Field evaluation of the use of select entomopathogenic fungal isolates as microbial control agents of the soil-dwelling life stages of a key South African citrus pest, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae)

The control of false codling moth (FCM), Thaumatotibia leucotreta (Meyrick, 1912) (Lepidoptera: Tortricidae), in citrus orchards is strongly reliant on the use of integrated pest management as key export markets impose stringent chemical restrictions on exported fruit and have a strict no entry policy towards this phytosanitary pest. Most current, registered control methods target the above-ground life stages of FCM, not the soil-dwelling life stages. As such, entomopathogenic fungi which are ubiquitous, percutaneously infective soil-borne microbes that have been used successfully as control agents worldwide, present ideal candidates as additional control agents. Following an initial identification of 62 fungal entomopathogens isolated from soil collected from citrus orchards in the Eastern Cape Province, South Africa, further laboratory research has highlighted three isolates as having the greatest control potential against FCM subterranean life stages: Metarhizium anisopliae G 11 3 L6 (Ma1), M. anisopliae FCM Ar 23 B3 (Ma2) and Beauveria bassiana G Ar 17 B3 (Bb1). These isolates are capable of causing above 80% laboratory-induced mycosis of FCM fifth instars. Whether this level of efficacy was obtainable under sub-optimal and fluctuating field conditions was unknown. Thus, this thesis aimed to address the following issues with regards to the three most laboratory-virulent fungal isolates: field efficacy, field persistence, optimal application rate, application timing, environmental dependency, compatibility with fungicides and the use of different wetting agents to promote field efficacy. Following fungal application to one hectare treatment blocks in the field, FCM infestation within fruit was reduced by 28.3% to 81.7%. Isolate Bb1 performed best under moderate to high soil moisture whilst Ma2 was more effective under low soil moisture conditions. All isolates, with the exception of Ma2 at one site, were recorded in the soil five months post-application. None of the wetting agents tested were found to be highly toxic to fungal germination and similar physical suspension characteristics were observed. Fungicide toxicity varied amongst isolates and test conditions. However, only Dithane (a.i. mancozeb) was considered incompatible with isolate Ma2. The implication of these results and the way forward is discussed. This study is the first report of the field efficacy of three laboratory-virulent fungal isolates applied to the soil of conventional citrus orchards against FCM soil-dwelling life stages. As such, it provides a foundation on which future research can build to ensure the development and commercialisation of a cost-effective and consistently reliable product

Entomopathogenic fungi for control of soil-borne life stages of false codling moth, Thaumatotibia leucotreta (Meyrick) (1912) (Lepidoptera: Tortricidae)

False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essentia

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 moth's 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)

Dynamic and redundant regulation of LRRK2 and LRRK1 expression

<p>Abstract</p> <p>Background</p> <p>Mutations within the <it>leucine-rich repeat kinase 2 </it>(<it>LRRK2</it>) gene account for a significant proportion of autosomal-dominant and some late-onset sporadic Parkinson's disease. Elucidation of LRRK2 protein function in health and disease provides an opportunity for deciphering molecular pathways important in neurodegeneration. In mammals, LRRK1 and LRRK2 protein comprise a unique family encoding a GTPase domain that controls intrinsic kinase activity. The expression profiles of the murine LRRK proteins have not been fully described and insufficiently characterized antibodies have produced conflicting results in the literature.</p> <p>Results</p> <p>Herein, we comprehensively evaluate twenty-one commercially available antibodies to the LRRK2 protein using mouse <it>LRRK2 </it>and human <it>LRRK2 </it>expression vectors, wild-type and <it>LRRK2</it>-null mouse brain lysates and human brain lysates. Eleven antibodies detect over-expressed human LRRK2 while four antibodies detect endogenous human LRRK2. In contrast, two antibodies recognize over-expressed mouse LRRK2 and one antibody detected endogenous mouse LRRK2. LRRK2 protein resides in both soluble and detergent soluble protein fractions. <it>LRRK2 </it>and the related <it>LRRK1 </it>genes encode low levels of expressed mRNA species corresponding to low levels of protein both during development and in adulthood with largely redundant expression profiles.</p> <p>Conclusion</p> <p>Despite previously published results, commercially available antibodies generally fail to recognize endogenous mouse LRRK2 protein; however, several antibodies retain the ability to detect over-expressed mouse LRRK2 protein. Over half of the commercially available antibodies tested detect over-expressed human LRRK2 protein and some have sufficient specificity to detect endogenous LRRK2 in human brain. The mammalian LRRK proteins are developmentally regulated in several tissues and coordinated expression suggest possible redundancy in the function between <it>LRRK1 </it>and <it>LRRK2</it>.</p

An evaluation of detection methods for large lariat RNAs

Ty1 elements are long terminal repeat (LTR) retrotransposons that reside within the genome of Saccharomyces cerevisiae. It has been known for many years that the 2′-5′ phosphodiesterase Dbr1p, which debranches intron lariats, is required for efficient Ty1 transposition. A recent report suggested the intriguing possibility that Ty1 RNA forms a lariat as a transposition intermediate. We set out to further investigate the nature of the proposed Ty1 lariat branchpoint. However, using a wide range of techniques we were unable to find any evidence for the proposed lariat structure. Furthermore, we demonstrate that some of the techniques used in the initial study describing the lariat are capable of incorrectly reporting a lariat structure. Thus, the role of the Dbr1 protein in Ty1 retrotransposition remains elusive

Entomopathogenic fungi associated with cultivated honeybush, Cyclopia spp., in South Africa and their pathogenicity towards a leafhopper pest, Molopopterus sp. (Hemiptera: Cicadellidae)

The southern and eastern parts of the African Fynbos region favour the production of honeybush tea. Honeybush biomass and extracts are used to prepare a beverage both locally and internationally, mainly as herbal tea with health benefits. Honeybush tea is mostly grown organically requiring natural control measures for pests and diseases. The leafhopper, Molopopterus sp., is one of the most important pests of cultivated honeybush in South Africa, as its feeding compromises the quality and quantity of the yield through leaf discolouration and reduction of the photosynthetic area. Local entomopathogenic fungi (EPF) can provide a pool of potential biocontrol agents for this pest. Therefore, a total of 98 soil samples were collected from organically grown honeybush fields and vegetation surrounding the honeybush fields in the Western Cape province of South Africa. Entomopathogenic fungi were isolated using the insect bait method and were characterised using molecular techniques. Twenty fungal isolates of Metarhizium anisopliae and Fusarium oxysporum were recovered from soil samples, of which 70% were from honeybush fields and 30% were from surrounding vegetation. Fusarium oxysporum isolates comprised 20% of the recovered isolates; M. anisopliae the remainder. Laboratory bioassays of the recovered isolates against adults and nymphs of the leafhopper, showed that F. oxysporum isolates caused 10–45% mortality and M. anisopliae isolates 30–80% mortality. Metarhizium anisopliae isolates J S1, KF S3, KF S11, KF S13, LS1 and LS2 were the most virulent and induced over 60% mortality in both nymphs and adults at a concentration of 1 × 107 conidia/ml.

The influence of citrus orchard age on the ecology of entomopathogenic fungi and nematodes

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

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

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

The influence of citrus orchard age on the ecology of entomopathogenic fungi and nematodes

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