Efficacy of Various Low Temperature and Exposure Time Combinations for Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) Larvae.

A systems approach was developed as an alternative to a standalone quarantine disinfestation treatment for Thaumatotibia leucotreta in citrus fruit exported from South Africa. The systems approach consists of three measures: pre and postharvest controls and measurements, postpacking inspection, and postharvest exposure to low temperatures. Different cold treatment conditions with a range of efficacy levels can be used for this last measure. A series of trials reported here evaluated the efficacy of seven temperatures ranging from 0 to 5°C for durations from 14 d to 26 d. Mortality of the most cold-tolerant larval stages of T. leucotreta was determined. Temperatures of 0, 1, 2, and 3°C for 16, 19, 20, and 24 d respectively, induced 100% mortality of the tested populations. Probit 9 level treatment efficacy was achieved at 0 and 1°C for 16 and 19 d respectively. Mortalities higher than 90% were obtained with temperatures of 4, 4.5, and 5°C, after exposure for the longer durations. We demonstrated a significant difference in cold-induced insecticidal efficacy between 1, 2, 3, and 4°C. There was no significant difference in insecticidal efficacy between 4 and 4.5°C, but both of these temperatures were more efficacious than 5°C. The results of this study are valuable to support the use of cold treatment conditions with lower risk of fruit chilling injury in an effective systems approach, where the cold treatment efficacy can be augmented with other components of the systems approach.

First report of 'Candidatus Liberibacter africanus' associated with African Greening of Citrus in Angola.

Huanglongbing (HLB, Asian Citrus Greening), the most devastating disease of citrus has not been detected in southern Africa (Gottwald, 2010). HLB is associated with 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium vectored by Diaphorina citri Kuwayama (Hemiptera: Liviidae), the Asian Citrus Psyllid (ACP). African Citrus Greening, associated with 'Candidatus Liberibacter africanus' (CLaf) and its vector the African Citrus Triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), are endemic to Africa, although not previously reported from Angola. African Greening is less severe than HLB, largely due to heat sensitivity of CLaf and its vector. Introduction of HLB into southern Africa would be devastating to citrus production in commercial and informal sectors. Concern was raised that CLas or ACP might hae inadvertently been introduced into Angola. In July 2019, a survey was conducted in two citrus nurseries in Luanda and Caxito and in different orchards on 7 farms surrounding Calulo and Quibala. Yellow sticky traps for insects were placed at the various localities and collected after c. 3 weeks. Breeding signs of T. erytreae (pit galls) were observed on citrus in some locations, but no insect vectors were detected on traps. Trees were inspected for signs and symptoms of citrus pests and diseases, particularly those that resemble HLB (foliar blotchy mottle, shoot chlorosis, vein yellowing and corking, lopsided fruit with aborted seeds and colour inversion) and its vectors (pit galls on leaves or waxy exudates). Leaves and shoots with suspect symptoms were sampled for laboratory analysis (43 samples). DNA was extracted from petiole and midrib tissue of leaves using a modified CTAB extraction protocol of Doyle and Doyle (1990). Real-time PCR was done using universal Liberibacter primers of Roberts et al. (2015), CLaf specific primers of Li et al. (2006) and CLas specific primers of Bao et al. (2019). All real-time PCR protocols indicated the presence of CLaf in 6 samples (Tab. S1). CLas or other citrus Liberibacter species were not detected. The presence of CLaf in sample 37 was confirmed by constructing a library (NEXTFLEX® DNA Sequencing Kit, PerkinElmer) with extracted DNA and performing high-throughput sequencing on an Ion Torrent™ S5™ platform (Central Analytical Facility, Stellenbosch University). To improve the quality of the reads, all 233,617,700 obtained reads were trimmed from the 3' end to a maximum length of 240 nt using Trimmomatic (Bolger et al. 2014). The high quality reads were mapped to the Citrus sinensis reference genome (NC_023046.1) using Bowtie 2.3.4 (Langmead and Salzberg 2012) to subtract all the reads that had high identity to the host plant (number of mismatches allowed in the seed was set to 1). The 14,691,369 unmapped reads (6.2% of original data) were mapped to the CLaf reference genome NZ_CP004021.1 using CLC Genomics Workbench 10.1.1 (Qiagen) (Length fraction = 0.8; Similarity fraction = 0.9). A CLaf consensus genome was generated that spanned 99.7% of the reference genome and the 163001 mapped reads had a 22.9 mean read coverage. The consensus sequence was 99.7% identical to NZ_CP004021.1 and was submitted to Genbank as accession: CP054879. The positive CLaf detections were from trees with typical HLB or African Citrus Greening symptoms, viz. lopsided fruit with green stylar ends, aborted seed and stained columella at base of fruit button; yellow shoots with leaves showing symptoms of blotchy mottle and vein yellowing and corking (Fig. S1) in a commercial citrus farm outside Calulo and included 2 'Ponkan' mandarin (C. reticulata), 2 Valencia and 1 'Navelina' tree (C. sinensis), and a citrus nursery in Luanda (1 lime tree; C. aurantifolia) (Tab. S1). This first report of CLaf in Angola highlights the need to prevent spread by removing infected trees and managing the insect vector, as well as the need for further surveys to determine the occurrence of African Greening and its vectors in other provinces and to confirm the absence of exotic citrus pests and diseases. References Bao, M. et al. 2020. Plant Dis. 104:527 Bolger, A. M. et al. 2014. Bioinformatics. 30:2114-2120. Doyle, J.J. and Doyle, J.L. 1990. Focus 12:13 Gottwald, T.R. 2010. Annu. Rev. Phytopathol. 48:119 Langmead, B. and Salzberg, S. 2012. Nature Methods. 9:357-359. Li, W. et al. 2006. Jnl. Microbiol. Methods 66:104 Roberts, R. et al. 2015. Int. J. Syst. Evol. Micr. 65:723.

An Improved Systems Approach as a Phytosanitary Measure for Thaumatotibia leucotreta (Lepidoptera: Tortricidae) in Export Citrus Fruit From South Africa.

A systems approach was previously developed for mitigating phytosanitary risk of Thaumatotibia leucotreta (Meyrick) in citrus fruit exported from South Africa, as an alternative to a standalone cold disinfestation treatment. The present study first tested the original systems approach by applying it on a semicommercial scale in 10 Nova mandarin orchards. Fruit were inspected at points in the production, packing, and simulated shipping process, to assess performance of the systems approach. Additional data were obtained from 17 Valencia orange orchards and six packinghouses. In the second part of this study, the systems approach was accordingly revised and improved, consisting of three measures: 1) preharvest controls and measurements and postpicking sampling, inspection, and packinghouse procedures; 2) postpacking sampling and inspection; and 3) shipping conditions. The model quantifying the effectiveness of the systems approach was improved by correcting errors in the original version, updating parameter values and adding a component that provides for comparison with the risk mitigation provided by a standalone disinfestation treatment. Consequently, the maximum potential proportion of fruit that may be infested with live T. leucotreta after application of the improved systems approach is no greater than the proportion of fruit that may be infested after application of a Probit 9 efficacy postharvest disinfestation treatment to fruit with a 2% pretreatment infestation. The probability of a mating pair surviving is also determined. The model enables a priori determination of the required threshold levels for any of the three measures, based on quantification of the other two measures.

Development of a Postharvest Cold Treatment for Cryptophlebia peltastica (Lepidoptera: Tortricidae) for Export of Litchis From South Africa.

The litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae), is endemic to sub-Saharan Africa and certain Indian Ocean islands. It is an important pest of litchis and to a lesser extent macadamias. Litchis are exported to certain markets that consider C. peltastica as a phytosanitary pest. Consequently, an effective postharvest phytosanitary treatment is required. This study sought to develop a cold disinfestation treatment for this purpose. First, it was established that the fifth instar was the most cold-tolerant larval stage, as it was the only instar for which there was still some survival after 12 d at 1°C. It was then determined that cold treatment trials could be conducted in artificial diet, as there was no survival of fifth instar C. peltastica in litchis after only 9 d at 1°C, whereas it took 15 d at this temperature before no survival of fifth instar C. peltastica was recorded in artificial diet. Consequently, cold susceptibility of fifth instar C. peltastica and the most cold-tolerant larval stages (fourth and fifth instar) of false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), were compared in artificial diet. There was no survival of C. peltastica after 13 d at 1°C, whereas this was only so for T. leucotreta after 16 d. Consequently, it can be concluded that any cold treatment that has been proven effective against T. leucotreta would be as effective against C. peltastica. Finally, it was confirmed that the cold susceptibility of T. leucotreta in artificial diet did not overestimate the effect of cold on T. leucotreta larvae in litchis.

The Cryptophlebia leucotreta granulovirus-10 years of commercial field use.

In the last 15 years, extensive work on the Cryptophlebia leucotreta granulovirus (CrleGV) has been conducted in South Africa, initially in the laboratory, but subsequently also in the field. This culminated in the registration of the first CrleGV-based biopesticide in 2004 (hence, the 10 years of commercial use in the field) and the second one three years later. Since 2000, more than 50 field trials have been conducted with CrleGV against the false codling moth, Thaumatotibia leucotreta, on citrus in South Africa. In a representative sample of 13 field trials reported over this period, efficacy (measured by reduction in larval infestation of fruit) ranged between 30% and 92%. Efficacy was shown to persist at a level of 70% for up to 17 weeks after application of CrleGV. This only occurred where the virus was applied in blocks rather than to single trees. The addition of molasses substantially and sometimes significantly enhanced efficacy. It was also established that CrleGV should not be applied at less than ~2 × 1013 OBs per ha in order to avoid compromised efficacy. As CrleGV-based products were shown to be at least as effective as chemical alternatives, persistent and compatible with natural enemies, their use is recommended within an integrated program for control of T. leucotreta on citrus and other crops.