucotreta for 7days after application on leaves, after which degradation of the active ingredient resulted in a marked reduction in efficacy after 14days and rendering them harmless. Mortality was found to be similar for irradiated and non-irradiated male T.leucotreta after treatment. Consequently, even though these insecticides might have an effect on moths in the field, ratios of sterile:wild moths should not be altered. Supporting field data from six sites in the Sundays River Valley over a season of sterile insect releases showed the conventional chemical crop protection programme to be as effective as an integrated pest management programme in facilitating effective control of T.leucotreta through sterile insect releases. The study also confirmed that the ratios of sterile:wild male moths in the commercial citrus orchards were not affected by the application of insecticides. These findings confirm the high potential of sterile insect releases for control of T.leucotreta in citrus.

False codling moth (FCM), Thaumatotibia leucotreta (Lepidoptera: Tortricidae) is an important pest of various fruit crops in South Africa. Current FCM control strategies include the use of chemical insecticides. However, FCM has developed resistance to some of the insecticides, and stringent chemical residue restrictions have been imposed by some foreign markets. Thus, the demand for high-quality fruit has translated into a need for new, efficient and effective integrated pest management (IPM) strategies. One such strategy is the control of the soil-dwelling life stages of FCM, using entomopathogenic nematodes (EPNs) and entomopathogenic fungi (EPF). Both of the biocontrol agents concerned have individually been shown to be effective against FCM. However, it is possible that, if they are applied simultaneously, a synergistic relationship might be observed between EPNs and EPF that could serve to enhance their efficacy against the target pest. In addition to reviewing previous and current control options against FCM in South African fruit crops, this study investigates the potential for using EPNs and EPF individually, and in combination, as biological control agents against FCM within an IPM system.

Markets importing citrus fruit including lemons, Citrus limon (L.) Burman f., from South Africa require that the fruit be free of fruit fly pests (Diptera: Tephritidae). Historically there has been no fruit fly infestation recorded on lemons destined for export from SouthAfrica. In this study, we assessed the host status of commercial export grade Eureka lemons, Citrus limon (L.) Burmanf. cv. Eureka, for four fruit fly pest species of economic importance in South Africa: Ceratitis capitata (Wiedemann), Ceratitis rosa Karsch, Ceratitis quilicii De Meyer, Mwatawala & Virgilio, and Bactrocera dorsalis (Hendel). Trapping was conducted in 10 Eureka lemon orchards in two major citrus production regions over two citrus seasons between 2016 and 2017 to determine the level of fruit fly abundance in the sampled orchards. Lemons were collected at harvest over the two seasons in the same orchards where trapping was conducted. Fruit fly infestation of the sampled lemons was determined by dissection. Additionally, infestation of lemons was determined under forced exposure to mature mated females of C. capitata and B. dorsalis. Trapping data showed the presence of adults of all four fruit fly species in the sampled lemon orchards. Nofruit fly infestation was detected in 43 222 Eureka lemons sampled at harvest. There was also no infestation of lemons under forced exposure conditions. The results of this study provide evidence with 99.99 % efficacy and a 99% confidence level that SouthAfrican commercial export grade Eureka lemon fruit is not a host for C. capitata, C. rosa, C. quilicii or B. dorsalis.

Ectomyeolis ceratoniae Zeller (Lepidoptera: Pyralidae), carob moth, is a pest of several crops in South Africa. A laboratory culture was established from field-collected larvae infesting mummified pecan nuts. Biological parameters of larvae reared on an artificial diet were measured. The insect goes through five larval instars, and the head capsule sizes of the five instars were determined to be ≤0.34 mm, 0.35–0.64 mm, 0.65–0.94 mm, 0.95–1.14 mm and ≥0.15 mm for the five instars, respectively. The insect was reared individually and communally in glass vials, the latter to develop a mass-rearing technique. Developmental time from neonate to pupa was significantly slower when larvae were individually reared (38.18 ± 1.2 days) compared to when they were communally reared (24.6 ± 0.65 days).Amicrosporidian infection (Nosema sp.) was recorded in the culture, causing significantly (F1,6 = 14.99, P = 0.0082) higher mortality of communally reared larvae (76.25 % ± 11.87) than individually reared larvae (24.9 % ± 9.6).

Citrus is grown commercially in several different regions throughout the world, both for juicing and for fresh fruit consumption. All of these regions experience attack by a range of insect and mite pests. Although the exact species present in each pest complex differs, there are certain key pests that are common among most citrus-producing regions, such as armored scales, mealybugs, thrips, mites, and fruit flies. It is extremely rare to find an insect or mite pest that is not at least susceptible to attack by an entomopathogen; therefore, all citrus pests should be seen as potential targets and investigated as such. We review the entomopathogens (fungi, nematodes, viruses, and bacteria) recorded on citrus pests and focus on those microbes that have been tested and developed as biological control agents. Due to market requirements, future prospects for microbial control in citrus are bright. However, products will have to demonstrate similar efficacy to chemical alternatives. This may be achievable through integration with other modes of control, synergism and utilization of pathogen genes. Regulatory and market pressures on industries that supply discerning Western markets are likely to be the strongest driver in the growth of nonchemical pest control solutions, including microbial control.

'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.

Entomopathogenic fungal isolates Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) strain G Ar 17 B3 and Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) strain FCM Ar 23 B3 have been identified as effective control agents of the important citrus pest Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) wandering fifth instars under laboratory conditions. This study is the first report on the ability of these isolates to persist and reduce T. leucotreta infestation in commercial citrus orchards in South Africa. A reduction in pest infestation of between 28 and 82 % upon trial completion was reported. Both isolates were recovered from soil samples collected five months post-application with high host density and moderate to high soil moisture favouring recovery. Low soil moisture negatively influenced the persistence of both isolates and the control efficiency of B. bassiana . These results provide evidence and support for the future use of these isolates against T. leucotreta thus warranting further investigation.

The complete genomes of two novel South African betabaculovirus isolates, namely Phthorimaea operculella granulovirus (PhopGV-SA) and Plutella xylostella granulovirus (PlxyGV-SA), were sequenced and compared to the respective reference isolates PhopGV-1346 and PlxyGV-K1. For both isolates, the genome size and guanine-cytosine (GC) content were similar to those of the respective reference genomes. However, numerous-single nucleotide polymorphisms (SNPs) and several insertions/deletions were observed, revealing the novelty of the isolates. Focus was placed on analysing the observed insertion/deletion events by conducting amino acid sequence alignments for all ORFs of each isolate against all respective ORFs in the corresponding reference isolate. Certain ORFs in each granulovirus genome contained significant insertion/deletion events. In addition, the PlxyGV-SA genome had single-nucleotide insertions/deletions in ORFs 38 and 49 that resulted in the extension and complete overlap of these two ORFs with the neighbouring ORFs 39 and 48, respectively. These novel isolates have significant potential for development and application as biopesticides in South Africa, and the genetic variations observed may have important implications for the biological activity and management of host resistance in the field.

Agathis bishopi (Nixon) (Hymenoptera: Braconidae) is a koinobiont larval endoparasitoid of false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a pest of economic importance on citrus in South Africa. In the field Agathis bishopi was found to parasitise up to 34 % of FCM larvae in fruit, reflecting reasonable biocontrol potential. Improving the rearing of A. bishopi would therefore complement the existing biocontrol strategies for FCM. In several parasitic wasps, sugar concentration and feeding duration has been shown to influence parasitism and longevity. However, their effect on parasitism and longevity of A. bishopi is unknown. In the present study a rearing protocol for A. bishopi is described, including evaluation of the effects of honey concentration on parasitoid longevity. On average, 18.2 % of FCM larvae in rearing containers were parasitised under the rearing. protocol described. Cotton wool, instead of paper towelling, as honey carrier for feeding parasitoids in rearing containers significantly increased parasitism and yield of offspring. Furthermore, longevity significantly increased with higher concentrations of honey. Maximum lifespan duration for male and female parasitoids was achieved when parasitoids were fed on 36 % (w/v) honey. Results from this study indicate that A. bishopi requires a sufficient concentration of sugar, coupled with frequent and prolonged feeding on a cotton wool substrate, in order to achieve maximum parasitism and longevity. Such information provides a basis for optimising mass-rearing and longevity of A. bishopi and parasitism of FCM in orchards.

Planococcus citri (citrus mealybug) is a common and damaging citrus crop pest which has proven difficult to control using conventional methods, such as chemical pesticides and insect growth regulators, particularly late in the citrus growing season. The virulence of two entomopathogenic fungal species was studied in laboratory bioassays against the crawlers and adults of P. citri. Isolates of Metarhizium anisopliae and Beauveria bassiana, collected from citrus orchards in the Eastern Cape Province in SouthAfrica, were verified using and molecular techniques. Mealybug bioassays were performed in 24-well plates. Beauveria bassiana (GAR 17 B3) and M. anisopliae (FCM AR 23 B3) isolates both resulted in 67.5 % mortality of mealybug crawlers and B. bassiana (GB AR 23 13 3) resulted in 64 % crawler mortality with concentrations of 1 x 10(7) conidia/ml. These three isolates were further tested in multiple-dose bioassays to determine the median lethal concentration (LC50), which were 5.29 x 10(5) conidia/ml for the M. anisopliae isolate (FCM AR 23 B3), 4.25 x 106 conidia/ml for B. bassiana (GAR 17 B3), and 6.65 x 10(7) conidia/ml B. bassiana (GB AR 23 13 3) for crawlers, respectively. The results of this study suggested that two isolates (M. anisopliae FCM AR 23 B3 and B. bassianaGAR 17 B3) showed potential for further development as biological control agents against citrus mealybug. Further research would be required to determine their ability to perform under field conditions.

Agathis bishopi (Nixon) (Hymenoptera: Braconidae)is an arrhenotokous larval endoparasitoid of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) (Gendall 2007; Hofmeyr et al. 2015) commonly known as false codling moth (FCM), a major pest of citrus in South Africa (Moore et al. 2004; Malan et al. 2011). Under field conditions, A. bishopi was identified attacking more than 34% of FCM larvae in fruit, showing good biocontrol potential (Gendall 2007). Preference by A. bishopi for parasitising the early instars of its concealed host suggests that the parasitoid has strong natural host location ability (Sishuba 2003; Gendall 2007).

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 degrees 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 degrees 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 underestimating the effect of cold on feral larvae in citrus fruit.

False codling moth (FCM), Thaumatotibia leucotreta, is one of the most important insect pests of citrus in South Africa. No treatment is currently directed towards the soil stages of FCM, with entomopathogenic nematodes (EPNs) having the potential to fill the niche. Laboratory bioassays in orchard soil, using Heterorhabditis bacteriophora, showed the LD50 to be ≈3 and the LD90 to be ≈58 infective juveniles (IJs) per FCM larva. In a field trial, after application of three concentrations (20, 40 and 80 IJs/cm2) of H. bacteriophora, mortalities of >90 %were obtained for FCM larvae, with significant difference between the lower concentrations, but not with higher concentrations. Twenty-one days after application, there was no further FCMcontrol.With the field application of H. zealandica after 6 days, no significant differences were found in FCM mortality of >80 % between three nematode concentrations (5, 10 and 20 IJs/cm2). After 21 and 35 days no significantly different FCM mortality was found for all three treatments compared to the untreated trees. In a field trial using three nematode species, treatment with H. zealandica resulted in significant control for each evaluation day, up to day 49. Results from field trials showed local EPN species to have great potential for control of the soil stages of FCM, with the added possibility of good persistence.

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 degrees C cold treatment was significantly more effective against fourth and fifth instars (the most cold-tolerant instars) than treatments at 3 degrees C and 4 degrees C for a duration of 18 d. Secondly, it was determined that 2 degrees C for 18 d and 1 degrees C for 16 d were similarly effective, but both treatments were significantly more effective than 1 degrees C for 14 d. Mean mortality of fourth and fifth instars treated with 2 degrees 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 degrees C for 18 d treatment to 99.96%. Inclusion of reproductive nonviability of survivors increased mortality to 99.99%.