R-677 English

11/8/02

10:14 AM

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Important

R-677 / R-677F MICROWAVE OVEN WITH TOP AND BOTTOM GRILL OPERATION MANUAL WITH COOKBOOK

800 W (IEC 60705)

ENGLISH
OPERATION MANUAL This operation manual contains important information which you should read carefully before using your microwave oven. IMPORTANT: There may be a serious risk to health if this operation manual is not followed or if the oven is modified so that it operates with the door open.

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CONTENTS

OVEN AND ACCESSORIES.2 CONTROL PANEL.3 IMPORTANT SAFETY INSTRUCTIONS.4-6 INSTALLATION.6 BEFORE OPERATION.7 SETTING THE CLOCK.7 ENERGY SAVE MODE.8 MICROWAVE POWER LEVELS.9 MICROWAVE COOKING.10 GRILL COOKING.11 DUAL COOKING.12 OTHER CONVENIENT FUNCTIONS.13-14 AUTOMATIC OPERATION.15-16 PIZZA CHART.16 AUTO COOK CHART.17 AUTO DEFROST CHART.18 RECIPES FOR AUTOCOOK AC-5.19 CARE AND CLEANING.20 SERVICE CALL CHECK.21 SERVICE ADDRESSES.22-27 SPECIFICATIONS.28

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OVEN AND ACCESSORIES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Grill heating element (top grill) Oven lamp Control panel Door opening button Waveguide cover Oven cavity Turntable motor shaft Grill heating element (bottom grill) Door seals and sealing surfaces Ventilation openings Power cord Outer cabinet
ACCESSORIES: Check to make sure the following accessories are provided: 13. Turntable 14. Rack Place the turntable over the turntable motor shaft on the floor of the cavity. Before first using the turntable and the rack, clean with mild soapy water. Do not cut or scratch the turntable. WARNING: The accessories (e.g. turntable) will become very hot during Grill, Dual and Automatic operation (except Auto Defrost) modes. Always use thick oven gloves when removing the food or turntable from the oven to prevent burns.
NOTE: When you order accessories, please mention the part name and model name to your dealer or SHARP authorised service agent.

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CONTROL PANEL
Digital Display and Indicators
1 LESS/MORE indicator 2 AUTO indicator - will light during automatic operation 3 WEIGHT (KG) indicator 4 MICROWAVE (WATT) indicator 5 CLOCK SET/TIMER indicator 6 COOKING IN PROGRESS indicator
Operating buttons 7 LESS/MORE buttons 8 PIZZA button 9 AUTO DEFROST button 10 COOKING MODE dial Rotate the dial so that indicator points to appropriate symbol: Microwave Microwave and Top Grill
Microwave and Bottom Grill Top Grill Bottom Grill
Top and Bottom Grills 11 WATT button 12 TIME/WEIGHT dial 13 STOP button

(START) +1min button

15 CLOCK button

16 AUTO COOK button

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IMPORTANT SAFETY INSTRUCTIONS
IMPORTANT SAFETY INSTRUCTIONS: READ CAREFULLY AND KEEP FOR FUTURE REFERENCE To avoid the danger of fire To make popcorn, use only special microwave popcorn makers. The microwave oven should not be left Do not store food or any other items inside the oven. unattended during operation. Power Check the settings after you start the oven to ensure levels that are too high or cooking times the oven is operating as desired. that are too long may overheat foods See the corresponding hints in the cookery book resulting in a fire. section. This oven is not designed to fit into a kitchen unit. The electrical outlet must be readily accessible so To avoid the possibility of injury that the unit can be unplugged easily in an WARNING: emergency. Do not operate the oven if it is damaged or The AC power supply must be 230 V, 50 Hz, with malfunctioning. Check the following before use: a minimum 16 A distribution line fuse, or a a)The door; make sure the door closes properly minimum 16 A distribution circuit breaker. and ensure it is not misaligned or warped. It is recommended that a separate circuit serving b)The hinges and safety door latches; check to only this appliance be provided. make sure they are not broken or loose. Do not place the oven in areas where heat is c) The door seals and sealing surfaces; ensure generated. For example, close to a conventional that they have not been damaged. oven. d)Inside the oven cavity or on the door; make Do not install the oven in an area of high humidity sure there are no dents. or where moisture may collect. e)The power supply cord and plug; ensure that Do not store or use the oven outdoors. they are not damaged. If food being heated begins to smoke, Never adjust , repair or modify the oven DO NOT OPEN THE DOOR. Turn off and yourself. It is hazardous for anyone unplug the oven and wait until the food other than a competent person to carry has stopped smoking. Opening the door out any ser vice or repair operation while food is smoking may cause a fire. which involves the removal of a cover Use only microwave-safe containers and which gives protection against exposure utensils. See Page X. to microwave energy. Do not leave the oven unattended when using disposable plastic, paper or other combustible food containers. Clean the waveguide cover, the oven cavity and the turntable after use. After cooking fatty foods without a lid, always clean the cavity and especially the grill heating elements thoroughly. These must be dry and free from grease. Built-up grease may overheat and begin to smoke or catch fire. Do not place flammable materials near the oven or ventilation openings. Do not block the ventilation openings. Remove all metallic seals, wire twists, etc., from food and food packages. Arcing on metallic surfaces may cause a fire. Do not use the microwave oven to heat oil for deep frying. The temperature cannot be controlled and the oil may catch fire. Do not operate the oven with the door open or alter the door safety latches in any way. Do not operate the oven if there is an object between the door seals and sealing surfaces. Do not allow grease or dirt to build up on the door seals and adjacent parts. Follow instructions for Care and Cleaning, Page 20. Individuals with PACEMAKERS should check with their doctor or the manufacturer of the pacemaker for precautions regarding microwave ovens. To avoid the possibility of electric shock Under no circumstances should you remove the outer cabinet. Never spill or insert any objects into the door lock openings or ventilation openings. In the event of a spill, turn off and unplug the oven immediately, and call an authorised SHARP service agent.

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Do not immerse the power supply cord or plug in water or any other liquid. Do not let the power supply cord hang over the edge of a table or work surface. Keep the power supply cord away from heated surfaces, including the rear of the oven. Do not attempt to replace the oven lamp yourself or allow anyone who is not an electrician authorised by SHARP to do so. If the oven lamp fails, please consult your dealer or an authorised SHARP service agent. If the power supply cord of this appliance is damaged, it must be replaced by an authorised SHARP service agent. To avoid the possibility of explosion and sudden boiling: WARNING: Liquids and other foods must not be heated in sealed containers since they are liable to explode. Never use sealed containers. Remove seals and lids before use. Sealed containers can explode due to a build up of pressure even after the oven has been turned off. Take care when microwaving liquids. Use a widemouthed container to allow bubbles to escape. Microwave heating of beverages can result in delayed eruptive boiling, therefore care has to be taken when handling the container. To prevent sudden eruption of boiling liquid and possible scalding: 1. Stir liquid prior to heating/reheating. 2. It is advisable to insert a glass rod or similar utensil into the liquid whilst reheating. 3. Let liquid stand for at least 20 seconds in the oven at the end of cooking time to prevent delayed eruptive boiling. Do not cook eggs in their shells, and whole hard boiled eggs should not be heated in microwave ovens since they may explode even after microwave cooking has ended. To cook or reheat eggs which have not been scrambled or mixed, pierce the yolks and the whites, or the eggs may explode. Shell and slice hard boiled eggs before reheating them in the microwave oven. Pierce the skin of such foods as potatoes, sausages and fruit before cooking, or they may explode. To avoid the possibility of burns Use pot holders or oven gloves when removing food from the oven to prevent burns. Always open containers, popcorn makers, oven cooking bags, etc., away from the face and hands to avoid steam burns. To a v o i d b u r n s , a l w a y s t e s t f o o d temperature and stir before serving and pay special attention to the temperature of food and drink given to babies, children or the elderly. Temperature of the container is not a true indication of the temperature of the food or drink; always check the food temperature. Always stand back from the oven door when opening it, to avoid burns from escaping steam and heat. Slice stuffed baked foods after heating to release steam and avoid burns. Keep children away from the door to prevent them burning themselves. Do not touch the oven door, outer cabinet, rear cabinet, oven cavity, ventilation openings, bottom grill, accessories and dishes during GRILL, DUAL and AUTOMATIC operation (except AUTO DEFROST) as they will become hot. Before cleaning make sure they are not hot. To avoid misuse by children WARNING: Only allow children to use the oven without super vision when adequate instructions have been given so that the child is able to use the oven in a safe way and understands the hazards of improper use. Do not lean or swing on the oven door. Do not play with the oven or use it as a toy. Children should be taught all important safety instructions: use of pot holders, careful removal of food coverings; paying special attention to packaging (e.g. self-heating materials) designed to make food crisp, as they may be extra hot. Other warnings Never modify the oven in any way. Do not move the oven while it is in operation. This oven is for home food preparation only and may only be used for cooking food. It is not suitable for commercial or laboratory use.

In Energy Save Mode, if you do not operate the oven for 2 minutes or more (i.e. closing the door, pressing the STOP key, or at the end of cooking), you will not be able to operate the oven until you open and close the oven door.

ENERGY SAVE MODE

Your oven has two operating modes, Energy Save Mode and Clock Set Mode. The difference between them is that when you are not using the oven, in Energy save Mode nothing will appear on the digital display and in Clock Set Mode the time will be shown. In Energy Save Mode, if you do not operate the oven for 2 minutes or more (i.e. closing the door, pressing the STOP key, or at the end of cooking), you will not be able to operate the oven. To restore power on, open the door. If you set the clock, energy save mode will be cancelled. To start energy save mode manually, follow the instructions below. Example: To start the energy save mode, (the current time is 23:35): 1. Make sure the correct time appears on the display. 2. Press the CLOCK button once. 3. Adjust the display to 0 by rotating the TIME/WEIGHT dial. 4. Press (START) +1min button. The power will be off and the display will show nothing.

Display:

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SETTING THE CLOCK

There are two setting modes: 12 hour clock and 24 hour clock. 1. To set the 12 hour clock, press the CLOCK button once, as shown: Example: To set the 24 hour clock to 23:35: 1. Choose the 24 hour clock by pressing the CLOCK button twice. 2. Set the hours. Rotate the TIME/ WEIGHT dial clockwise until the correct hour is displayed. 3. Change from hours to minutes by pressing the CLOCK button once. 2. To set the 24 hour clock, press the CLOCK button twice, as shown:
x2 4. Set the minutes. 5. Start the clock. Check the display.
x1 NOTES: 1. You can rotate the TIME/WEIGHT dial clockwise or counter clockwise. 2. Press the STOP button if you make a mistake during programming. 3. If the oven is in cooking mode and you wish to know the time of day, touch the CLOCK button. As long as your finger is touching the button, the time of day will be displayed. 4. If the electrical power supply to your microwave oven is interrupted, plug in the oven again, then open and close the door. The display will show:.0.

If this occurs during cooking, the programme will be erased. The time of day will also be erased. 5. When you want to reset the time of day, follow the above example again. 6. If you do not set the clock, press the STOP button once.0 will appear on the display. When the operation of the oven is finished,.0 will reappear on the display instead of the time of day. 7. If you set the clock, energy save mode does not work.

USING THE STOP BUTTON

Use the STOP button to: 1. Erase a mistake during programming. 2. Stop the oven temporarily during cooking. 3. Cancel a programme during cooking, press the STOP button twice.

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MICROWAVE POWER LEVELS
Your oven has 5 power levels. To choose the power level for cooking, follow the advice given in the recipe section. Generally the following recommendations apply: 800 WATT (800 P) 100 % used for fast cooking or reheating e.g. soup, casseroles, canned food, hot beverages, vegetables, fish, etc. 560 WATT (560 P) 70 % used for longer cooking of dense foods such as roast joints, meat loaf and plated meals, also for sensitive dishes such as cheese sauce and sponge cakes. At this reduced setting, the sauce will not boil over and food will cook evenly without over cooking at the sides. P = POWER To select Microwaving, rotate the COOKING MODE dial to the MICROWAVE setting. Select the desired microwave power setting by pressing the WATT button. If the WATT button is pressed once, 800P (100 %) will be displayed. If you miss your desired level, continue pressing the WATT button until you reach the level again. If the level is not selected, the level 800P (100 %) is automatically set. 400 WATT (400 P) 50 % for dense foods which require a long cooking time when cooked conventionally, e.g. beef dishes, it is advisable to use this power setting to ensure the meat will be tender. 240 WATT (240 P) 30 % (Defrost setting) to defrost, select this power setting, to ensure that the dish defrosts evenly. This setting is also ideal for simmering rice, pasta, dumplings and cooking egg custard. 80 WATT (80 P) 10 % For gentle defrosting, eg. cream gateaux or pastry.

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MICROWAVE COOKING
Your oven can be programmed for up to 90 minutes. (90.00). The input unit of cooking (defrosting) time varies from 10 seconds to five minutes. It depends on the total length of the cooking (defrosting) time as shown on the table opposite: Cooking Time: 0-5 minutes 5-10 seconds 10-30 minutes 30-90 minutes Increasing unit: 10 seconds 30 seconds 1 minute 5 minutes
Example: Suppose you want to heat soup for 2 minutes and 30 seconds on 560 W microwave power. 1. Rotate the COOKING MODE dial to the MICROWAVE setting. 2. Press the WATT button twice for 560 W microwave power. 3. Enter desired cooking time by rotating the TIME/WEIGHT dial clockwise. 4. Press the (START) +1min button once to start cooking.

NOTES: 1. When the door is opened during the cooking process, the cooking time on the digital display stops automatically. The cooking time starts to count down again when the door is closed and the (START) +1min button is pressed. 2. If you wish to know the power level during cooking, press the WATT button. As long as your finger is pressing the WATT button, the power level will be displayed. 3. You can rotate the TIME/WEIGHT dial clockwise or counter-clockwise. If you rotate the dial counterclockwise, the cooking time will decrease from 90 minutes by degrees. 4. If you have not used the oven for over 2 minutes, open and shut the door before programming.

GRILL COOKING

Your oven has 2 grill heating elements, and a combination of 3 grill cooking modes. Select the desired grill mode by rotating the COOKING MODE dial to the desired setting. COOKING MODE DIAL GRILL HEATING ELEMENT IN USE Top Grill

Bottom Grill

Top and Bottom Grill together

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Example: Suppose you want to cook cheese on toast for 5 minutes using the top grill only: (Place toast on the rack.) 1. Rotate the COOKING MODE dial to the TOP GRILL setting. 2. Enter the desired cooking time by rotating the TIME/WEIGHT dial clockwise. 3. Press the (START) +1min button to start cooking.

x1 Display:

1. The rack is recommended when grilling. 2. You may detect smoke or a burning smell when using the grill for the first time, this is normal and not a sign that the oven is out of order. (Please see heating without food below.)
3. After cooking using TOP GRILL and BOTTOM GRILL the display may show Hot.
WARNING: The oven cavity, door, outer cabinet, turntable, rack, dishes and especially the bottom grill will become very hot, always use thick oven gloves when removing the food or turntable from the oven to prevent burns.

HEATING WITHOUT FOOD

You may detect smoke or a burning smell when using the grill(s) or dual grill for the first time. This is normal and not a sign that the oven is out of order. To avoid this problem, when first using the oven, operate both top and bottom grills without food for 20 minutes. IMPORTANT: During grill operation, to allow smoke or smells to disperse open a window or switch the kitchen ventilation on. Make sure there is no food in the oven. 2. Enter the required 1. Rotate the COOKING heating time. (20 min.) MODE dial to the TOP & BOTTOM GRILL setting. 3. Start cooking by pressing the (START) +1min button. 4. The oven will count down. When the oven has finished cooking, open the door to cool the oven cavity.
x1 WARNING: The oven door, outer cabinet & oven cavity will become hot. Take care to avoid burns when cooling the oven down after operation.

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DUAL COOKING
Your oven has 2 DUAL cooking modes to combine the Microwave with the Grill(s). To select the DUAL cooking mode, rotate the COOKING MODE dial to the desired setting. Generally, dual cooking time shortens the total cooking time. Setting COOKING MODE dial Initial Microwave power Grill heating element

DUAL W Top grill

DUAL W Bottom grill
* If you want to adjust the microwave power, press the WATT button. You can select up to 800 W power. Example: Suppose you want to cook for 20 minutes on DUAL 1, combining 80 W (10 %) with the TOP GRILL: 1. Rotate the COOKING MODE dial to the DUAL 1 setting, combining microwaving with the TOP GRILL. 2. Press the WATT button twice for 80 W microwave power. 3. Enter the desired cooking time by rotating the TIME/WEIGHT dial clockwise. 4. Press the (START) +1min button to start cooking.
NOTE: After cooking, the display may show Hot. WARNING: The oven cavity, door, outer cabinet, turntable, rack, dishes and especially the bottom grill will become very hot, always use thick oven gloves when removing the food or turntable from the oven to prevent burns.

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OTHER CONVENIENT FUNCTIONS
1. LESS / MORE button. The LESS ( ) and MORE ( ) buttons allow you to easily decrease or increase programmed setting times, (for a less well or more well cooked result), used in automatic operations or cooking time while in operation. a) Changing the pre-programmed time setting. Example: Suppose you want to cook 0.9 kg Grilled Chicken using the AUTO COOK button and MORE ( ) button. 1. Select AUTO COOK (Grill Chicken) by pressing the AUTO COOK button 4 times. The AUTO indicator light will come on. 2. Enter the amount by rotating the TIME/WEIGHT dial clockwise. 3. Choose the desired result (well cooked) by pressing the MORE ( ) button once. 4. Press the (START) +1min button to start cooking.

x4 Display:

NOTE: To cancel LESS or MORE, press the same button again. To change MORE to LESS simply press the LESS ( ) button. To change LESS to MORE simply press the MORE ( ) button.
b) Adjusting the heating time while oven is operating. The cooking time can be decreased or increased in 1 minute steps each time the LESS ( ) and MORE ( ) buttons are pressed. NOTE: You can use this function for manual cooking only.

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2. MINUTE PLUS function. The (START) +1min button allows you to operate the two following functions: a) 1 minute cooking You can cook on your desired cooking mode for 1 minute without entering the cooking time. Example: Suppose you want to cook for 1 minute at 400 W (50 %) microwave power. 1. Rotate the COOKING MODE dial to the MICROWAVE setting. 2. Press the WATT button 3 times for 400 W microwave power. 3. Press the (START) +1min button to start cooking.
NOTE: 1. You can use this function for manual cooking only. 2. When the COOKING MODE dial is on microwave ( ) and you press the (START) +1min button, the microwave power is always 800 W. When the COOKING MODE dial is on dual ( or ) and you press the (START) +1min button, the microwave power is always 240 W. 3. To avoid misuse by children the 1 minute cooking function can be used only within 3 minutes after the preceding operation, ie closing the door or pressing the STOP button. b) Extend the cooking time You can extend the cooking time in multiples of one minute if the button is pressed while the oven is in operation. NOTE: You can use this function for manual cooking only.
3. TO CHECK THE POWER LEVEL To check the microwave power level during cooking press the WATT button. As long as your finger is pressing the WATT button, the power level will be displayed. The oven continues to count down, although the display shows the power level.

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AUTOMATIC OPERATION
These functions automatically work out the correct cooking mode and cooking time to get the best results. You can choose from the 3 PIZZA, 7 AUTO COOK and 2 AUTO DEFROST menus. PIZZA button AUTO COOK button AUTO DEFROST button
WARNING: For all menus except AUTO DEFROST: The oven cavity, door, outer cabinet, turntable, rack, dishes and especially the bottom grill will become very hot. Use thick oven gloves when removing food or the turntable from the oven to prevent burns. What you need to know when using these automatic functions: 1. Menu can be input by pressing the PIZZA, AUTO COOK or AUTO DEFROST button until the desired menu number is displayed. 2. The weight of the food can be input by rotating the TIME/WEIGHT dial until the desired weight is displayed. Enter the weight of the food only. Do not include the weight of the container. For food weighing more or less than weights/ quantities given in the cooking chart, cook using manual operation. 3. The programmed cooking times are average times. If you want to alter cooking times pre-programmed in the automatic operations, use the LESS ( ) or MORE ( ) buttons. For best results follow the cooking chart instructions, pages 16 - 18. 2. 1.

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AUTO COOK CHART

BUTTON

MENU AC-1 Cook French Fried Potatoes (recommended for conventional ovens) e.g Standard and thick type AC-2 Cook Frozen Baguettes e.g. Baguettes with Pizza topping
WEIGHT (Increasing Unit) / UTENSILS 0,2 - 0,3 kg (50 g) (initial temp -18 C) Directly on the turntable
PROCEDURE Remove the deep frozen French fried potatoes from the packaging and place on the turntable. For thick French fried potatoes, use the MORE ( ) button. After cooking, place on a plate for serving. Remove the deep frozen baguettes from the packaging and place on the turntable. After cooking, place on a plate for serving.
0,15 - 0,4 kg (50 g) (initial temp -18 C) Directly on the turntable
0,2 - 0,4 kg (50 g) AC-3 Cook (initial temp -18 C) Fish Fingers, Directly on the turntable Poultry meat pieces (e.g. Chicken Nuggets) AC-4 Cook Grilled Chicken 0,9 - 1,4 kg (100 g) (initial temp 5 C) Flan dish Saucer
Remove the deep frozen fish fingers from the packaging and place on the turntable. After cooking, place on a plate for serving.
Ingredients for 1,0 kg grilled chicken: 1/ tsp Salt and Pepper, tsp sweet paprika, 2 tbsp oil
Mix the ingredients and spread on the chicken. Pierce the skin of the chicken with a fork. Place a saucer upside down in a flan dish and place the chicken on the saucer. When audible signals sound, turn the chicken over. After cooking, leave for approx. 3 minutes in the oven, remove and put on a plate for serving. See recipes for Gratinated Fish Fillet on page 19. * Total weight of all ingredients.
AC-5 Cook Gratinated Fish Fillet
0,6 - 1,2 kg* (100 g) (initial temp 5 C) Gratin dish

AC-6 Cook Roast Pork

Ingredients for 1,0 kg rolled lean pork: 1 garlic clove, crushed, 2 tbsp oil, 1 tsp sweet paprika, a little cumin powder 1 tsp salt
0,6 - 1,5 kg (100 g) (initial temp 5 C) Flan dish
Lean roast pork is recommended. Mix all ingredients and spread on the pork. Place the pork in a flan dish on the turntable. When audible signals sound, turn the food over. After cooking, let the food stand wrapped in aluminium foil for approx. 10 minutes.
AC-7 Cook Grill Skewers See recipes on page XX.
0,2 - 0,6 kg (100 g) (initial temp 5 C) Rack
Prepare the grill skewers. Place on the rack and cook. When the audible signal sounds turn over. After cooking, place on a plate for serving.

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AUTO DEFROST CHART
MENU Ad-1 Defrost Poultry
WEIGHT (Increasing Unit) / UTENSILS 0,9 - 1,5 kg (100 g) (initial temp -18 C) (See note below for utensils)
PROCEDURE Put a plate up side down on the turntable and place the poultry on it. When audible signal sounds, turn over. When audible signal sounds, turn over again. After defrosting, let stand wrapped in aluminium foil for 30-90 minutes, until thoroughly defrosted. Put the chicken legs on a plate. When the audible signal sounds, turn the chicken legs over and re-arrange. When the audible signal sounds, turn the chicken legs over again. After defrosting, let the chicken stand, wrapped in aluminium foil for 10-30 minutes, until it has thoroughly defrosted. Position the food on a plate in a single layer with thinner parts to the centre. If portions are frozen together, try to separate before defrosting. When the audible signal sounds, turn the food over and re-arrange. When the audible signal sounds turn the food over again. After defrosting, let the food stand, wrapped in aluminium foil for 10-30 minutes, until it has thoroughly defrosted. Position a plate upside down on the turntable and place the meat on it. When the audible signal sounds, turn the meat joint over. When the audible signal sounds, turn the meat joint over again. After defrosting, let the food stand, wrapped in aluminium foil for 30-90 minutes, until it has thoroughly defrosted.

Ad-1 Defrost Chicken Legs
0,2 - 0,8 kg (100 g) (initial temp -18 C) (See note below for utensils)
Ad-1 Defrost Steak, Chops and Fish Fillets

Ad-2 Defrost Meat Joint

0,6 - 1,5 kg (100 g) (initial temp -18 C) (See note below for utensils)
NOTES: 1 Steaks, Chops, Fish fillets and Chicken legs should be frozen in one layer. 2 After turning the food over, shield the defrosted portions with small, flat pieces of aluminium foil. 3 The poultry should be cooked immediately after defrosting. 4 Arrange the food in the oven as shown below: Chicken legs, Steaks, Chops and Fish fillets. Food Dish Turntable Poultry and Meat joints.

11/18/02

3:07 PM

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RECIPES FOR AUTO COOK AC-5
GRATINATED FISH FILLET (AC-5) Fish Gratin Italian Style Ingredients: 600 g Rose Fish Fillet 250 g Mozarella. approx 250 g tomatoes 2 tbsp. anchovy butter salt and pepper 1 tbsp chopped basil lemon juice of 1/2 lemon 2 tbsp chopped mixed herbs 75 g grated Gouda (45 % fat) sauce thickening powder Procedure: Wash the Fish and dry. Sprinkle with lemon juice and salt and grease with the anchovy butter. Place in a round gratin dish (25 cm). Sprinkle the gouda over the fish. Wash the tomatoes and remove the stalks. Cut into slices and place on top of the cheese. Season with salt, pepper and the mixed herbs. Drain the mozarella, cut into slices and place on the tomatoes. Spread over the basil. Place the gratin dish on the turntable and cook on AUTO COOK AC-5 Gratinated Fish Fillet (1,2 kg). Hint: After cooking remove the fish from the gratin dish and stir in some sauce thickening powder. Cook again for 1-2 minutes on 800 W power. Procedure: Wash the leeks and divide in 2 parts from top to bottom. Cut into thin strips. Peel the onions and carrots and cut into thin strips. Put the vegetables, butter and spices into a casserole dish and mix well. Cook for 5-6 minutes on 800 W power. Stir once in-between cooking. In the meantime wash the fish fillet, dry and sprinkle with lemon juice and salt. Mix the Crme frache under the vegetables and season again. Put half of the vegetables in a round gratin dish (25 cm). Place the fish on top and cover with the remaining vegetables. Spread over the Gouda and place on the turntable. Cook on AUTO COOK AC-5 Gratinated Fish Fillet (1,2 kg).

GRATINATED FISH FILLET (AC-5) Gratinated Rose Fish Fillets Esterhazy Ingredients: 600 g Rose Fish Fillet 250 g Leeks 50 g onion 100 g carrot 1 tbsp. butter salt, pepper and nutmeg 2 tbsp. lemon juice 125 g crme frache 100 g grated Gouda (45 % fat)
GRATINATED FISH FILLET (AC-5) Gratinated Rose Fish - Broccoli. Ingredients: 500 g Rose Fish Fillet 2 tbsp. lemon juice 1 tbsp. butter 2 tbsp flour 300 ml milk chopped dill, pepper and salt 250 g frozen broccoli 100 g grated Gouda (45 % fat)
Procedure: Wash the fish fillet and dry. Sprinkle with lemon juice and salt. Heat together butter and flour in a casserole dish without cover for 1-11/2 mins on 800 W power. Add the milk and stir well. Cook again without cover for 3-4 minutes on 800 W power. After cooking stir and season with dill, salt and pepper. Defrost the broccoli in a casserole dish for 4-6 mins on 800 W power. Once defrosted, place the broccoli into a round gratin dish (25 cm) and put the fish on top and season. Pour the sauce over and sprinkle over the cheese Place on the turntable and cook on AUTO COOK AC-5 Gratinated Fish Fillet (1,1 kg).

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CARE AND CLEANING
Oven Interior 1. For cleaning, wipe any splatters or spills with a soft damp cloth or sponge after each use while the oven is still warm. For heavier spills, use a mild soap and wipe several times with a damp cloth until all residues have been removed. Do not remove the waveguide cover. 2. Make sure that mild soap or water does not penetrate the small vents in the walls which may cause damage to the oven. 3. Do not use spray type cleaners on the oven interior. Do not use a steam cleaner. 4. Heat up your oven regularly by using both heating elements, refer to Heating without food on page 11. Remaining food or fat splashed on the oven interior can cause smoke or a bad smell. Turntable Remove the turntable. Wash the turntable in mild soapy water. Dry with a soft cloth. The turntable is dishwasher safe. Rack The rack should be washed in a mild washing up liquid solution and dried. The rack is dishwasher safe. Door To remove all trace of dirt, regularly clean both sides of the door, the door seals and adjacent surfaces with a soft, damp cloth.
CAUTION: DO NOT USE COMMERCIAL OVEN CLEANERS, ABRASIVE, HARSH CLEANERS OR SCOURING PADS ON ANY PART OF YOUR MICROWAVE OVEN. WARNING! After GRILL, DUAL and AUTOMATIC operation (except Auto Defrost) modes, the oven cavity, door, oven cabinet and accessories will become very hot. Before cleaning, make sure they have cooled down. Oven exterior The outside of your oven can be cleaned easily with mild soap and water. Make sure the soap is wiped off with a moist cloth, and dry the exterior with a soft towel. Oven controls Open the door before cleaning to de-activate the oven controls. Care should be taken when cleaning the oven control. Using a cloth dampened with water only, gently wipe the panel until it becomes clean. Avoid using excessive amounts of water. Do not use any sort of chemical or abrasive cleaner.

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XXXXXXXXXXXXXXXX SERVICE CALL CHECK
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INVERTEBRATE PATHOLOGY

Journal of Invertebrate Pathology 82 (2003) 7583 www.elsevier.com/locate/yjipa

Journal of

Strain-specic detection of introduced Beauveria bassiana in agricultural elds by use of sequence-characterized amplied region markers
L.A. Castrillo,* J.D. Vandenberg, and S.P. Wraight
US Department of Agriculture, Agricultural Research Service, US Plant, Soil and Nutrition Laboratory, Tower Road, Ithaca, NY 14853, USA Received 7 March 2002; accepted 4 November 2002
Abstract Field studies on the ecacy and persistence of an introduced strain of Beauveria bassiana for insect control require detection assays to dierentiate the non-native strain from indigenous populations. In this study we developed strain-specic molecular markers based on polymerase chain reaction amplication of sequence-characterized amplied regions (SCAR) in combination with dilution plating on semi-selective medium to detect and estimate density of propagules of a commercial strain of B. bassiana (strain GHA) in eld samples. Using random amplied polymorphic DNA (RAPD) analysis, unique fragments that distinguished GHA from other strains of B. bassiana were obtained. Three amplicons, OPA-140:44 , OPA-150:44 , and OPB-90:67 , generated with RAPD primers were cloned and sequenced and used as bases for designing SCAR primers OPA14 F=R445 , OPA15 F=R441 , and OPB9 F=R677 , respectively. All three SCAR primers were highly sensitive, capable of detecting 100 pg B. bassiana GHA genomic DNA, and thus could be used to detect varying levels of the fungus in the eld. 2003 Elsevier Science (USA). All rights reserved.
Keywords: Beauveria bassiana; Entomopathogenic fungus; Microbial control; Molecular markers; Sequence-characterized amplied region markers; Strain detection
1. Introduction Field studies on the ecacy and persistence of an introduced microbial control agent require detection assays to dierentiate the non-native strain from indigenous populations. Such techniques are critical for the entomopathogenic fungus Beauveria bassiana because this fungus has a wide insect host range and is common in nature (McCoy et al., 1988). There are also several B. bassiana-based mycoinsecticides currently registered or under commercial development worldwide for agricultural pests (Hajek et al., 2001). Field releases of these mycoinsecticides add to fungal populations in the eld and may become established over time. The presence of indigenous populations that can produce an outbreak in the target pest population and mask or enhance the effects of an introduced fungal strain makes it necessary
Corresponding author. Fax: 607-255-1132. E-mail address: lac48@cornell.edu (L.A. Castrillo).
for surveys of B. bassiana to be conducted prior to and after eld applications. These surveys identify the etiological agent(s) attacking target insects and verify the impact of the introduced fungal strain on insect pest populations. Molecular markers have been utilized to assess genetic variation among isolates of B. bassiana and other entomopathogenic fungi, thereby providing means to identify strains of interest, determine origin of isolates, or study population structure. One technique that has been used to dierentiate strains of B. bassiana is polymerase chain reaction (PCR)-based random amplied polymorphic DNA (RAPD) (Bidochka et al., 1994; Castrillo et al., 1999; Maurer et al., 1997). This technique utilizes short primers of arbitrary sequence that anneal to multiple target sequences producing diagnostic patterns (Williams et al., 1990). Because RAPD analysis does not require prior knowledge of target site sequence, it can be easily adapted to study various entomogenous fungi, even those with poorly studied genomes.
0022-2011/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0022-2011(02)00190-8
L.A. Castrillo et al. / Journal of Invertebrate Pathology 82 (2003) 7583
RAPD analysis has also been utilized to generate unique PCR products or amplicons in lamentous fungal species or strains of interest to be converted into species- or strain-specic sequence-characterized amplied region (SCAR) markers (Abbasi et al., 1999; Lecomte et al., 2000; Li et al., 1999; Schilling et al., 1996). SCAR markers dier from RAPD markers in that SCAR primers are designed based on known DNA sequences of the organism in study. This allows for the development of sensitive and diagnostic assays to amplify specic fungal DNA in eld samples containing mixed DNA because primers anneal specically to fungal sequences. This is in contrast to RAPD analysis that requires the establishment of single spore isolates for strain identication. We are currently evaluating B. bassiana strain GHA as a microbial control agent against the Colorado potato beetle and other agricultural insect pests. In this study we utilized RAPD-PCR technique to screen for markers that would dierentiate B. bassiana GHA from other strains of the fungus. These RAPD markers were converted to SCAR markers for development of a sensitive diagnostic assay for the selective detection of B. bassiana GHA. Additionally, we combined SCAR assays with dilution plating to estimate density of fungal propagules present in foliage and soil samples from eld plots.

liquid medium following the method of Pfeifer and Khachatourians (1993) with modication: 100 mg Penicillium funiculosum cellulase and 100 mg Trichoderma viridae cellulase were replaced with 100 mg Aspergillus niger cellulase and 50 mg Trichoderma harzianum cellulase (Sigma), respectively. DNA concentration was determined by use of a spectrophotometer (Pharmacia Biotech) or by running aliquots of DNA extracts against a molecular mass ladder (Life Technologies). 2.2. RAPD analysis and PCR conditions A preliminary screening was conducted using nine B. bassiana strains, including strain GHA, against 10-nucleotide random primers obtained from Operon Technologies. A total of 88 RAPD primers were screened, from which four primers producing robust, reproducible, and unique amplicons of less than 1 kb in B. bassiana GHA were selected. The four primers selected were OPA-5 (50 -AGGGGTCTTG), OPA-14 (50 -TCTGTGC TGG), OPA-15 (50 -TTCCGAACCC), and OPB-9 (50 TGGGGGACTC). These primers were screened against 43 strains of B. bassiana and seven strains of B. amorpha, B. brongniartii, and M. anisopliae (Table 1). Four B. bassiana GHA isolates were included in the assays to test the homogeneity of their source and to ensure consistency of amplicons generated. PCR mixtures (25 ll volume) contained 1 PCR buer with 1.5 mM MgCl2 (Qiagen); 200 lM each of dATP, dCTP, dGTP, and dTTP (Qiagen); 0.5 lM RAPD primer; 10 ng fungal DNA; and 2.5 U Taq PCR enzyme (Qiagen). PCR amplication was performed in a PTC-200 thermal cycler (MJ Research) programmed for initial denaturation at 94 C for 4 min; 35 cycles of denaturation at 94 C for 60 s, annealing at 37 C for 60 s, and extension at 72 C for 2 min; and a nal extension step at 72 C for 5 min. Reaction tubes were held at 4 C prior to visualization of PCR products in a 1.0% agarose gel stained with ethidium bromide. Each RAPD assay was done three times to ensure reproducibility. 2.3. Cloning and sequencing of strain-specic RAPD amplicons The selected diagnostic RAPD marker for B. bassiana GHA from each of the four primers was excised from agarose gels and the DNA fragment was recovered using QIAquick gel extraction kit (Qiagen) following the manufacturers protocol. An aliquot of the recovered DNA fragment was reamplied using the corresponding primer to verify that only a single band was excised. The RAPD markers were cloned using a Perfectly Blunt Cloning kit with pSTBlue-1 vector (Novagen). Then, NovaBlue competent cells (Novagen) were transformed with ligated DNA following the manufacturers instructions. Plasmid DNA from recombinants was

2. Materials and methods 2.1. Fungal isolates and DNA extraction Strains of B. bassiana, Beauveria amorpha, Beauveria brongniartii, and Metarhizium anisopliae were obtained from the USDA-ARS Entomopathogenic Fungi (ARSEF) Culture Collection in Ithaca, NY, USA. B. bassiana strain GHA, the active ingredient in commercial products registered for control of several agricultural insect pests, was isolated from a technical product (Lot No. 980528) provided by Mycotech (now Emerald BioAgriculture, Lansing, MI). Strains of B. bassiana isolated from infected Colorado potato beetles collected in July 1997 and in July 2000 from non-GHA-treated potato elds in Freeville, NY, were included in the study to represent naturally occurring strains at the test site. Information on insect host, collection site and year of isolation for each fungal strain are listed in Table 1. Single spore isolates were established for each strain and were cultured on Sabouraud dextrose agar supplemented with 0.1% yeast extract (SDAY) for 10 days at room temperature ($24 C). Cultures were maintained on SDAY plates stored at 4 C or agar blocks (0:cm2 ) cut from growing cultures were stored in cryogenic vials with 10% glycerol at )80 C (Humber, 1997). Fungal genomic DNA for screening RAPD and SCAR markers was isolated from blastospores grown in
L.A. Castrillo et al. / Journal of Invertebrate Pathology 82 (2003) 7583 Table 1 Strains of B. bassiana and other entomopathogenic fungi investigated in this study Species and strains Beauveria sp. ARSEF 32 B. bassiana GHA ARSEF 1 ARSEF 201 ARSEF 252 ARSEF 292 ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF ARSEF NY 1 NY 2 NY 4 NY 5 NY 9 NY 11 NY Host origin (Orthoptera: Acrididae) From technical product (Mycotech Industries) Malacosoma americanum (Lepidoptera: Lasiocampidae) Diabrotica undecimpunctata (Coleoptera: Chrysomelidae) Leptinotarsa decemlineta (Coleoptera: Chrysomelidae) Reisolate of Boverin L. decemlineata (Coleoptera: Chrysomelidae) (Coleoptera: Cerambycidae) Not specied L. decemlineata (Coleoptera: Chrysomelidae) (Orthoptera: Acrididae) (Orthoptera: Acrididae) L. decemlineata (Coleoptera: Chrysomelidae) L. decemlineata (Coleoptera: Chrysomelidae) Blissus leucopterus (Hemiptera: Lygaeidae) Strinia nubilalis (Lepidoptera: Pyralidae) L. decemlineata (Coleoptera: Chrysomelidae) (Orthoptera: Acrididae) L. decemlineata (Coleoptera: Chrysomelidae) (Orthoptera: Acrididae) L. decemlineata (Coleoptera: Chrysomelidae) From formulated product (Abbott Laboratories) L. decemlineata (Coleoptera: Chrysomelidae) (Orthoptera: Acrididae) Lymantria dispar (Lepidoptera: Lymantriidae) Blissus leucopterus (Hemiptera: Lygaeidae) Coccinella septempunctata (Coleoptera: Coccinelidae) Schizaphis graminum (Homoptera: Aphididae) Diuraphis noxia (Homoptera: Aphididae) O. nubilalis (Lepidoptera: Pyralidae) Anthonomus musculus (Coleoptera: Curculionidae) Diabrotica virgifera (Coleoptera: Chrysomelidae) (Homoptera: Aphididae) Plutella xyllostella (Lepidoptera: Plutellidae) L. decemlineata (Coleoptera: Chrysomelidae) L. decemlineata (Coleoptera: Chrysomelidae) GHA reisolated from Locusta migratoria (Orthoptera: Acrididae) L. decemlineata (Coleoptera: Chrysomelidae) L. decemlineata (Coleoptera: Chrysomelidae) L. decemlineata (Coleoptera: Chrysomelidae) L. decemlineata (Coleoptera: Chrysomelidae) L. decemlineata (Coleoptera: Chrysomelidae) L. decemlineata (Coleoptera: Chrysomelidae) L. decemlineata (Coleoptera: Chrysomelidae) Coleoptera Solenopsis sp. (Hymenoptera: Formicidae) Melolontha melolontha (Coleoptera: Scarabaeidae) M. melolontha (Coleoptera: Scarabaeidae) Popillia japonica (Coleoptera: Scarabaeidae) Rhizotrogus majalis (Coleoptera: Scarabeaidae) P. japonica (Coleoptera: Scarabaeidae) Collection site Montana Year 1987 1987

Dennis, Massachusetts Corvalis, Oregon Orono, Maine Commonwealth of Independent States Rhode Island Rhode Island Orono, Maine New South Wales, Australia New South Wales, Australia Ithaca, New York Ithaca, New York Riley, Kansas Tully, New York Tully, New York Parana, Brazil Washington Pyrenees-Orientales, France Tully, New York Tully, New York Bahia, Brazil Dexter, Oregon Wakeeld, Kansas Niles, Michigan Parma, Idaho Parma, Idaho Centre, Pennsylvania Carver, Massachussetts Mead, Nebraska Ontario, New York Ontario, New York Freeville, New York Freeville, New York Freeville, Freeville, Freeville, Freeville, Freeville, Freeville, Freeville, New New New New New New New York York York York York York York
B. amorpha ARSEF 2251 ARSEF 2641 B. brongniartii ARSEF 617 ARSEF 1068 Metarhizium anisopliae ARSEF 1280 ARSEF 2547 ARSEF 2548
Para, Brazil Sao Paulo, Brazil France Thurgau, Switzerland Clinton Corners, NewYork Syracuse, New York Sleepy Hollow, New York
puried using Qiagen anion-exchange resins, followed by restriction digest with EcoR1 to recover the insert DNA. The cloned fragments were sequenced by the BioResource Center at Cornell University, Ithaca, NY using BIG Dye Terminator chemistry and AmpliTaq-FS DNA polymerase (Applied Biosystems). The nucleotide sequence of the cloned RAPD fragments from OPA-5, OPA-14, OPA-15, and OPB-9 have been deposited in the GenBank database under Accession Nos. BH190223, BH190224, BH190225, and BH190226, respectively. 2.4. SCAR primers and PCR conditions The nucleotide sequence of each of the cloned RAPD fragment was used to design pairs of SCAR primers (Table 2). These primers were synthesized by the BioResource Center at Cornell University, Ithaca, NY. PCR mixtures (50 ll volume) for SCAR assays consisted of 1 Expand High Fidelity buer (10 mM TrisHCl, 50 mM KCl at pH 8.3) with 1.5 mM MgCl2 (Roche); 200 lM each of dATP, dCTP, dGTP, and dTTP (Roche); 0.5 lM of each primer (forward and reverse primers); 20 ng fungal DNA; and 2.6 U of Expand High Fidelity PCR System enzyme mix (Taq and Pwo DNA polymerases) (Roche). The amplication prole was 2 min initial denaturation at 94 C; 10 cycles of denaturation at 94 C for 15 s, annealing at 63 C for 30 s, and elongation at 72 C for 45 s; followed by 15 cycles of denaturation at 94 C for 15 s, annealing at 63 C for 30 sec, and elongation at 72 C for 45 s, with an additional 5 s for each successive cycle; and a nal elongation at 72 C for 7 min. PCR products were visualized in 1% agarose gels stained with ethidium bromide.

Table 2 Sequence of SCAR primers derived from RAPD markers diagnostic for B. bassiana strain GHAa SCAR markersb SCA14445 SCA15441 SCB9677 Primersc OPA14 F445 OPA14 R445 OPA15 F441 OPA15 R441 OPB9 F677 OPB9 R677 Sequence ()d TCT GTG CTG GCC CTT ATC G TCT GTG CTG GGT ACT GAC GTG TTC CGA ACC CGG TTA AGA GAC TTC CGA ACC CAT CAT CCT GC TGG GGG ACT CGC AAA CAG TGG GGG ACT CAC TCC ACG
Specicity of each SCAR primer pair was tested by PCR assays against fungal strains used in the RAPD screening. Isolates of B. bassiana collected from mycosed Colorado potato beetles from GHA-treated plots in September 2000 in Freeville, NY, were also included in SCAR assays to identify the B. bassiana strain(s) infecting these beetles. Using a sterile loop, conidia from infected beetles were collected, suspended in 600 ll sterile distilled water containing 0.2% Tween 80, and 200-ll aliquots were plated on three oatmeal dodine agar (ODA) plates for isolation of B. bassiana colony forming units (CFU) (Chase et al., 1986; Costa and Gaugler, 1989). ODA plates were incubated as described above. Six B. bassiana-like CFUs on ODA plates were cultured for DNA extraction and assayed with SCAR primers to detect presence of B. bassiana GHA. All SCAR assays were replicated thrice. SCAR primers pairs derived from OPA-5 amplicon were found to be highly polymorphic, and, consequently, were eliminated for further screening. PCR products generated by SCAR primers derived from OPA-14, OPA-15, and OPB-9 amplicons were recovered from agarose gels using QIAquick gel extraction kit (Qiagen) and were digested with restriction enzymes HaeII or HindIII, BsrF1, and BsrF1 or DraI, respectively, to verify whether DNA fragments obtained from other strains of B. bassiana or from isolates from infected beetles in GHA-treated plots were identical to the B. bassiana GHA amplicons. The sensitivity of devised SCAR assays was tested by setting up PCR with variable quantities (1 pg to 100 ng) of B. bassiana GHA genomic DNA. A possible inhibitory eect on PCR amplication by contaminant DNA was tested by spiking a separate set of these PCR with 50 ng B. bassiana NY1, a strain found to be distinct from GHA in preliminary assays. Sensitivity assays were replicated at least twice. 2.5. Detection of B. bassiana strain GHA in eld sample: plating method Soil and potato foliage samples from experimental plots in Freeville, NY, were collected in late June 2001, prior to and immediately after spraying of GHA-based Mycotrol ES (Emerald BioAgriculture) in the summer of 2001. Samples were collected prior to spraying to establish background levels of indigenous B. bassiana strains present, as well as the strain GHA since these plots were used for experiments on eld ecacy of GHA formulated products during previous years (Wraight and Ramos, 2002). Using a soil corer (8 cm diameter), three soil samples (approximately 5 cm in depth) were collected along a transect of each of three plots. From each sample, 25 g soil (loam, pH 5.45.8) was mixed with 25 ml sterile distilled water with 0.2% Tween 80 for 5 min using a vortexer. Serial dilutions () of the soil slurry were prepared, and a 200-ll aliquot was

a SCAR primer pairs derived from RAPD amplicon OPA-50:49 were highly polymorphic and were not included for further analysis. b Subscript refers to the size of the amplied product from B. bassiana strain GHA genomic DNA. c Primer nomenclature is according to Paran and Michelmore (1993). Primer name refers to the Operon Technologies primer kit and number, followed by the letters F and R to indicate forward and reverse primer, respectively, and a subscript denoting size of amplication product. Each primer set is based on the sequence of both ends of the cloned RAPD fragment. d Underlined nucleotides indicate sequences of the corresponding RAPD primers.
plated on each of three ODA plates. Fungi from potato foliage were sampled by immersing three leaf disc samples (2 cm diameter) from individual leaves of a given potato plant in 20 ml sterile distilled water containing 0.2% Tween 80 followed by vortexing for 5 min to dislodge conidia. Three leaves were sampled per plant, with three plants sampled from each of three plots. Dilutions were prepared and aliquots plated as described above. Another extraction method was adopted for isolating genomic DNA for the detection of B. bassiana GHA from eld samples. A loopful of conidia from individual B. bassiana-like CFUs on ODA plates was cultured in 2 ml PD broth (10 g bacto peptone and 20 g dextrose/L) in a 15-ml round-bottomed polypropylene tube with shaking (150 rpm) at room temperature ($24 C). After 3 days of growth, 2 ml YPD broth (PD broth plus 2 g yeast extract/L) were added, and the culture was incubated for another 24 h. A 1.5-ml aliquot of fungal culture was transferred into a 2-ml microcentrifuge tube for DNA extraction. A small-scale extraction protocol using Wizard Genomic kit (Promega) was adopted following the manufacturers yeast protocol with the following modication: 150 lg lyticase was replaced with 10 mg each of A. niger cellulase and T. harzianum cellulase. 2.6. Detection of B. bassiana strain GHA in eld sample: direct culture method An alternative procedure to the plating method for processing environmental samples was tested. Undiluted soil wash was directly inoculated into the liquid medium to detect any B. bassiana propagule present. Four milliliters of PD broth supplemented with dodine (Cyprex, 65% AI, 100 mg/L) and antibiotics (70 mg penicillin and 100 mg streptomycin sulfate/L) was inoculated with one milliliter of soil washing from the same soil suspension sampled for CFUs on ODA plates and incubated at room temperature ($24 C) with shaking (150 rpm). After 7 days, 5 ml YPD broth was added and the fungal culture was incubated for another 24 h. A 1.5-ml aliquot was collected for DNA extraction following the modied Wizard yeast protocol (Promega). In cultures with sparse fungal growth, an additional 1.5-ml aliquot was collected. Five microliters of DNA solution (from total volume of 20 ll) extracted from liquid cultures of either CFUs or soil washings was used in SCAR assays as described in this paper.

strains used for initial screening. Among the 55 primers, four were selected for generating well-resolved and reproducible bands that were less than 1 kb and thus easily amenable to cloning and sequencing. The selected RAPD primers OPA-5, OPA-14, OPA-15, and OPB-9 produced unique amplicons of 494 (OPA-50:49 ), 445 (OPA-140:44 ), 441 (OPA-150:44 ), and 677 (OPB-90:67 ) bp in B. bassiana GHA (Fig. 1). Further screening against other strains of B. bassiana and other entomopathogenic fungi revealed that RAPD primer OPA-14 generated a DNA fragment in ARSEF 350 of similar size to the GHA amplicon (Fig. 1B). With primers OPA-5, OPA15, or OPB-9 PCR products of similar size to the GHA amplicon were generated by two or more B. bassiana strains: for OPA-5 or OPB-9, ARSEF strains 32 and 792 (data not shown); and for OPA-15, ARSEF strains 318, 2430, 2860, and 2861 (Fig. 1C). There was, however, no B. bassiana strain among the 43 tested in this study that showed all four diagnostic RAPD markers similar to those in strain GHA other than ARSEF 6444, a reisolate of B. bassiana GHA from Locusta migratoria. ARSEF 6444 also had a RAPD prole identical to GHA for all the RAPD primers tested (data not shown). All four single spore isolates of GHA from the technical product had identical RAPD proles (Fig. 1). Although dierentiation of GHA from 41 other strains of B. bassiana was readily accomplished using the RAPD markers, this technique required the establishment of single spore isolates, making detection in eld samples very laborious and time consuming. Conversion of RAPD amplicons that distinguished GHA into molecular probes specic for this strain would facilitate detection in mixed eld samples whether from mycosed insects or environmental samples such as soil and foliage. 3.2. Development of SCAR markers To develop a diagnostic assay for B. bassiana GHA the four selected RAPD markers were converted to SCAR markers. A pair of SCAR primer, forward and reverse primers, was designed based on the sequence of both ends of each of the four cloned RAPD markers. Primer pair sequences, except for those derived from OPA-50:49 amplicon, which were found highly polymorphic, are listed in Table 2. Primer sequence consisted of the 10-nucleotide sequence of the corresponding RAPD primer, followed by a variable number of bases of the amplied sequence. Primer length was determined by compatibility of melting temperatures of the forward and reverse primers. PCR assays of B. bassiana GHA with SCAR primers resulted in DNA fragments of the same size as the cloned RAPD amplicons as expected based on primer design. PCR assays of 42 other strains of B. bassiana, two strains of B. amorpha, two strains of B. brongniartii,

3. Results and discussion 3.1. RAPD analyses of B. bassiana GHA and other entomopathogenic fungi Of the 88 RAPD primers screened, 55 generated PCR products unique to GHA among the nine B. bassiana
Fig. 1. PCR amplication of strains of B. bassiana, B. amorpha (Ba), B. brongniartii (Bbr), and Metarhizium anisopliae (Ma) with RAPD primers (A) OPA-5, (B) OPA-14, (C) OPA-15, and (D) OPB-9. Arrows indicate diagnostic amplicons of 495, 445, 441, and 677 bp for strain GHA with primers OPA-5, OPA-14, OPA-15, and OPB-9, respectively. Fungal strains other than GHA are listed by their ARSEF culture collection number. Lane M, molecular weight markers (EcoRI and HindIII digested bacteria phage Lambda DNA; Life Technologies).
three strains of M. anisopliae, and six B. bassiana isolates from infected Colorado potato beetle cadavers showed that SCAR primer OPA14 F/R445 was specic to B. bassiana GHA. A 445-bp DNA fragment was generated only in GHA, in ARSEF 6444, and in isolates collected from eld samples from GHA-treated plots (Fig. 2A). Identity of DNA fragments was veried by use of restriction enzymes HaeII or HindIII (data not shown). The restriction enzymes HaeII and HindIII each recognize one site in the 445 bp DNA sequence, sites 60 and 136 bp, respectively, generating two fragments upon cleavage. All GHA-based isolates were observed to generate these DNA fragments (data not shown). SCAR primers OPA15 F/R441 and OPB9 F/R677 , however, were polymorphic, generating bands of similar size to the GHA amplicons in other B. bassiana strains (Figs. 2B and C) even after several attempts to optimize PCR conditions and increase specicity. Bands generated in other B. bassiana strains were veried to be non-
specic by use of restriction enzyme BsrF1 and BsrF1, or DraI for the 441 and the 677 bp amplicons generated by OPA15 F/R441 and OPB9 F/R677 , respectively. DNA fragments in other B. bassiana strains used in this study showed dierent restriction patterns compared to strain GHA (data not shown). It is noteworthy that two of six B. bassiana isolates, A1-a and A1-b, recovered from mycosed beetles from experimental plots sprayed with GHA in Freeville, NY, in September 2000, and included in SCAR assays, were not GHA (Fig. 2A). RAPD analysis of single spore isolates from A1-a and A1-b revealed that they were identical to the most common indigenous strain in Freeville, NY, represented by NY1 (L. Castrillo, unpublished data). In a separate RAPD analysis of isolates collected in July 1997 and in July 2000, data revealed the presence of this common genotype among strains associated with Colorado potato beetles in Freeville, NY (L. Castrillo, unpublished data). The presence of indigenous

Fig. 2. PCR assays of B. bassiana strain GHA (G) and other strains of B. bassiana (Bb), B. amorpha (Ba), and B. brongniartii (Bbr) with SCAR primers (A) OPA14 F/R445 , (B) OPA15 F/R441 , and (C) OPB9 F/R677. Primer OPA14 F/R445 was specic to B. bassiana GHA, generating the 445 bp amplicon only in GHA, ARSEF 6444 (a reisolate of GHA), and isolates from mycosed Colorado potato beetle (CPB) cadavers collected from GHAtreated plots (isolates A3-c, E3-c, L4-c, and L4-g). No PCR product was generated in any of the non-GHA based isolates by primer OPA14 F/R445. Primers OPA15 F/R441 and OPB9 F/R677 were polymorphic, generating the 441 and the 677 bp amplicon, respectively, in GHA and GHA-based isolates as well as in a few other strains of B. bassiana. Lane M, molecular weight markers (1 kb Plus DNA ladder; Life Technologies).
strains of B. bassiana among those collected from treated plots underscores the importance of identifying B. bassiana isolates collected after eld application to corroborate ecacy studies of an introduced strain. The presence of indigenous strains also raised the question of whether the limit of detection of B. bassiana GHA propagules in mixed eld samples by use of devised SCAR assays will be aected by non-target or contaminant DNA. Non-target DNA may cause reduced sensitivity of detection resulting in false-negative reactions (Wilson, 1997). Sensitivity assays with variable quantities of GHA genomic DNA showed that as little as 100 pg GHA genomic DNA was sucient to generate easily visible bands with each of the three SCAR primers whether GHA genomic DNA was pure or contaminated with strain NY1 (data not shown). The sensitivity of devised SCAR assays for B. bassiana strain GHA even in the presence of contaminant DNA indicated that these assays could be used to detect GHA in mixed cultures from environmental samples. However, the possibility of non-specic bands generated by SCAR primers OPA15 F/R441 and OPB9 F/R677 limits their use to cases where it has been shown that these primers do not anneal to any of the indigenous B. bassiana strains. With primer OPA14 F/R445 , the specicity and sensitivity of devised SCAR assay permit diagnostic detection of variable levels of GHA propagules in mixed eld samples. The exact limit of detection of propagules in terms on conidia per gram of soil, however, will be dicult to estimate because eld samples
obtained for SCAR detection need to be cultured on selective medium prior to obtaining fungal DNA for analysis. 3.3. Detection of B. bassiana GHA in eld samples Beauveria bassiana propagules were recovered from soil and foliage samples collected from potato plots after application of a GHA-based mycoinsecticide, but not from samples collected prior to spraying in the summer of 2001. The absence of indigenous strains of B. bassiana may be attributed to the limited number of samples collected and the very low number of Colorado potato beetles observed during this sampling period. Previous samples of indigenous strains of B. bassiana were collected from mycosed beetles. The absence of GHA from these plots, with a history of GHA application, may be due to factors relating to eld persistence of GHA and also to the limited number of samples collected. The number of B. bassiana CFUs observed on ODA plates ranged from 5:to 2:CFUs/g soil sample and 7:to 2:CFUs/cm2 leaf sample. All representative CFUs assayed with SCAR marker OPA14 F/R445 revealed the presence of B. bassiana GHA. The use of SCAR markers facilitated the detection of GHA in CFUs from environmental samples without the need for establishment of single spore isolates. SCAR assays of CFUs also provided additional information on eld collected isolates compared to dilution plating alone, which provided propagule

Fig. 3. PCR amplication with SCAR primers OPA14 F/R445 , OPA15 F/R441 , and OPB9 F/R677 of genomic DNA extracted from fungal cultures from soil suspensions inoculated into a semi-selective liquid medium for B. bassiana. Shown are PCR products from liquid cultures from four soil samples with variable amounts of B. bassiana GHA propagules (lanes 13 with 103 CFUs/g soil; lane 4 with 102 CFUs/g soil) and from a liquid culture inoculated with Mycotrol ES containing $106 conidia of B. bassiana GHA (lane 5). Lane M, molecular weight markers (1 kb Plus DNA ladder; Life Technologies).
estimates only. Thus, a combination of SCAR assays and dilution plating can be used for studies verifying identity of the etiological agent and studies tracking the level of inoculum persisting in the environment after application. Although CFUs cannot be directly equated with number of spores present because a given CFU can be based on more than a single conidium or mycelial fragments, the number of CFUs sampled over time provide an estimate of and show trends in fungal population numbers. Direct culture of soil washings in liquid medium provided an alternative method of detecting the presence of GHA in soil samples. SCAR assays of genomic DNA from fungal cultures from soil washings all showed DNA fragments corresponding to the specic product for each SCAR primer (Fig. 3). Intensity of bands varied and correlated with the results of dilution plating method. Soil inoculum that revealed 103 CFUs/g soil generated more intense bands than soil samples with 102 CFUs. Direct correlation of band intensity with counts of CFUs cannot be used, however, because SCAR assays utilize a small sample of the genomic extract and inherent variability in the sequence of steps entailed to obtain these samples can confound sampling errors. Thus, quantication of a PCR product cannot be directly equated with the number of fungal propagules present in the soil sample (Wilson, 1997). Nonetheless, SCAR assays of cultures from soil washings allow for simple testing of environmental samples for absence or presence of GHA and can be used for studies involving large number of samples. Both methods described for detection of GHA in eld samples still required culture in selective media prior to extraction of genomic DNA. The development of DNA extraction methods for fungi in soil samples (Kuske et al., 1998; Volossiouk et al., 1995) would enhance and
hasten detection by obviating the need for culture in articial media. However, these methods do not ensure viability of detected propagules, making these methods of little value in persistence studies because the presence of a propagule cannot be assumed to count as a source of inoculum for infection. In summary, the conversion of RAPD markers that dierentiated B. bassiana GHA from other strains of the fungus into GHA-specic SCAR markers facilitated the detection of GHA propagules from environmental samples. SCAR assays obviated the need for establishment of single spore isolates and the combination of SCAR assays with dilution plating method allowed for the detection and estimation of GHA propagules in eld samples for ecacy and persistence studies.

Acknowledgments We thank Mycotech (now Emerald BioAgriculture, Lansing, MI) for providing B. bassiana GHA-based technical and formulated products, Richard Humber and Karen Hansen (USDA-ARS) for providing fungal isolates from the ARSEF collection, and the following USDA-ARS personnel for technical assistance: Michael Griggs, Mark Ramos, Jennifer McManus, and Carolyn Lipke. This paper reports the results of research only. Mention of a proprietary product does not constitute a recommendation or endorsement by the US Department of Agriculture for its use.

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