Sean D. Moore

Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential against false codling moth

A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50IJs/FCM larva caused 100% mortality and 74% at a concentration of 200IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20IJs/cm(2). This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal.

Overcrowding of false codling moth, Thaumatotibia leucotreta (Meyrick) leads to the isolation of five new Cryptophlebia leucotreta granulovirus (CrleGV-SA) isolates.

False codling moth, Thaumatotibia leucotreta (Meyrick) is a serious pest of economic importance to the South African fruit industry. As part of sustainable efforts to control this pest, biological control options that involve the application of baculovirus-based biopesticides such as Cryptogran and Cryptex (both formulated with a South African isolate of Cryptophlebia leucotreta granulovirus, CrleGV-SA) are popularly used by farmers. In order to safeguard the integrity of these biopesticides as well as protect against any future development of resistance in the host, we conducted a study to bioprospect for additional CrleGV isolates as alternatives to existing ones. Using overcrowding as an induction method for latent infection, we recovered five new CrleGV isolates (CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit, CrleGV-SA MixC and CrleGV-SA Nels). Single restriction endonuclease (REN) analysis of viral genomic DNA extracted from purified occlusion bodies showed that isolates differed in their DNA profiles. Partial sequencing of granulin and egt genes from the different isolates and multiple alignments of nucleotide sequences revealed the presence of single nucleotide polymorphisms (SNPs), some of which resulted in amino acid substitutions in the protein sequence. Based on these findings as well as comparisons with other documented CrleGV isolates, we propose two phylogenetic groups for CrleGV-SA isolates recovered in this study.

An improved larval diet for commercial mass rearing of the false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae)

False codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera:Tortricidae), is endemic to sub-Saharan Africa (Newton 1998). It is a pest of citrus (Newton 1998), stone fruit (Daiber 1978), macadamias (La Croix & Thindwa 1986), avocados (Erichsen & Schoeman 1992) and various other agricultural crops. All available control methods were recently reviewed by Moore & Hattingh (2012). Included amongst these is the use of granulovirus (Cryptophlebia leucotreta granulovirus (CrleGV)) sprays, inundative releases of egg parasitoids and the sterile insect technique (SIT). All of these require production of large numbers of T. leucotreta. CrleGV and the egg parasitoid, Trichogrammatoidea cryptophlebiae (Nagaraja) (Hymenoptera: Trichogrammatidae), are produced in vivo (Moore et al. 2011) and for SIT, the sterilized adult male moth is the product (Hofmeyr et al. 2005).

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.

Investigation of native isolates of entomopathogenic fungi for the biological control of three citrus pests

The pathogenicity of 15 isolates of Beauveria bassiana (Balsamo) Vuillemin, five isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and one isolate of M. flavoviride (Gams and Rozsypal) were tested under laboratory conditions against the subterranean life stages of the citrus pests, Ceratitis rosa Karsch, C. capitata Wiedemann (Diptera: Tephritidae) and Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae). When these citrus pests were treated with a concentration of 1×107 conidia mL−1, fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult and pupal mycosis were considered. Depending on fungal isolate, the percentage of T. leucotreta adults which emerged from fungal treated sand ranged from 5 to 60% and the percentage of pupae with visible signs of mycosis ranged from 21 to 93%. The relative virulence of the four most promising fungal isolates, as well as the commercially available B. bassiana product, BroadBand® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against T. leucotreta which exhibited a dose-dependent response. The estimated LC50 values of the three most virulent B. bassiana isolates ranged from 6.8×105 to 2.1×106 conidia mL−1, while those of the least pathogenic ranged from 1.6×107 to 3.7×107 conidia mL−1.

Baculovirus-based strategies for the management of insect pests: a focus on development and application in South Africa

There is growing concern among governments, scientists, agricultural practitioners and the general public regarding the negative implications of widespread synthetic chemical pesticide application for the control of crop pests. As a result, baculovirus biopesticides are gaining popularity as components of integrated pest management (IPM) programmes in many countries despite several disadvantages related to slow speed of kill, limited host range and complex large scale production. In South Africa, baculoviruses are incorporated into IPM programmes for the control of crop pests in the field, and recent bioprospecting has led to the characterisation of several novel isolates with the potential to be formulated as commercial products. This contribution will provide an overview of the use of baculoviruses against insect pests in South Africa, as well as research and development efforts aimed at broadening their application as biocontrol agents. Challenges faced by researchers in developmental projects as well as potential users of baculoviruses as biopesticides in the field are also discussed.

Persistence and virulence of promising entomopathogenic fungal isolates for use in citrus orchards in South Africa

Abstract Thaumatotibia leucotreta (Meyrick) (1912) (Lepidoptera: Tortricidae) is a key economic pest of citrus in South Africa. Although a variety of control options are available, none currently registered target the soil-dwelling life stages of the pest. Three fungal isolates, two Metarhizium anisopliae sensu lato Metschnikoff (Sorokin) isolates (G 11 3 L6 and FCM Ar 23 B3) and one Beauveria bassiana sensu lato Balsamo (Vuillemin) isolate (G Ar 17 B3) have been identified as showing the greatest potential against T. leucotreta soil-dwelling life stages. Since environmental persistence is an important factor in the success of entomopathogenic fungi against soil pests, the isolates, along with two commercial isolates (B. bassiana Eco-Bb® and M. anisopliae ICIPE 69), were subjected to a six-month field persistence trial. Every month after burial of the fungal isolates, inside net bags, in orchard soil, the number of colony forming units (CFUs) per gram of soil was measured and the viability of the conidia was assessed using bioassays. All isolates investigated were capable of persisting in the soil for the duration of the trial period and in addition, were capable of initiating infection in T. leucotreta late fifth instar larvae and subsequent pupae. However, with the exception of the commercial isolates which showed some correlation, no correlation was found between the number of CFUs recorded and percentage T. leucotreta mycosis. Persistence of entomopathogenic fungi in the field is affected by a series of factors, and although the effect of these factors was not measured, the data have provided preliminary evidence towards the persistence capability of these candidate biopesticides against T. leucotreta.

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

Abstract Some of South Africas 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 underestimating the effect of cold on feral larvae in citrus fruit.

Beauveria and Metarhizium Against False Codling Moth (Lepidoptera: Tortricidae): A Step Towards Selecting Isolates for Potential Development of a Mycoinsecticide

False codling moth, Thaumatotibia leucotreta Meyrick (1912) (Lepidoptera: Tortricidae), can cause both pre- and post-harvest damage to citrus fruit. Not only can this result in reduced crop yield, but more importantly because of the moths endemism to sub-Saharan Africa, it is classified as a phytosanitary pest by many export markets. An entire consignment of citrus may be rejected in the presence of a single moth (Moore 2012). Since the bulk of citrus fruit production in South Africa is exported, the control of T. leucotreta is critical (Citrus Growers Association, South Africa 2012). Traditionally, control has been achieved through the use of chemical insecticides; however, residue restrictions, resistance development and concerns about environmental pollution have substantially reduced the dependence on chemical pesticides in citrus. Research on T. leucotreta control has therefore focused on the use of biological organisms (e.g. parasitoids and viruses), which are used as control agents within an integrated pest management (IPM) programme in citrus. These biological control agents, however, only targeted the aboveground life stages of the pest, not the soil-dwelling life stages (late fifth instars, prepupae, pupae), which is the subject of this contribution (Moore 2012).

Thaumatotibia leucotreta and the Navel orange: ovipositional preferences and host susceptibility

False codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), presents a significant threat to the South African citrus industry. To limit income loss due to direct larval damage or from fruit rejection due to the phytosanitary status of this pest, additional pre‐harvest control techniques are required for Navel oranges, which are known to be susceptible to T. leucotreta damage. A number of Navel orange cultivars have been developed, and differences in female T. leucotreta ovipositional preference and susceptibility of cultivars to larval penetration are known to exist. Navel orange cultivars were grouped according to time of maturity (early, mid‐ and late season). Female T. leucotreta were subjected to choice and no‐choice tests with these cultivars, measured by oviposition. Host susceptibility was tested by allowing neonate T. leucotreta larvae to penetrate the different Navel cultivars. In the early maturing group, Fischer Navels were least preferred for oviposition and the least susceptible to larval penetration. The mid‐ and late season maturing groupings showed limited differences in oviposition preference, although host susceptibility did appear to be an important factor in assessing the vulnerability of fruit to T. leucotreta. Despite being widely planted in South Africa, the mid‐season Palmer Navels were highly susceptible to larval penetration, while for the late season cultivars, Cambria and Glen Ora Late were the least susceptible to T. leucotreta. As a result of these laboratory trials, it is recommended that farmers increase cultivation of Fischer Navels as the principal early season cultivar, avoid Palmer Navels in favour of other mid‐season maturing cultivars and give preference to the late maturing Cambria and Glen Ora Late cultivars, to limit T. leucotreta damage.