Aschersonia aleyrodis as a microbial control agent of greenhouse whitefly

Abstract

Various aspects of the development of the entomopathogenic fungus Aschersoniaaleyrodis as a control agent of greenhouse whitefly, Trialeurodesvaporariorum , were investigated. For control of greenhouse whitefly in tomato crops the parasitoid Encarsiaformosa has been successful, but in cucumber crops a successful suppression of the whitefly population is often not achieved. Therefore, an additional selective control method is needed. The attention was focused on the fungal pathogen Aschersoniaaleyrodis (Chapter 1).The spores of A. aleyrodis germinated on the integument of whitefly larvae. Penetration of the cuticle took place after formation of an appressorium. The haemolymph and insect tissues were colonized by the fungus and the insect changed in colour from transparent yellow green to clear or opaque orange. Under favourable conditions the mycelium protruded from the insect and orange-coloured spore masses were produced in a mucilaginous layer (Chapter 2).Information on the susceptibility of greenhouse whitefly at the various life stages is of importance for application of A. aleyrodis . Eggs of the host did not become infected. First instar, second instar and third instar larvae were highly susceptible to infection. Fourth instar larvae were susceptible to a lesser extent. When these larvae developed into the so-called prepupal and pupal stage the cuticle changed and the whitefly became more resistant. Generally, adults did not become infected (Chapter 3).Dose-mortality responses were determined for the first, second, third and fourth instar larvae. Several experiments over time were carried out which gave consistent results. The dosage of spores on leaves needed to obtain 50% mortality (LC50) of first, second and third instar larvae was 19.53 spores/mm 2, 21.03 spores/mm2 and 33.81 spores/mm 2, respectively. This represents a dose of about 0.77 spores per first instar larva, 1.44 per second instar larva and 4.39 spores per third instar larvae. The LC95 values, expressed as number of spores per amount sprayed, were 1.98 X 10 7spores/2ml for first instar larvae, 2.34 x 10 7spores/2ml for second instar larvae and 3.27 X 10 7spores/2ml for third instar larvae. The LC95 for fourth instar larvae was outside the dosage range tested. The LC50 varied with the age of the fourth instar larvae, from 6.0 x 10 6spores/2ml to 2.6 x 10 8spores/2ml in two different bioassays. The period before 50% of the larvae showed signs of infection (orange colouration) (LT50) at 20°C, was 11.8 days for first instar larvae, 9.5 days for second instar larvae and 7.0 days for third instar larvae, after application of 5.0 x 107 spores in 2 ml. The LT25 for fourth instar larvae was 5.6 days (Chapter 4).Another bioassay method was tested using cucumber leaf discs (6.5 cm, diameter). The presence of free water on the leaf surface enhanced the infection to such a degree that 77 to 90% of the larvae became infected after application of 1.0 x 10 6spores in 2 ml per leaf disc. No LC50 values could be derived. After exposure of larvae to dosages of 5.0 x 10 7and 1.0 x 10 8spores in 2 ml a delay in the development of infection was noticed. After treatment of fourth instar larvae the final percentage infection was lower at the higher dosages than at the lower dosages. This was different from the linear relationship found in previous bioassays on plants. on water agar the spores of A. aleyrodis showed reduced germination at high densities (3100 spores/mm2). This density-dependent effect of the spores on the germination was apparently also present when leaf discs were used under conditions of high humidity and free water on the leaf surface. This may indicate the presence of a self-inhibitor (Chapter 5).Impressions of cucumber leaves treated with A. aleyrodis spore suspension on water agar showed that only a low percentage of the spores (4.5%) germinated on the leaf surface. However, the ungerminated spores remained viable and infective for a long time. Spores from leaves treated 43 days before showed 78% germination after incubation on water agar for 24 hr. whitefly treated in the egg stage became infected when young larvae contacted spores on the leaf surface after hatching. Nearly all young larvae (96%) contacting spores present on the leaf surface for about 22 days became infected (Chapter 6).When aiming to apply A. aleyrodis in a glasshouse environment knowledge on the influence of temperature and relative humidity (RH) is wanted. over 90% of the A. aleyrodis spores germinated within 48 hr on water agar in the temperature range of 15 to 28°C. Larvae were infected at 15, 20, 25 and 30°C, with the most rapid development at 30°C, (LT50: 3.3 days), though the final mortality rates of whitefly at the different temperatures were the same. Spores on cellophane sheets were exposed to various relative humidities. Germination was fastest at 100% RH and 20°C (78% within 24 hr), but after 168 hr 88% of the spores germinated at 93.9% RH. In experiments using cucumber plants it was found that successful infection of the larvae occurred at a RH of 50% and 20°C A period of 100% RH for 24 hr enhanced the development of infection (LT50: 7.1 days). After exposure of the plants bearing treated larvae to 0, 3, 6, 12 or 24 hr 100% RH a linear relationship between these periods of high humidity and the LT50 values was not observed. The LT50 value amounted to 8.9, 8.6, 10.4 and 10.1 days for periods of 0, 3, 6 and 12 hr high RH, respectively. It is suggested that germinating spores are in a vulnerable phase after the periods of 6 and 12 hr at 100% RH, and are then highly susceptible to the decrease in RH from 100% to 50% (Chapter 7).Serial in vitro passages of the fungus influenced the rate in which signs of infection became apparent. one in vivo passage of A.aleyrodis on greenhouse whitefly did not influence the infection rate but this needs further investigation (Chapter 7).The interaction between A. aleyrodis and the parasitoid Encarsiaformosa was studied in relation to the introduction of both natural enemies for control of whitefly (Chapters 8 and 9). From behavioural observations it could be concluded that the parasitoid was able to distinguish infected hosts from noninfected hosts if the fungus is present in the haemolymph of the host. Infected larvae were rejected for oviposition after the ovipositor penetrated the host. From four days onwards after inoculation of the spores the parasitoid could detect the fungus in the host. By distinguishing between infected and noninfected hosts the parasitoid is able to complement the fungal pathogen. E. formosa was able to transmit A. aleyrodis from infected hosts to noninfected hosts by the contaminated ovipositor. The transmission of the fungus was restricted to one or two healthy hosts (Chapter 8).whitefly larvae parasitized by E. formosa more than three days before were not susceptible to infection by the fungus. Nonparasitized larvae, however, were still infected by the fungus. The regulation of this phenomenon of reduced susceptibility in parasitized larvae is yet unknown. It may be related to the hatching of the parasitoid larva from the egg in the host (Chapter 9).From the presented results of the experiments it can be concluded that A. aleyrodis shows promise as a microbial control agent of greenhouse whitefly in glasshouses (Chapter 10). Further research should be concentrated on the development of mass production, formulation of a product and application strategies.</TT