Part II.Facilities and techniques for biological detection of CGTPs

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The plant laboratory
The greenhouse
Soil mixes for plant growth
Production and care of indicator plants
Techniques for graft-transmission in citrus

 

The plant laboratory

The greenhouse

A greenhouse, screenhouse or a controlled environment structure is necessary for the production of index plants and for indexing. This structure need not be expensive or elaborate. It should provide lighting, heating and cooling, and be sufficiently well built to prevent insect intrusion. An entrance with two doors and a darkened vestibule between them is desirable as a preventive measure against insect invasion. The exterior can be of glass or plastic. Excel lent plants can be grown in a simply designed and inexpensive wooden structure covered with heavy fibreglass and containing a good system for heating and cooling. Modern structures of aluminium framing are now available.

Three greenhouse layouts are shown in Figure 106. A number of greenhouse structures in use worldwide are shown in Figures 107a to 107j. In areas where hailstorms occur, the use of glass should be avoided but, if used, it should be shielded by wire mesh (Figure 107i). Corrugated fibreglass rather than glass is recommended where hail is a problem, and in many respects is preferable since it may be cheaper, unbreakable and easier to erect and maintain.

The size of the greenhouse will depend upon the amount of indexing and research to be carried out. There should be at least three compartments: a cool room for indexing citrus graft-transmissible pathogens (CGTPs) which are best expressed in plants grown under relatively cool temperatures of 24 or 27C to a maximum not to exceed 30C during the day, and 18-21 C at night; a relatively warm temperature room primarily used for growing plants which can be held at flexible temperatures ranging from 30 to 35C maximum day and 20 to 24C night; and a hot room which may be used for preconditioning budwood prior to thermotherapy, and for indexing of diseases that require hot temperatures for the best symptom expression. Temperatures in this room should be maintained as warm as possible without inducing plant injury or leaf distortion. A recommended maximum day temperature is 32-40C with minimum night temperatures of about 24-27C.

Benches

Benches for holding plant containers can be made of wood, concrete, wire mesh, plastic or any appropriate material (Figures 109a to 109g). A satisfactory bench system in use at the Rubidoux laboratory at the University of California, Riverside, uses 2 x 6-in (5 x 15-cm) Douglas fir boards spaced about 2 cm apart. The wood is painted or sprayed with a 2 percent copper naphthenate solution (Roistacher and Baker.1954), which acts both as a wood preservative and disinfectant (Figure 109h). Wooden benches can be placed on concrete blocks or on a metal frame or other foundation (Figures 107e, 108d, 109a and 109b) at a convenient working height, usually about 80 cm from the ground. An excellent new design in plastic bench tops is shown in Figure 109g. It is a one-piece, semirigid perforated top mounted on a wooden frame.

Flooring

Flooring can be of concrete with provisions for drainage. However, gravel flooring with concrete walkways is highly recommended. Gravel of 1-2 cm should be spread over the ground about 8-10 cm thick (Figures 109a and 109c). This provides good drainage and aids in maintaining sanitation. The greenhouse should be constructed on a well-drained soil base. If this is not possible, supplementary drainage tiling should be provided during construction.

Containers

Plastic containers are recommended for growing citrus plants. A tapered container approximately 15 cm in diameter and 15 cm deep has been found satisfactory over many years of use at Riverside and elsewhere (see Figures 109a, 109b, 109d, 109e, 109g and 109h). Containers measuring 18-20 cm in diameter, but preferably no larger, can also be used. Such a single small lightweight container filled with the proper soil mix will grow three plants to 1 m height readily and without nutritional or other problems (Figure 109a). A relatively large number of these small containers can be placed on each bench but should be adequately spaced to avoid overcrowding (Nauer, Holmes and Boswell,1980).

Plastic containers must be tested for their ability to withstand steam (permitting their reuse) because steaming is the preferred method of sterilization. Clay pots are not recommended as they accumulate salts, are heavier and breakable, and must be soaked and washed after each use, a process requiring hard labour.

Temperature control

Each room must have a recording thermograph (Figure 116). These should be periodically calibrated against two thermometers for accuracy. At the end of each week, when charts are changed, the maximum and minimum temperature readings for each day should be recorded in a special book. This provides a record for research and is also a means of noting changes which may give warning of heating or cooling unit failure or breakdown.

Supplemental lighting

Supplemental lighting used during winter months will enhance symptom expression in the plant laboratory. For example, at Riverside, California, at 34N latitude, the addition of five hours of 4050-foot candles of supplemental light (at the plant level) from October to April induced symptoms of oak-leaf patterns in 207 leaves on plants of four indicator varieties, compared with only 60 leaves on plants grown without lights (Roistacher,1963). In addition to the more than threefold increase in the number of leaves showing symptoms under artificial lights, there was a 32 percent increase in the total number of leaves produced.

Recent studies have shown that growth of certain citrus seedlings could be significantly enhanced during the winter months with light from 25-watt bulbs, which induced the same growth response as light from 100-watt bulbs (placed 1 m above the bench tops). This has resulted in lower electricity bills (Roistacher and Nauer,1985).

Heating

Heating can be provided by gas heaters with fans, by steam heat using radiators, or by steam pipes placed along the sides of the structure. Heat may also be distributed from gas heaters using supplementary fans blowing the heat through perforated plastic tubes (Figure 108c). Most gas heaters are placed inside the structure. However, ethylene released by faulty heaters can be very damaging to plants. Preferably, gas heaters should be placed outside the structure (Figures 108a and 108b), and the warmed air circulated inside the greenhouse by a fan that forces the air through large-diameter perforated plastic tubes, as illustrated in Figures 108c to 108e.

Catalogues of the larger companies manufacturing greenhouse structures and heating and cooling equipment are available in many countries and should be consulted for ideas and costs. The construction of a greenhouse is best carried out through local builders, with design and equipment suggested by those in charge of indexing.

Cooling

There are three methods for cooling a greenhouse: introducing air from the outside when the temperature is cooler than the temperature inside the house; use of evaporative coolers if the relative humidity is low enough to make such cooling effective; and refrigeration. Other innovative methods can be used, such as a double-layered plastic bubble which acts as an insulator and through which cool (or warm) air can be forced between the sandwiched layers, as in Figure 107j. Combinations of these methods may be used for economy and efficiency.

Air cooling. The simplest and most economical means of cooling a greenhouse is by bringing in outside air to replace the warm air within. This is best accomplished by the use of fans (Figure 119) and thermostatic control (Figure 120). When temperatures rise, the thermostat is activated, the fans turn on and cooler air is drawn through the greenhouse. A greenhouse designed to utilize the cooling ability of outside air will save energy, money and wear on cooling equipment. Air brought in from outside must be screened or filtered to keep out insects. A protective screen at the air intake is illustrated in Figure 107d, showing a screened area covered with 32-mesh plastic screen surrounding the water evaporation cells. Figure 121 shows an air-filtering system at Riverside, containing both 32-mesh plastic screen and glass wool filters. This system also has charcoal trays to filter out air pollutants.

The thermostatic controls shown in Figure 120 are designed to control both heating and cooling. As the temperature rises inside the greenhouse, the thermostat will activate the fan (Figure 119) thus bringing outside air into and through the house, forcing out the warm air. When the temperature increases further, the thermostat switches on the pump to send water to the cooling cells to begin evaporation cooling (Figure 124).

One method of using outside air to cool a greenhouse is that of providing vents at the peak of the roof. Vents may be activated mechanically by hand or by a thermostatically-controlled motor. When the vents are opened, they permit the warmed inside air to rise and bring in the cooler outside air through filtered vents at the lower sides of the structure. There are many problems associated with this method of air cooling and, though it is present in many older installations, it cannot be recommended for a plant laboratory greenhouse.

Evaporator coolers. The use of evaporator coolers is recommended for most greenhouses where humidity during the summer months is low. An engineering study can calculate the cooling ability of evaporator coolers where humidity is moderate or high during the warm months. Evaporative coolers may prove uneconomical and unsound if the relative humidity is too high. However, in some areas evaporative coolers can be combined with refrigeration for efficient cooling.

Equipment and methods for cooling by evaporation are shown in Figures 122- 125. Figure 122 shows a standard commercial evaporator cooler available in most countries where humidity is low and where houses and buildings are cooled by this means. Figure 123 shows the inside of this cooler with the panel removed exposing the squirrel-cage fan, water reservoir at the bottom, water pump, water dripping down from two outlets at the top, and the pads made of wood fibre or glass wool housed inside the panel door. Such units have been used at the Riverside laboratory for many years and are very efficient in their cooling ability and in screening out insects. They should be carefully serviced each year by cleaning and painting and by changing the cooling pads. A standby cooler should be available for emergency replacement, as well as spare water pumps, fan belts and drive motor.

A more efficient apparatus for cooling is shown in Figures 124 and 125. Figure 124 shows cooling cells consisting of rectangular units of specially treated cardboard placed together to form a solid block. Water is pumped from a reservoir tank shown in the lower left of Figure 124, to a trough above the cells. The water then drips down by gravity over the cardboard cells. The outside air is forced through the moistened cells by the diminished pressure induced by the fans located at the opposite end of the greenhouse (Figure I19). The operation of the fans and water pump is controlled by a thermostat as shown in Figure 120. Figure 125 illustrates a greenhouse at Lake Alfred, Florida, with this cooling system but without an insect filter screen. The cooling cells occupy the full length of the outside wall of the greenhouse.

Refrigeration. Refrigeration can be used to supplement evaporator coolers where the relative humidity is too high during the warmer months, where extra cooling capacity is needed as a supplement for the plants in a cool-temperature indexing room or for cooling small individual rooms in a design similar to that shown in Figure 106c. Small plastic chambers can be built inside a large glasshouse to give areas of controlled cooling using refrigeration. Figure 126 shows refrigeration units used to cool a grape-indexing building in South Africa, where electrical energy was relatively inexpensive. Refrigeration is recommended in smaller greenhouses for those compartments to be held at cooler temperatures. These units should be designed so that they are easily removable for repair and replacement, and a spare unit should be held in reserve in case of breakdown.

Soil mixes for plant growth

Since most symptoms of graft-transmissible citrus diseases are seen primarily in plants as shown in Tables 1 and 3 in the Introduction, plants should be of the highest quality. Therefore, the soil mixture with its balanced supply of micro- and macronutrients is of prime importance, and much emphasis will be given to the soil mixture and plant growth in this section. The University of California (or UC) system for producing healthy container-grown plants was developed by Baker and co-workers (1957) and published as Manual 23. This system was based on the John Innes system of soil mixes developed in England. The original objective was to provide a rapidly growing nursery industry in California with the means of producing uniform and healthy plants. This was done by developing a soil mixture of readily available ingredients and nutrients, incorporating a rigid sanitary programme at all levels of production, including clean nursery stock, sanitary greenhouse practices and soil disinfection. The system was modified by Nauer, Roistacher and Labanauskas (1967,1968) for growing citrus by the addition of micronutrients to the artificial mixture. Micronutrients were found to be absolutely essential for the successful growth of most citrus cultivars.

Through strict sanitation practices, the UC system provides a means for the total prevention of soil diseases (especially Phytophthora species). It permits fertility control, provides for a renewable, consistent and dependable set of soil ingredients and assists in salinity control. It meets the objective in the production of uniform., healthy, vigorous citrus and herbaceous plants free of deficiency symptoms, and the production of plant growth flushes producing the clear, large young developing leaves necessary for observing many symptoms.

The modified UC system containing micronutrients for growing citrus and other herbaceous plants has been in use at Riverside for over 30 years. By practicing rigid sanitation, as recommended by the system, not a single case of soil-contamination by Phytophthora has ever appeared in that laboratory. Since plants are the "eyes" with which we see most graft transmissible citrus pathogens, a successful indexing programme using plants would be very difficult to maintain if Phytophthora were present. Many of the specific index plants recommended as seedling indicators or as rootstocks under specific indicator scions are very susceptible to Phytophthora. Since citrus plants take six to 12 months to reach buddable size, the destruction of these plants by soil borne diseases cannot be tolerated. Every precaution should be taken to prevent Phytophthora a infection in the plant laboratory. One such precaution is the use of a soluble copper compound in a foam pad, or Bordeaux mixture as a powder, placed at the entrance of the greenhouse.

The UC mix

Ingredients. The basic soil mixture consists of 50 percent Canadian peat moss and 50 percent fine sand, with macro- and micronutrients added to the mix (Figure 110). Canadian peat moss is recommended as the prime ingredient. A trial mixture should first be prepared using equal parts of Canadian peat moss and fine sand. Comparative tests can then be made, substituting other local or more readily available types of peat. Canadian peat has been tested and found to be superior to other peat or sphagnum mosses in nutrient retention and chelating ability. Comparative tests can be made to find a substitute for part of the more specific Canadian peat moss. Ingredients such as redwood shavings (if available), other wood shavings complemented with extra nitrogen, other peat mosses, perlite or vermiculite can be tried. The recommended mixture of 50 percent Canadian peat plus 50 percent fine sand should always be used as the standard for comparison.

It is recommended that a fine sand or silt, with a particle size ranging from 0.05 to 0.5 mm, be used. Beach sand 0.5-1 mm in diameter or clay is not recommended. Fine sands can be found in wind-blown deposits or as the fine silt separated out as waste material from a sand-and-gravel company processing pit. A quick and simple test for determining the presence of clay in a proposed sand source is to shake a sample of the test soil in a jar with water. If the sand settles fairly rapidly and the water remains relatively clear, it is satisfactory. If clay is present, the water will have a muddy appearance and that source should not be used.

The sand should be inert and preferably siliceous. Calcareous or limestone sands should not be used since they may affect the pH. If a good grade of silicate sand is not available, try substituting the sand fraction of the mix with vermiculite and perlite in a proportion of 1/2 peat, 1/4 vermiculiteand 1/4 or 1/3 of each other ingredient. The objective is to obtain an artificial / medium which is consistently reproducible, will absorb and release macro- and micronutrients, and will maintain pH of the drainage water at 5.5 to 6.5.

The ingredients can be mixed together with a shovel on a flat concrete surface. However, a small or medium-sized electric or gasolinepowered concrete mixer is the preferred mixing device. The procedure at Riverside using a medium-sized concrete mixer is as follows:

• Specific numbers of uniform, standard shovel-scoops of soil, peat and redwood shavings (or other substitutes for part of the peat moss) are counted and shovelled into the apron of the concrete mixer.
• In the apron, a weighed quantity of macronutrients, i.e. phosphate, calcium and magnesium as shown in Figure 110, is sprinkled on top of the unmixed ingredients.
• The soil ingredients plus macronutrients are then dumped into the concrete mixer and thoroughly tumbled.
• The micronutrients, weighed and mixed together in a package, are first dissolved in water and then poured into the turning mixer.
• A small quantity of water can be added to the soil while the mixer is turning to bring the soil mixture to a friable, moist level if the soil or peat is too dry.
• After about 20 minutes of tumbling and mixing, the soil is emptied from the mixer into a trailer fitted with steam pipes on the bottom, as shown in Figures 111 a and 111b. The trailer top is covered with a cloth tarpaulin. Containers and flats may be placed on top of the soil in the trailer before covering, or steamed separately (Figure 111b).
•The soil mixture is then steamed. The steaming time will depend on the quantity of steam produced; this will depend on the size and capacity of the boiler. A good criterion is to continue to steam for about 15 minutes after the steam puffs up the covering tarpaulin. Soil thermometers placed in the corners of the trailer will determine the correct period for steaming. One minute at 100C or 10 minutes at 83C is usually sufficient for controlling soilborne pathogens. Any longer is a waste of energy and is unnecessary. Steaming has never been found toxic or harmful to plants grown in a UC mix using a fine siliceous sand, Canadian peat moss, redwood shavings, perlite or vermiculite.

Fertilization. The initial mix contains both macro- and micronutrients added during mixing. The micronutrients are tied up in the peat moss. The peat, which acts as a chelating agent, releases sufficient small amounts of micronutrients to the plant for periods of up to one or two years (Nauer, Roistacher and Labanauskas,1967,1968).

Liquid fertilizer is applied with each watering using a device which injects fertilizer in proportion to the water used. An effective, simple and very inexpensive device is a Venturi-type siphon (Figure 117). Immediately after purchase, the siphon should be calibrated. Many siphons will vary considerably from the advertised ratio of concentrate to water as printed on the instructions. Calibration is done by putting a measured amount of water (500 or 1 000 ml) into a graduated cylinder, then placing the suction end of the siphon into the cylinder and measuring the final amount of water exiting from the hose. Allow water to flow and fill a container until the 500 or 1 000 ml of measured liquid is siphoned up. Then measure the water in the container and convert the results to a ratio.

Another device which injects a given quantity of liquid fertilizer into the water system at a uniform rate in direct proportion to the water flow is the Smith Measuremix proportioner (Figure 1 18). This is a precision instrument and highly reliable. This device has been in use at the Riverside greenhouses for 30 years with a minimum of upkeep or repair problems. The proportioner is set to deliver at a ratio of 1 to 100. However it should be calibrated in the same manner as the Venturi siphon.

A liquid fertilizer mix based on that given by Nauer et al. (1968) is: (dry) parts by weight, 9 parts NH4NO3 (Ammonium nitrate) + 3.75 parts Ca(NO3)2 (Calcium nitrate) + 2.75 parts KNO3 (Potassium nitrate). (If KNO3 is difficult to obtain, substitute KCl.) This fertilizer should be applied at the rate of 67.5 g of mixture to 1001 water (9 oz per 100 gallons). Calculate the amount to use and put the proper amount of fertilizer mix in the concentrate tub, add the correct amount of water and stir well. With the UC system of soil mix, fertilize with every watering. The soil should be fertilized directly after mixing as well as before and immediately after planting since the basic UC soil mix contains no nitrogen or potassium.

As a general practice, potted plants in a UC system need to be watered with enough volume periodically to flush out any accumulated salts and prevent salinity buildup. It is important that the soil not be filled to the top of the container. A space of 2-3 cm should be left between the top of the container and the soil level. This will allow a sufficient volume of water to flush the soil in the container.

Production and care of indicator plants

The indicator plants recommended for indexing are given in Tables 1 and 3 of the Introduction. Seedlings are recommended for indexing for most of the CGTPs. However, in some cases where seedlings are difficult to obtain, or other seedlings are readily available, a clonal propagation can be made with a bud from a selected indicator plant grafted to a vigorously growing rootstock seedling. The rootstock is then inoculated, and the indicator bud forced as a scion. This procedure has been successfully used for detection of a number of pathogens. However, a comparison should be made between the seedling and a clonal propagation of the seedling to be sure that the clonal budline will induce clear positive symptoms equal to those of inoculated seedlings. In some trials, clonal budlines have not performed as well as seedlings, but in other trials they have performed equally well.

Where tristeza is endemic, the use of clonal propagations may be necessary to filter out the tristeza virus in order to detect or see other pathogens. For example, trifoliate or citrange may be used to filter the citrus tristeza virus so that other pathogens are not masked.

For any consistent long-range index programme, a block of seed-source trees containing the desired indicator varieties should be planted as soon as possible to obtain a reliable and consistent source of seed. In the meantime, small or large quantities of seed can be obtained from commercial outlets or small quantities can be requested from other research stations (Table 4).

Seed treatment

All fruit collected for seed extraction should be picked as high on the tree as possible, and picking up fruit from the ground should be avoided because of the danger of Phytophthora infection. After extraction, the seed should be routinely treated against possible contamination from Phytophthora by a hot-water dip for 10 minutes at 52C (125F), followed by a short dip in cool water to return the seed to normal temperatures (Klotz et al.,1960). In addition to the hot-water treatment, the seed should be disinfected with a fungicide to aid in preserving the seed during storage and to prevent albinism when the seedlings emerge. The commercial fungicide Thiram as a 75 percent powder can be used to dust the seed after drying, or the seed can be dipped for three minutes in a 1 percent solution of 8-hydroxy quinoline sulfate, available from most chemical supply houses. The seed is then spread out on paper or on a fine-mesh screen and allowed to air-dry. It should be fumed frequently during drying. Be careful to avoid overdrying. As soon as the last moisture has disappeared and the surface appears dry, the seed should be packaged in small polythene bags. The bags should be dated, labelled and sealed with a rubber band, and then placed in a second small polythene bag with a small piece of slightly moist tissue paper placed between the bags. The second bag is also sealed with a rubber band. Seeds which have been treated, packaged and sealed in this manner and stored at refrigeration temperatures of +5 to +6C have maintained excellent viability for as long as three years (Nauer and Carson,1985).

Seed planting

Seed can be planted in flats or containers of wood or plastic. Redwood, if available, is ideal since it is easy to use, will not decay, lasts a long time and can be steam-sterilized. Other woods can be used but should be dipped or painted with a relatively non-toxic preservative such as copper naphthenate (Roistacher and Baker,1954). Plastic containers with drainage holes are also quite satisfactory. They should be tested to see if they will withstand steam sterilization so that they can be reused. The sizes of the two types of redwood flats used at Riverside for growing seedlings are approximately 40 x 40 cm, and 40 x 20 cm by 14 cm deep (Figures 111b, 112 and 113).

The sterilized soil is placed in the flat and compacted with a flat metal tamper (Figure 112). It is important that the soil be levelled at about 3 cm from the top of the flat to permit uniform distribution of water. If the soil is not level, water will settle at one corner of the flat and the other corner will usually remain dry, possibly resulting in poor seed germination in that corner. A planting board (Figure 113) made of thin Masonite or plastic with 1.5-cm holes drilled 2.5 cm apart is placed on top of the soil and the individual seed placed in each hole. The seed is then lightly pressed into the soil with a dowel (Figure 113). After seeding is complete, the planting board is removed and the seed covered with about 1 cm of soil and tamped lightly. Watering should be done by using a soft-spray sprinkler nozzle on the hose end or a watering can with a perforated sprinkler head until the seedlings emerge.

TABLE 4a. Commercial nurseries that sell citrus seed and virus-free budwood. Catalogues and price lists are available on request

Adams Citrus Nursery Inc.
PO Box 1505
State Road 544 East
Haines City FL 33840
United States of America

AVASA (Agrupacin de Viveristas de Agrios SA)
Reina Doa Germana 6-10-2a
Valencia 46005, Spain

Thermal Plaza Nursery
68035-P Highway 86
Thermal, CA 92274
United States of America

Willets and Newcomb Inc.
PO Box 428
Arvin, CA 93203
United States of America

TABLE 4b. Citrus research stations where small quantities of citrus seed or virus-free budwood may be obtained

Arizona: Arizona Cooperative Citrus Registration-Certification
Program
Department of Plant Pathology
University of Arizona
Tucson, AZ 85721, United States of America

California: Citrus Clonal Protection Program
Department of Botany and Plant Sciences
University of California
Riverside, CA 92521, United States of America

Florida: Florida Citrus Budwood Registration Program
3027 Lake Alfred Road
Winter Haven, FL 33880, United States of America

Texas: Texas A. and 1. University Citrus Center
Weslaco, TX 78596, United States of America

Spain: I.V.I.A.
Apartado Oficial
46071 Moncada, Valencia
Spain

Corsica: Station de Recherches Agronomiques de Corse
San Giuliano
20230 San Nicolao
Haute Corse
France

Seedling trays, as illustrated in Figure 114, are a satisfactory means of growing individual seedlings. Such trays are available through greenhouse and nursery suppliers.

The seeded flats or seedling trays are best kept in the warm growing room but can also be kept in the hot room if rapid forcing is desired. Most seedlings will reach transplantable size in 11-19 weeks depending on the variety (Table 5). Sweet orange seedlings take much longer to grow, averaging 29 weeks. The seedlings must be periodically and critically examined for off-type, gametic or non-nucellar variants; these must be culled and should not be used since they can be poor indicators. By permitting seedlings to reach 8-15 cm of growth rather than transplanting when they are too small, the offtype variants are more readily detected and can be pulled from the flat.

Insect control

If possible, the laboratory should be located away from citrus groves to lower the infestation pressure from insects. Ornamental landscape plants should not be grown too close to the laboratory since they may provide a host for the growth of insects. Personnel should avoid insect infested plants in the field before entering the greenhouse. A balanced and thorough insect control programme is of the utmost importance. Damage to indicator plants by insects or insecticidal spray can make symptom-reading very difficult.

Any insect-control programme that reduces the amount of insecticide spraying is worthwhile. Insecticide application, although necessary, should be limited and carefully controlled. Spray damage to leaves is usually in the form of circular translucent spots and may confuse symptom readings as well as damage young growth. The presence of adequate non-inoculated control plants should verify any damage to leaves done by insects or sprays or other non-viral effects. The insect-control programme used at Riverside is as follows:

• All plants are critically examined at least once each week for any signs of insects. If insects are found, the area surrounding the infestation is treated by spot spraying with an appropriate insecticide at concentrations usually below those recommended by the manufacturer to avoid chemical damage to the tender young emerging leaves.
• At least once each week all plants are sprayed with water using a standard pressure hose nozzle with a fine-spray attachment. The objective of this water spray programme is to control small infestations of mites. A few undetected mites are readily washed off the leaves by this water spray and usually do not return. This is a preventive measure, and it has been found very effective in reducing the number of times the entire greenhouse must be sprayed. The combination of periodic inspection, spot spraying and water spraying have been highly successful in keeping insect infestation under control. The importance of routine periodic preventive inspection for detecting new low-level insect infestations cannot be overemphasized.
• The use of the two-door vestibule entryway for the critical inspections of clothing for the presence of insects is important. Green-coloured clothes should be avoided during the aphid season. Only one door at a time should be opened when bringing in or taking out plants or materials.
• When spraying with an insecticide becomes necessary, select an insecticide and a dilution that will not spot or injure plants. New insecticide sprays should first be tested on a few plants. Dilutions should be carefully calculated and double-checked to be sure they are correct. A number of effective miticides should be kept on hand and their use rotated to prevent build-up of insect resistance.

In general, aphid infestation at the Riverside greenhouse has been rare and never serious.

TABLE 5. The time for growth of seedlings from seed to one metre at the Riverside laboratory

Seedling cultivar No. of seedlings per container No. of weeks to reach 1 m
  Range Average
Citron 1 19-28 25
Eureka lemon 1 22-34 27
Rough lemon 1 23-34 28
Citrus excelsa 3 25-30 29
Sour orange 3 26-38 32
Troyer citrange 1 24-42 33
Mexican lime 3 24-44 35
Dweet tangor 3 29-41 36
Duncan grapefruit 3 28-48 40
Pineapple sweet orange 3 48-56 51

Mites are the major pest problem. Soft brown and other scales, whitefly, mealy bugs or thrips can be serious problems. Again, the importance of periodic inspection and good insect control cannot be overstated in the maintenance of an efficient plant laboratory.

The introduction of plant material from other areas or from the field into the greenhouse should be avoided. If the plants must be brought in, they should be carefully examined for pests, then cut back to a minimum number of leaves, given a preventive insecticidal spray and isolated in a separate location until they are shown to be free of pests.

If spraying with insecticides becomes necessary, it may be wise to consider spraying all rooms or houses simultaneously. If only one room or house is sprayed, reinfestation may readily occur from other areas not sprayed.

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