Control of Locusts and Grasshoppers Development of Metarhizium as a Mycoinsectide

A report for the Rural Industries Research and Development Corporation by Dr Richard Milner, CSIRO Entomology

December 2000 RIRDC Publication No 00/190 RIRDC Project No CSE-71A

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© 2000 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 58221 1 ISSN 1440-6845 Control of Locusts and Grasshoppers - Development of Metarhizium as a Mycoinsecticide Publication No. 00/190 Project No. CSE-71A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other inquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Dr Richard Milner CSIRO Entomology GPO Box 1700 CANBERRA ACT 2601 Phone: 02 6246 4169 Fax: 02 6246 4042 Email: <richardm@ento.csiro.au>

RIRDC Contact Details

Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4539 Fax: 02 6272 5877 Email: rirdc@rirdc.gov.au. Website: http://www.rirdc.gov.au

Published in December 2000 Printed on environmentally friendly paper by Canprint

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Foreword Locusts and grasshoppers are major pests of pastures in Australia and alternatives to hard chemicals for their control have been extremely limited. The objective of this project was to develop a mycoinsecticide based on oil formulations of the hyphomycete fungus Metarhizium anisopliae var. acridum for this purpose. Promising results have stimulated further research being continued by the researchers with the assistance of the Australian Plague Locust Commission. This project was funded from RIRDC Core Funds which are provided by the Federal Government. This report, the latest addition to our diverse range of over 600 research publications, forms part of our Resilient Agricultural Systems sub-program which aims to enable agricultural production systems that have sufficient diversity, flexibility and robustness to be resilient and respond to challenges and opportunities. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at www.rirdc.gov.au/reports/Index.htm

purchases at www.rirdc.gov.au/eshop

Peter Core Managing Director Rural Industries Research and Development Corporation

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Acknowledgments This work would not have been possible without additional financial support from the Tablelands Wingless Grasshopper Committee, ACIAR, APLC and the Queensland DNR. Considerable assistance was provide by Mr Graeme Baker (formerly NSW Agriculture) for field trials on wingless grasshopper, and by Dr David Hunter and Mr Peter Spurgin of the APLC for large-scale field trials. Mr Laurie Lloyd (Tablelands Wingless Grasshopper Committee) has been a very effective advocate for the project and has been responsible for obtaining additional funding. Kevin Strong and Dr Joe Scanlon have provided support for field trials on spur-throated locusts. Finally both the present and former directors of the APLC, Dr Gordon Hooper and Dr Graeme Hamilton have provided valuable support and have been excellent collaborators.

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Contents

FOREWORD III

ACKNOWLEDGMENTS IV

CONTENTS V

LIST OF FIGURES vi

LIST OF TABLES vii

EXECUTIVE SUMMARY VIII

INTRODUCTION 1

MAJOR OBJECTIVES OF THE PROJECT 3

RESEARCH METHODOLOGY 4

RESULTS 11

PROGRESS BY MILESTONE 47

IMPLICATIONS AND RECOMMENDATIONS 50

PUBLICATIONS 51

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List of Figures

Figure 1: Austraacris guttulose infected with metarhizium anisopliae var. Acridum (FI-985) showing external sporulation 6

Figure 2: Austracris guttulosa infected with metarhizium anisopliae var. Acridum (FI-985) showing internal sporulation 7

Figure 3: Twelve isolates of metarhizium anisopliae var. Acridum grown on Sabouraud’s Dextrose Agar for 2 weeks at 25°c, showing similar growth patterns. 12

Figure 4. Mortality of field-treated wingless grasshoppers collected 4 days after treatment with one of 4 formulations of FI-985. 23

Figure 5. Effect of two formulations of RI-985 on population density of wingless grasshoppers at Llangrove 25

Figure 6. Effect of two formulations of FI-985 on population density of wingless grasshoppers at Dimboola 26

Figure 7. Mortality of field-treated wingless grasshoppers collected 1 day after spraying with either a mineral oil or a vegetable oil formulation at Llangrove. 27

Figure 8: Mortality of field-treated wingless grasshoppers collected 1 day after spraying with either a mineral oil or a vegetable oil formlation at Dimboola 28

Figure 9: Effect of spraying with fenitrothion and metarhizium as well as an unsprayed control plot on changes in population density at Dalgety 33

Figure 11: Changes in population density at amaroo following aerial spraying with metarhizium 37

Figure 12: Changes in population density at dalgety and cooma on untreated control plots and those sprayed twice with either fenitrothion or metarhizium. Results are for all plots combined. 38

Figure 13: Persistence of the metarhizium spray deposit as shown by mortality of healthy grasshoppers incubated with field-treated vegetation collected 1, 4 and 6 days after spraying 39

Figure 14. Persistence of the fenitrothion spray deposit as shown by mortality of healthy grasshoppers incubated with field-treated vegetation collected 1, 4 and 6 days after spraying 40

Figure 15. The % control in all plots at Cooma for all species combined 4 weeks after spraying 43

Figure 16. The % control in all plots at Cooma for all wingless grasshopper 4 weeks after spraying 43

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List of Tables

Table 1: List of isolates of metarhizium anisopliae used in this project 4

Table 2: Summary of results of screening isolates against wingless grasshoppers 13

Table 3: Summary of results of screening isolates against Australian plague locust 13

Table 4: Summary of results of a detailed comparison of 4 isolates against wingless grasshopper 15

Table 5: Probit analysis based on final mortality data 15

Table 6: Comparison of 3 selected isolates using adult Australian plague locusts; results after 7 days incubation at 30oc 16

Table 7: Comparison of 4 selected isolates using adult Australian plague locusts; results after 7 days incubation at 25oc 16

Table 8: Effect of isolate on the % dead grasshoppers forming conidia of metarhizium. 17

Table 9: Size of colonies (mm diameter) after 3 weeks on SDAYE 17

Table 10: Effect of temperature regime on % mortality in Australian plague locust nymphs inoculated with FI-985 sprayed onto grass at 2 x 109/ml. 18

Table 11: Effect of temperature on % mortality after 15 days incubation. Dose 1 was 2 x 109 condia/ml, while the other doses are decimal dilutions. 19

Table 12: Combined results from two experiments on migratory locust showing the effect of incubation temperature on rate of mortality (dose = 50,000 conidia/insect) 19

Table 13: Effect of suspending oil on mortality of wingless grasshopper 21

Table 14: Effect of dose and oil on mortality of wingless grasshopper at 25oc 22

Table 15: Persistence of two formulations in the field at Dimboola 29

Table 16: Viability of undried conidia stored in various ways 30

Table 17: Viability of dried conidia stored in various ways 30

Table 18. Summary of field trials on efficacy 31

Table 19: Summary of final mortality (and sporulation) in live grasshopper samples from Cooma and Dalgety 41

Table 20: Mortality and sporulation of spur-throated locusts field treated with metarhizium at Emerald In 1997. 44

Table 21: Effect of dose and days after spraying on mortality and sporulation of Australian plague locusts at Broken Hill 46

Table 22: Relative susceptibility of different species of locust and grasshopper to FI-985 47

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Executive Summary This research project has shown that the Metarhizium fungus is promising as a mycoinsecticide for use against locusts and grasshoppers in Australia. The major findings were: 1. That most (and probably all) acridid pests are highly susceptible to the fungus requiring about

1000 spores or fewer to infect and kill 50% of a population in 10 days at 28oC. 2. That the fungus can be produced on rice such that in excess of 5 x 1012/kg spores can be

produced in three weeks using a sterile rice/milk powder substrate. These spores can be harvested from the rice by sieving, use of a cyclonic extractor or by fluidised bed dryer. The rice can be reused to produce a further batch of spores by resterilising and then adding protein and inoculating with FI-985.

3. That the spores can be dried to 5-10% and these dried spores store well for at least 1 year cold.

On vegetation in the field, they have a half-life of two or more days depending on weather conditions.

4. That FI-985 is the more effective of the two Australian isolates and that no exotic isolate is

sufficiently more virulent to warrant further study. 5. That a number of oils can be used for formulating the spores and that the type of oil used has

very little effect on virulence. However, Propar and probably soyabean oil are the oils of choice as they are relatively cheap, have a viscosity suitable for ULV spraying, are non-toxic to plants and are non-flammable.

6. That the time taken to kill insects is temperature and dose dependant but is likely to take 7-21

days in the field. Constant temperatures over 35oC and below 20oC inhibit disease development with the optimum being 28-30oC. Temperatures at least up to 40oC are tolerated by the fungus in vivo.

7. That the fungus is effective under field conditions when sprayed at a rate of 1 - 5 X 1012

conidia/ha using a oil-based ULV spray or a oil/water emulsion using a boom sprayer. There is commercial interest now in developing a product and a “Locust and Grasshopper Biocontrol Committee” consisting of representatives from CSIRO, APLC, NSW Ag, Qld. DNR, and Tablelands Wingless Grasshopper committee has been established to advise on further development in particular registration with the NRA.

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Introduction

The 3 main locust and grasshopper pests in Australia are the Australian plague locust, Chortoicetes terminifera, the spur-throated locust, Austracris guttulosa, and the wingless grasshopper, Phaulacridium vittatum. Control of these pests is undertaken by the Australian Plague Locust Commission (APLC), State Governments and landholders. In the main, control relies on the use of a broad-spectrum chemical insecticide, fenitrothion, used either as an ultra-low volume concentrate sprayed from fixed-wing aircraft, or as a water-based high volume spray from tractor mounted boom sprayers. Other chemicals are always being tested and one of these, fipronil, produced by Rhone-Poulenc, is likely to start replacing fenitrothion in a few years time when it is registered for this use. The APLC strategy for control is based on prevention of outbreaks. Thus by modelling, monitoring and forecasting, the APLC can determine the extent of the locust populations at any one time and initiate control to prevent build up and migration from source areas in the drier parts of Australia into the cropping areas. Wingless grasshopper is non-migratory and has an annual life-cycle. It breeds in pastures adjacent to, or even within, high value crops such as lucerne, grapes, cereals and soft-fruit. Wingless grasshopper is of increasing concern to landholders attempting to establish stands of eucalyptus trees on their properties as when the surrounding pastures hay off in summer, the grasshoppers will start feeding on young trees causing defoliation leading in some cases to death of the tree. Wingless grasshopper is under partial biological control from naturally occurring nematode parasites and when conditions are wet these parasites can cause high mortality of nymphs and adults. However, over the past few years, conditions have been very dry in the major outbreak area of the southern tablelands of New South Wales and consequently, the number of these insects have remained high during the period of this project. Locusts have many natural enemies but they are not usually effective in controlling or preventing outbreaks. Thus suggestions that locusts and grasshoppers could be controlled biologically have been regarded with scepticism. In October 1989, a project commenced based at the International Institute of Biological Control laboratories in Ascot, UK, to develop biological control of locusts for Africa. Very quickly the scientists on this project realised that the most promising strategy was to use the conidia of certain specialised isolates of Metarhizum flavoviride (now regarded as Metarhizium anisopliae var. acridum, Driver and Milner 1998) formulated in oil as a mycoinsecticide. This project is still continuing and is now known by its acronym LUBILOSA. Three commercial companies, two in Africa and one in France, have been licenced and hope soon to be able to start selling their product “Green Muscle” for locust and grasshopper control in Africa. In 1993, CSIRO commenced a project in collaboration with LUBILOSA to undertake parallel development of a mycoinsecticide for locust and grasshoppers in Australia. This project received the current funding from RIRDC in 1995 following preliminary work funded by the Tablelands Wingless Grasshopper Committee which showed the promise of the approach for use against wingless grasshoppers and Australian plague locusts (Milner and Prior, 1994). In the last 2 years of operation, the project developed increased significance because of the increasing demands in Australia for a non-chemical method for control of locusts and grasshoppers. Some of the reasons for this change are:

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1. A rapid increase in “organic” production especially beef production in SE Queensland.

2. An increased concern on the part of growers of produce such as rice and grapes over the use of chemical pesticides.

3. A desire on the part of State governments to avoid the use of chemical pesticides (for example, the Queensland government has recently provided a large amount of funding for development of biological control of spur-throated locust in the state)

4. Concern of the grazing industry over residues in produce.

At the time this report was written, the only viable alternative to the use of chemical pesticides for control of locusts and grasshoppers is the use of a Metarhizium-based mycoinsecticide.

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Major Objectives of the Project The objective of the research was to develop a mycoinsecticide based on oil formulations of the fungus Metarhizium for control of locusts and grasshoppers. It was hoped that by the end of the 3 year program, substantial progress would have been made towards a registered product being commercially available in Australia. The main elements of the research were seen as selection of the best isolates, understanding their response to environmental conditions, optimising production, storage and formulation, obtaining data on host specificity and establishing effective dose rates and spraying strategies for operational use.

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Research Methodology Fungal Isolates

A list of the main isolates used in this study is given in Table 1. Some of these isolates, such as those from Brazil, were obtained recently and have been used in our studies on the taxonomy of Metarhizium and have not been bioassayed against locusts or grasshoppers. The names are as proposed by Driver et al. (in press). Molecular studies using RAPDs and sequence analysis of the ITS region have shown that isolates belonging to M. anisopliae var. acridum are all very closely related and only small genetic differences can be found. These differences do not correlated with the geographical origin. In nature, M. anisopliae var. acridum isolates have only been found infecting acridid hosts while laboratory studies by the LUBILOSA team have shown that these isolates are generally more virulent for acridids than isolates belonging to M. anisopliae var. anisopliae (Bateman et al., 1996). We have found many grasshoppers in nature infected with Metarhizium, however only in one case (FI-1155) was this an isolate of M. anisopliae var. acridum. Thus, while M. anisopliae var. acridum does occur naturally in Australia, it is extremely rare.

Table 1 – List of isolates of Metarhizium anisopliae used in this project

isolate species host country source code FI-0610 M. anisopliae var. anisopliae termite mound material Australia none FI-0974 M. anisopliae var. anisopliae acridid (Qld.) Australia none FI-0983 M. anisopliae var. acridum acridid Benin I91-659 FI-0984 M. anisopliae var. acridum acridid Benin I91-658 FI-0985 M. anisopliae var. acridum Austracris guttulosa Australia ARSEF 324 FI-0986 M. anisopliae var. acridum acridid Benin I92-715 FI-0987 M. anisopliae var. acridum Ornithracris cavroisi Niger IMI 330189 FI-1028 M. anisopliae var. acridum Zonocerus elegans Tanzania IMI 324673ss FI-1034 M. anisopliae var. anisopliae acridid Thailand I94-614 FI-1066 M. anisopliae var. anisopliae acridid Benin Ben 4 FI-1067 M. anisopliae var. acridum acridid Benin I92-701 FI-1155 M. anisopliae var. acridum Chortoicetes terminifera Australia none FI-1189 M. anisopliae var. acridum Schistocerca pallens Brazil CG429 FI-1190 M. anisopliae var. acridum S. pallens Brazil CG423 FI-1191 M. anisopliae var. acridum S. pallens Brazil CG431 FI-1192 M. anisopliae var. acridum S. pallens Brazil CG430 FI-1193 M. anisopliae var. acridum S. pallens Brazil CG288 FI-1216 M. anisopliae var. acridum acridid Galapagos ARSEF 2023

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For isolation of Metarizium from cadavers and the soil, Veen’s medium has normally been used. This is a semi-selective medium consisting of mycological agar with 0.10% chloramphenicol and 0.05% actidione autocalved complete. For production of small number of conidia and for general maintenance, SDAYE consisting of Sabouraud’s Dextrose Medium with 1% yeast extract added prior to autoclaving. Colonies on SDAYE can be stored for up to 6 months at 4oC. For longer term storage, conidia are stored as a dry powder at –80oC, and also as growing slopes at –20oC. Isolates of fungi are often unstable when frequently subcultured on a rich medium like SDAYE and therefore many workers passage isolates of Metarhizium through a host periodically to maintain sporulation capacity and virulence. We have not done this and have found the M. anisopliae var. acridum isolates to be remarkably stable in these characteristics. For field trials, large numbers of conidia are produced on rice (see section on mass production). Insects

Acridids are notorious for inducing allergies in susceptible people. While these may be only a mild irritation when handling locusts and grasshoppers, in other cases the symptoms are more severe and may lead to individuals seeking work away from these insects. It is therefore essential when culturing these insects to have adequate facilties which ensure good air circulation and an extraction system to remove the contaminated air away from the workers. Funding was inadequate for such a facility to be built and therefore we did not culture grasshoppers or locusts during this project. The insects we used were all field collected and kept only for the duration of a particular experiment. The insects were generally kept in groups of 20 to 100 in “bread-bin” cages. These were constructed from plastic boxes sold for keeping bread. They measure about 28 X 21 X 21 cm and large holes were made in the sides which were covered with metal gauze for ventilation. A plastic sleeve was provided at one end to allow access. Insects were fed on green vegetation, usually wrapped in moist paper towelling, and replaced daily. This was supplemented with dry rolled oats. The cages were kept in a glasshouse running between 20 and 30oC. To check for sporulation, dead insects were removed daily and placed in Petri dishes with moist filter paper to ensure 100% RH, and incubated for a few days at 28oC. Infected insects normally produced profuse outgrowth and typical green spores (Figure 1) often concentrated between segments. Locusts, especially spur-throated Austracris guttulosa, were found to produce large numbers of conidia internally (Figure 2) under dry conditions, while at high humidities both internal and external sporulation. Laboratory Bioassays

To bioassay insects, conidia produced on SDAYE plates were suspended in Propar oil (Ampol, Sydney) and sonicated briefly to break up the clumps. The concentration of conidia was estimated using a Petroff-Hausser particle counter and adjusted with oil to the required dose. Insects were inoculated using a 1 ml syringe mounted on a foot operated micro-applicator (ISCO, USA). Small species such as wingless grasshopper, Phaulacridium vittatum and Australian plague locust, Chortoicetes terminifera, were dosed with a droplet of 0.5µl on the mouth-parts, while larger insects such as the migratory locust, Locusta migratoria, the droplet was placed under the pronotum. The insects were incubated in groups of 20-30 in bread-bin cages and mortality assessed daily. Unless otherwise stated the insects were kept in a heated glasshouse under a

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fluctuating regime from 24-28oC. LD50 values were computed using the POLO-PC computer programme.

Figure 1: Austracris guttulose infected with Metarhizium anisopliae var. acridum (FI-985)( showing external sporulation 0

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Figure 2: Austracris guttulosa infected with Metarhizium anisopliae var. acridum (FI-985) showing internal sporulation

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Mass Production and Formulation

For field trials, conidia were mass-produced on moist, autoclaved, rice using the two-phase system. During the project numerous small experiments were undertaken to improve the mass production method and this resulted in higher yields, lower labour input and fewer problems with contamination. The details of these experiments will \not be presented, however the present method is as follows: A stock culture of FI-985 is stored on a slope at –20oC. The conidia from this stock is used to inoculate Petri dishes of Sabouraud's dextrose agar with 1 % yeast extract added and the plates are incubated at 25oC for 2-3 weeks. The conidia from these plates are used to inoculate 250 ml flasks containing 100 ml of sterile nutrient broth (10 g mycological peptone, 40 g dextrose, 10 g yeast extract and 1 litre tap water, sterilized by autoclaving for 15 mins.) which are then incubated on a shaker at 25oC for 5 - 7 days. By this time a thick mass of mycelium has been produced. This is used to inoculate the rice. Whole parboiled rice (Sungold) is used. Autoclavable Nalgene bottles are each filled with 800 g of dry rice and this is autoclaved dry for 90 minutes. The rice is allowed to cool and then 700 ml of sterile water plus 1% whole milk powder is added to each bottle together with 50 ml of the broth culture as inoculum. The mixture is shaken and left to allow the rice to absorb the liquid. The mixture is then shaken again and the plastic top replaced by a double layer of autoclaved Kimwipes attached with 2 elastic bands. The bottles are then incubated on their sides for 3 weeks. As the mycleium develop they tend to clump the rice inhibiting gas exchange and resulting in very poor sporulation. Therefore the bottles are shaken twice per day for the first 3-4 days by which time sporulation has started. After that the bottles are more gently shaken (or turned) every few days. A small proportion (< 5 %) of bottles become contaminated, usually with an Aspergillus or Penicillium fungus, and is discarded. These contaminated bottles are easily detected by the odour, the pronounced heating and the development of large amounts of liquid water in the bottles. At the end of the 3-week fermentation period, the rice/conidia mixture is removed from the bottles and placed in exposed plastic trays for 2- 3 days to partially dry. The conidia are then removed from the rice using an electrically vibrated set of sieves. The rice is discarded and the conidial powder placed 500 g to a 4 litre plastic screw-capped bottle with 2 kg of silica gel granules at 4oC. This dries the conidia to about 9-10 % moisture. These dry conidia are stored, in contact with silica gel at 4oC until required (1 to 6 months later). The dry spores are removed from the silica gel by dry sieving and formulated by mixing with Propar 12 mineral oil (Ampol). The concentration of conidia in the oil is adjusted to 5 X 1012 conidia/litre by adding more Propar or allowing to settle and then decanting off the excess oil. The concentration of conidia in oil is estimated using a Petroff-Hausser bacteria counting chamber, or by centrifuging the conidia down and then estimating the volume or weight compared to a standard.

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Field Trials

Application: The Metarhizium has been applied as an oil-concentrate by ultra-low volume (ULV) and as an oil/water emulsion by high volume nozzle sprayers. The current preferred method is to apply the material at 1 litre/ha (or less) using a fixed wing aircraft fitted with Micronair AU8000 rotary atomiser set to deliver droplets averaging 70 µm in diameter. This type of application was also undertaken from the ground using a vehicle mounted sprayer fitted with a single Micronair AU5000 rotary atomiser and with a Micro-Ulva or Micro-Ulva plus hand-held ULV sprayer. For high volume application, the oil concentrate was mixed with an emulsifier such as Teric 215 (ICI) or Wetter (Monsanto) and water added to enable about 30 litres of emulsion to be applied per hectare. Application was generally by a vehicle drawn trailer-mounted GreenAcres boom sprayer. A “Solo” motorised back-pack sprayer was also used successfully. All these apparatuses and methods were used successfully and the material did not block nozzles or filters except when the spray formulation inadvertently contained particles of silica gel used for drying. Population Assessments: For wingless grasshopper, the populations were usually assessed soon after spraying and then at weekly intervals for 4 weeks. A standard 50 cm sweep net was used and the density per square metre estimated from the total count divided by 6 of 30 sweeps of 120-180 degrees. The population was assessed either at fixed areas in the centre of plots or by taking separate transects of lower, middle and upper slopes. No attempt has been made to quantitatively assess population densities of locusts in the field. Visual assessments have been made using the APLC’s standard procedures of recording adult locusts as isolated; scattered; numerous; concentrated; or swarms. Disease Incidence: Two methods were used for assessing the level of infection in the treated field populations. For both assessments, live insects were sampled using a sweep net at various intervals after spraying. These insects were then incubated either in large field cages or in the standard bread-bin cages in the Canberra glasshouse. Field cages were considered important as this more closely represented the field temperature conditions while the constant (ideal) conditions in the glasshouse served to establish the best possible level of infection and rate of kill. With both types of cages fresh green food was provided as required and dead insects were removed to moist chambers daily, or as near to daily as practical. Confirmation of infection was provided by the cadavers sporulating after a few days in a moist chamber.

Persistence of the Spray Deposit: Samples of the treated vegetation were taken at intervals after spraying and were exposed to live untreated grasshoppers in breadbin cages in the glasshouse. They were fed untreated food after 2 –3 days and the dead removed to moist chambers as for the disease incidence evaluations. In one experiment, small plots were sprayed in the field and then insects cages over this treated vegetation for 2 days before the cage was moved and the insects incubated in the glasshouse to assess the level of infection. Non-target Effects: For one field trial non-target effects were determined by using a trasect of 10 pitfall traps in the centre of each plot and recording the number of different insects groups caught. A comparison was made between untreated plots, Metarhizium-treated and fenitrothion treated

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plots. In other instances, when there were non-target insects present these were collected and incubated in the glasshouse as for locusts to determine the level of infection.

Survival in Soil: In the large-scale trial at Cooma in 1987, samples of litter and surface soil were taken to determine if the applied conidia would persist in these sites. The samples were homogenised in a dilute aqueous solution of Tween-80 and plated out onto Veens medium plates. The plates were incubated at 25oC for 2 weeks and then the number of Metarhizium colonies counted.

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Results Selection of Isolate

The main isolates of Metarhizium used in this study are listed in Table 1. Associated taxonomic studies showed that the most effective isolates against acridoid insects all belonged to a relatively homogenous taxon for which the name Metarhizium anisopliae var. acridum has been proposed (Driver et al., in press). However most publications still use the name Metarhizium flavoviride for these isolates. A comparison of the colony morphology is shown in Figure 3. The two main target pests are the wingless grasshopper, P. vittatum, and the Australian plague locust, C. terminifera, and these were used to screen a range of isolates. The first two experiments screened 10 isolates at a single dose for each species, while the next two experiments provided a more detailed comparison over a range of doses for the best 4 isolates. Finally these 4 isolates were compared in a single field trial.

Screening 10 isolates of Metarhizium against the wingless grasshopper

The virulence of 10 isolates for wingless grasshopper was compared using target insects were field collected in the Cooma region as third and fourth instar nymphs. The isolates of Metarhizium were grown on SDAYE at 25oC for 2 weeks and the conidia scraped off into 0.05% Tween-80. The concentration in the suspension was adjusted to 2 X 107/ml and 0.5µl from a syringe mounted on an ISCO microapplicator applied to the mouthparts of each insect. This gave a dose of 10,000 conidia/insect. The conidia were almost all viable as indicated by a germination check on SDAYE which gave over 90% germination after 24 hrs at 25oC. For each isolate, 24 grasshoppers were treated and 24 were treated with Tween-80 only as controls. Each group was placed in a bread-bin cage and incubated in a cabinet at 27oC constant. The insects were fed fresh green food each day and any dead removed to a moist chamber to promote sporulation after 1 and 5 days and then daily. The experiment was run for a total of 9 days. All isolates tested killed grasshoppers with the first Metarhizium deaths on day 5. Only isolate FI-1029 had killed less than 80% of the grasshoppers by day 8. Control mortality was minor reaching 8% by day 9.

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Figure 3: Twelve isolates of Metarhizium anisopliae var. acridum grown on Sabouraud’s dextrose agar for 2 weeks at 25°C, showing similar growth patterns.

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Table 2. – Summary of results of screening isolates against wingless grasshoppers

isolate day 1 day 5 day 6 day 7 day 8 day 9 FI-0974 0 25 42 75 83 92 (58)*

FI-0983 4 21 58 87 96 100(67)

FI-0984 8 16 54 100 (100)

FI-0985 0 17 33 58 92 96 (67)

FI-0986 0 17 54 96 96 96 (58)

FI-0987 0 4 46 71 92 96 (67)

FI-1028 8 8 46 87 92 92 (67

FI-1029 4 12 25 37 50 62 (37)

FI-1038 0 4 12 58 92 96 (67)

FI-1067 0 12 96 100 (92)

control 4 4 4 4 4 8

% mortality (% sporulation)

FI-984 and FI-1067 were the most effective isolates causing 100% mortality after 7 days. The Australian isolate FI-985 was almost as pathogenic giving 96% mortality after 9 days. Screening 10 isolates of Metarhizium against the Australian plague locust

The methods used were the same as used in the previous experiment. The locusts were 3rd and 4th instar nymphs field collected by the Australian Plague Locust Commission. All isolates gave over 90% kill by day 9, with FI-984 and FI-987 giving 100% kill by day 6. There were differences in the proportion of killed locusts sporulating with the best being FI-974, FI-984, FI-985 and FI-1029. Table 3 - Summary of results of screening isolates against Australian plague locust

isolate day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9

FI-0974 8 8 12 32 40 48 88 100 100 (92)*

FI-0983 8 24 36 48 96 96 100 100 100 (36)

FI-0984 8 8 8 52 92 100 100 100 100 (76)

FI-0985 12 12 16 28 60 76 100 100 100 (76)

FI-0986 16 24 27 40 88 96 96 96 96 (52)

FI-0987 4 4 12 32 92 100 100 100 100 (60)

FI-1028 8 8 12 16 76 88 100 100 100 (68)

FI-1029 16 20 24 32 44 76 92 100 100 (76)

FI-1038 16 20 28 44 64 80 88 88 92

FI-1067 16 20 24 40 68 92 100 100 100 (56)

control 4 4 4 16 20 20 20 20 20 (0)

* % mortality (% sporulation)

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This experiment showed that all the tested isolates were effective and therefore provided no data to assist selecting isolates for further testing. The results also indicated that, in general, the Australian plague locust is more susceptible than wingless grasshopper. Detailed comparison of 4 isolates for wingless grasshopper

To determine the LD50 for the Australian isolate FI-985 in comparison with the 2 selected exotic isolates, FI-984 and FI1067. The fourth isolate, FI-1155, was recently discovered on an infected Australian plague locust in the Hunter valley and so was included in the comparison. The methods used were similar to those for the previous experiment except that the insects were adults and the incubation temperature was 25oC. Twenty-five insects were each treated on the mouthparts with one of 4 doses: 275 conidia/insect, 833, 2500 and 7500. The controls were treated with Tween-80 only. The experiment was continued for 11 days. The results were analysed using Polo PC a computer programme for probit analysis based on the method of Finney (1971). The results are summarised in Tables 4 and 5. All isolates were virulent and with all 4 isolates most mortality due to Metarhizium occurred between days 6 and 9. The relatively proportion of controls dying and the low proportion of sporulation probably reflected the fact that the adults were quite old when use for this experiment. Probit analysis revealed that the slope of the mortality dose relationship was similar for all isolates and therefore the data were analysed using a common slope. The LD50 values ranged from 1,581 conidia/insect for FI-984 to 5,495 conidia for FI-1067. However the confidence intervals were quite wide and these differences were not significant (P< 0.05). Other experiments (data not given) have shown that FI-1155 is less pathogenic than the other Australian isolate FI-1155. These results show that FI-985 our preferred isolate is equally virulent to FI-984 the most virulent exotic isolate.

15

Table 4: Summary of results of a detailed comparison of 4 isolates against wingless grasshopper

dose isolate day 2 day 5 day 6 day 7 day 8 day 9 day 10 day 11

7500 FI-984 4 4 40 60 76 80 80 80 (68)*

FI-985 0 4 24 48 68 92 92 92 (88)

FI-1067 0 4 20 44 52 72 76 84 (67)

FI-1155 0 8 8 12 24 52 52 72 (56)

2500 FI-984 0 0 0 24 56 76 84 84 (80)

FI-985 0 4 20 36 52 60 60 68 (32)

FI-1067 0 8 8 20 28 36 36 44 (24)

FI-1155 4 4 4 16 36 48 48 56 (40)

833 FI-984 4 12 16 28 48 52 52 56 (36)

FI-985 8 16 24 28 36 40 48 48 (16)

FI-1067 4 4 4 24 32 48 52 56 (28)

FI-1155 8 8 8 12 16 40 44 48 (48)

275 FI-984 0 12 12 12 20 24 24 28 (8)

FI-985 0 12 20 32 40 52 52 64 (16)

FI-1067 0 4 4 12 20 28 32 32 (4)

FI-1155 0 4 4 16 16 28 28 40 (40)

control 0 8 8 20 20 24 24 32 (0)

* % mortality (% sporulation)

Table 5: Probit analysis based on final mortality data

isolate LD50 (conidia/insect)* 95% confidence limits

FI-984 1,581 555 - 4034

FI-985 1,597 428 - 4355

FI-1067 5,495 1620 - 27996

FI-1155 4,456 1364 -16561

* analysed on a common slope of 1.27 + 0.24

Comparison of 3 isolates for the Australian plague locusts

The insects were a mixture of 3rd, 4th instars and adult locusts field collected by APLC. Twenty insects per dose were inoculated on the mouthparts and incubated in bread-bin cages at 30oC for 7 days. First insects to die due to Metarhizium occurred after 4 days incubation with most mortality between day 5 and day 6. By day 7, most of the treated insects had died (Table 6) All isolates are highly virulent with FI-984 and FI-985 more virulent than FI-1067.

16

Table 6: Comparison of 3 selected isolates using adult Australian plague locusts; results after 7 days incubation at 30oC

dose (conidia/insect) FI-984 FI-985 FI-1067

7500 100 (60)* 95 (35) 95 (25)

3750 100 (45) 100 (60) 95 (65)

1875 95 (65) 100 (20) 95 (45)

930 80 (30) 100 (45) 90 (55)

control 10 (0)

* % mortality and in brackets % sporulation after 7 days at 30oC

Detailed comparison of 4 isolates for the Australian plague locust

Adult locusts were field collected by the APLC and sent to Canberra. These were dosed in groups in 20 as for the wingless grasshoppers and incubated in breadbin cages at 25oC for 10 days. As with wingless grasshopper, all isolates were virulent (Table 7). Unfortunately survival of the control insects was poor and this reduces the value of the data. Nevertheless, the trend is similar to that with wingless grasshopper with FI-984 and FI-985 being the two best isolates. The relative susceptibility of the Australian plague locust to Metarhizium isolates is similar to wingless grasshopper. Thus FI-984 and FI-985 are the most virulent isolates. It is interesting that FI-1155 despite being isolated from Australian plague locust, was less virulent than FI-985. Table 7: Comparison of 4 selected isolates using adult Australian plague locusts; results after 7 days incubation at 25oC

dose FI-984 FI-985 FI-1067 FI-1155

75,000 100 (10) 100 (50) 95 (5) 95 (15)*

7,500 100 (15) 100 (40) 90 (10) 95 (5)

750 95 (15) 85 (15) 75 (5) 90 (0)

control 60 (0)

* % mortality (% sporulation)

Field Comparison of Isolates

The two best exotic isolates, FI-984 and FI-1067, were compared with the two Australian isolates using small field plots. The site was a lucerne field near Bredbo, and 5 contiguous plots each 0.5ha were sprayed with a dose equivalent to 2 X 1012 conidia/ha in 2 litres of Propar oil using a Micro-Ulva Plus. Live samples of grasshoppers were taken 1, 4 and 7 days after spraying and the grasshoppers incubated for 14 days breadbin cages in the glasshouse. Dead insects were removed and placed in moist chambers to check for sporulation. All isolates caused 100% mortality of all samples after 14 days incubation in the glasshouse and therefore the data on sporulation were used to compare isolates (Table 8). FI-985 and FI-984 were

17

not significantly different (Tukey’s test p=0.05) and were more effective than the other two isolates. All isolates caused most mortality between day 5 and day 8. Table 8: Effect of isolate on the % dead grasshoppers forming conidia of Metarhizium.

sample time (days)

FI-984 FI-985 FI-1067 FI-1155

1 56.8* 56.8 39.4 30.3

4 62.8 56.7 52.8 55.0

7 51.5 70.6 43.0 38.9

* number is % of insects forming conidia

General Conclusion

These experiments showed that FI-985 was more pathogenic than the other Australian isolate FI-1155 for both wingless grasshopper and the Australian plague locust. The two most promising exotic isolates were FI-94 and FI-1067, but these were not significantly better than FI-985. It was thus concluded that FI-985 be selected as the isolate for further development and commercialisation.

Effect of Temperature

Growth In Vitro

The rate of growth of FI-985 was compared with that of the other Australian isolate on 90 cm Petri dishes of SDAYE. The isolates had a very similar profile (Table 9) with FI-985 growing rather faster overall. Despite being from a cooler area (Hunter valley, NSW) FI-1155 grew faster at 36oC and slower at 13.5oC than FI-985 (Rockhampton, Queensland). Other studies have shown that FI-985 will grow slowly at 37oC and will not grow at 40oC, and that other isolates of M. anisopliae var. acridum have similar temperature profiles. The optimum temperature for both isolates is about 28oC. Table 9: Size of colonies (mm diameter) after 3 weeks on SDAYE

temperature FI-985 FI-1155 10.0 0 0 13.5 10 0 16.5 30 10 20.0 32 25 22.5 45 40 24.0 56 55 28.5 70 50 31.5 60 50 36.0 0 22

Effect of Incubation Temperature on Mortality of Australian Plague Locusts

A mixture of field collected second and third instar locust nymphs were inoculated by being fed grass sprayed with FI-985 in Propar at a range of doses. The highest dose was 2 X 109 conidia/ml which is similar to that used in field trials. The other doses were decimal dilutions. The insects were placed 10 to a 500 ml plastic jar with 3 replicates of 10 per dose/temperature regime

18

combinations. Three constant temperatures were used: 20, 25 and 30oC and two alternating (12:12) 25-30 and 25-40oC. At the highest dose, the disease developed fastest at 25-30oC and slowest at 20oC (Table 10). However at all doses mortality was close to 100% after 14 days. The disease killed a similar proportion of locusts at all temperatures when all the doses are considered together (Table 11). The LD50 values were determined by probit analysis and the differences with temperature were not significant (P= 0.05%). The LD50 values ranged from 1.9 X 106 conidia/ml for the 25-30oC range to 2.4 X 107 conidia/ml at 30oC constant. Table 10: Effect of temperature regime on % mortality in Australian plague locust nymphs inoculated with FI-985 sprayed onto grass at 2 X 109/ml.

days 20oC 25oC 30oC 25-30oC 25-40oC

0.0 0.0 0.0 0.0 0.0 0.0

4.0 24.0 29.0 52.0 73.0 42.0

5.0 28.0 43.0 72.0 88.0 46.0

6.0 32.0 64.0 89.0 92.0 50.0

7.0 32.0 78.0 96.0 92.0 54.0

8.0 44.0 78.0 96.0 96.0 61.0

9.0 64.0 89.0 96.0 100.0 96.0

10.0 84.0 96.0 100.0 96.0

11.0 88.0 100.0 96.0

12.0 92.0

13.0 96.0

14.0 96.0

19

Table 11: Effect of temperature on % mortality after 15 days incubation. Dose 1 was 2 X 109 condia/ml, while the other doses are decimal dilutions.

treatment

20oC 25oC 30oC 25-30oC 25-40oC

D1 86.0 100.0 100.0 100.0 96.0

D2 83.0 77.0 86.0 86.0 75.0

D3 57.0 68.0 71.0 85.0 71.0

D4 43.0 34.0 32.0 68.0 35.0

D5 35.0 14.0 17.0 23.0 30.0

oil only 19.0 28.0 8.0 8.0 35.0

untreated 16.0 13.0 13.0 15.0 13.0

Effect of Incubation Temperature on Mortality of Migratory Locusts

Two additional experiments were carried out to assess the effects of high temperatures on development of FI-985 in locusts. These were with adult migratory locusts, Locusta migratoria, obtained from the Australian National University where they have a permanent culture. In the first of these experiments, the locusts were inoculated behind the pronotum with 0.5µl of a 1 X 108 conidia/ml suspension (50000 conidia/insect) and incubated at 25, 30 and 35oC constant for 8 days. In the second experiment, the insects were treated in the same way but incubated at 15, 20, 25 and 40oC. Table 12: Combined results from two experiments on migratory locust showing the effect of incubation temperature on rate of mortality (dose = 50,000 conidia/insect)

days 15oC 20oC 25oC (1) 25oC (2) 30oC 35oC 40oC 3 0 7 7 0 0 0 13

4 0 7 7 0 57 100 13

5 0 14 27 8 71 13

6 0 14 67 38 86 13

7 0 21 93 92 86 13

8 0 21 100 100 93 13

9 0 29 13

10 0 64 13

11 0 64 13

12 0 86 13*

13 3 86

14 15 93

15 31 100

21 100

* experiment stopped

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There was only 13% mortality and no evidence of any infection at 40oC, while at the other temperatures the infection caused 90-100% mortality. The disease developed very slowly below 20oC, and killed 100% of insects after just 4 days at 35oC. None of these locusts, despite being killed by the Metarhizium, formed new conidia in a moist chamber, while the majority of locusts dying at the lower temperatures formed new conidia. Mathematical Model

In collaboration with Dr David Hunter and Gunter Maywald, a model has been developed based on the above data to predict the time to death under field conditions. While the model was developed for the Australian plague locust, it has been tested using other targets as well as the Australian plague locust. Only one field trial has been undertaken against Australian plague locust and this was under very hot conditions in January 1998. Here the model predicted the mean time to death as 11 days for 5th instars and 10 days for adults, while the observed mortality in the field was mainly after 8-10 days for both ages. The model was also accurate in predicting the time to death for migratory locusts in a field trial in November 1998 at Cleremont (model: 12-13 days; actual 8-14 days). However for a trial on spur-throated locusts at Normanton in June 1998 the model predicted 16 days to kill while the actual time in the filed was only 7-10 days. This discrepancy may be due to the fact that spur-throated locusts are more susceptible than the other two species. Overall the model does provide a useful prediction of time to death under a range of field conditions.

Laboratory Comparison of Formulations

Effect of Oil on Wingless Grasshopper

Initial studies suggested that vegetable oils such as peanut oil might enhance the effectiveness of Metarhizium against locusts (Milner et al., 1997), while studies on fly control with Metarhizium suggested that oils with a high viscosity were more effective (see Barson et al., Ann. Appl. Biol., 1994). Therefore 10 oils were selected to cover a range of viscosities and including both mineral and vegetable oils. The oils selected were: soyabean, canola, linseed and a range of mineral oils of increasing viscosity: Protea 10, 15, 21, 40 and 45 (Ampol). Our “standard” mineral oil “Propar” which is the same as Protea 10 was also included. In this first experiment 4th and 5th instar wingless grasshopper nymphs were treated with 0.5 µl of the oil/conidia suspension on the mouthparts. Thirty insects were inoculated with each oil and incubated at 25oC for 8 days. For comparison a treatment with conidia suspended in an aqueousTween-80 was included. A single high dose of 100,000 conidia/insect was used. There were 4 controls: water only, linseed oil, Propar and Protea 45. The mineral oils generally killed insects faster than the vegetable oils, however all oils except Protea 45 (and Tween-80) gave 100% mortality by day 8. Protea 45 was the most viscous mineral oil. The best oils were Protea 10/Propar, and Protea 15. These caused about 100% mortality after 6 days. Canola oil and linseed also caused 95% mortality after 6 days.

21

Table 13: Effect of suspending oil on mortality of wingless grasshopper

suspending medium day 5 day 6 day 7

untreated 7 10 10

linseed control 7 7 7

Protea 45 control 12 25 25

Propar control 10 16 20

Tween-80 14 25 37

Protea 10 63 100 100

Propar 73 96 100

Protea 15 65 100 100

Protea 21 58 90 100

Protea 40 23 96 100

Protea 45 0 12 42

soyabean 30 90 100

canola 30 95 100

peanut 35 85 100

linseed 40 95 100

These results suggested that both mineral and vegetable oils were better than Tween-80 and that low viscosity oils were better then high viscosity oils, however this was only with extremely viscous oils such as Protea 45. Low viscosity oils are better for aerial spraying and thus Propar or low viscosity vegetables oils such as canola and soya are to be preferred. Effect of Dose on Infectivity of Oil Formulations for Wingless Grasshopper

The previous experiment was at a single high dose, and so the best oils were retested using a range of doses. The methods were similar with the highest dose being 100,000 conidia/insect and the decimal dilutions of this high dose. There was a high level of control mortality which reduced the value of the results (Table 14). At 7 days after treatment the soyabean and Propar formulations gave 100% mortality at the highest dose and 50-60% mortality at the ten-fold dilution. The linseed oil was less effective giving 43% and 67% mortality at the two highest doses. By day 11, mortality was similar with all 3 oils, except that the linseed oil dose 1 was inexplicably low at 82% compared to 97% mortality at the 10-fold dilution. These results confirmed that the oil had only a small effect on mortality and that mineral and vegetable oils of suitable low viscosity were likely to be suitable for ULV spraying in the field. The results do not support the suggestion that the high viscosity vegetable oil, linseed, enhanced the efficacy of the conidia.

22

Table 14 – Effect of dose and oil on mortality of wingless grasshopper at 25oC

dose soyabean oil linseed oil Propar oil

100,000 100 (100)* 43 (82) 100 (100)

10,000 53 (96) 67 (97) 61 (96)

1000 24 (48) 43 (65) 27 (62)

100 20 (63 40 (73) 17 (59)

control 26 (64) 20 (48) 24 (72)

* % mortality after 7 days (and 11 days)

Field Comparison of Formulations

Three experiments have been carried out against wingless grasshoppers to compare formulations in the field.

Comparison of 4 formulations against early instar nymphs

In this first experiment, the opportunity was taken of comparing a mineral oil (Propar) with a vegetable oil (canola), a water formulation and using the rice on which the conidia were produced as a bait formulation. Small plots were treated in a berry farm near Dalgety. The plots were each 48 X 11m (0.05 ha) and consisted of 3 rows of raspberries. The middle of the 3 rows was sampled to minimise migration in and out of the plot. A single dose equivalent to 5 X 1012 conidia per ha was used. The liquid formulations were sprayed at 1 litre/ha using a Micro-Ulva Plus hand sprayer while the rice granules were scattered by hand. The target grasshoppers were 1st and 2nd instars. The efficacy was assessed by taking a live grasshopper sample from each plot 4 days after treatment and incubating 4 replicates of 50 insects in bread-bin cages in the glasshouse at 26oC for 14 days. The two oil formulations caused over 90% mortality after 11 days (Figure 4). The water formulation was surprisingly effective giving 80% mortality after 14 days. This high level of efficacy was probably due to the cool humid conditions experienced during the 4 days after application and the lush vegetation. The rice granule formulation was least effective but did cause almost 50% mortality after 14 days. The results suggest that both canola and Propar would be equally effective formulations.

23

Figure 4. Mortality of field-treated wingless grasshoppers collected 4 days after treatment with one of 4 formulations of FI-985.

24

Comparison of vegetable and mineral oil formulations for field population reduction

In order to further compare vegetable and mineral oil formulations and to assess the effects on actual population reductions in the field, two sites, Dimboola and Llangrove, were selected in the Cooma area. Both were lucerne-based pastures but had been grazed hard so that the vegetation was quite sparse at the start of the experiment. Conditions were hot and dry during the trial and so the pasture conditions deteriorated further inducing the grasshoppers to disperse. The two formulations chosen were Propar and soyabean oil. The later was preferred to canola oil as it has a lower viscosity and is thus more suitable for ULV spraying with fixed wing aircraft. Application was at a rate of 4 X 1012 conidia/ha with 2 litres of oil concentrate being emulsified with water and applied using a trailer mounted boom sprayer. At Dimboola two 5 ha plots were treated while at Llangrove the plots were 3 ha. An equivalent area adjacent to the treated plots was left untreated as a control.

The efficacy was assessed in three ways: 1. Sweep net sampling at day 0, 8, 15, 22 and 29 to assess effect on population density

2. Vegetation samples were taken at Dimboola only on day 0, 1, 4 and 8 to assess persistence of the spray deposit

3. Live grasshopper samples were taken of 100 insects on days 0,1 and 4 to assess the level of infection in the population. These insects were incubated in the glasshouse for 14 days

At both sites, the control population density was greater than either treatment from day 7 onwards (Figures 5 and 6). The two formulations were always not significantly different from each other (P<0.005). The treatments were apparently more effective at Dimboola than at Llangrove, however this probably was due to the very sparse vegetation at Llangrove which resulted in greater dispersal of grasshoppers.

25

0 10 20 30 40

days after spraying

0

10

20

30

40

50

60nu

mbe

r/m

2

controlsoyabeapropar

Figure 5. Effect of two formulations of RI-985 on population density of wingless grasshoppers at Llangrove

26

-10 0 10 20 30 40

days after spraying

0

10

20

30

40

50

60

70

80

num

ber/m

2

Figure 6. Effect of two formulations of FI-985 on population density of wingless grasshoppers at Dimboola

27

Figure 7. Mortality of field-treated wingless grasshoppers collected 1 day after spraying with either a mineral oil or a vegetable oil formulation at Llangrove.

28

Figure 8: Mortality of field-treated wingless grasshoppers collected 1 day after spraying with either a mineral oil or a vegetable oil formulation at Dimboola

29

There was no difference in the persistence of the two formulations on the vegetation with most of the spray deposit being inactive by day 1 (Table 15). Table 15 – Persistence of two formulations in the field at Dimboola

treatment day 0 day 1 day 4 day 8

soyabean oil 84 (75) 93 (51) 20 (3) 32 (13)

Propar 82 (77) 83 (52) 28 (12) 30 (5)

control 44 (40) 48 (23) 28 (12) 48 (5)

The rate of mortality and sporulation in the live insect samples taken on Day 0, Day 1 and Day 4 were very similar for the two formulations (Figures 7 and 8). Samples taken on Day 0 and Day 1 gave 90-100% mortality with the majority of cadavers sporulating. At Day 4, the level of infection was reduced especially at Llangrove again suggesting that the grasshoppers at this site were actively dispersing. Comparison of oils for persistence of spray deposit

The final field trial compared the persistence of the spray deposit for Propar and soyabean formulated conidia. The dose used was 2 X 1013 conidia/ha. The persistence was assessed using field cages moved every two days on the treated vegetation. At each time about 100 healthy wingless grasshoppers were added and then removed after the 2 day exposure period. The insects were incubated in the glasshouse to determine the level of infection.

With both oils, the incidence of infection declined rapidly from the 0-2 to the 2-4 days exposure. The level of infection continued to decline slightly with soyabean oil but with Propar the infection rate remained at 30-40% until the final time interval which was 6-8 days. This experiment suggests that the persistence of the mineral oil formulation may be better than that of the vegetable oil. General Conclusions

These experiments have shown that there is only a small effect of the suspending oil on the efficacy of the fungus. Thus it is suggested that where there is a need for a fully organic product, a soyabean formulation is likely to be as effective as the standard Propar formulation. There is a suggestion that the persistence of the conidia is better in the mineral oil, perhaps because it is not as good a food source for the fungus. It was noticed that the conidia germinating from the vegetable oils generally grew longer germ tubes than those from the mineral oil, suggesting the fungus was able to obtain nutrition from the vegetable oil. In order to check this, we have grown the fungus in liquid medium to which either Propar or soyabean oil has been added. The fungus grew more rapidly at first with the vegetable oil but in the end both oils were utilized by the fungus and resulted in greater growth than in the oil-less controls. Storage

For experiments on storage the conidia of FI-985 were produced on rice. Half the conidia were not dried and were 40 % moisture. The other were dried using silica gel to a moisture content of about 6%. The conidia were then suspended in oil or kept as dry powder and stored at 4oC and 25oC. The viability was determined by % germination after 48 hrs incubation at 25oC.

30

Table 16 - Viability of undried conidia stored in various ways

temperature treatment 0 m 1m 2m 3m 4m 5m 7m 9m 12m

5oC propar 97 90 90 80 70 5 6 1 0

soya 91 62 2 0.5 0 0 0 0 0

canola 95 55 5 0.5 0 0 0 0 0

powder 99 94 96 85 63 7 1 0 0

25oC propar 97 0 0

soya 91 0 0

canola 95 0 0

powder 98 4 0

Table 17 - Viability of dried conidia stored in various ways

temperature treatment 0 m 1m 2m 3m 4m 5m 7m 9m 12m 18m

5oC propar 70 44 46 29 33 62 59 63 59 45

soya 70 47 56 53 42 74 62 72 61 43

canola 70 69 63 53 47 77 72 73 61 45

powder 90 83 45 57 53 58 65 54 65 60

25oC propar 70 50 47 36 22 26 23 20 2 0

soya 70 66 66 52 28 36 41 41 2 0

canola 70 70 63 64 41 50 42 58 2 0

powder 90 67 62 47 26 34 36 36 14 0.5

After 18 months storage the results show:

1. dried spores store better than undried spores whether held as powder or in oil

2. spores store better at 5oC than at 25oC

3. undried spores store better in mineral oil or a powder than in soyabean or canola oil

Dried spores at 5oC as powder or in any of the 3 oils germinated well after 12 months storage. The initial germination varied from 70-90%. Thus spores have stored well for 18 months in the cold. At 25oC, these same spores stored well for 9 months but are now giving less than 1 % after 18 months. Other studies have shown that a small proportion of dried spores survive for several weeks at 50oC while undried spores do not survive more than 1 day.

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Field Experiments on Efficacy

Twelve field trials have been undertaken to assess efficacy since 1993 (Table 18). The first two were preliminary and were prior to the current grant. Each field trial generates a large amount of data and consequently only a summary is presented here.

Table 18. Summary of Field Trials on Efficacy

Site Date Sprayed

Target Treatments Dose (conidia/ha)

Ground(G) or Aerial (A)

Number of treated plots

plot size

1 Coleambally 10/1993 C. terminifera 2L oil/ha 3 X 1012 G 7 bands of nymphs

2 Dalgety 12/1993? P.vittatum 2L oil/ha 25L oil/water /ha fenitrothion

4 X 1012 G 7 0.25 ha

3 Tarrago 16/3/94 P.vittatum 4L oil/ha 4 X 1012 G 2 5 ha 4 Amaroo 13/12/94 P.vittatum 2L oil/ha 4 X 1012 A 1 50 ha 5 Dalgety/

Carabost 29/11/94 P.vittatum 6L oil in 34L

oil/water emulsion 7 X 1012 G 1 ha

6 Clermont 7/1995 A. guttulosa 1.3-2.0L oil/ha 5-7 X 1012 G 2 0.5-0.75 ha 7 Broken Hill 11/101995 C. terminifera 500ml – 2L/ha 1 X 1011 –

4 X 1012 G 12 bands of

nymphs 8 Cooma/

Dalgety 23/11/95 plus 10/1/96

P. vittatum 6L oil concentrate as oil/water emulsion

6 X 1012 2 X 1012

G 6 1 ha

9 White Cliffs 1996 C. terminifera 1 L oil/ha 5 X 1012 A 1 100 ha 10

Emerald 21/3/97 A. guttulosa 1.3L oil/ha 4 X 1012 A 2 70-80 ha

11

Trangie 6/1/96 C. terminifera

1L/ha 5 X 1012 A 1 100 ha

12

Cooma 25/11/1997 P. vittatum

1L oil/ha 34 L oil/water emulsion

5 X 1012 A + G 9 1 ha (G) 50-80 ha (A)

13

Coonamble 26/1/1998 C. terminifera

1L oil/ha 5 X 1012 G 1 0.5 ha

14

Normanton 14/6/1998 A. guttulosa

1L oil/ha 5 X 1012 A 3 80-100 ha

15

Emerald 6/11/1998 L. migratoria

1L oil/ha 3 X 1012 A 3 60-80 ha

16

Peak View 3/12/1998 P. vittatum

1L oil/ha 5.5 X 1012 A 3 30-60 ha

Note: Trials 14, 15 and 16 were undertaken after the completion of this project and so the results are not presented in this report

32

Field Trials against Wingless Grasshopper

Four trials have been undertaken on this grasshopper during the project period. In the first trial, which was on 3 replicate plots on both Dalgety and Carabost. Each plot was 1 ha and was sprayed using a trailer-mounted boom sprayer. In addition plots were left untreated or sprayed with fenitrothion as a positive control. Consequently there were 18 plots at each site. The conidia of FI-985 were formulated, undried, in Propar and applied as 6 litres of oil concentrate emulsified in 34 litres of water per 1 ha plot. The Dalgety plots were sprayed on 21 November while the Carabost plots were sprayed 1 week later. The weather was cool and wet for the week after spraying at Dalgety while it was much warmer and drier after the spraying at Carabost. At Dalgety, pitfall traps were used to monitor non-target effects (results in next section). Table 19 - The mortality of the live grasshopper samples from the treated and control plots

days post spraying

% mortality Dalgety treated

% mortality Carabost treated

% mortality Dalgety controls

% mortality Carabost controls

0 98.9 (19.2)* 99.5 (68.1) 68.9 (0) 44.1 (0)

1 99.1 (38.3 90.1 (68.5) 62.2 (0) 26.1 (0)

7 71.6 (41.0) 40.5 (35.8) 31.9 (0) 36.1 (0)

14 63.9 (36.0) 85.5(31.4) 44.1 (0) 32.4 (0)

21 79.3 (12.5) 64.1(17.7) 31.6 (0) 29.6 (0)

30 26.8 (5.5) 31.0 (5.2) 18.9 (0) 23.6 (1.5)

* figure in brackets is % sporulation

Mortality not related the fungus was high in most samples, especially the early ones from Dalgety where the insects were only 1st and 2nd instars. There was very little infection in the controls except at 30 days, suggesting that few infected insects were able to move across the 200 m buffer strips between plots. There was a reduction in the % infection between 14 and 21 days at Dalgety suggesting that this was the time when most mortality occurred in the field. At Carabost the reduction occurred sooner and was probably in part to the invasion of untreated insects from outside the plots. At both sites, there was only a small incidence of infection after 30 days. This was probably due to grasshoppers picking up a low dose from the residual conidia on the vegetation and there was no suggestion that the disease was recycling in the population. Grasshoppers were sampled in transects away from the plots and at no time did the incidence of infection exceed 10% when the grasshoppers were sampled from outside the plots. This suggests that infected grasshoppers disperse very little and that invasion of treated plots by untreated grasshoppers is more important. A sample of treated vegetation was taken from each plot on the day of spraying and untreated grasshoppers incubated with the vegetation to assess the potential for pick-up of the infection. The insects were incubated for 14 days and at Dalgety, 80% of the grasshoppers died with 65% sporulating, while at Carabost, 95% of the grasshoppers died and 40% sporulated. This showed that the spray deposit could be an important source of infection by secondary pick-up.

33

Figure 9: Effect of spraying with fenitrothion and Metarhizium as well as an unsprayed control plot on changes in population density at Dalgety

34

Figure 10. Effect of spraying with fenitrothion and Metarhizium as well as an unsprayed control plot on changes in population density at Carabost

35

The effect of the treatments on population densities are shown in Figures 9 and 10 . To allow for the differences in initial density which ranged from 10 to 70 with most plots around 40-50/m2, the density is expressed as % initial density. At Dalgety, fenitrothion caused over 90% mortality after 1 day and the density remained low for 3 weeks. However these plots were then reinvaded to the extent that the density was over 40% of the initial density by 9 weeks. The control plots did decline a little over the first 9 weeks probably due to dispersal away from the plots and then declined further as the adults died off due to natural causes. The Metarhizium treated plots decline rapidly between day 7 and day 21. By Day 21 there had been a 70% reduction. After day 21 there was a further gradual decline with the Metarhizium-treated plots having the lowest population for the rest of the sampling period which ceased after 16 weeks. At Carabost, the density changes were similar with Metarhizium giving a maximum of 80% control after 3 weeks. These plots were then re-invaded to some extent but they all remained lower than the controls and similar to the fenitrothion plots for the rest of the 15 week sampling period. There is no evidence that the Metarhizium recycled in the population. Very few infected cadavers were found in the field and none of these were seen to sporulate. A second trial, at Amaroo near Humula, tested the efficacy of Metarhizium when applied as a ULV oil concentrate using a fixed wing plane. A single plot of 50 ha was sprayed on the 13 December 1994 using a Cesna fitted with 2 Micronair AU5000 nozzles. The application was supervised by Mr Peter Spurgin of the Australian Plague Locust Commission. The dose applied was 2 X 1012/h a in 2 L oil. A similar plot about 1 Km away was left untreated as a control. The weather during and shortly after spraying was very hot (max. 37oC), sunny and dry. Eighty to ninety percent of grasshoppers sampled at 0, 7 and 14 days after spraying died when caged in the glasshouse. The day 3 sample was inexplicably low at 62% mortality. Control mortality was always less than 25%. However the proportion of grasshoppers from the treated plot which sporulated declined from 70% at day 0 to only 10% at day 14. Subsequent studies have shown that insects which die in the field under temperatures of 30-40oC often do not sporulate. Since temperatures did exceed 35oC on several days during this 2 week period, it is likely that these high temperatures are the reason for the low level of sporulation. Treated vegetation caused 100% mortality after 14 days and 42% of these insects sporulated. Population density changes were determined at 9 points along a transect in the treated plot and 8 points in the control on days 1,6 and 16 post spraying. In the centre of the control plot the density remained at 10-20 grasshoppers/m2 over this sampling time, while in the treated plot the density reduced from 90-120 down to 20 to 25 grasshoppers/m2. Overall the control plot population density declined by about 15% and the treated by 55% (Figure 11). The very hot weather caused considerable dispersal of grasshoppers including a probable invasion into both plots by grasshoppers further up the slope of the valley. However the landholder reported seeing large numbers of pink (Metarhizium-killed) grasshoppers were seen on the treated plot 12 days after spraying. The following conclusions can be drawn from these results:

1. Mortality due to FI-985 in the field takes 2-3 weeks with little further mortality after 30 days post-treatment. There was no suggestion of recycling.

36

2. A dose of 2-7 X 1012 conidia/ha caused 75-90% kill in the field.

3. The oil concentrate can be applied from the air using standard ULV equipment and 2 litres of concentrate per ha.

4. Plots treated with fenitrothion gave a high initial kill but is then re-invaded after 1-2 weeks. As a result the Metarhizium treated plots usually had fewer grasshoppers than either the control or the fenitrothion plots from day 14 onwards.

5. There is very little spread of the infected grasshoppers away from the treated plots. In 1995, one field trial was undertaken in order to confirm the findings of the previous season and to evaluate the potential for repeat spraying at 30 day intervals to control wingless grasshoppers over the whole season. The field trial was replicated over 2 sites in the Cooma and Dalgety regions. In all there were 11 plots. Two strips of a “protected area” with 3 plots on one side (Cooma) and on both sides (Dalgety). These 3 plots were either untreated control, or sprayed with fenitrothion or Metarhizium. These plots were about 1 ha. The plots were sprayed with a boom sprayer using 6 litres of oil in 34 litres of oil/water emulsion/ha. The dose of conidia was 4 X 1012 /ha. The plots were first sprayed on 22/23 November 1995 and then repeat sprayed on 10 January 1996 with 2 X 1012 conidia/ha. The initial spray reduced the populations in the Metarhizium treated plots from 15-20 grasshoppers/m2 to less than 5/m2 after 3 weeks (Figure 12). The population density did then increase due to reinvasion up to 10-15 before the respray on 10 January which again resulted in a decrease to about 2 grasshoppers/m2 by day 62. A similar trend was shown by the insecticide treated plots however the effect of the spray was much more rapid. The control plots remained over 15 grasshoppers/m2, until they dropped to about 8/m2 by day 62 due to natural mortality. The incidence of disease in live grasshopper samples (Table 20) confirmed the fact that the Metarhizium sprays were effective with high levels of infection for over 15 days after spraying. By 28 days after spraying, the fungus had caused mortality in the field and so the level of infection dropped to 0-7% for spray 1 and 9-22% for the repeat spraying. Persistence of the spray deposit on the vegetation was only assessed for the first spray at Dalgety. The spray persisted quite well for 4 days but was largely gone by day 6 (Figure 13). The fenitrothion showed a similar decline with much of the activity being lost by day 4 (Figure 14). These results confirmed those of the previous year in suggesting that the infection would take 14-21 days to kill in the field and that repeat spraying at about 30 day internals would give season-long control. The reduction of the dose by ½ for the repeat spray did not seem to affect the efficacy. This suggests a strategy of 2-3 low dose sprays over the season might be more effective than a single high dose spray.

37

Figure 11: Changes in population density at Amaroo following aerial spraying with Metarhizium

38

Figure 12: Changes in population density at Dalgety and Cooma on untreated control plots and those sprayed twice with either fenitrothion or Metarhizium. Results are for all plots combined.

39

Figure 13: Persistence of the Metarhizium spray deposit as shown by mortality of healthy grasshoppers incubated with field-treated vegetation collected 1, 4 and 6 days after spraying

40

Figure 14. Persistence of the Fenitrothion spray deposit as shown by mortality of healthy grasshoppers incubated with field-treated vegetation collected 1, 4 and 6 days after spraying

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Table 19. Summary of final mortality (and sporulation) in live grasshopper samples from Cooma and Dalgety

days after spraying

Met. Cooma

Met Dalgety 1

Met. Dalgety 2

Control Cooma

Control Dalgety 1

Control Dalgety 2

1 88 (71) 99 (62) 97 (61) 35 (2) 26 (0) 24 (1)

5 59 (41) 84 (73) 79 (57) 32 (2) 36 (3) 30 (4)

7 65 (57) 62 (56) 76 (67) 30 (1) 8 (1) 8 (4)

15 30 (27) 70 (53) 70 (44) 24 (1) 8 (0) 27 (0)

28 25 (7) 20 (1) 15 (0) 16 (0) 9 (2) 22 (0)

42 40 (6) 13 (3) 7 (1) 31 (0) 10 (1) 10 (0.5)

repeat spray

1 75 (42) 85 (78) 89 (95) 55 (0) 4 (0) 4 (0)

8 94 (10) 88 (59) 92 (46) 32 (0) 18 (3) 33 (1)

15 56 (24) 56 (56) 39 (35) 21 (1) 24 (5) 20 (2)

28 47 (9) 42 (22) 35 (15) 47 (2) 14 (0) 14 (1)

A large-scale field trial was established in the Cooma/Dalgety area in November 1997. Previous trials had been on small plots and because grasshoppers and locusts move about a lot, it is not possible to fully assess reductions in population density on these plots. Nine plots of 40-80ha each were aerially sprayed from 25-27 November 1997. The plots were in the Dalgety, Cooma and Peak Hill areas of the Monaro. Two other plots of 1ha were sprayed from the ground with the same material in an oil/water emulsion using a boom sprayer. Two plots were sprayed with the standard. The Metarhizium sprayed plots were treated at 1 litre/ha with a mineral oil concentrate formulation containing 5 X 1012 conidia. The weather during the spraying on 25, 26 and 27 November was very hot (max ca 37oC). The plots were sprayed early in the morning. Because of the prevailing drought the plots mostly were covered with sparse vegetation and became more sparse as the trial progressed. Also the grasshopper populations were very dense and often were dominated by species such as Austracoitetes and Brachyexana rather than wingless grasshopper. The plots were assessed using the standard methods. Changes in population density were assessed using sweep net counts, while other tests measured the persistence of the spray on the vegetation and the level of infection in the grasshopper population. Soon after the plots had been sprayed it was found that a high proportion of the spores had not survived the spraying and those that had reached the grasshoppers also did not live much longer. One measure of this was the half-life on the vegetation which was less than ½ a day rather than several days as normally achieved. Nevertheless, the initial spray deposit was capable of killing 80-100% of the grasshoppers as revealed by our sampling soon after spraying. In the field the treated populations had declined considerably by 14 days after spraying and continued to decline. At several sites, numbers of characteristic pink Metarhizium-killed cadavers were seen. The fenitrothion sprayed plots gave a

42

high initial kill but were rapidly invaded so that after 2 weeks the only populations to be reduced significantly were the Metarhizium treated plots. However some Metarhizium treated plots had more grasshoppers than at the start while at others the population had been reduced by up to 80% (Figure 15). Detailed analysis of the data has shown:

1. That the Metarhizium spray had been more effective in the very sparsely vegetated plots, and 2. Some plots had been invaded by grasshoppers from the untreated surrounding populations

leading to an apparent lack of efficacy. The major target is the wingless grasshopper, and all plots showed a reduction of over 40% in the density of this species (Figure 16). Overall the population reduction was about 50% with 2 plots giving over 80% reductions. The ground-treated plots gave a similar result suggesting that it is the dose of conidia and not the suspending medium that is important. The overall level of grasshopper control in this trial was disappointingly low. Subsequent studies have shown that this was due to the environmental fragility of the conidia used. Methods for stabilizing the conidia by drying have now been developed and subsequent trials have been with conidia which survive high temperatures and high UV exposure much better. Field Trials against Spur Throated Locusts

With the assistance of the Queensland DNR, two field trials have been completed against spur-throated locusts, Austracris guttulosa, in the Clermont/Emerald region of the Central Highlands in Queensland. In the first trial, 2 plots of 0.5 to 0.75 ha infested with adult spur-throated locusts were sprayed in July 1995. One plot was the corner of a large sorghum field and the other an adjacent area of open woodland. A large swarm of adult locusts was present in the area and were feeding on the sorghum heads during the day and roosting in the trees at night. The plots were sprayed using a hand-held MicroUlva Plus delivering 2 litres/ha of a suspension of 3.7 X 1012 conidia/l. Both plots were sprayed in the late morning. A third area was left untreated as a control. Samples of the treated locust were collected with a sweep net later on the day of spraying. The next day a second sample was taken from this plot, however no locusts were present in the sorghum crop. These insects were returned to Canberra and incubated in breadbin cages in the glasshouse. The insects from the sorghum crop started dying after 7 days incubation and reached100% kill by Day 9. Almost all these insects sporulated. From the woodland, the insects started dying after day 2 and died consistently until by Day 8 there was 100% kill. However only about 65% of these sporulated. The second woodland sample gave 30% mortality and these all sporulated. This trial showed that Metarhizium has the potential to induce a high proportion of mortality in spur-throated locusts. However these large insects are strong fliers and are highly mobile, thus much larger plots will be needed to show any effect in the field.

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Figure 15. The % control in all plots at Cooma for all species combined 4 weeks after spraying

Figure 16. The % control in all plots at Cooma for all wingless grasshopper 4 weeks after spraying

-80-60-40-20

020406080

100

FV2 GU2 SP2 AR1 GU1 SP1 MP1 FV1 DP1

0

20

40

60

80

100

FV2 GU2 GU1 AR1 SP2 SP1 MP1 FV1 DP1

44

A second trial, with much larger plots was undertaken in March 1997 at Emerald. Two plots of 70 ha and 35 ha were sprayed with a Cesna fitted with 2 micronairs on 21 and 22 March 1997. Both sites were sorghum fields with broad headlands of tall open grassland. The adult spur-throated locusts abundant at both sites and were thought moving within an area of 50 m diameter. Another similar area was left untreated as a control. The dose used was 2 litres/ha of oil formulation containing 3.5 X 109 conidia/ml. 1. Four methods were used to assess the efficacy of the treatment:

2. changes in density in the actual plots using APLC transect scores

3. mortality of insects collected soon after spraying and placed in field cages

4. mortality of field sprayed insects returned to the glasshouse in Canberra, and

5. mortality of healthy locusts placed on treated vegetation.

The changes in population density were monitored by Qld. DNR for a period of 21 days post-treatment using APLC transect methods. By 10 days post-spraying the treated locusts were seen to be inactive and mortality was observed in the field and in the field cages from day 10 to 21. The field populations were reduced in density by day 17 and by day 21 the drop was about 80% in the field plots. In the field cages, the final mortality (dead or missing) was 92% with 68% sporulation while in the control cages the mortality was 46% with non sporulating. Sampling of live locusts to return to Canberra and of vegetation for persistence studies were curtailed because very heavy rain prevented access to the plots after the second day of spraying. However, the sampling which was done indicated that over 80% of the target insects were infected (Table 19) and that there was substantial opportunity for secondary pickup from the vegetation. Table 20 – Mortality and sporulation of spur-throated locusts field treated with Metarhizium at Emerald in 1997.

plot sample % mortality % sporulation

1 day 1 live locusts (rep. 1) 100 10

1 day 1 live locusts (rep.2) 100 58

1 day 13 live locusts 100 81

1 day 1 vegetation (rep. 1) 100 74

1 day 1 vegetation (rep. 2) 100 80

2 day 0 live locusts 100 83

2 day 0 vegetation 100 85

control live locusts 55 0

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General Conclusions

The two field trials, supported by laboratory data on the high level of innate susceptibility of spur-throated locusts, suggest that FI-985 is effective for control of spur-throated locusts. Field Trials against Australian Plague Locust

Three field trials have been carried out against the Australian plague locust, C. terminifera, at Broken Hill, Trangie and White Cliffs. Two of these trials, unfortunately, did not produce any useful data. At Trangie, a single plot of 100ha of nymphs were aerially treated on a field of wheat stubble with a small amount of lucerne on which the locusts were feeding. However the insects rapidly moved away from the plot and mixed with other untreated insects. Live locust samples returned to the glasshouse and incubated in breadbin cages, gave a disappointingly low level of infection – about 40%. Subsequent studies showed that the conidia used in this trial had deteriorated during storage and were significantly less infectious than fresh conidia (Figure 18). At White Cliffs, about 100 ha of recently fledged adults were aerially treated. Unfortunately the plane had recently been used to spray fipronil and had not been properly cleaned. Consequently many of the treated insects died from fipronil poisoning rather than Metarhizium. At Broken Hill, individual bands of Australian plague locust nymphs were sprayed from the ground with the APLC vehicle mounted Micronair sprayer on 11 October 1995. This experiment was used to assess the relative significance of volume of formulation/ ha and the number of conidia/ha. Consequently 4 treatments were tested:

1. 2.2 X 109/ml applied at 2 litres/ha = 4.4 X 1012/ha (Dose 1)

2. as above but 500 ml/ha = 1.1 X 1012/ha (Dose 2)

3. 2.2 X 108/ml applied at 2 litres/ha = 4.4 X 1011/ha (Dose 3)

4. as above but 500 ml = 1.1 X 1011/ha (Dose 4)

In addition, 2 control plots were treated with oil only. Each plot was between 0.5 and 1.0 ha and contained several bands. Assessment of field mortality was not possible as the treated bands merged together before any mortality occurred. Therefore the assessment was based on sampling of treated bands and incubation of treated vegetation with healthy insects. The field treated insects were incubated in Canberra in breadbin cages in an outside enclosure. They were exposed to very cool conditions and mortality was delayed. For this reason, the 4 week mortality data is used. Both mortality and sporulation was directly correlated with dose (linear regression r2= 0.95) (Table 21). With the 1 day sample, the final % mortality at dose 1 corrected for control mortality was 98.1%, 68.2% at dose 2, 60.9% at dose 3 and 41.3% at dose 4. The mortality and sporulation of the locusts declined with the locusts sampled after 3 and 5 days. This is thought to be due to the mixing of treated and untreated bands. The vegetation samples indicated that the spray deposit declined over the 5 day sampling period (Figure 20).

46

Table 21 – Effect of dose and days after spraying on mortality and sporulation of Australian plague locusts at Broken Hill

dose replicate day 1 day 3 day 5

dose 1 1 100 (87) 63 (43) 62 (35)

2 100 (25) 86 (67) 53 (30)

3 100 (57) 95 (71) 51 (19)

dose 2 1 85 (67) 46 (15) 48 (11)

2 87 (67) 66 (42) 52 (20)

3 80 (66) 39 (18) 35 (28)

dose 3 1 80 (70) 52 (14) 74 (44)

2 79 (38) 74 (30) 51 (26)

3 82 (44) 45 (23) 58 (19)

dose 4 1 77 (26) 39 (15) 52 (23)

2 69 (7) 32 (15) 18 (10)

3 63 (30) 54 (29) 60 (16)

oil only 1 40 (16) 36 (24) 32 (7)

2 46 (14) 20 (10) 45 (17)

3 43 (20) 36 (21) 42 (22)

control 1 38 (3) 38 (14) 36 (8)

2 29 (12) 37 (29) 49 (9)

3 40 (4) 29 (14) 39 (15)

Effects on Non-Targets

Pitfall traps were used to assess the effects on non-targets of the Metarhizium spray in comparison with fenitrothion and the untreated controls at Dalgety in November 1994. These were sampled each week and the catches sorted into orders and counted. Since fenitrothion causes rapid mortality in a day or so, and Metarhizium rarely kills in less than 1 week, it might be expected that any adverse effects of the insecticide would be seen after 7 days and the Metarhizium after 2 or 3 weeks. Catches of arachnids and Dipper were quite variable with no obvious trends suggesting an effect of either treatment (Figure 21). The Coleopteran and Lepidoptera were relatively consistent again suggesting not effects due to the Metarhizium or the fenitrothion (Figure 22 and 23). The number of Hymenoptera and Hemiptera were consistently lowest in the fenitrothion plots suggesting an adverse effect here, however in the Metarhizium plots numbers increased between weeks 2 and 3 (Figure 24 and 25). The most dramatic effect was on Collembola which were reduced to almost zero by the fenitrothion spray and were unaffected by the Metarhizium. The number of Orthoptera (many wingless grasshoppers) was reduced by the fenithrothion treatment and by the Metarhizium treatment as expected (Figure 26).

47

In conclusion these pitfall trap catch data do not suggest that the Metarhizium spray is affecting any insects other than grasshoppers. Other trials have collected data on particular non-targets when they were particularly abundant. No infections in crickets or asilids have been detected and the level in tettigonids has been extremely low (<1%). In one trial, where bees were foraging, some mortality of worker bees, was observed but this is not likely to have caused any problems in the hive. Susceptibility of different species of grasshopper and locust

Nine species of acridoid grasshoppers and locusts have been bioassayed against FI-985 and all have been found to be highly susceptible (Table 23 ). Table 22 Relative susceptibility of different species of locust and grasshopper to FI-985

Target Days LD50 95% confidence limits

Chortoicetes terminifera 6 417 220 - 721

Phaulacridium vittatum 7 1212 689 - 1900

P. vittatum (short winged) 7 1548 545 - 4390

P. vittatum (long winged) 7 1626 533 – 4954

Locusta migratoria 9 4363 987 – 16464

Austracris guttulosa 9 413 227 - 867

Austracoites sp. 7 657 206 - 2090

Brachyexana sp. 7 952 290 – 2919

Praxybulus sp. 7 1089 285 – 4094

Oedaleus sp. 7 928 226 - 3556

Progress by Milestone By November 30 1995

1. assemble library of isolates

2. initiate commercial negotiations

A library of isolates was assembled and are listed in Table 1. Commercial negotiations were commenced and a product is now under development by Inoculant Services (SGB an IAMA company)

By May 31 1996

1. screen at least 6 isolates against plague locust and wingless grasshopper

2. compare at least 2 isolates in small scale field trials

Ten isolates were screened and 4 compared in a field trial

48

By November 30 1996

1. choose one or two isolates for detailed investigation

2. initiate laboratory studies on ecological factors such as temperature

3. initiate storage experiments FI-985 was selected for further development and detailed studies undertaken on temperature relationships. Storage experiments with dried and undried conidia as powder and in various oils at two temperatures were set up. These have now been running for over 18 months. By May 31 1997

1. develop models to predict field activity

2. test various formulations in the laboratory

3. establish further field trials with chosen isolate(s)

4. collect some data for registration In collaboration with Dr David Hunter and Dr Bob Sutherst, a model has been developed to predict the likely time to death in the field. This has been tested and found to be accurate for wingless grasshopper and Australian plague locusts. Eleven oil formulations as well as a rice granule formulation and a water-based spray have been tested in the laboratory and the field. Field trials have been undertaken with FI-985 against wingless grasshopper, Australian plague locust and spur-throated locusts. A considerable amount of data on field efficacy and non-target effects have been collected for registration. By November 30 1997

1. continue storage experiments

2. produce large amounts of fungus for field trials

Storage experiments have continued. Conidia of FI-985 were produced for almost 400 ha for a large-scale field trail against wingless grasshopper. By May 31 1998

1. complete large-scale field trials

2. compare best formulations under field conditions

3. establish reliable data on storage

4. collect most of the efficacy data needed for registration package

5. initiate writing final report

Large-scale field trials have been completed against wingless grasshopper, Australian plague locust and spur-throated locust. Soyabean and Propar formulations have been compared in the field and the storage experiment continued. Further efficacy data has been collected, however the

49

environmental fragility of the conidia used in the 1997 Cooma trials has led to experiments on drying of conidia to stabilise them. These experiments are still underway but show that conidia can be dried to 5% and viability maintained, and that these conidia have enhanced field persistence. Large amount of efficacy data has been collected for registration. The writing of the final report was considerably delayed due to the demands of continuing field trials and investigations of the drying problem.

50

Implications and Recommendations The research described in this report has confirmed that Metarhizium is promising as a biopesticide for use against locusts and grasshoppers in Australia. Major potential users have formed a committee to coordinate further development towards a registered commercially available product in about 2000. This committee consists of representatives from the Australian Plague Locust Commission, Qld Department of Natural Resources, NSW Agriculture, CSIRO, and the Tablelands Wingless Grasshopper Committee. Production of conidia for field trials is gradually being transferred from CSIRO to Inoculant Services, a commercial company based in Albury. There are a number of factors are currently driving this research:

the expansion of organic agriculture such as the production of organic beef in SW Queensland. Metarhizium has been certified as “organic” by NASSA

the need for a product to control locusts and grasshoppers in environmentally sensitive areas such as National Parks and near wetlands

the increasing concern over residues and withholding periods

It is recommended that further research on this biopesticide is directed at the following:

the development of an effective and low cost drying systems to ensure a stable conidial

product

further understanding of the effects of hot sunny conditions on the efficacy of the product

further data on effects of the infection on fat deposition and ovarian development

collaborate with commercial companies to develop large-scale economical methods for mass production

obtaining NRA registration

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Publications The following papers relevant to this project have been published: Baker, G.L., Milner, R.J., Lutton, G.G. and Watson, D.M. (1994) Preliminary field trial on control

of Phaulacridium vittatum (Sjostetd) (Orthoptera: Acrididae) populations with Metarhizium flavoviride Gams and Roszypal (Deuteromycetina: Hyphomycetes). J. Aust. Ent. Soc. 33, 190-192.

Milner, R.J., Hartley, T.R., Lutton, G.G. and Prior, C. (1994) Control of Phaulacridium vittatum

(Sjostedt) (Orthoptera: Acrididae) in field cages using an oil-based spray of Metarhizium flavoviride Gams and Rozsypal (Deuteromycetina: Hyphomycetes). J. Aust. Ent. Soc. 33, 165-167.

Milner, R.J., Driver, F., Curran, J., Glare, T.R., Prior, C., Bridge, P.D. and Zimmermann, G.

(1994) Recent problems with the taxonomy within the genus Metarhzium, and a possible solution. VIth Int. Coll. Invert. Pathol. , Montpellier, August 1994, p. 109-110.

Milner, R.J. and Prior, C. (1994) Susceptibility of the Australian plague locust, Chortoicetes

terminifera, and the wingless grasshopper, Phaulacridium vittatum, to the fungi Metarhizium spp. Biological control 4, 132-137.

Hooper, G.H.S., Milner, R.J., Spurgin, P.A. and Prior, C. (1995) Initial field assessment of

Metarhizium flavoviride Gams and Rozsypal (Deuteromycetina: Hyphomycetes) for control of Chortoicetes terminifera (Walker) (Orthoptera: Acrididae). J. Aust. Ent. Soc. 34, 83-84.

Milner, R.J. (1995) Future prospects for fungal biopesticides. In: Monsour, C.J., Reid, S.R. and

Teakle, R.E. (Eds.) Biopesticides: Opportunities for Australian Industry, pp. 28-34. [Brisbane: Queensland University Press]

Milner, R.J. (1995) Towards a biopesticide for locusts and grasshoppers - Recent developments

with Metarhizium. In: Monsour, C.J., Reid, S.R. and Teakle, R.E. (Eds.) Biopesticides: Opportunities for Australian Industry, pp. 103-106. [Brisbane: Queensland University Press]

Glare, T.R., Milner, R.J. and and Beaton, C.D. (1996) Variation in Metarhizium: Is phialide morphology a useful taxonomic criterion? Journal of Orthopteran Research 5, 19-27.

Milner, R.J., Staples, J.A. and Prior, C. (1996) Laboratory susceptibility of Locusta migratoria

(L.), Austracris guttulosa (Walker) and Valangra irregularis (Walker) (Orthoptera: Acrididae) to an oil formulation of Metarhizium flavoviride Gams and Rozsypal (Deuteromycotina: Hyphomycetes). Aust. J. Ent. 35, 355-360.

Milner, R.J. and Jenkins, K. (1996) Metarhizium: a versatile mycoinsecticide of the future.

Professional Pest Manager 1, 32-36. Milner, R.J., Baker, G.L., Hooper, G.H.S. and and Prior, C. (1997) Development of a

mycoinsecticide for the Australian plague locust. New Stratagies in Locust Control , p.177-183. [Birkhauser, Basel]

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Milner, R.J. (1997) Metarhizium flavoviride (FI985) as a mycoinsecticide for Australian acridids.

Mem. Can. Ent. 171, 287-300. Driver, F. and Milner, R.J. (1998). Applications of PCR to research on the taxonomy of

entomopathogenic fungi. Application of PCR in Mycology, edit. P.Bridge, D.K. Arora, C. A. Reddy, and R.P. Elander, pp.153-186. [CAB International, Wallingford, UK]

Milner, R.J. and Staples, J.A. (1998) The effect of formulation on field efficacy of Metarhizium

flavoviride for control of wingless grasshopper, Phaulacridium vittatum. J. Orthopt. Res. 7 (in press)

Milner, R.J. (1999). Biological control of locusts and grasshoppers: the Australian experience.

Proceedings of a Workshop on Locusts and Grasshoppers, Brasilia, April 1997. (in press) Driver, F., Milner, R.J. and Trueman, J.W.H. (1999) A taxonomic revision of Metarhizium based

on sequence analysis of ribosomal DNA. Mycological Research (accepted subject to revision).