Control of Plant-Parasitic Nematodes (1968)

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CHAPTER 8 Prevention of Spread Plant-pathogenic nematodes move only relatively short distances under their own power; therefore, the most usual means of nematode spread is the trans- portation of infested soil and infected vegetative plant parts by man. Nema- todes may also be carried by wind, water, and domestic or wild animals and birds. Active stages of most nematodes are susceptible to desiccation; thus, the resistant or resting stages are the ones most important in long-distance spread. Establishment in a new area occurs only when sufficient numbers of viable nematodes are transported to a location where susceptible hosts are subsequently planted and where the environment is suitable for reproduction of the nematode species. MEANS OF DISSEMINATION SOIL AND PLANT TISSUE Soil and vegetative plant parts, because they often protect nematodes from desiccation and are frequently transported by man, are important carriers of nematodes over both short and long distances. Soil is also important because most plant-pathogenic nematodes spend at least part of their lives in soil, and soil thus infested is commonly transported along with plant materials. Nematodes are frequently present on the surface of true seed and in asso- ciated debris or soil. For example, cysts of the sugar-beet nematode (Heter- odera schachtii) have been found on sugar-beet seeds. Only a few nematode species, such as seedgall nematodes (Anguina spp.) and the stem nematode 85

86 BASIC PRINCIPLES OF CONTROL (Ditylenchus dipsaci), infect true seed, but many kinds of plant-pathogenic nematodes infect vegetative plant materials used for propagation, such as transplants, ornamentals, nursery stock, bulbs, and corms. Since these high- acre-value plants are cultivated on the best soil available, one crop is grown frequently or continuously on the same land, often resulting in damaging nematode populations in plant materials and in the soil. Infected propagation materials are particularly important in nematode spread, because materials from a relatively small area are used to plant much larger and often widely separated areas. A pathogenic nematode introduced into a field in this manner is likely to spread throughout the field (Figure 39), to adjacent fields on the same farm, and to adjacent farms. Although experimental data indicate that plant-parasitic nematodes passing through the digestive tracts of animals are killed, infected plant parts and soil associated with manure are important in spreading nematodes. MACHINERY, REUSABLE CONTAINERS, AND FERTILIZER Nematode-infested soil and infected plant materials may be transported along with machinery and reusable containers, such as burlap bags and crates. Nema- todes moved by these means are often deposited in soil in which susceptible crops are grown later. Nematodes may also be carried from infested to clean FIGURE 39 Areas of poor growth shown in aerial view of sugar- beet field indicate pattern of spread of sugar-beet nematode (Heter- odera schachtii).

PREVENTION OF SPREAD 87 fields on automobiles, especially in soil clinging to tires and fenders. The pea- nut lesion nematode (Pratylenchus brachyurus) is spread by the use of in- fected peanut hulls as a conditioner in fertilizers. The golden nematode (Heterodera rostochiensis), an introduced pest, was found in more than 30 fields of one grower on Long Island, New York. These fields were on various farms located among uninfested fields. It was con- cluded that nematode spread was associated with the movement of machinery and used burlap bags. ANIMALS Undoubtedly, nematodes are disseminated in mud or plant debris clinging to birds and other animals, but there is little published information on the extent of this means of spread. Potentially, stages of plant-pathogenic nematodes resistant to drying could be carried for long distances by migrating birds. Some animals, such as rodents and insects, live in the soil and thus are often con- taminated with infested soil. Horses and other animals used to pull farm im- plements may transport infested soil within and between fields. Many kinds of nematodes may be spread on clothing, shoes, hand tools, or hands. Rhadi- naphelenchus cocophilus, the causal agent of red ring of coconut, is dissemi- nated by the palm weevil and other insects. WATER Although nematodes may be carried for relatively long distances in irrigation water, only local spread generally occurs in surface water. Nematodes may be transported short distances by spattering raindrops. Living nematodes may be carried by the movement of underground water, but dead or inactive nema- todes are not carried through soil, even by water at high flow rates. In coarse-textured soils of Florida citrus orchards, spread of the burrowing nematodes (Radopholus similis) downhill in surface-drainage water was eight times the rate of uphill movement. Also, in Florida citrus orchards this nema- tode moved under a hard-surface road in underground water. In an experiment in which a column of coarse-textured Florida soil 42 inches high and 3 inches in diameter was watered intermittently from the top, the burrowing nematode was moved from top to bottom in 30 hours. Nematode spread by water, particularly long-distance spread, depends on resistance of the nematode to submersion in water, and resistance varies among species and among stages of a species.

88 BASIC PRINCIPLES OF CONTROL Irrigation, which is used more in crop production every year, effectively transports nematodes. More information of the importance of this means of spread is urgently needed. WIND Although wind is often mentioned as an important factor in nematode dis- semination, there is little evidence of long-distance spread by wind or of ex- tensive local spread in the direction of prevailing winds. Large quantities of soil and small pieces of plant debris are blown from field to field, farm to farm, and for even greater distances, but only nematodes resistant to drying survive such dissemination, and most stages of the majority of nematodes are killed easily by desiccation. Large numbers of cysts of the golden nematode, which are highly resistant to drying, were found in snow along roads adjacent to Long Island, New York, potato fields infested with this nematode. It ap- pears doubtful that these relatively heavy cysts would be carried by wind for long distances. NATURAL BARRIERS Natural barriers such as mountains and oceans reduce nematode spread by water, wind, animals, and by soil adhering to farm machinery. However, the use of refrigerated ships and trucks and of airplanes makes possible the trans- portation of infected plant materials over or around practically all natural barriers. Infested soil may be carried along with both host and nonhost plants. If the climate is unsuitable, an introduced nematode species either will not survive or its population increase will be so slow that it will not become an economically important pest. Climatic factors not only influence nematode survival directly but also influence it indirectly by their effect on kinds of crop plants that can be grown. Although a nematode is transported to a new area with favorable environ- ment, it will not become established unless one or more cultivated or wild hosts is present. If a cultivated host is grown infrequently in a crop rotation and wild hosts are absent, the introduced nematode may die from starvation. However, any species of plant-parasitic nematode should be considered a potential threat to agriculture in areas free of that species, and all means should be taken to prevent its entry.

PREVENTION OF SPREAD 89 PRACTICES TO RESTRICT SPREAD SANITATION It is particularly important to control nematodes in nurseries, because plant materials from them are widely distributed. All soils in nurseries should be treated with steam or nematocides. Floors, benches, containers, tools, and storage areas of buildings in which plant materials are handled or stored should be thoroughly cleaned or fumigated. Splashing of water should be avoided, and hose nozzles should be kept off the floor. Clothing and shoes worn by workers in nematode-infested areas should not be worn in nematode- free areas. The identity of new plant material should be maintained and the material isolated until it is known to be free of plant-parasitic nematodes. Nematode- contaminated plant material should be isolated and either discarded or treated, if satisfactory treatments are available. NEMATOCIDAL TREATMENTS The control of plant-pathogenic nematodes in nurseries by the use of heat and nematocidal chemicals results in the production of high-quality nursery stock and reduction of nematode spread. Moist heat, when properly used, will eliminate nematodes from soil, but it is expensive and thus practical only for relatively small areas or quantities of soil. Nematocidal chemicals, when used commercially as preplan! treatments, will not eradicate nematodes from soil but will kill a high percentage of them. Because of application problems and phytotoxicity of most nematocidal chemicals, the treatment of soil around living plants is generally less effective than preplant treatments. Control, which approaches eradication of nematodes inside roots of some plant species, has been achieved by dipping bare roots in aqueous solutions of nematocidal chemicals. Although extensive plant damage often results, some hot-water treatments kill nematodes in plant tissues. For example, hot water is used to treat garlic bulbs infected with stem nematodes, grape rootstocks infected with root-knot and lesion nematodes, and citrus rootstocks infected with burrowing nematode and citrus nematode (Tylenchulus semipenetrans) (Figure 40). To increase the effectiveness of nematocidal treatments, research should be directed toward developing inexpensive, effective, nonphytotoxic chemical-dip treatments for bare-rooted stock; safe and effective drenches or fumigants for established plants and container-grown or balled stock; and effective systemic nematocides.

90 BASIC PRINCIPLES OF CONTROL FIGURE 40 Hot-water treatment of grapevine rootings for eradica- tion of root-knot (Meloidogyne spp.) and lesion (Pratylenchus spp.) nematodes: right tank for presoaking; center tank for treatment, 51.5°C for 5 minutes; left tank for cooling. (Courtesy of the Depart- ment of Nematology, University of California.) CERTIFIED PLANT MATERIALS The production of vegetative plant-propagation material that is certified to be nematode free or to have a specified level of infection is accomplished by growing clean plants in clean soil or other media. Strict sanitation must be practiced, and both the material and the rooting medium must be periodically checked for the presence of pathogenic nematodes. Vegetative seed of banana free of the burrowing nematode, potato seed pieces free of the golden nema- tode, garlic cloves free of the stem nematode, and strawberry plants free of root-knot and lesion nematodes are produced commercially. The use of resistant or immune plant varieties also reduces nematode spread. Of course, resistant varieties, which are symptomless hosts of a patho- genic nematode, reduce chances of nematode detection and thus increase spread. QUARANTINES AND REGULATIONS Practically all countries and subdivisions of countries have some type of plant disease and pest act under which exclusionary measures are promulgated.

PREVENTION OF SPREAD 91 Quarantine measures are fundamentally regulatory and prohibitory. They prohibit the introduction into a specified area of a particular plant or possible carrier of a pest, whether or not it is known to be carrying the pest, but nearly every quarantine includes provisions that allow for the introduction of plants or possible carriers that in some manner have been protected against or freed from contamination by the pest against which the quarantine is estab- lished. These exceptions to total exclusion regulate rather than prohibit the movement of the pest and of plant parts with which it is associated. For example, the United States federal soybean cyst-nematode (Heterodera glycines) quarantine prohibits the movement of root crops from the regulated areas where they are grown, but it also provides that root crops (except sugar beets) may be exempted "if cleaned free of soil." The prohibition of move- ment is a quarantine measure, while the provision for cleaning the root crops is a regulation allowing free movement of plant materials freed of the pest. The New York State golden-nematode quarantine prohibits the movement of plants of tomato or eggplant grown on infested or dangerously exposed fields. Quarantine acts may be either local or general. A local or district quaran- tine, of which the soybean cyst-nematode quarantine is an example, prohibits introduction of plants or other potential carriers from specified districts in which the nematode pest is known to occur. A general quarantine forbids the importation of plants or carriers from any area, regardless of whether or not the pest is known to occur there. The California quarantine against the burrowing nematode is not a general quarantine, as it prohibits introduction of soil and rooted plants from Florida, Hawaii, and Puerto Rico, with various specified exceptions and regulatory provisions. However, administrative instructions added as an appendix to the quarantine require that host plants in nine specified genera must be intercepted and inspected for burrowing nematodes by laboratory methods, regardless of their origin. These latter provisions, in effect, extend the quarantine into a general one in regard to certain plants. Provisions included in quarantines, and other regulatory measures, may in- volve a wide variety of measures aimed at giving assurance that the pest is not being introduced with the exempted plants or articles. Certification of origin is one of the most common provisions. The California burrowing-nematode quarantine provides, for example, that plants may be exempted if they bear an official certificate stating that they were grown where surveys failed to detect the pest. The same quarantine includes another type of provision that deals with the conditions under which the plants were produced, viz., "above ground in sterilized soil or other suitable material prepared or treated to assure freedom from burrowing nematode." Another common regulatory measure requires that plants or carriers be subjected to a specified treatment for destruction of the pest. Several counties

92 BASIC PRINCIPLES OF CONTROL in California's central valley restrict the entrance of sugar-beet harvesting machinery unless it has been thoroughly steam-cleaned, and similar require- ments are in effect in soybean cyst-nematode and golden-nematode quaran- tines. United States federal plant-quarantine officials at ports of entry require methyl bromide fumigation of used bags that may be contaminated with golden-nematode cysts. Regulations may place stringent controls over every aspect of the growing of crops to prevent the possibility of spread of nematode pests. The New York State Golden Nematode Act of 1947 placed the planting, growing, and harvest- ing of potatoes under the direction of the project administrator and included regulations relating to crop rotation, topsoil movement, and soil treatment. In addition to compulsory regulations issued in direct connection with quarantines, other types of regulatory measures may be used on a voluntary basis to exclude nematode pests or restrict their spread. These include various programs mentioned earlier for certification, registration, or inspection of nursery stock, bulb crops, seed potatoes, seed garlic, or other types of agri- cultural seed. While these programs may contribute to the purposes of quar- antine and are often enforced by quarantine officials, their principal object is improvement of quality of the planting stock, and they are usually instigated by the growers themselves. Accurate knowledge of the distribution of a nematode is essential before the promulgation of a quarantine or regulation involving that nematode. Be- fore the distribution can be determined, the nematode species must be cor- rectly identified by a taxonomic specialist. In general, a nematode is quarantined or regulated only when it is of known economic importance in one area and unknown, or occurs as a localized or incipient infestation, in the area to be protected. Occasionally, as a safeguard, a quarantine or regulation is established against a nematode believed to be new to an area and suspected of being economically important. When the barley root-knot nematode (Meloidogyne naasi) was identified in a small area in the Tulelake Basin of northern California, regulations on the movement of root crops and machinery were established. The economic im- portance of the nematode was not well known, but it was the only known infestation of this nematode in North America, and safeguards against spread appeared to be warranted. Inspections for the presence of nematode pests, for enforcement of quaran- tines and regulations, may be made at either the point of origin or destination. Inspection at origin offers several advantages over destination inspection: nematodes are more readily detected in fresh soil or plant samples collected from the growing crop; infested areas may be effectively delimited; duplica- tion of sampling of material shipped to many destinations is avoided; and shipping costs of contaminated materials are avoided. Bulb crops and other

PREVENTION OF SPREAD 93 ornamentals grown in the Netherlands are inspected annually for presence of the golden nematode before they are shipped to the United States. In the enforcement of the U.S. Plant Quarantine Act and subsequent regu- lations and quarantines, federal port and border inspections annually intercept numerous important plant-pathogenic nematodes. During the fiscal year 1964- 1965, the golden nematode was intercepted 101 times; the oat cyst nematode (Heterodera avenae), 42 times; and root-knot nematodes, 66 times. State regulatory agencies also make quarantine or regulatory inspections of plant materials in interstate shipments. In 1965, the nematology laboratory of the California Department of Agriculture examined about 1,250 soil and plant samples taken from shipments originating outside the state: 31 of the samples contained nematodes not known to be established in California, and nematode pests of significant economic importance to agriculture were found in 275 of the samples. Although careful inspections and surveys are of great value, all nematodes are not detected by present methods. Thus, populations below the detectable level must be considered in an over-all control program. The detection of nematodes by quarantine and regulatory workers is diffi- cult, because plant symptoms are not diagnostic for most of the important root-feeding nematodes. Although visual inspection for nematodes is widely used, it is recognized that usually only heavy infections of root-knot and lesion nematodes can be detected in this manner. Despite the limitations of quarantines and regulatory measures and the difficulties in measuring their effectiveness, they have played an important role in limiting the spread of plant-pathogenic nematodes. Furthermore, they encourage growers to produce clean plant materials and discourage the ship- ment of nematode-infected materials. Basic information from future research in such areas as nematode taxon- omy, host-range studies, pathogenicity, and soil- and plant-sampling is urgently needed for improving methods used to prevent nematode spread. BIBLIOGRAPHY McCubbin, W. A. 1950. Plant pathology in relation to federal domestic plant quaran- tines. Plant Dis. Rep. SuppL 191:67-91. McCubbin, W. A. 1954. The plant quarantine problem. Chronica Botanica Co., Waltham, Mass. 255 pp.

CHAPTER 9 Reduction of Nematode Populations through Land-Management and Cultural Practices Most plant-parasitic nematodes can be controlled to varying degrees by land- management and cultural practices. These include fallow, the practice of keep- ing the land free of all plant growth; flooding; growing cover crops; crop rota- tion; time of planting; organic manuring; removal or destruction of infected plants; trap and antagonistic crops; nutrition and general care of host; and sanitation and the use of nematode-free planting stock. The specific principles involved in control of nematodes by land- management and cultural practices differ; however, all are based on the inability of nematodes to survive, multiply, and cause disease under the con- ditions imposed on them by the use of these practices. Most of these practices reduce nematoui, populations gradually over a period of weeks, months, or even years, as opposed to rapid kill such as that obtained with heat or toxic chemicals. Furthermore, as control is relative, satisfactory economic control may not be achieved by any single practice but by a combination of several practices. The fact that a practice that reduces the nematode population con- siderably may not be economically effective at present does not preclude the possibility that it may be economically feasible in the future. With the advance of knowledge of the effect of specific practices on nematode populations and with more efficient implementation of practices, for example, through im- proved machinery, a high degree of control may be possible. Because of this possibility, those practices that are known to reduce nematode populations to a measurable extent are discussed even though they may not be economically feasible or widely used at this time. 94

LAND-MANAGEMENT AND CULTURAL PRACTICES 95 FALLOW Fallow is the practice of keeping land free of all vegetation for varying periods by frequent tilling of the soil by disking, plowing, harrowing, or by applying herbicides to prevent plant growth. At least two principles of nematode con- trol are represented by this practice. The first principle, and perhaps the most important, is starvation of the nematode. Plant-parasitic nematodes are obligate parasites, depending on living hosts for the food necessary to develop to maturity and to reproduce. There- fore, in the absence of a host plant, the nematode will die after the stored food in the body has been depleted. Some of the cyst nematodes (Heterodera spp.) can survive as unhatched eggs or dormant larvae in cysts in the soil in the absence of a host for at least 14 years, but these are exceptions. In upper soil layers, most plant-parasitic nematodes probably do not survive for more than 12 to 18 months, and many do not survive the first 6 months. Compared with upper soil layers, soil at lower depths is more constantly cool and moist, increasing the length of nematode survival. Survival is also influenced by the amount of infected root debris remaining in the soil from the previous crop. The second principle involved in fallow is death through desiccation and heat. With some exceptions, nematodes of most species, depending on stage of development, will die if exposed to the drying action of the sun and wind. When fallow land is tilled frequently to destroy vegetation, the surface strata of soil are exposed to the drying and heating effects of wind and sun. Fallow is especially effective in areas of low rainfall and high soil temperatures or in areas where rainfall is seasonal, thus resulting in long periods, perhaps 6 months or more, of dry conditions. There are several objections to the practice of fallow: the operations nec- essary to maintain lands completely free of vegetation are difficult and ex- pensive; fallow in areas of high rainfall is a poor soil conservation practice and is likely to impair the physical structure of the soil; and fallow land does not contribute to farm income. FLOODING Flooding of fields to control nematodes is not widely accepted. Results of early investigations indicated that flooding for 12 to 22 months is required to rid soil of root-knot nematodes (Meloidogyne spp.). Where water is plentiful and level land can be taken out of production for long periods, flooding may be a useful control practice. Certain crops, such as rice, can grow under flooded conditions. Experiments showed that rice seeded in water and kept flooded for 4 to 6 weeks had only a trace of the white tip disease caused by

96 BASIC PRINCIPLES OF CONTROL the white-tip nematode (Aphelenchoides oryzae), whereas rice drilled and flooded after the seedlings were 3 to 4 inches tall was 60 percent diseased. In this case, the nematodes were seed-borne rather than soilborne, but this is a special situation, since flooded fields are not generally planted. The principles of control involved in flooding are not completely under- stood. Presumably, flooding eliminates all host plants, and the nematodes die from starvation. In addition, flooding decreases the oxygen content of the soil and may kill nematodes by asphyxiation. It has been shown, however, that stored foods of some nematodes are not used as rapidly under conditions of low oxygen, and this may actually extend the length of survival. Chemicals lethal to nematodes, such as butyric and propionic acids, hydrogen sulfide, and perhaps others, often develop in flooded soils of low pH containing large amounts of rapidly decomposing organic matter. Flooding of rice fields in Louisiana gives good control of certain nematodes that are parasitic on rice. It should be remembered, however, that nematodes are essentially aquatic, and some species may persist but will not reproduce in saturated soils. Some dis- advantages of flooding include the possibility of introducing new pests, as well as changes in structure, fertility, and pH of the soil. COVER CROPS Cover crops are grown in the winter as a soil-conservation measure and to pro- vide forage for livestock or in the summer between rows of widely-spaced crops such as fruit trees. Populations of some nematode species may decline on cover crops, but others undoubtedly increase. A reduction in population is probably caused by resistance of the cover crop to the particular nematode, and, conversely, any increase is due to susceptibility of the crop to the species that increased. Low winter temperatures also may limit populations of para- sitic species so that they do not increase substantially even though the cover crop is susceptible. With some sedentary endoparasites, the "trap-crop princi- ple" may be operative. In trap-cropping, the larvae enter the roots, and many develop to an immobile stage but fail to develop into adults; thus, they are trapped within the root tissues. The addition of organic matter resulting from the plowing under of green-manure crops increases the population and pre- dacious activity of nematode-trapping fungi, predacious nematodes, and of the internal parasites of nematodes. Also, nematocidal substances, such as butyric acid, form during the decomposition of cover crops such as rye and timothy. The importance of these substances in biological control of plant- parasitic nematodes, however, is little understood. Although some investiga- tors are optimistic about the potential of biological control, no practical con- trol of nematodes by predacious fungi, toxic substances resulting from decomposition of crop residues, or other biological agents is known. This subject is discussed more fully in Chapter 10.

LAND-MANAGEMENT AND CULTURAL PRACTICES 97 CROP ROTATION The use of crop rotation to reduce nematode populations is without question the most effective and most widely used land-management practice. This practice was used by growers long before its significance as a means of nema- tode control was recognized. To be an effective control practice, crops that are unfavorable hosts for the nematode must be included in the rotation se- quence. Some of the more serious nematode pathogens, such as the golden nematode of potatoes (Heterodera rostochiensis), the soybean cyst nematode (H. glycines), the stem nematode of alfalfa (Ditylenchus dipsaci), and some species of the root-knot nematode, are comparatively host specific, which makes selection of unfavorable hosts relatively easy. Furthermore, in areas where one of these nematodes occurs, it is usually the predominant species, and growth of a resistant crop for two to four years will greatly decrease the population of that species by starvation. Growth of a resistant crop for one year is generally inadequate. Two resistant crops between susceptible crops may give fair control, but three or four years and, with some nematodes, seven or eight years are necessary for effective control (Figures 41-44). Although crop rotation is widely used and is effective in nematode control, it has important limitations. First, the degree of control is based on the level of resistance of the rotation crops and on the number of years between sus- ceptible crops. Also, populations of other species of nematodes may occur on the alternate crop. Furthermore, the nonhosts or resistant crops grown in the rotation may be of low acre value and consequently contribute little to the farm income. TIME OF PLANTING Certain pathogenic nematodes are inactive during the winter months because low temperatures inhibit their activities. For example, California sugar-beet yields in fields infested with the sugar-beet nematode {Heterodera schachtii) are much higher if the beets are planted in January or February than if planted in March or April. Similarly, the root-knot nematode seldom damages the spring potato crop in North Carolina, since the crop grows at temperatures lower than those at which the nematodes are very active. Potatoes planted in the late spring, however, grow most during the hot summer months and are harvested in the fall. Under these conditions, root knot can be a serious prob- lem unless otherwise controlled. In Britain, the golden nematode disease of potatoes may be controlled effectively by early planting of early potato varieties; potato roots develop and grow at a lower temperature than is favorable for hatching, movement, and development of the nematodes.

98 BASIC PRINCIPLES OF CONTROL FIGURE 41 The effect of crop rotation on the control of a root- knot nematode (Meloidogyne incognita) on tobacco. Above, 3 years of tobacco; below, tobacco after 2 years of fescue. (Courtesy of C. J. Nusbaum.)

LAND-MANAGEMENT AND CULTURAL PRACTICES 99 FIGURE 42 The effect of crop rotation on the control of a root- knot nematode (Meloidogyne hapla) on peanut. Above, 3 years of peanuts; below, peanuts after corn and tobacco. (Courtesy of C. J. Nusbaum.)

100 BASIC PRINCIPLES OF CONTROL • I FIGURE 43 The effect of crop rotation on the control of the soybean cyst nematode (Heterodera glycines) on soybeans. Above, 3 years of soybeans; below, soybeans after 2 years of corn.

LAND-MANAGEMENT AND CULTURAL PRACTICES 101 I FIGURE 44 The effect of crop rotation on the control of the sugar- beet nematode (Heterodera schachtii) on sugar beets. Left, sugar beets following sugar beets; right, sugar beets following 1 year of grain. ORGANIC MANURING Several investigators have found a reduction in the population levels of plant- pathogenic nematodes following the addition of organic manures to soil. In most cases, increased activity of microorganisms in the soil followed these treatments, and reductions in nematode populations were assumed to be caused by the buildup of nematode-destroying organisms in the soil. However, in few cases were the actual factors responsible for killing the nematodes determined. REMOVAL OR DESTRUCTION OF INFECTED PLANTS Because of sucker growth, some annual crops, such as tobacco, continue to live for several weeks after harvest is completed. This sucker growth is suffi- cient to keep the root systems alive, and, consequently, parasitic nematodes present in the roots continue to reproduce. One or two additional generations may develop between the end of harvest and the time the plant is killed by frost. Experiments show, however, that if the stalks are cut soon after harvest and the root system of the plant is then turned out and exposed (Figure 45), the population of nematodes, especially of the root-knot nematode, is reduced

102 BASIC PRINCIPLES OF CONTROL FIGURE 45 Field showing tobacco roots exposed to the drying effects of the sun and wind. (Courtesy of F. A. Todd.) through the drying action of the sun and wind. Two control principles are utilized in this practice: the destruction of the host plant by cutting the stalk and uprooting the plant, thus preventing further reproduction of the nema- tode; and the killing by desiccation of large numbers of nematodes concen- trated in the soil around the root system and inside the roots. TRAP AND ANTAGONISTIC CROPS Early investigators, employing the trap-crop principle in attempts to control certain species of the cyst and root-knot nematodes, planted highly suscep-

LAND-MANAGEMENT AND CULTURAL PRACTICES 103 tible crops in infested fields and allowed the crop to grow only long enough for the second-stage larvae to enter the roots and begin their development into adults. Since only the second-stage larvae of nematode species in both these genera are infective, any development beyond the second stage renders the nematode immobile, and death occurs if the crop is destroyed prior to ma- turity of the nematodes. This practice is undoubtedly effective in reducing the populations of certain species, but it can result in even higher populations if destruction of the crop is not accomplished before nematodes complete their development and reproduce. In fact, the population can increase several- fold above the original infestation if reproduction occurs. In addition, the grower has the expense of planting and destroying a crop that brings in no revenue. The trap-crop method of nematode control, while theoretically feasible, is rarely used commercially. A more effective approach to the use of trap crops is to plant crops that are highly susceptible to invasion by the nematode but are resistant to the de- velopment of larvae into adults. In this case, the crop does not have to be destroyed but can be harvested or used as a cover crop and turned under as green manure. Crotalaria has been successfully used to reduce populations of certain species of root-knot nematodes. Roots of certain plant species have recently been found to exude toxic chemicals, thus reducing the soil-population levels of some nematode species. Marigolds and asparagus are examples of such plants. While the use of antago- nistic crops reduces populations of certain nematode species under some conditions, little is understood concerning principles involved, and such practices are not yet developed to the point of practical control of nematodes. NUTRITION AND GENERAL CARE OF HOST The deleterious effects of nematode damage to certain crops can be offset to some degree by proper nutrition, moisture, and protection from adverse con- ditions, such as cold, which place the plant under stress. For this reason, greenhouse plants can usually tolerate much higher populations of nematodes than can field-grown plants. Practices tending to offset the damage caused by nematode attack are irrigation, conservation of moisture by mulching, fertilization, protection of plants on cold nights, and control of root and foliar diseases caused by other pathogens. It should be pointed out, however, that these are only delaying tactics, and, if susceptible crops are grown con- tinuously, the nematode population will reach proportions that will cause serious damage. The rapidity of disease development and the magnitude of the damage will depend on the host and nematode species involved, the re-

104 BASIC PRINCIPLES OF CONTROL sistance or tolerance of the host, and on various factors of the environment that favor or deter development of the disease. Results of some recent investigations showed that soil-population levels of several nematode species may be differentially altered by host nutrition and, similarly, that disease development and severity are more pronounced in in- fected plants that are deficient in one or more essential nutrients. Also, nema- tode infection caused an increase or decrease in concentration of one or more minerals in leaf or root tissue. The interactions among host, parasite, and nu- trition are complex, and the application of such information to fertilization programs designed to minimize damage caused by nematodes to crop plants is just beginning. SANITATION AND USE OF NEMATODE-FREE PLANTING STOCK The land-management and cultural practices discussed above reduce nema- tode populations in fields to varying degrees. Most of these measures have limitations: the degree of control is erratic, and sometimes those factors actually responsible for the reduction in nematode populations are not fully understood. However, sanitation and the use of nematode-free planting stock are sure and effective means of nematode control. Cost of these practices is small, yet many growers continue to use nematode-infected transplants or seed pieces of crops such as tomato, pepper, strawberry, peach, sweet potato, tobacco, and potato, as well as infected bulbs, corms, rhizomes, and tubers of many other plants. Examples of nematode-infected seeds and plants are alfalfa seed infected with stem nematode, wheat seed with wheat nematode (Anguina tritici), and rice seed and strawberry and chrysanthemum plants with species of bud and leaf nematodes (Aphelenchoides spp.). Nursery planting stock harbor- ing nematode parasites is shipped all over the world. Although pathogenic nematodes are already widespread, indiscriminate use of nematode-infected plants and plant parts introduces new species into many fields and conse- quently complicates control measures. Furthermore, nematodes introduced in this manner are in a favorable position for survival, since they are already in or close to host-plant tissues. The greatest yield loss, however, is probably not in the plants on which the nematodes were introduced but in plants grown subsequently in the newly infested field. FUTURE RESEARCH NEEDS Reducing nematode populations through land-management and cultural practices is dependent on two approaches: the prevention or depression of

LAND-MANAGEMENT AND CULTURAL PRACTICES 105 reproduction of the nematode on host crops and the acceleration of popula- tion decline of resting stages of nematodes through practices tending to shorten the survival of forms that persist for several years in the absence of a host. The first of these objectives can be accomplished by fallow, which removes any source of food for the nematode, and by planting resistant host plants which may range from completely resistant (immune) to slightly resistant. Since living host plants are necessary for nematode development and repro- duction, elimination of a suitable food source will effectively stop population increase. The net result is a lower nematode population than would occur through the use of susceptible plants, the degree of reduction being dependent on the level of resistance of the crop planted. Therefore, tests of relative re- sistance or susceptibility of as many crops as possible to the pathogenic species of nematodes in a given area are urgently needed. Without this, selec- tion of crops to use in a rotation sequence designed to reduce the nematode population will mostly be guesswork. The second objective, the acceleration of the death rate in the soil of nematodes neither feeding nor reproducing, but merely persisting, will depend on knowledge of the effect on nematode survival of soil environment or cultural practices applied to the soil. Therefore, studies should be conducted to determine the influence of soil environment and cultural practices on the survival rate of nematodes. For example, factors responsible for nematode mortality when fields are flooded, or when the over-all microflora is changed due to one or more of the above practices, should be determined. Such in- vestigations are necessary before practical application of these practices can be used to accelerate the death of nematodes in soil.

CHAPTER 10 Biological Control of Nematodes USE OF BIOTIC AGENTS TO CONTROL NEMATODES There is abundant empirical evidence that plant-parasitic nematodes are at- tacked by numerous and varied soil organisms, but the activities of such or- ganisms and their effects on nematode populations in agricultural or non- agricultural soils are little understood. Most soils are inhabited by some microorganisms that are parasites and predators on many different kinds of soil nematodes, including plant-parasitic species. In addition, there are soil microfauna that may be predacious on nematodes. The sedentary plant- parasitic nematodes protruding from roots may be especially vulnerable to all these parasites and predators. FUNGI Over 50 species of fungi capture and consume nematodes. Of these fungi, only those included in several genera of the Hyphomycetes and the Zoopa- gales have received attention as biological control agents. All the known fungal parasites of plant-parasitic nematodes also attack other widely diversi- fied types of soil nematodes. Spores of some parasitic fungi are ingested by the nematode, while other fungi trap nematodes by various devices, such as a sticky material adhering to fungal mycelium. The trapping of nematodes is apparently only through chance contact with the fungal traps. Large, robust nematodes may escape, but most soil nematodes are small enough to be held fast by trapping fungi (Figure 46). Large quantities of fungal mycelium and a 106

BIOLOGICAL CONTROL OF NEMATODES 107 FIGURE 46 Citrus nematode larvae (Tylenchulus semipenetrans) trapped and penetrated by Arthrobotrys dactyloides. This predacious fungus occurs commonly in the rhizosphere of nematode-infested citrus plants. high specificity between host and parasite, or predator, are necessary if fungal traps are to be important in controlling nematodes. The physiology of predacious fungi, especially those factors inducing trap formation, has been studied by mycologists and microbiologists. A morpho- genic substance believed to induce trap formation in Arthrobotrys conoides has been purified. However, some isolates of this fungus produce traps spon- taneously in pure culture, as do many other predacious fungi; therefore, other mechanisms of trap induction must also be involved. Different predacious fungi have widely varied reactive thresholds and responses to trap-inducing substances. The various species of nematode-trapping fungi are divided into two ecological groups, based on the pattern of germination of conidia exposed to soil: a sensitive group, where conidia or tubes quickly give rise to trapping organs; and an insensitive group, in which little trap formation occurs. Fungi belonging to the sensitive group are efficient predators and poor saprophytes compared with the inefficient predators and able saprophytes of the insensi- tive group. The nutritional requirements of A. conoides, a member of the in- sensitive group, are similar to the requirements of other soil fungi; biotin, thiamine, and zinc are required for growth in a glucose-inorganic salts medium. However, very little is known about the nutrition and physiology of those species belonging to the sensitive group. Further information on the preda- cious fungi is needed.

108 BASIC PRINCIPLES OF CONTROL Despite some research in this area, possible ways to utilize these biotic agents in effectively controlling nematodes have not yet been discovered. Greatest emphasis has been on the use of nematode-trapping fungi. While their effectiveness in vitro and in pot tests has been encouraging, attempts to use them for nematode control on a field scale have been unsuccessful. An- nual cultivation reduces the density of these fungi in agricultural soil, but they are apparently able to persist under these conditions. In citrus orchards, three or four species of nematode-trapping fungi are frequently intimately as- sociated with infestations of the citrus nematode (Tylenchulus semipenetrans). Artificial application of nematode-trapping fungi to soil would seem ap- propriate only if they were absent from the soil in question, if it were known that a given fungus species was more effective than fungi already present against a particular nematode pest, or if greater population densities of the fungi would result. The latter may be difficult to attain in view of demon- strated antagonisms to such introduced predacious fungi. Very little is known about the trapping activity in the natural soil environment. Some evidence in- dicates that trapping increases for a short time after the addition of organic matter to the soil and then declines. Evidence from laboratory tests shows that various species of nematode-trapping fungi differ considerably in their ability to trap nematodes in soil. This may account for the inconsistent results ob- tained with these organisms. Certain little-known phycomycetous fungi, which develop internally in their hosts, parasitize nematodes. Those phycomycetes studied are only weakly parasitic on nematodes and are probably saprophytic on dead or in- jured fauna of the soil. The efficiency of fungal predators and parasites is probably limited by lack of mobility, inability to seek out prey or hosts, and by their low population densities. In the confined biotic community of the soil, the production of large quantities of motile spores capable of spreading in soil-water films would be a desirable characteristic of an effective biological control agent. More in- formation is needed about the occurrence and biology of these microorga- nisms and the susceptibility of nematodes attacked by them. BACTERIA AND VIRUSES Bacteria are often found attacking nematodes maintained in the laboratory, but the deteriorated condition and unfavorable environment of the nematodes generally preclude judging the significance of such associations. A widespread bacterial infection of soil populations of a dagger nematode (Xiphinema americanum) was reported, but, as with other reports of bacterial diseases of nematodes, the observations are not unequivocal. A disease of a root-knot

BIOLOGICAL CONTROL OF NEMATODES 109 nematode (Meloidogyne sp.), transmissible and apparently caused by a virus, was noted, but the observation was never confirmed. Certain myxobacters, or protozoa-like bacteria, isolated from soil, produced lytic enzymes that dissolved or lysed some bacterium-feeding nematodes in the genera Caenorhabditis, Rhabditis, and Panagrellus. The lytic substances from these myxobacters did not lyse the fungus-feeding nematode (Aphelen- chus avenae) or the plant-parasitic clover cyst nematode (Heterodera trifolii); thus, differences in the chemical composition of the nematodes are implied. Although bacterial enzymes attack certain nematodes in a simple laboratory system, it is unlikely that their effectiveness in soil, with its physical, chemical, and biological complexity, would be specific for plant-parasitic nematodes. Obtaining and developing bacterial or viral pathogens of nematodes should re- ceive increased attention from nematologists studying plant-parasitic nematodes. PROTOZOANS A sporozoan parasite, generally referred to as Duboscquia penetrans, is often found parasitizing plant-parasitic and other soil nematodes, but its small size and complex life cycle make it difficult to study. The effectiveness of sporo- zoans as biological control agents is unknown. Their widespread occurrence and ability to destroy the reproductive organs of nematodes or to kill their nematode hosts indicate the potential control value of these organisms; con- sidering this, the biology and ecology of such sporozoans merit further study. A large amoeboid proteomyxan organism, Theratromyxa weberi, although frequently observed ingesting nematodes, is not considered of practical im- portance in the control of plant-parasitic nematodes. Other soil protozoa probably have only an incidental predatory relationship to nematodes. OTHER NEMATODES Certain carnivorous nematode species, including many of the Mononchidae, comprise one of the most important and least studied groups of organisms predacious on soil and plant-parasitic nematodes. The small predatory species ofSeinura feed voraciously on nematodes and may, in some cases, be of con- siderable value in nematode control. Various widespread predators in the nematode superfamily Dorylaimoidea may play a significant role in the bio- logical balance of soil organisms (Figure 47). Because information on the effectiveness of these predacious nematodes is derived almost entirely from observation rather than experimentation, further investigation is needed.

110 BASIC PRINCIPLES OF CONTROL FIGURE 47 Predacious Eudorylaimm sp. feeding on larva of Aphelenchus avenue in agar culture. OTHER INVERTEBRATES Tardigrades, small animals found chiefly in water films surrounding leaves of terrestrial mosses and lichens, but sometimes numerous in the soil, will kill nematodes. Despite recent study of the role of tardigrades in soil-nematode population dynamics, relatively little is known of the biology of these curious organisms. Under laboratory conditions, a soil-inhabiting turbellarian flatworm has been observed ingesting large numbers of root-knot nematodes. Since parasites and predators of nematodes are widespread, biological- control studies should be primarily ecological and should aim at modifying both the physical and biological characteristics of their environment in an

BIOLOGICAL CONTROL OF NEMATODES 111 effort to reduce nematode populations. Despite increasing studies in the special area of soil biology dealing with nematodes, knowledge of fundamental factors affecting the biological equilibrium and biotic potential of nematode pests is inadequate. INFLUENCE OF ORGANIC MANURING ON NEMATODE CONTROL In some instances, the addition of large amounts of organic materials to soil results in reduced populations of plant-parasitic nematodes and in higher crop yields. The reduction in numbers of plant-parasitic nematodes is thought to be caused, at least in part, by an increase in natural enemies of nematodes. In addition, the presence of decomposing organic materials in the soil apparently provides host plants with some tolerance to nematode attack. Decomposition products of organic matter and plant residues may also be detrimental, directly or indirectly, to plant-parasitic nematodes. Certain highly concentrated volatile fatty acids produced by decomposing rye residues in soil are toxic to a root- knot (Meloidogyne incognita) and a lesion nematode (Pratylenchus penetrans) but not to the saprophagous species tested. It may be significant that certain saprophagous nematodes appear to be more tolerant than plant-parasitic nematodes to the halogenated hydrocarbon nematocides. PLANT-ROOT EXUDATES TOXIC TO NEMATODES Roots of several plants contain chemicals that, on leaching into the soil, are toxic to plant-parasitic nematodes. A compound found in asparagus roots and tentatively identified as a glycoside is toxic to several species of plant- parasitic nematodes. The French marigold, when grown on soil infested with lesion nematodes (Pratylenchus spp.), suppresses the population of these nematodes and re- duces the numbers found in the roots of susceptible host plants. The African marigold behaves similarly. A population of stunt nematode (Tylenchorhyn- chus dubius) also was suppressed, but populations of spiral nematode (Roty- lenchus robustus) and certain other Tylenchida were unaffected. Three com- pounds of ana-terthienyl type, toxic to nematodes, were identified in root exudates from these plants. There is little doubt that the roots of many plants release chemicals toxic to nematodes into the soil. Possible uses and the economic value of plants such as the marigold should be studied. The majority of plant-parasitic nematodes inhabit soil during all or part of their life cycles. The impossibility of directly observing a phenomenon

112 BASIC PRINCIPLES OF CONTROL occurring in soil, coupled with the physical and biological complexity of the soil, make research in biological control of plant-parasitic nematodes ex- tremely difficult. However, results of research conducted to date demonstrate the great diversity of natural control agents. The combining of chemical and biological control measures offers advantages such as reduced cost for nema- tocidal chemicals and reduced problems of pesticide residues. The search should continue for more specific parasites of injurious nema- todes: parasites able to persist in soil, parasites able to attack endoparasitic nematodes and continue their lethal activities after the nematodes escape the soil environment and enter the host roots, and, finally, effective parasites obtainable by man in large numbers for wide dissemination in agricultural soils. BIBLIOGRAPHY Adams, R. E., and J. J. Eichenmuller. 1963. A bacterial infection of Xiphinema ameri- canum. Phytopathology 53:745. (Abstract) Roosalis, M. G., and R. Mankau. 1965. Parasitism and predation of soil microorganisms, pp. 374-391. In K. F. Baker and W. C. Snyder (eds.). Ecology of soil-borne plant pathogens. Univ. of California Press, 571 pp. Cooke, R. C. 1962. Behaviour of nematode-trapping fungi during decomposition of organic matter in the soil. Trans. Brit. Mycol. Soc. 45:(3):314-320. Cooke, R. C. 1964. Ecological characteristics of nematode-trapping Hyphomycetes. II. Germination of conidia in soil. Ann. Appl. Biol. 54:375-379. Coscarelli, W., and D. Pramer. 1962. Nutrition and growth of Arthrobotrys conoides. J. Bacteriol. 84:60-64. Duddington, C. L. 1957. The friendly fungi. Faber and Faber, London. 188 pp. Katznelson, H., D. C. Gillespie, and F. D. Cook. 1964. Studies on the relationships be- tween nematodes and other soil microorganisms. III. Lytic action of soil myxobacters on certain species of nematodes. Can. J. Microbiol. 10:699-704. Loewenberg, J. R., T. Sullivan, and M. L. Schuster. 1959. A virus disease of Meloidogyne incognita incognita, the southern root-knot nematode. Nature (London) 184:1896. Mankau, R., and O. F. Clarke. 1959. Nematode-trapping fungi in southern California citrus soils. Plant Dis. Rep. 43:968-969. Oostenbrink, M., K. Kuiper, and J. J. S'Jacob. 1957. Tagetes als feindpflanzen von Pratylenchus-arten. Nematologica II, Suppl.: 424-433. Pramer, D. 1964. Nematode-trapping fungi. Science 144:382-388. Sayre, R. M., Z. A. Patrick, and H. J. Thorpe. 1965. Identification of a selective nema- tocidal component in extracts of plant residues decomposing in soil. Nematologica 11:263-268.

CHAPTER 11 Plant Resistance Potentially, the most economical and effective method of controlling nema- todes is the use of nematode-resistant plant varieties. At present, largely be- cause of limited research in this area, few nematode-resistant varieties of plants are available to the commercial grower. Plant breeders and nematolo- gists have developed cotton, cowpeas, lespedeza, tobacco, lima beans, soy- beans, peppers, tomatoes, and grape and peach rootstocks resistant to root- knot nematodes (Meloidogyne spp.) (Figure 48); clover and alfalfa resistant to the stem nematode (Ditylenchus dipsaci); potatoes resistant to the golden nematode (Heterodera rostochiensis); barley resistant to the cereal root nema- tode (//. major); soybeans resistant to the soybean cyst nematode (H. glycines) (Figure 49); citrus rootstocks resistant to the citrus nematode (Tylenchulus semipenetrans); and corn resistant to a stunt nematode (Tylenchorhynchus claytoni). Commercial varieties of cotton, lima beans, and soybeans resistant to the root-knot nematode, and varieties of alfalfa, oats, and barley resistant to the stem nematode are grown extensively. Undoubtedly, some commercial varieties of a number of crops developed primarily for high yield and quality of product are tolerant to one or more nematodes. Nematode resistance has been discovered in both cultivated and wild plant species. For example, root-knot resistance was found in wild species of Lyco- persicon from South America, in small-fruited hot peppers, in Chilean and African alfalfa varieties, in selections from alfalfa varieties cultivated in the United States, and in phylloxera-resistant grape hybrids and pure species of American origin. Resistance to the golden nematode is present in wild species of Solarium and in selections from commercial potato varieties from South America. A lima bean variety, selected over a number of years by the Hopi 113

114 BASIC PRINCIPLES OF CONTROL FIGURE 48 Young peach trees of the same age growing in soil in- fested with a root-knot nematode (Meloidogyne incognita). Left, on susceptible Lovell rootstock; right, on Rancho-resistant rootstock. Another rootstock now available, Nemaguard, is also resistant to another root-knot nematode (M. javanica). (Courtesy of the Depart- ment of Nematology, University of California.) FIGURE 49 Comparison of cyst nematode-resistant and susceptible soybean varieties in soil infested with soybean cyst nematodes (Heter- odera glycines). Left, Picket; right, Lee. (Courtesy of Phytopathology. After Brim and Ross.)

PLANT RESISTANCE 115 Indians of Arizona for adaptability to their soils, was found to be resistant to the root-knot nematode. Few plants are immune to nematode attack. Resistance is usually relative or incomplete; therefore, selection of resistant and susceptible plants, either by nematode increase or by degree of plant damage, is often difficult in breeding programs. DEVELOPMENT OF NEW VARIETIES Although a few nematode-resistant plant varieties have been developed by selection from commercial varieties, such as a Swedish red clover resistant to the stem nematode, the most common method of obtaining resistant varieties is to cross plants having desirable commercial characters with those possessing nematode resistance. An original cross between a commercial variety and a nematode-resistant source, followed by repeated back crosses to commercial varieties, is generally used in breeding programs to incorporate resistance into a commercially acceptable plant variety. Unrelated plant species, especially those with different chromosome num- bers, are difficult to hybridize. Some crosses can be made only by using specialized culture techniques; a root-knot resistant tomato hybrid obtained by this method has been used as a source of resistance by plant breeders. Undesirable horticultural characters are often linked to nematode resistance, making transfer of resistance without these characters difficult. A wild cotton (Gossypium barbadense var. darwinii) is highly resistant to a root-knot nema- tode (M. incognita), but this resistance is inherited recessively and is probably polygenic. More time and work are required to transfer such resistance to commercial varieties than to transfer resistance owing to a single gene. In developing nematode-resistant varieties, a rapid and accurate method of determining the degree of resistance is necessary. With the exception of nema- todes from a few genera, such as Meloidogyne, Heterodera, and Ditylenchus, difficulty in obtaining large quantities of uniformly viable nematode inoculum is an important limiting factor in breeding for resistance to many plant dis- eases caused by nematodes. Roots infected with root-knot nematodes are commonly used as inoculum in breeding experiments. Infected roots containing many egg masses are chopped and equal quantities are added to the soil in which the test plants are to be grown. The success of this method depends on a high percent of nema- todes remaining viable in the plant roots during the inoculation procedure. Inoculum of most of the cyst nematodes (Heterodera spp.) is relatively easy to obtain. Cysts (dead bodies of swollen females containing eggs and larvae) are washed from soil by flotation and sieving. These cysts are freed of

116 BASIC PRINCIPLES OF CONTROL accompanying debris, proportioned by counting or weighing, and added to soil. The number of larvae present is estimated by counting the number of eggs or larvae contained in samples of cysts. Adding approximately equal numbers of larvae hatched from cysts is more precise but is time consuming and difficult to adapt to routine tests. Furthermore, free larvae are more sus- ceptible to adverse environmental conditions than larvae in cysts and thus are more likely to die before reaching plant roots. Large quantities of stem nematodes for use in breeding programs are grown routinely on host tissue. As these nematodes are generally found inside stems or bulbs, their recovery is relatively easy. Stem nematodes for testing resistance of rye seedlings are obtained by inoculating potato tubers with this nematode and storing them at room temperature for several months. Aseptic inoculum of this nematode also may be produced on callus-tissue cultures. Infected roots are often used as sources of inoculum of migratory endo- parasitic nematodes. Large quantities of some species, primarily lesion nema- todes (Pratylenchus spp.), are grown on callus-tissue or excised-root cultures. Although this method is not now used extensively to produce inoculum for breeding programs, it probably will be widely used in the future. Inoculum of ectoparasitic root feeders, nematodes that spend most of their lives in the soil feeding on outer root cells, is particularly difficult to obtain because of the time required to extract these nematodes from soil and to separate the test species from other species. Although large numbers of a stunt nematode (T. claytoni) can be produced on callus-tissue cultures under aseptic conditions, for most ectoparasitic root feeders the only practical method is to test resistance in the field or greenhouse in soil infested by more than one nematode species. Unfortunately, natural soil populations of most of these nematodes fluctuate greatly over relatively short time periods and thus are unreliable sources of inoculum. When it is possible to produce large numbers of most ectoparasitic forms on callus tissue or excised roots or in vitro, breed- ing for resistance to those species will be facilitated. In testing for resistance, nematode-infected plant material is uniformly mixed with soil or placed in the vicinity of the test-plant roots, or extracted nematodes are added by pipettes, hypodermic needles, or other devices to the root zone or to soil at the time of transplanting or sowing. Whichever method is used, the unprotected nematodes must be placed in the vicinity of plant tissue. Suitable moisture and temperature and other requirements of the par- ticular nematode species should be considered. The active stages of most nema- todes are particularly sensitive to drying. Nematodes from more than one source should be included in the inoculum, because different collections of nematodes of a single species may vary in pathogenicity. Although such variability has not been demonstrated for all plant-parasitic nematodes, it occurs in a high percent of the species tested.

PLANT RESISTANCE 117 Generally, nematode resistance or tolerance is tested by measuring plant performance and rating disease symptoms, such as root galls. Because growers are primarily interested in yield and quality of product, these are very im- portant criteria. However, the rate of nematode reproduction also should be determined. Good plant performance in the presence of pathogenic nematodes may re- sult from either plant resistance or tolerance. A resistant variety is a poor host and does not support high nematode populations. Many levels of plant re- sistance occur, varying from immune plants on which no nematodes develop to those supporting populations almost as high as susceptible varieties. A tolerant variety, however, is a good host, possessing low susceptibility to the disease caused by the nematode. If a tolerant crop is grown frequently in a rotation, the nematode population may increase above the tolerance level of that or other crops in the rotation. When available, resistance is preferable to tolerance. Occasionally, crop damage results from the feeding of large soil populations of nematodes on immune or highly resistant plants. NATURE OF RESISTANCE Substances given off by plant roots, which stimulate hatching of nematode eggs or attract nematodes to roots, are not related to the resistance of plants to nematodes. Stimulants or attractants from roots of immune or resistant plants are sometimes more potent than such substances from susceptible ones. Nematodes enter roots of most resistant plants, but often in smaller num- bers than they enter roots of susceptible plants. An exception is that larvae of a root-knot nematode (M. hapla) do not penetrate roots of rye and oat plants. The number of endoparasitic nematodes penetrating plant tissues may be influenced by age of the tissues. Although young chrysanthemum leaves are entered by chrysanthemum foliar nematodes (Aphelenchoides ritzemabosi), the nematodes apparently cannot move among the mesophyll cells because of the compactness of the tissue at that stage of growth; therefore, the nema- todes cannot feed and reproduce. In older leaves, the mesophyll cells have intercellular spaces sufficiently large for easy movement and colonization by the nematodes; therefore, the nematodes can live and reproduce successfully. It is unknown whether morphological features are solely responsible for lack of or limited penetration of plant parts. Perhaps biochemical or physiological factors also are involved in these phenomena. The most common reaction of nematodes to resistant plants may be failure of all or a high percent of females to develop to maturity even though infective stages of nematodes enter plant tissue. In most cases, development of females does not proceed further than the third stage. Although females fail to

118 BASIC PRINCIPLES OF CONTROL develop, or develop at a slower rate, males may mature normally. Plant re- sistance may cause not only slower nematode development but also produc- tion of fewer eggs by females. It has been postulated that resistance may, in many cases, be related to the failure of the host to supply some nutrients necessary for rapid reproduction or even for survival of the nematode. The formation of few or no giant cells at feeding sites of maturing female nematodes is associated with plant re- sistance to cyst and root-knot nematodes. The cytoplasm in these giant cells is less dense than that in giant cells induced in susceptible varieties. Perhaps these nematodes can obtain food suitable for maturation from only these en- larged cells. Infection by stem nematodes causes conspicuous enlargement and separation of parenchymatous cells in susceptible plants but only slight cell enlargement and separation in resistant plants. Nematode populations around the roots of resistant plants sometimes de- cline at a more rapid rate than can be explained by starvation, and it is pre- sumed that toxins of plant origin are responsible. In two instances, specific biochemical substances, which were present as inherent constituents of the plants and were thought to be responsible for plant resistance to nematodes, were identified or partially identified. a-Terthienyl compounds isolated from some species of marigold and a glycoside isolated from asparagus were identified as the toxic factors. These compounds have a wide spectrum of activity against nematodes. Substances specific in their toxicity towards nema- tode species, or even races or biotypes, are responsible for varietal resistance, rather than compounds such as those mentioned above. Perhaps phytoalexins, substances formed only after infection of plant tissue by the nematode, are also involved in plant resistance to nematodes. Results of reciprocal grafting studies indicate that resistance factors are associated with individual plant cells attacked by the nematode and are not generally translocated in the plant. In one case, however, a resistance factor from a resistant Lycopersicon scion was translocated to a susceptible stock, as evidenced by lowered penetration and egg production by a root-knot nematode (M. incognita) infecting the stock roots. Death of cells hypersensitive to nematode feeding and browning of tissues around infecting nematodes occurs in many resistant plants in response to in- fection by several different nematode species. Death of cells usually occurs within a day or two after infection. Larvae of cyst and root-knot nematodes die in reacting tissues of a number of plant species, but stem nematodes do not die in tissues of resistant pea despite death of plant cells. Death of hyper- sensitive cells may cause extensive damage to a particular plant species even though the nematode species causing the damage cannot reproduce on the plant. Hypersensitive reactions have been postulated for resistance of chrysan- themum to the chrysanthemum foliar nematode; red clover, alfalfa, and pea

PLANT RESISTANCE 119 to the stem nematode; citrus to the citrus nematode; lima bean to the lesion nematode; and soybean to root-knot nematodes. Browning of plant tissues as a result of feeding of a lesion nematode (P. penetrans) appears to be caused by accumulation and oxidation of phenolic compounds. In apple and peach rootstocks, lesion formation, as a result of feeding of lesion nematodes, is roughly correlated with the amygdalin content of rootstocks. Whether such compounds are basically responsible for resistance is unknown. Formation of wound periderm or corky layers, which wall off and retard the development of nematodes in plant tissues, has been reported. Cork layers in old yam tubers retard entrance of root-knot nematode larvae, and cork layers around developing females retard their development. Wound periderm is also present around feeding sites of developing citrus nematode larvae in roots of resistant citrus plants. Altered mineral nutrition may change factors such as organic constituents in cells and physiology of the plant and may influence nematode reproduction and pathogenicity. For example, soybean and lima bean are more resistant to root-knot nematodes when grown under low-potassium fertilization than when grown under medium or high levels of potassium. When potassium is deficient, nematodes on lima bean develop slowly and egg production is delayed, but the pathogenic effects of the nematode are severe; when potassium is high, the nematodes develop rapidly and lay many eggs, but the pathogenic effects are less severe than those when potassium is deficient. However, a negative corre- lation exists between potassium levels and development of lesion nematodes in cherry. The apparent discrepancy may be explained by the levels of potassium used, duration of the experiment, plant species used, or nematode species used. Levels of several elements, rather than a single one, and relative levels of several elements interacting, may influence nematode reproduction and pathogenicity in plant tissues. The vigor of a plant influences resistance to nematodes. For example, populations of a lesion nematode (P. penetrans) on Wando pea plants increase as root development and plant growth are restricted by low but not deficient nutrition, low light intensity, fruiting of the plants, and late defoliation. Treatments that greatly reduce root development and plant growth, such as girdling the stem by scalding and defoliating plants after two days, reduce population levels of P. penetrans. INFLUENCE OF ENVIRONMENT Although resistance is a characteristic of the host plant, environmental factors may alter symptom expression of plants and development of the nematode. Temperature may affect the rate of nematode entry into the plant host,

120 BASIC PRINCIPLES OF CONTROL nematode reproduction, and pathogenicity. Factors such as moisture, pH, and other microorganisms also may be important. Some kinds of plant-pathogenic nematodes apparently cause greater crop losses in coarse-textured soils than in fine-textured soils. This effect may be caused partly by greater nematode movement in the coarse-textured soils. GENETIC BASIS FOR RESISTANCE Resistance to nematodes of several genera has been found in a wide variety of plant species, and the mode of inheritance of this resistance has been deter- mined. Inheritance in both diploid and tetraploid plants has been described. In general, resistance to nematodes is caused by one or a few completely or incompletely dominant genes. The resistance of grape to a root-knot nematode (M. incognita acritd) is caused by a single dominant gene, whereas resistance of cowpea to one root-knot nematode (M. incognita) involves two dominant genes. In addition to major genes, modifying genes or those with minor effects may be involved in resistance to some nematodes, such as in resistance of oats to the stem nematode. However, resistance of soybean to the soybean cyst nematodes is governed by three independently inherited re- cessive genes, and resistance of corn to the ectoparasitic stunt nematode (T. daytoni) also appears to be recessive. The resistance of wild cotton (G. barba- dense var. darwinii) to a root-knot nematode (M. incognita) is inherited reces- sively and appears to be polygenic. Resistance caused by a specific plant gene has been found responsible for resistance to one to four nematode species. RESISTANCE TO COMPLEX DISEASES In plant-breeding programs to control disease caused by nematodes and micro- organisms such as fungi or bacteria, it is important to breed for resistance to all organisms but particularly for nematode resistance. For example, although root-knot nematodes damage cotton in the absence of other microorganisms, these nematodes further depress cotton yields by increasing the severity of fusarium wilt, a disease caused primarily by a fungus, Fusarium oxysporum f. vasinfectum. RESISTANCE-BREAKING BIOTYPES Potato varieties resistant to the golden nematode, if repeatedly grown in some infested fields, bring about a change in the pathogenicity of the nematode

PLANT RESISTANCE 121 population from one that is almost entirely composed of nonresistance- breaking individuals to one composed almost entirely of those capable of breaking resistance. Furthermore, in a rotation including potatoes every 4 to 5 years, and no other hosts of the golden nematode, it is estimated that within 20 years there will be a complete change from nonresistance-breaking to resistance-breaking populations. Because movement of nematodes from field to field and area to area is usually more restricted than that of most fungi, spread of resistance-breaking biotypes of nematodes is probably slower than that of most fungi. Resistance-breaking biotypes have been reported for many but not all nema- todes. Oat varieties resistant to the stem nematode have been widely grown in Wales, but no resistance-breaking population of the nematode has appeared. FUTURE RESEARCH More effective methods for testing resistance must be devised, and the im- portance of correctly identifying nematodes must be emphasized. Progress in breeding for resistance to root knot increased rapidly after the root-knot nematodes were separated into a number of species differing in host range. The high cost of direct control measures, and persistence of nematodes in the soil, point to a need for emphasis on breeding resistant varieties of plants. The development of many plant varieties with nematode resistance will result in untold benefits, especially to growers of low-acre-value and of perennial crops, such as tree fruits and forage crops. Before major progress is possible in this field, there must be a marked increase in research workers and research teams, such as nematologists and plant breeders, actively engaged in coopera- tive programs. The biochemical basis of resistance in plants to nematodes has been little studied. Research in this area should aid in developing resistant varieties of plants and, perhaps, in the discovery of new chemical means of nematode control. BIBLIOGRAPHY Bingefors, S. 1954. Resistens mot nematoder hos vara kulturvaxter. Svensk Jordbruks- forskning. Year 1954:174-180. Cotten, J. 1964. Eelworm pests of cereals and their control. J. Agr. Soc., Univ. Coll. of Wales, Aberystwyth 45:4-7. Hare, W. W. 1965. The inheritance of resistance of plants to nematodes. Phytopathology 55:1162-1167.

122 BASIC PRINCIPLES OF CONTROL Kehr, A. E. 1966. Current status and opportunities for the control of nematodes by plant breeding, pp. 126-138. In Pest control by chemical, biological, genetic, and physical means. (A symposium.) Agr. Res. Serv., USDA, ARS 33-110. Kirkpatrick, J. D., S. D. Van Gundy, and W. F. Mai. 1964. Interrelationships of plant nutrition, growth and parasitic nematodes. Plant Anal. Pert. Probl. 4:189-225. Krusberg, L. R. 1963. Host response to nematode infection. Ann. Rev. Phytopathol. 1:219-240. Rohde, R. A. 1960. Mechanisms of resistance to plant-parasitic nematodes, pp. 447-453. In J. N. Sasser and W. R. Jenkins (eds.). Nematology, fundamentals and recent ad- vances. Univ. North Carolina Press, Chapel Hill. 480 pp. Rohde, R. A. 1965. The nature of resistance in plants to nematodes. Phytopathology 55:1159-1162. Wallace, H. R. 1963. The biology of plant-parasitic nematodes. Edward Arnold Ltd., London, 280 pp.

CHAPTER 12 Control by Physical Factors The application of heat, a widely used pest and pathogen control practice, is the most important of the various physical factors used in nematode control. Other physical factors, such as irradiation, plasmolysis, or electricity, are harmful or lethal to nematodes, but their potential for effective nematode control is not yet realized; further research and advanced technology may make these physical factors important for control in the future. CONTROL BY HEAT-BASIC CONSIDERATIONS With control by heat, time of exposure to a specific temperature is a most important relationship; for every temperature there is a minimum exposure time required for the heat to produce a particular effect on an organism. In addition to this time-temperature combination, specific heat and heat ex- change are used in calculating the heat energy involved and the subsequent cost, an important practical factor. Engineering computations for cost and efficiency are readily adaptable to pathogen control methods utilizing heat and are very useful if the biological aspects are given due consideration. Heat affects nematodes in many ways, ranging from immediate destruction by burning or searing at extremely high temperatures to the coagulation of cytoplasm at approximately 65°C, a relatively low temperature. Tempera- tures below this coagulation point, but above normal, cause lethal or sub- lethal injury to nematodes; however, the exact nature of the injury is not known. Such temperatures are effective for nematode control despite the fact that the nematodes may not be killed immediately. As with other control 123

124 BASIC PRINCIPLES OF CONTROL methods, the immediate death of the nematode is not necessary for disease control, a point sometimes overlooked in evaluating new control methods and materials. Each developmental stage of a nematode has its own level of heat sensi- tivity, its own time-temperature curve. These curves differ for each species, for certain temperature-tolerant life-cycle stages or states of being, and under conditioning influences of moisture content and rates of temperature change. These biological details are especially significant in disinfecting a living host of nematodes when the heat susceptibilities of parasite and host differ only slightly. They are also important in efficiently disinfesting nonliving objects, so that areas of inadequate heating are eliminated and reinfestation is pre- vented. The term "disinfestation" refers to the control of nematodes asso- ciated with nonliving materials; "disinfection" refers to the control of nema- todes associated with living materials. Heat treatments that kill other pests and pathogens of plants are also effec- tive and practical for nematode control, enabling the use of a single treatment for control of several pests. The following figures, illustrating the lethal tem- peratures for various organisms, can serve as a guide for disinfestation treat- ments with heat. A few highly resistant seeds and some plant viruses require 30 minutes of exposure at 100°C under moist conditions to be killed. Most seeds and viruses, all plant-pathogenic bacteria and fungi, various worms and mollusks, and all arthropods are killed when held at 82°C for 30 minutes. Most nematodes are destroyed when held at 49°C for 30 minutes. In practice, when infested soils are heated thoroughly and uniformly by tumbling in a continuous-flow treatment system, using dry heat or steam, a temperature of 82°C for 30 minutes is satisfactory for nematode control. If treated soils are in containers or in a stationary mass, a final temperature of 100°C for 30 minutes is recommended. DISINFESTATION BY HEAT Heat disinfestation usually involves soil and other substrates, containers, and inert surfaces that become infested with undesirable nematodes. Heat must be present long enough to control the nematodes, usually by causing their death. Nematodes on exposed surfaces are easily reached by heat, so that main- taining the lethal temperature on the surface for a sufficient time is all that is required. Nematodes inside porous substrates can be reached by transferring heat throughout the substrate by agents such as hot water, steam, or perhaps hot air, or by crumbling and spreading the substrate into a thin layer to obtain exposed surfaces and then applying heat.

CONTROL BY PHYSICAL FACTORS 125 In heat disinfestation, it is advantageous to conserve the heat after appli- cation to the substrate, thus allowing maximum time for heat penetration and thereby increasing the effectiveness of the treatment. Heat retention also provides the possibility of nematode control at lower temperatures because of longer exposure time. It is necessary to apply heat carefully to avoid leaving relatively cool areas where nematodes can survive. DRY HEAT Dry heat may be applied in a quick searing manner, as when surface-borne organisms are contacted briefly by flame, such as the use of a weed burner to disinfest the surfaces of a potting bench or soil-mixing base. Efficiency de- pends largely on accessibility of the organisms to the direct heat. In applying flash-heat to soil or sand, the use of an open flame moving over a thin, loose layer of the substrate is necessary. Dry heat may also be applied so that the entire mass being heated is brought to an over-all desired temperature. Any ovenlike enclosure, heated by a suitable source, serves for dry-heat disinfestation. Heat may be applied ex- ternally, internally, or in combination. Convenience in loading, with minimum effort and damage to enclosed heating elements, is important in the oven design. A continuous-flow soil system is particularly useful in applying dry heat. In some systems, soil is fed into one end of a heater unit and collected at the other end. The rate of flow through the heater and the distance traveled in contact with the heat source are regulated by the machine design to give the desired amount of heat. The equipment provides for exposure to open flame, passage over heated metal plates, or both. In another continuous-flow system, a machine picks up soil to a desired depth in a path, passes the soil in a thin layer through the heat chamber, and then deposits the treated soil behind as the machine slowly advances. The burning of wood or brush on the surface of an intended planting site, although wasteful of fuel, is effective if sufficient heat is generated to obtain penetration. The convenience of the continuous-flow system in treating soil as it moves past a heat source is obvious, but the intense heat required in this type of system destroys organic materials in the soil. An oven permits the use of a lower temperature, thereby avoiding the destruction of organic materials. Treatment of a moving and tumbling soil mass by dry heat assures uniform heat distribution. However, none of these methods is widely used.

126 BASIC PRINCIPLES OF CONTROL MOIST HEAT Disinfestation by moist heat utilizes hot water, steam, or steam plus chemi- cals. Direct exposure to boiling water or steam is used to disinfest open sur- faces. Scouring by flowing water, the force of steam, and brushing can be used with this method. Soil can be disinfested by moist heat, either as hot water or steam. Precau- tions to ensure uniform heat distribution and penetration throughout the sub- strate are necessary. Soils should be pulverized to eliminate clods and should not be waterlogged. Soil moist enough for planting is ideal for steam treat- ment, since the organisms are hydrated and pore spaces are open. Because of its greater caloric content, steam is a more efficient heat carrier than hot water; a pound of water at the boiling point of 100°C requires 970 BTU to be converted to a pound of steam at 100°C, and this heat is released when steam is converted back to water. At atmospheric pressure, the maximum temperature reached is 100°C, so that the organic material in the soil is not destroyed. Autoclaving at elevated temperatures and pressures is sometimes used in laboratory experiments. HOT WATER Drenching or soaking objects in hot water effectively controls nematodes. The duration of heat exposure is easily regulated. Vigorously boiling water in adequate volume is necessary to attain and maintain the desired temperature. Since hot water has low heat energy, an excessive amount is necessary to pro- duce sufficient heat to destroy other types of plant pathogens. Hot water is impractical for soil treatment, since the soil becomes waterlogged and unde- sirable leaching may occur. STEAM Steam disinfestation is adaptable to the needs of home growers, to the exten- sive operations of commercial nurserymen or mushroom growers, and to many other types of plant production where large acreages are not involved. Mate- rials to be treated are brought to the equipment, or suitable equipment is moved to the growing site. Due to its lethal effect, free-flowing steam is widely used for disinfesting growing beds, containers, and media. Precautions such as breaking of soil clods, drainage of waterlogged soil, adequate disper- sion of steam outlets, and elimination of cool spots in treatment beds are necessary to ensure thorough uniform penetration of the steam and effective heating.

CONTROL BY PHYSICAL FACTORS 127 Steam treatment of soils has many advantages. The treatment is short, easy to apply, and nontoxic to man. In many cases, planting can be done after the soil cools. A broad spectrum of kill, including weeds, is obtained. Treatments can take place near living plants. Lethal heat can penetrate unrotted crop refuse that may harbor pathogens. Steamed, fine-textured soils become more granular, with resulting improvement in aeration and drainage. Increased plant growth sometimes results from increased availability of nutrients, improved physical structure of the soil, and, perhaps, from favorable biological changes, in addition to the benefits of pest and pathogen destruction. Steam treatment of soils also has disadvantages. Treated soils, especially those high in organic matter, may be toxic to certain kinds of plants, and seed germination may be reduced. In nurseries, the choice of proper ingredi- ents in preparing soil mixtures can eliminate the problem. As the toxic effects may be temporary or may last for several months, postponing planting for several weeks after treatment is advisable when toxicity is suspected. Leaching the soil reduces toxicity, but it waterlogs the soil, is inconvenient, and costs time and money. The steam-generating equipment is cumbersome and expen- sive, and steaming large amounts of soil is laborious. STEAM PLUS CHEMICAL Combining volatile toxicants, such as formaldehyde at the rate of 0.2 to 0.4 fluid ounce per gallon of water, with steam may lower the cost of effective treatments by reducing the amount of heat required. A low-temperature steam-air mixture has been used without loss of effectiveness, and with re- duced fuel costs. The application of a combination of a low-temperature steam-air mixture with volatile nematocides and other pesticides may offer a solution for problem situations. ENDOGENIC HEAT Heat generated by decomposition in compost piles or bins is sufficient to destroy plant-parasitic nematodes. Checking compost temperatures and turn- ing any relatively cool compost materials into the pile will assure uniform nematode kill. The variability in the heat produced is dependent not only on location in the compost pile but also on the composition of the compost. Some control of the plant parasites present in composting materials can also result from the biological agents present. Saprophagous nematodes invariably are abundant; their high populations appear to induce increased populations of other organisms that prey upon the nematodes.

128 BASIC PRINCIPLES OF CONTROL DISINFECTION BY HEAT Control of nematodes associated with living plant materials requires removal or destruction of the nematodes without harming the living material. When certain ectoparasitic nematodes are present on the root system or other underground plant parts, mechanical removal may be effective and economical. Washing the plant parts in a tank or with a stream of water, aug- mented by agitating, light brushing, or rubbing, may be sufficient. Such meth- ods are applied to many ornamental plants and turf grasses that are bare-rooted at some time in their processing for distribution or replanting. A hot-water treatment or nematocidal dip is needed for plants contaminated with ecto- parasitic nematodes, such as Hemicycliophora spp. or Criconemoides spp., that are difficult to dislodge by mechanical means because their long stylets are inserted in plant cells. When endoparasitic nematodes are present within plant tissues or enclosed within protective layers of plant parts, they require a penetrating chemical or physical agent to kill them. Heat is most commonly used. Heat applied ex- ternally is absorbed by the plant propagule and spread within to reach patho- gens. When a differential in heat susceptibility exists between plant tissue and nematode, with the latter being more sensitive, effective heat treatment is possible. To develop satisfactory treatments, the thermal-time-death curves must be determined for all nematode stages present in the infected plant parts. The plant parts, also, are tested for tolerance to temperature-time combinations that kill the nematodes. Because different stages in the development and dormancy of the plant materials may have different heat tolerances, the most tolerant stage is selected for treatment. The procedure is thoroughly tested on a small scale before large lots of plants are treated. Because water transfers heat rapidly and excludes any possibility of harm- ing plant tissue by desiccation, it is preferable to air as the medium for effect- ing moderate temperature changes in treated materials. A pretreatment soak of plant parts is desirable to rehydrate nematodes and make them more sus- ceptible to heat injury, to bring the plant tissues to a uniform starting tem- perature, and to dispel insulating air bubbles trapped by the plant parts. The presoaked materials are drained, then transferred to the hot-water bath, and the treatment timing is begun. The temperature of this bath is held constant within about 0.3°C. This is achieved by having present a large volume of water compared with the volume of plant materials being treated, having adequate heater capacity, or having additional hot water available to add to the bath for quick correction of temperature drops (see Figure 40, Chapter 8). Thor- ough mixing and agitation of the water in the treatment tank also are neces- sary for accurate temperature control. If necessary for plant survival, the

CONTROL BY PHYSICAL FACTORS 129 treatment temperature can be brought down to normal rapidly by a post- treatment immersion in another tank of water held at a suitable temperature. Otherwise, the plant parts are cooled to the ambient temperature on removal from the treatment tank. Some plant propagules may require drying. Even forced drying may be necessary to avoid other disease hazards likely to be in- volved in subsequent storage. Some plant parts require protection from ex- cess drying. Plants in a dormant state are most likely to withstand heat treatment. The effectiveness of hot-water treatments for control of nematodes and other pathogens is increased by adding suitable chemicals to the treatment baths. In some instances, this may be necessary for effective treatment. A few examples of recommended hot-water treatments indicate the general range of temperature-time combinations found useful: narcissus bulbs infected with stem nematode (Ditylenchus dipsaci), treat 4 hours at 43.5°C in 0.5 percent formalin in water; iris bulbs with the potato rot nematode (D. destructor), 3 hours at 43.5°C in water and formalin; Easter lily bulbs with spring crimp nematode (Aphelenchoidesfragariae), 1 hour at 44°C in a water and formalin bath; citrus rootstock with burrowing nematode (Radopholus similis), 10 minutes at 50°C; seed with bentgrass seedgall nematode (Anguina agrostis), 15 minutes at 52.2°C in water containing a wetting agent; wheat seed with wheat nematode (A. tritici), 30 minutes at 49°C or 10 minutes at 50°C; begonia plants with spring crimp nematode, treat by submerging pot and con- tents for 1 minute at 49°C, 2 minutes at 47.8°C, or 3 minutes at 46.8°C; sweet potatoes with root-knot nematodes (Meloidogyne spp.), 65 minutes at 46.8°C; and grape rootings with root-knot nematodes, 30 minutes at 47.8°C, 10 minutes at 49°C, 5 minutes at 51.6°C, or 3 minutes at 53°C. Hot-water disinfection may be the only practical way to control nematodes infecting some plants. Heat treatments can also be valuable in controlling viruses and other plant pathogens, thus giving multiple benefits for one treat- ment. The treatment can often be worked into the normal processing of the plants. The disadvantages are the lack of protection against reinfection, the possibility of spreading other pathogens by water, the rather precise condi- tions required, and the expensive and cumbersome equipment employed. CONTROL BY LOW TEMPERATURE Little information is available on either the effect of cold on nematodes or the possible use of cold in controlling nematodes. Cold storage of tubers or bare-root nursery stock does not normally result in disinfection. Study of cold in relationship to nematode survival offers possibilities for research that may lead to new control methods for nematode diseases.

130 BASIC PRINCIPLES OF CONTROL CONTROL BY ELECTRICITY Electrocution, electrotaxis (galvanotaxis), heating, and diathermy are possible ways electricity might be used in controlling nematodes. In practice, it is difficult to electrocute small organisms such as nematodes, even at electrical potentials that are hazardous to man. The lethal effect of electricity on a small organism within an electrical field depends on the dif- ference in potential (voltage) existing from one side of the organism to the other and on the amount of current flow (amperage) passing through the organism. In addition to the small size of the nematodes in an electrical field making the resultant voltage differential and current flow in the organism small, nematodes usually are in an environment containing electrical conduct- ing pathways of lesser resistance than the nematodes. For example, the elec- trolytes in moisture films of soil or in tissues of a plant may conduct electricity more readily than the nematodes. Electricity is known to influence the direction of nematode movement, and it may be possible to devise some practical means of applying electricity to the plant or surrounding soil to ward off nematodes. Resistance to the flow of electricity through a substrate, such as soil, pro- duces heat. Containers with metallic plates to serve as terminals distributing the flow throughout the soil within the container are easily constructed. With suitable moisture content and source of electrical power, usually changed to high voltage with a transformer, the soil can be brought to a temperature lethal to nematodes. Wire-mesh grids have been placed at two levels in green- house benches to accomplish the same result. These techniques do not elec- trocute nematodes, but they destroy them with heat. Electrical energy applied at radio-wave frequencies, similar to diathermy, killed encysted golden nematodes (Heterodera rostochiensis) located within bales of burlap bags. Such use of electrical energy for nematode disinfestation deserves further investigation. CONTROL BY IRRADIATION The few studies concerning lethal effects of irradiation on nematodes have been discouraging. Published data indicate a remarkably high tolerance of nematodes to radiation. Cathode rays (electron-beam radiation) such as those produced by electron accelerators have limited power of penetration but can sterilize on very brief exposure. No information is available on their possible use for nematode control.

CONTROL BY PHYSICAL FACTORS 131 Radiation sterilization techniques used in food-processing, or of steriliza- tion of surgical supplies and drugs, may be adaptable to disinfestation treat- ments of objects involved in plant production. Ultraviolet rays, particularly in the shorter wavelengths, are harmful to some types of organisms. Since these rays are unable to penetrate matter, only surface-borne organisms exposed directly to the rays are affected. It is com- monly assumed that the ultraviolet light of longer wavelengths, filtered through the earth's atmosphere, is harmful to exposed nematodes. Although prospects of using irradiation to control nematodes do not appear promising, the different kinds of radiation, such as x rays, gamma rays, ultra- violet rays, and cathode rays, should be tested. CONTROL BY MISCELLANEOUS PHYSICAL FACTORS Physical methods, such as ultrasonics, osmotic concentration, mechanical destruction, pressure, desiccation, and mechanical seed-cleaning, have been insufficiently tested or have been found unfeasible. Sugar, used to increase the osmotic concentration of the soil solution, was found to kill all nematodes within 24 hours when applied at 1 to 5 percent of soil by weight, but 10 to 50 tons of sugar per acre would be required. Ultrasonics are ineffective for killing nematodes in soil but may be effec- tive in water. Modern mechanical seed-cleaning methods, although not widely used at present, are used to remove infected wheat kernels (cockles) or galls, which contain large numbers of the wheat nematode, from wheat seed. Seed-cleaning methods might be further developed to remove small particles of debris in- fected with the stem nematode (D. dipsaci) from seed of crops such as clover and alfalfa.

CHAPTER 13 Control by Chemicals Practical control of plant-parasitic nematodes with nematocidal chemicals is a relatively recent development. The discovery of the nematocidal properties of a l,3-dichloropropene-l,2-dichloropropane mixture (1,3-D or D-D Soil Fumi- gant) and EDB (1,2-dibromoethane or ethylene dibromide) in 1943 and 1945, respectively, had a profound influence on progress in the field of nematology. Initial trials with these nematocides resulted in economically effective control under field conditions and strikingly demonstrated the destructive potential of certain plant-parasitic nematodes, particularly the root-knot nematode. Since then, the use of nematocides has developed rapidly from a few hundred pounds in 1943 to an annual total exceeding 60 million pounds in 1963. Chemical control has limitations and cannot completely replace crop rota- tion, fallow, and the use of resistant varieties. Interactions among soil, chemi- cals, and nematode pests are not yet well understood. Despite these problems, a rapid increase in the use of chemicals has occurred, and an even more rapid increase in their use is expected. CHARACTERISTICS OF NEMATOCIDES TYPES The most successful and effective nematocides in use today are volatile halo- genated hydrocarbons. It is generally considered that a material must have high vapor pressure to spread through the soil and contact nematodes in the water films surrounding soil particles. The first material used on a field scale 132

CONTROL BY CHEMICALS 133 was carbon bisulfide, but, because of the high dosage required for adequate nematode control (800 to 1,000 pounds per acre) and the explosive hazard involved, its use was never widespread. The use of chloropicrin, another of the early soil fumigants, is limited by high cost and the need for a surface seal be- cause of its relatively high vapor pressure. It is especially valuable where serious fungus and nematode problems occur in the same field. Methyl bromide, al- though it is expensive and requires the use of soil covers, is used extensively in high-acre-value plant beds and nurseries. Weed control is an important added benefit from its use. A trend toward less volatile materials began with the development of DBCP(l,2-dibromo-3-chloropropane). The vapor pressure of this material at 21° C is 0.8 mm Hg, in contrast to 29 mm Hg for 1,3-D and 17 mm Hg for EDB. Recently, promising new compounds that are almost nonvolatile have been developed. These must be mixed with the soil or distributed through water in the soil or through the plant itself. None is widely used at present, but with further testing some may find commercial use. Since many nonvolatile com- pounds are relatively stable in soil, phytotoxicity and residues in plant and soil may be disadvantages. Compounds such as Vapam (sodiuirHV-methyldithiocarbamate), Trapex (20 percent methyl isothiocyanate), and propargyl bromide are nematocidal but are used principally to control disease complexes in which fungi, bacteria, insects, or weeds, in addition to nematodes, are involved. Combinations of nematocides and fungicides, such as Vorlex (1,3-D and methyl isothiocyanate) and Trizone (chloropicrin, methyl bromide, and propargyl bromide), are used to increase the activity of the pesticide. Several organic phosphates show some activity as nematocides but are primarily insecticides. Examples of these are: diazinon [0,0-diethyl 0-(2- isopropyl-4-methyl-6-pyrimidinyl) phosphorothioate]; Zinophos (0,0-diethyl 0-2-pyrazinyl phosphorothioate); Thimet or phorate {0,0-diethyl 5-[(ethylthio) methyl] phosphorodithioate}; and several experimental compounds not regis- tered for commercial use. PHYTOTOXICITY Phytotoxicity must always be considered when chemicals are added to soil to control nematodes, insects, or fungi. All the early commercial nematocides, such as carbon bisulfide, chloropicrin, EDB, and 1,3-D, can be phytotoxic and, depending on the dosage used, must be applied several weeks or months before the crop is planted. Phytotoxicity is influenced by soil type, temperature, moisture, soil tilth, and kind of plant grown. Some plants are severely injured

134 BASIC PRINCIPLES OF CONTROL by traces of certain nematocides in soil. The use of less-volatile, slower-acting chemicals increases the need for compounds with low phytotoxicity. Although the general phytotoxicity of these chemicals seems to be low, toxicity to specific plants has tended to increase. For example, many crops can be planted immediately following DBCP soil fumigation, and a number of crops will tol- erate a nematocidal dosage of DBCP to the root zone of the established plant. Certain crops, however, show sensitivity to DBCP; namely, red and sugar beets, Fordhook lima beans, garlic, onions, peppers, sweet and white potatoes, and tobacco. RESIDUES IN PLANTS AND SOIL With the trend to more stable, less volatile, and more persistent nematocides, the danger of excessive residues in the soil and plant is increased. Highly vola- tile nematocides usually present few residue problems, since a portion of the material passes out of the soil as vapor into the air and the remainder is quickly broken down or leached out, leaving only traces of degradation prod- ucts in the soil. Only occasional problems with residues have been found with EDB and DBCP. With these compounds, the uptake of bromine is a problem in certain crops, such as peanuts and citrus pulp, used for livestock feed. The burning quality of tobacco may be affected by the uptake of halogens when certain fumigants are used. Excessive chlorine can be controlled by reducing the amount of chlorine applied in the fertilizer. In those cases where bromine residues are a problem, they can be controlled by reduced use of bromine- containing chemicals. With increasing commercial use of stable compounds, the residue problem will become more serious. Long residual action may limit the use of some compounds that are highly toxic to nematodes. Chemical residues in plants grown in treated soil may be harmful or unpalatable to man or animals con- suming the plants. Isolated instances of "off taste" or "taint" in crops follow- ing the application of chemicals to soil are usually due to misuse, such as an overdose of chemical, or application when weather or soil conditions are un- favorable for escape or breakdown of the chemical. FACTORS INFLUENCING NEMATODE KILL IN SOIL Nematodes live in thin water films intimately associated with and surrounding soil particles. To be effective, nematocidal chemicals must penetrate and diffuse into pores or crevices in the soil to contact the nematodes and, in addi- tion, must penetrate the moisture films surrounding the nematodes. Highly

CONTROL BY CHEMICALS 135 water-soluble compounds are effective in penetrating these films but do not diffuse readily in soil. Rapid diffusion of the chemical through the soil occurs in the vapor phase rather than through the soil water. Many chemicals toxic to nematodes fail as nematocides because of limited penetration and diffusion or because of inactivation when applied to soil. As most nematocides in com- mercial use are volatile, their nematocidal activities in soil are influenced by factors such as soil type and condition, soil moisture and temperature, and chemical-application rate and depth. SOIL TYPE The diffusion of volatile compounds is definitely influenced by soil type. Clay particles or organic matter may adsorb the compound and reduce the dispersion of the material. In fine-textured clay soils, pore spaces are much smaller than those in sandy or sandy-loam soils. Such small pores are likely to be blocked by ex- cess moisture or compaction, resulting in noncontinuous passages into which vapors are unable to diffuse. As a consequence, fumigation may be incom- plete, especially if the nematocide is highly volatile or short-lived in the soil. Sandy soils, however, contain large pores that are less likely to be blocked by excess moisture or compaction; but a surface seal, necessary to prevent rapid loss of vapor, is more difficult with such coarse-textured soils. For effective nematode control, peat and other organic soils require two to three times the amount of nematocide needed for mineral soils. The high rates are necessary to compensate for the adsorption of the nematocides on the organic matter of the soil. SOIL CONDITION Soil preparation is very important for best results with volatile soil fumigants. Deep plowing or chiseling aids downward penetration of fumigant vapors. Where the soil has been tilled to the same depth by disking and plowing for many years, a compacted layer called a plow-sole layer results. This layer may be almost impervious to penetration by water, roots, and fumigants. If it is broken up by chisel or plow, the fumigant, depending on the dosage applied, is able to move down 2 to 6 feet. In addition to eliminating the plow-sole layer, the surface foot of soil should be well pulverized and smoothed before the chemical is applied. A drag or roller following the injection equipment will then effectively seal the surface and prevent the vapor from escaping too rapidly. The surface seal is

136 BASIC PRINCIPLES OF CONTROL important in increasing the concentration of the vapor in the surface inch or two of soil and thereby increasing nematode control in that zone as well as in the subsoil. The soil should not be disturbed for at least one week after treatment and for longer if deep penetration into the subsoil is desired. Since living nematodes are protected inside undecayed roots, crop residues should be incorporated into the soil and allowed to decompose before fumi- gant is applied. EDB, DBCP, and chloropicrin usually give poor control of nematodes inside plant residues, but fumigants containing 1,3-D are more effective in penetrating root tissue. Coarse residue or trash also can prevent an effective surface seal and create channels through which the chemical vapors escape. Alfalfa and corn stubble decrease effectiveness of fumigants unless several months are allowed between turning in the plant residue and applica- tion of the fumigant. Adsorption of the fumigant by undecayed organic matter in the soil also may decrease nematode kill by inhibiting complete diffusion of the chemical. SOIL MOISTURE AND TEMPERATURE Both soil moisture content and temperature are important to successful fumi- gation, especially of fine-textured and very-coarse-textured or sandy soils. In sandy-loam soils, these factors are not as critical. In general, most effective gas diffusion of nematocides occurs at a temperature of 18 to 24°C at an 8-inch depth in coarse-textured soil that is moist but is below field capacity in moisture content. If the moisture content is too high-approaching field capacity—a high proportion of the pore spaces are filled with water, and gas diffusion is retarded. Poor nematode control frequently results from fumiga- tion of fine-textured, cold, wet soil. With increased clay content, soil moisture and temperature conditions should be more favorable before fumigation is attempted. APPLICATION DEPTH The depth at which volatile fumigants are applied varies with vapor pressure, dosage, temperature, moisture, soil type, nematode species to be controlled, and depth of control desired. For minimum dosage rates under optimum soil conditions, application is generally made at a depth of 6 to 8 inches. If the application is too deep, the vapors will not reach the surface inch or two of soil; if it is too shallow, the vapors are lost through the soil surface.

CONTROL BY CHEMICALS 137 As dosage is increased, depth of application also should be increased. For example, 1,3-D at 20 gallons per acre should be injected at 8 inches, while 60 to 80 gallons per acre should be injected at 12 to 14 inches. Increased depth and rate of application are associated with high nematode kill to deep soil levels and little loss of vapors from the soil surface. If soil temperature is above 27°C and soil moisture is considerably below field capacity, depth of application should be increased and, to retain the vapors in the soil, the sur- face of the soil should be well compressed following application. In-the-row applications in beds should be injected at greater depth than flat or over-all applications. The usual practice is to inject to a depth of 12 to 13 inches in the bed. When cotton is planted in the bed after fumigation, the surface 2 to 3 inches of soil is pushed off by the planter. This removes the surface layer, which, due to the deep injection, may not have received an effec- tive dosage of fumigant. Recent studies indicate that, if injected at 18 to 22 inches in conjunction with deep tillage, the fumigant may be more effective than it would be with more-shallow injections. Where plow-sole layer or hard- pan occurs, deep tillage alone increases the growth of cotton. APPLICATION RATE The rate of chemical application is dependent on the properties of the particu- lar chemical and the crop to be grown. For annual crops, it is generally un- economical to apply a higher dosage of nonpersistent fumigant than that necessary to grow one profitable crop. For crops spaced wider than 2 feet, row application has been effective in many cases and may cost up to 50 per- cent less than over-all treatment. For perennial crops such as orchards and vineyards, recommended dosages are usually at least double those used for annual crops. A higher degree of control to a greater depth is needed for perennials than for annuals, because perennials are deep-rooted and grow for many years. Until compounds that could be used safely for treatment of living plants were developed, the need for highly effective preplant treatments was critical. With the advent of DBCP, which can be applied safely to the root zone of many living plants, nematodes on many perennial plants can be kept at low levels. The use of DBCP as a postplant treatment does not eliminate the need for preplant treat- ment, but, to keep the nematode population low, it should be utilized in con- junction with the preplant treatment. With present nematocides, eradication of nematode populations under field conditions is not practical.

138 BASIC PRINCIPLES OF CONTROL SEALING SOIL SURFACE AFTER APPLICATION Vapors of volatile soil fumigants may be lost rapidly through the soil surface if the material is not injected to a sufficient depth or if the surface of the soil is not well sealed. Vapor loss through the soil surface may be reduced by using various methods of surface-sealing, depending on the volatility of the com- pound, the type of application, and the result required. For highly volatile compounds such as chloropicrin and methyl bromide, a vapor-proof cover is necessary, especially if the material is used without dilution. The conventional method of applying methyl bromide is to release the vapor under a plastic film. The vapor is distributed over the soil surface under the cover and penetrates downward into the soil from several inches to several feet, depending on the dosage used. Chloropicrin may be applied by injection, but a seal is necessary. For best results with chloropicrin, a vapor-proof cover should be used, but a satisfactory seal can be obtained by sprinkling the soil surface immediately after injection with enough water to wet the soil to a depth of 0.5 inch. With less-volatile nematocides, such as 1,3-D, EDB, or DBCP, a roller or drag used immediately after application provides a satisfactory seal. A soil-surface seal is desirable following application of any volatile nema- tocide, regardless of soil type, temperature, moisture, or preparation. The seal improves nematode control in the surface inch of soil and, in addition, in- creases retention of vapors in the soil and penetration of vapors, both laterally and vertically. APPLICATION METHODS FORMULATIONS The method of applying a nematocide varies with the type of chemical. Some commercial nematocides can be formulated to suit the type of applicator available to the grower, but highly volatile compounds must be used as fumi- gants. The most common method of applying methyl bromide and chloropic- rin, which are gases under field conditions, is by injection, after which water is sprinkled on as a surface seal or the soil is covered with a vapor-proof film or cover. Equipment is available which efficiently injects these volatile com- pounds and covers the soil surface with polyethylene tarps in one operation. The gases may also be released directly under the cover. Ease in handling and effectiveness increase when these volatile materials are combined with other less volatile compounds, but a surface seal is still necessary.

CONTROL BY CHEMICALS 139 To reduce volatility and make handling easier, some volatile compounds are formulated as granules adsorbed on an inert carrier. Granules are not al- ways satisfactory, because the nematocide may be so tightly adsorbed to the carrier that the vapors cannot escape. In one case, 1,3-D was held so tightly that phytotoxicity resulted in two consecutive crops of sugar beets after a single application. A granular formulation of a chemical does not basically alter the method of application, except that the material can be metered into the soil with a fertilizer spreader rather than with a liquid injector. To obtain maximum nematode kill, the granules must be applied under the soil surface and allowed to diffuse from a point or band. Up to 50 percent of the activity is lost if the granules are spread on the soil surface and mixed into the soil. The lower the vapor pressure of the fumigant, the more successful it is in a granular formulation. Some of the carriers used are Attaclay (an attapulgite-type carrier), Hi-Sil (synthetic silicon dioxide), vermiculite, and carbon. Carbon is not satisfactory with some volatile compounds, because they are bound so tightly that the vapors are released too slowly to attain concentrations that provide satisfactory nematode control. Hi-Sil is reported to hold compounds tightly until the granules are moistened, at which time the fumigant is released. Additional work on granular formulations is needed, especially with the more volatile materials. Nonvolatile compounds are best used as granules or liquids that can be spread on the soil surface and then mixed into the soil by rototiller or disk harrow. Soil injection is not satisfactory for nonvolatile nematocides unless the toxicant is taken up by the roots, thus controlling nematodes by systemic activity. Granular formulations of nematocides can be mixed with fertilizer, or the nematocide can be adsorbed on the fertilizer granules. Although applying nematocides directly with fertilizer is a logical and economical method of application, it is not altogether satisfactory, because, to be most effective, the nematocide should usually be applied deeper and at an earlier date than the fertilizer. Combinations of nonvolatile, residual-type nematocides and fertilizers may prove satisfactory. However, such nematocides may require over-all application, with incorporation into the soil, to obtain effective nema- tode control, while the fertilizer may be used best when carefully applied in a specific location with respect to the roots of the plant. The same problems occur with combinations of nematocides and liquid fertilizers. Some materials, such as DBCP, are effective at such a low dosage rate that the equipment commonly available cannot be used. In such cases, the chemi- cal is diluted with solvent to increase the volume, or, when a suitable solvent is not available, an emulsifiable concentrate is used. Water can be used as a

140 BASIC PRINCIPLES OF CONTROL diluent with an emulsifiable concentrate, and the formulated material can be applied in irrigation water, by either flood or furrow. Nonvolatile compounds, which must be mixed with the soil to be effec- tive, may be formulated in a solvent or as an emulsion and sprayed on the soil surface before being mixed into the soil by disk or rototiller. The application of soluble or emulsifiable concentrates of nematocides in irrigation water has been attempted with a variety of compounds but is fea- sible with only a few. At this time, DBCP and Vapam are applied commercially in this way. The chemical may be metered into the irrigation water by gravity flow or through a centrifugal pump. The gravity-flow technique is successful if the nematocide is introduced into water with sufficient agitation for thorough mixing, such as a drop over a weir or head gate, or when the water comes from a pipeline with sufficient force for mixing. Pump application is needed when the material must be metered into very- slow-moving irrigation water. To obtain mixing, both the inlet and outlet hoses of the pump are placed in the stream of water. A portion of the water is pumped through the centrifugal pump and back into the stream. The chemical is metered into the inlet side of the pump, where it is mixed thoroughly with the water (Figure 50). Alternate-metering method with small dia. tubing Pipe fittings / Valve open Spraying-system orifice Syphon break meter with strainer \ Valve open Vent must be open when introducing into pump suction Union Mixing provided by centrifugal pump Treated-water discharge into [ditch or check Polyethylene tubing When sufficient agitation is present / meter directly into ditch FIGURE 50 Diagram of gravity-flow and centrifugal-pump application of DBCP in irrigation water. (Courtesy of Shell Development Company.)

CONTROL BY CHEMICALS 141 For sprinkler irrigation, the material may be metered into the pump or introduced into the sprinkler lines. The latter method requires some type of pressurized vessel with a pressure higher than that in the irrigation line in order to force the chemical into the system. Application of DBCP by sprinkler irrigation has not been as effective as application by flood irrigation. In areas where flood irrigation is not possible, sprinkler application is utilized to a limited extent, even though up to 80 per- cent of the material may be lost into the air as vapor. Less volatile materials could be more effectively used by sprinkler application, if sufficient penetra- tion into the soil could be achieved. Additional research on the effects of temperature, humidity, wind velocity, sunlight, and soil penetration, and on the application of chemicals in irrigation water is needed. APPLICATORS Injectors The first nematocidal chemicals used on a field scale were volatile liquids; consequently, the first applicators were adapted to apply these materials be- neath the soil surface to prevent vapor loss to the atmosphere. Commonly used applicators are hand injectors and shank or chisel injectors. Hand injectors are metering devices designed to apply a measured amount of material into the soil through a hollow tube or spike at a given depth. Several methods of metering have been used, but the displacement pump with a spring-loaded valve has been most successful. For small areas such as home gardens, planting sites for a limited number of trees, and experimental plots, the hand injector is very useful. The area to be treated is first marked off into squares varying from 10 to 18 inches in size, depending on chemical, soil type, temperature, and moisture. A measured amount of chemical is then injected at the intersection of each of the lines, but, for best coverage, injections should be made both at the intersections and at a point halfway between in- tersections on alternate rows. For most of the volatile compounds, 12-inch spacing is satisfactory. As the dosage rate is increased, the spacing also may be increased. The injection hole should be sealed with the foot immediately after the spike is withdrawn (Figure 51). For large-scale applications, the tractor-drawn or tractor-mounted shank or chisel applicator is used (Figure 52). With this type of applicator, the chemical is injected into the soil through tubes fastened to the back edge of the chisel or shank. The material is usually metered through orifices from a pressurized manifold. The manifold may be pressurized from a gear pump mounted on

142 BASIC PRINCIPLES OF CONTROL FIGURE 51 Application of volatile soil fumigants by hand injector. (Courtesy of Shell Development Company.) FIGURE 52 Tractor-mounted chisel-injection applicator. Cultipacker behind for sealing. (Courtesy of Shell Development Company.) the power takeoff of the tractor or by compressed gas, such as nitrogen, car- bon dioxide, or air. If compressed gas is used, a tank, properly constructed to withstand the pressures required for accurate metering of the nematocide, must also be used. Shipping drums or similar containers are not suitable. A simple, inexpensive method of metering liquid nematocides is by gravity flow through metering orifices or coils from a constant-head container. The constant-head container is a sealed vessel with the liquid outlet and air inlet located at the same level, near the bottom of the drum (Figure 50). By this

CONTROL BY CHEMICALS 143 arrangement, air is taken into the drum to displace the liquid removed. By in- troducing the air at the bottom of the container, a varying vacuum is produced over the liquid to maintain atmospheric pressure at the liquid outlet. There- fore, regardless of depth of liquid in the drum, the flow is constant. The pres- sure head on the orifice is dependent only on the vertical distance between the outlet and the metering orifice ("h," Figure 50). For this type of metering, the shipping drum is a satisfactory container. The gravity flowmeter may be used with the chisel method of application (Figure 53). To maintain a uniform rate of application with these devices, the tractor must be driven at a constant speed. More recently, land-driven dis- placement pumps, which eliminate the need for constant speed and are used FIGURE 5 3 Gravity-flow equipment for chisel injection. Copper- tubing coils meter the chemical into the soil. (Courtesy of Shell De- velopment Company.)

144 BASIC PRINCIPLES OF CONTROL with metering orifices for multiple-chisel application, have been devised. The pump is geared to the ground so that a given amount of material is passed through the pump per linear foot of travel, regardless of the speed of the tractor. Plow Application Some growers apply nematocides to small fields with a gravity-flow applicator mounted on a moldboard plow. The chemical is metered in the same manner as for chisel injection, but it is introduced directly in front of the plow in the bottom of the furrow and is covered immediately as the soil is plowed. This method may be used for a single plow or a gang of plows, providing the chem- ical is applied in the furrow in front of each plow (Figure 54). To seal the chemical in the soil, the soil should be harrowed and smoothed immediately following application. Nonvolatile materials must usually be incorporated into the soil by the use of either a disk harrow or a rototiller. Neither type of equipment will mix deeper than 4 to 6 inches, but water-soluble compounds may be carried deeper by rain or irrigation. With either method of mixing, the material must be distributed evenly over the soil surface. This can be accomplished with either a sprayer or a fertilizer distributor, depending on the formulation of the chemical. For liquid application, any pressure applicator with spray nozzles mounted on a boom serving as the metering device is suitable. For granular application, any spreader that distributes granules evenly over the soil surface is satisfactory. TYPES OF TREATMENT PREPLAN! TREATMENT As most of the commercial nematocides are phytotoxic, they must be applied to the soil before seeds are planted or plants are set. Over-all, row, or planting- site applications can be used, depending on the type of planting. In over-all (broadcast) application, the entire area is treated, usually by chisel application at 12- to 14-inch spacing or, if the compound is nonvolatile, by spreading it uniformly over the soil surface and mixing it with the soil by disk or rototiller. With high dosage rates, to obtain deep penetration, down to 8 feet, the chisels may be as far as 18 inches apart. Heavy equipment is necessary when the ap- plication depth is more than 14 inches. The 12-inch spacing and 8-inch depth of application are used for low dosage rates when controlling nematodes on fast-growing annual crops such as carrot, lettuce, and squash.

CONTROL BY CHEMICALS 145 FIGURE 54 Gravity-flow equipment for application by plow. Chem- ical applied directly in front of plow. (Courtesy of Shell Development Company.) An important way to reduce the cost of soil fumigation is by row rather than over-all application. For crops grown in rows more than 2 feet apart, row application is widely practiced. Depending on spacing, the cost of chemical is from one half to one tenth of that for over-all application. Not only is there a saving in the amount of chemical needed, but also a reduction in application cost, because the chemical can usually be applied during land preparation. Crops grown on beds can be treated when the beds are formed. For bed or row application, either one or two chisels are used per bed or row. If one

146 BASIC PRINCIPLES OF CONTROL chisel is used, which is common practice, the chemical dosage must be high enough to kill most nematodes in at least a 16- to 18-inch band of soil. The tomato root system in Figure 55 shows root-knot nematode (Meloidogyne spp.) galling of roots that have extended beyond the treated strip. An illustra- tion of reduction in dosage by row placement is the fumigation for control of root knot in cotton fields: 20 gallons per acre of 1,3-D-type nematocide is required for an over-all treatment, but with 38-inch row-spacing, 9 gallons per acre, a reduction of over 50 percent, is adequate. As the row-spacing is in- creased, the saving in chemical is correspondingly increased. For tomatoes, with rows 5 feet apart, a dosage of 5 to 6 gallons per acre of 1,3-D provides effective nematode control. A strip or row preplant treatment can also be used for some perennial crops, such as peach, grape, citrus, and walnut, saving as much as 50 percent of the chemical as compared with over-all application. A disadvantage of this procedure is that recontamination from cultivation or irrigation is almost cer- tain. Dosages of 40 to 100 gallons per acre of 1,3-D are required for deep- rooted perennials, but, where this is not practical, a high dosage applied in the planting rows is preferable to low dosage over the entire area. For example, it is better to treat the planting area, or one half of the total area, at the rate of 40 gallons per acre of 1,3-D rather than the total area at 20 gallons per acre. Despite the higher cost, the trend is toward over-all treatment on perennials rather than treatment of the planting strip only. FIGURE 55 Tomato root from plant grown in row-fumigated soil. Galled roots have grown out of treated zone. (Courtesy of the De- partment of Nematology, University of California, Riverside.)

CONTROL BY CHEMICALS 147 The same principles apply to planting-site application as to row or strip applications. The treatment of sites is complicated mechanically by the need to interrupt application between planting sites. However, this treatment, more adaptable to hand injectors than to power equipment, is commonly used for replant problems in established orchards or vineyards. Experimentally, it has been demonstrated to be feasible with wide-spaced annual crops such as melons. For melons, a treating site of 2 square feet is sufficient, while tree crops require a minimum area of 6 to 8 feet. Although the planting-site method reduces the amount of chemical used, the saving in chemical may be more than offset by the increased cost of application. TREATMENT AT PLANTING TIME Most commercial nematocides are too phytotoxic for use immediately before a crop is planted. Some materials are tolerated at nematocidal dosages by cer- tain plants, but none is completely nonphytotoxic to all plants. Peach is quite tolerant of DBCP and can be planted immediately following application, with- out phytotoxicity. Although cotton can be planted immediately following application of DBCP without serious injury, it is advisable to wait at least one week. Pineapple can be planted immediately following application of 1,3-D; however, it is not because the pineapple roots are exceptionally tolerant but because the fleshy slips do not start to put out new roots for a week or more following planting. In general, it is necessary or at least desirable to delay planting until the chemical has dissipated from the soil. A waiting period is beneficial even with relatively nonphytotoxic compounds, because nematode control is more effec- tive when planting is delayed than when planting immediately follows treat- ments. Evidently, in the latter situation, some of the larvae are able to invade roots before the chemical has had time to act. When the soil is wet or cold, phytotoxicity may result after an otherwise adequate waiting period, because volatilization, diffusion, degradation, and escape of the chemical are retarded. Nitrification may be depressed under these conditions, resulting in the accumulation of toxic levels of ammonia. POSTPLANT TREATMENT The application of chemicals to established plants for control of plant- parasitic nematodes, especially root parasites, is a recent development, used mostly with perennial crops such as trees, vines, and certain ornamentals. In- creased yields from postplant treatments usually occur only after the plant

148 BASIC PRINCIPLES OF CONTROL has had time to recover from nematode damage; thus, this treatment is more applicable to long-lived crops, such as those in orchards and vineyards, than to the short-lived annuals. As mentioned previously, DBCP is the compound used most widely in postplant treatments. Several other chemicals, which have been tested on turf and other living plants with varying success, are still in the experimental stage. Results of experiments with new chemicals on annual crops such as melons, cucumbers, and cotton showed yield increases, but preplant treatments with the same compound or standard commercial nematocides gave superior results. Two effective methods for applying nematocides to established plants in- clude side-dressing with a chisel applicator or hand injector and application in irrigation water. If the land is level and the soil type is satisfactory, application by irrigation is preferred, because it is easy, requires little equipment, and coverage is uniform, especially close to vines or trees. In nonirrigated areas or areas that are not level enough to flood-irrigate, the chisel method of applica- tion is used. Disadvantages of this method are the danger of root injury by the chisels and the difficulty of injecting close to the base of the tree or vine (Figure 56). More care is also necessary in preparing the land for injection than for irrigation application. Sprinkler irrigation has been used in areas where flood irrigation is not possible, but results are erratic because of ex- cessive loss, of the chemical to the atmosphere during application. FIGURE 56 Side-dress of citrus by chisel injection of DBCP for con- trol of citrus nematode (Tylenchulus semipenetrans). (Courtesy of the Department of Nematology, University of California, Riverside.)

CONTROL BY CHEMICALS 149 BARE-ROOT DIP Considerable effort has been directed toward finding a chemical that would control nematodes on bare-rooted nursery stock as a dip without the need for high temperatures. Although eradication has not been achieved, three mate- rials are reported to be highly effective: Zinophos, Dasanit (0,0-diethyl 0[-p-(methylsulfinyl) phenyl] phosphorodithioate}, and SD 4965 (1,6- hexanedithioldiacetate). The two phosphate materials used at 600 ppm as a 30-minute dip are re- ported to achieve near-eradication on some ornamentals. Extreme care must be exercised in handling these compounds, because they are highly toxic to mammals. SD 4965 is relatively nontoxic to mammals, but a 24-hour dip is recommended, and, even after such a long treatment, eradication is not always achieved. It is doubtful whether a material can be found that will be effective on all plants and toxic to all stages of all species of plant-parasitic nematodes and that will also readily penetrate plant tissue, roots, bulbs, rhizomes, and corms without injury to the plant. The most difficult nematode species to control are the migratory endoparasites (Pratylenchus spp. and Radopholus similis), the sedentary endoparasites (Meloidogyne spp.), bulb and stem nematodes (Ditylenchus dipsaci), and bud and leaf nematodes (Aphelenchoides spp.). These forms, present as larvae, eggs, or adults, may be deeply embedded in plant tissue. The surface feeders, such as Xiphinema spp., Trichodorus spp., and Belonolaimus spp., are easier to control, since they are present on the surface of the plant tissue or in the soil surrounding the roots. The most promising types of compounds are organophosphates or carba- mates, which appear to be readily taken up by the plant tissue in nematocidal dosages. This systemic-type material may be more specific in activity than a nonsystemic type such as SD 4965, but, for specific plants and nematode species, it may be more effective and less phytotoxic than a nonsystemic type. It appears that no one compound will be effective on all plants and nematode species, but several specific types or combinations of chemicals will be necessary in some situations. SEED TREATMENT There is no commercially available nematocidal chemical that is effective for treating seeds; such a chemical must be systemic, that is, taken into the root and distributed throughout the root system. For bud and leaf nematodes, the chemical must be translocated to aboveground plant parts. The mode of action may be as either a toxicant or a repellent to the attacking nematodes.

150 BASIC PRINCIPLES OF CONTROL Theoretically, repellency could result either from the chemical itself or from an alteration in plant metabolism, making the plant less attractive to the nematodes. It does not appear possible to apply enough nonsystemic material to the seed to protect the root zone of the plant for an extended time, because the root system soon grows beyond the treated zone. A volatile compound capa- ble of giving protection in the root zone could not be effective at the shallow depth of application necessary for a seed treatment. Volatile compounds must be applied 6 to 8 inches deep, while seed is usually planted less than 2 inches deep. Compounds effective as seed treatments would probably also be effective in hopper-box or in-the-row applications with the seed, since the mode of action would probably be identical. BASIC STUDIES ON NEMATOCIDES The limited information available on the mode of action of nematocides is supplemented by knowledge of insecticidal and fungicidal action that may apply to nematocides. In discussing the mode of action of nematocides, two different but re- lated phenomena must be considered. One is movement of the nematocide to the site of action. Movement to the site of action can be further separated into two distinct areas: movement in or through the medium harboring the nematode and penetration into or uptake by the nematode. MOVEMENT TO SITE OF ACTION IN SOIL Most nematodes of agricultural importance spend at least part of their life cycles in the moisture films surrounding soil particles. Thus, if a soil-contact nematocide is to function, it must be capable of passing through or persisting in the soil without being either adsorbed or degraded biologically or physically until it has had time to act on the nematodes. The nematocide must be suf- ficiently water-soluble to diffuse into the moisture film surrounding the nematode. The widely used nematocides 1,3-D, EDB, and DBCP possess these characteristics. The water solubilities of 1,3-D, EDB, and DBCP are approxi- mately 1,000; 2,700; and 930 ppm, respectively. For comparison, concentra- tions of 10, 5, and 2.5 ppm kill 50 percent of root-knot larvae in in vitro tests (LDso). These chemicals are sufficiently water-soluble to accumulate to lethal dosages in soil solution. Systemic nematocides, such as certain organo- phosphorus compounds, are taken up by plant roots and move to the site of

CONTROL BY CHEMICALS 151 action in the roots or in the aboveground parts of plants. In other cases, the nematocides are sprayed on the foliage to control foliar parasites. Sometimes nematode-infected plant parts are immersed in an aqueous solution of the nematocide, which then diffuses into the tissue. PENETRATION INTO THE NEMATODE Three principal layers can usually be distinguished in the cuticle of a nema- tode: the cortex, the matrix, and fiber. Some workers believe the cuticle is secreted collagen and the outer cortical layer is tanned protein. Others be- lieve the cuticle is not secreted but is condensed outer layers of the hypo- dermis. The outermost layer of the cuticle is a thin, thermolabile, lipoid mem- brane, which is believed to be the main barrier to penetration of drugs, stains, and other chemicals. The nematode cuticle occupies the same position toward nematocides as the insect cuticle does toward insecticides; thus, closer study of its chemical and physical properties might be rewarding with regard to mode of entry of nematocide into the nematode. Both water and lipoid solubility appear to be necessary characteristics of nematocides. It has been indicated that nematocidal properties of chlorinated hydrocarbons could be correlated with solvency of beeswax and cholesterol. The implication was that these properties were necessary for the nematocide to penetrate the nematode cuticle and cellular membranes. Penetration of alkyl halide nematocides, such as EDB and DBCP, is rela- tively fast when nematodes are exposed to aqueous solutions of these chemi- cals. A dynamic equilibrium can be established between the internal and ex- ternal concentration of the nematocide in as short a period as 15 to 30 minutes, depending on the specific nematocide and the nematode species. The internal concentration of the nematocide may be from 2.5 to 20 times the external concentration. The rate of accumulation of the nematocide is a function of the rate of penetration and the rate of release of the nematocide. At equilibrium, the rate of release equals the rate of penetration. Penetration appears to occur, for the most part, directly through the cuticle rather than through natural openings such as the amphids, mouth, anus, vulva, and ex- cretory pore. MODE OF ACTION OF THE NEMATOCIDE ON THE NEMATODE Halogenated Hydrocarbons Some possible mechanisms of the action of organic halide nematocides have been proposed: compounds that act by narcosis, that is, reversible inhibition,

152 BASIC PRINCIPLES OF CONTROL a physical mechanism; those that act by some chemical mechanism, that is, irreversible inhibition; and other compounds whose toxicities are a result of both mechanisms. One theory has been advanced to explain some of the relationships be- tween chemical structure and toxicity of organic halides. Those compounds that are highly reactive in bimolecular nucleophilic displacement reactions are the most toxic, and those having low reactivity in this reaction are the least toxic. Thus, toxicity is believed caused primarily by the inhibition of some essential enzyme system in the nematode by chemical reaction of a reactive halide with some required nucleophilic (basic) center, such as sulfhydryl, amino, or hydroxyl groups present in enzymes. The reactive halide may also react chemically with biologically important proteins or peptides or some regulator of cellular metabolism, inhibiting its function. A schematic repre- sentation of such a reaction follows: Protein or _Enzyme_ + R:X- Protein or Enzyme :R + \X (R:X = organic halide) Toxicity of organic halides parallels the ease of displacement of the halo- gens in the following order: I > Br > Cl. Toxicity to nematodes generally decreases with alkyl substitution, which is consistent with their rate of reac- tion in bimolecular nucleophilic substitution reactions. The 2,3-unsaturated allylic halides are usually more toxic than saturated forms, which correlates with their reactivity in both unimolecular and bimolecular nucleophilic substitution reactions. Because of the rapid rate of solvolysis in various solvent systems, including water, some organic halide nematocides show a lower order of toxicity than is consistent with their rate of reaction in bimolecular nucleophilic substitu- tion reactions. Fortunately, allyl alcohol and some of the halogen-substituted allylic alcohols are very good nematocides; therefore, solvolysis is not always detrimental. Another possible mode of intoxication of nematodes by alkyl halides may involve the interaction of halo-organic nematocides with an iron center in the respiratory chain of the nematode. This hypothesis is supported by the rapid oxidation of dilute solutions of Fe11 porphyrins to the corresponding Fe111 halide complexes (hemino) by alkyl halides such as DBCP and 1,3-D. Organophosphates A number of organic phosphate insecticides and nematocides are highly toxic to nematodes when applied to soil, roots, bulbs, or foliage. However, because

CONTROL BY CHEMICALS 153 of one or more factors, such as restricted movement in soil, selectivity, per- sistent residues, phytotoxicity, or high cost, they have not gained widespread use. An acetylcholine-splitting enzyme, sensitive to cholinesterase inhibitors, has been demonstrated in plant-parasitic nematodes. Histochemical techniques were utilized to demonstrate that Thimet and a glycoside from asparagus in- activated the enzymatic hydrolysis of acetylthiocholine. Positive reactions, indicating the presence of cholinesterase, were obtained in nematodes of several plant-parasitic genera. This is not complete evidence for a cholinergjc system in nematodes or for its inactivation by organic phosphate nematocides, but it is a strong indication that this is one mode of action of these com- pounds in nematodes. Certain other nematocides, such as the aliphatic carbamoyloxime, 2-methyl- 2-(methylthio)propionaldehyde-0-(methylcarbamoyl)oxime, may also kill nematodes by attacking the nervous system, since they are known to be potent inhibitors of acetylcholinesterase in man, animals, and insects. Carbamates Carbamate nematocides, such as Vapam, produce toxic volatile decomposition products such as methylisothiocyanate. The toxicity is believed to be caused by the chemical inactivation of biochemically important thiol groups within cells. The thiocyanate apparently reacts with enzymes containing free sulf- hydryl groups. Many organic compounds, including certain fatty acids, thiophenes, and a terthienyl compound from marigold, have been reported as toxic to plant- parasitic nematodes. In addition, several chemicals, including some of the organic phosphates, selenium compounds, maleic hydrazide, sodium fluoro- acetate and fluoroacetamide, act as systemic nematocides in plants. As with the commercial nematocides and other biocides, there is little experimental evidence concerning their mode of action as nematocides. Additional research on nematode physiology and biochemistry as related to toxicology and mode of action of nematocides is needed. SELECTIVITY OF NEMATOCIDES Field and laboratory data suggest that nematocidal chemicals display con- siderable selective toxicity against nematodes. Unfortunately, most of the data on this selectivity comes from field plots and consequently is confounded with many important physical and biological variables affecting the efficacy of nematocides. Differences occur in toxicity to various stages in the life cycle of a given nematode species and to species of various nematode genera.

154 BASIC PRINCIPLES OF CONTROL The common nematocides EDB, DBCP, and 1,3-D differ in their ovicidal properties. EDB and DBCP apparently are not as effective as 1,3-D in killing eggs of nematodes, and higher concentrations of all three nematocides are re- quired to kill eggs than to kill larvae. In the case of DBCP, 200 times as much chemical is required to kill eggs in egg masses of Meloidogyne javanica as compared with free second-stage larvae. Eggs within cysts of the sugar-beet nematode (Heteroder a schachtii) are highly resistant to nematocides. EDB and DBCP are more toxic to the root-knot nematode and the citrus nematode than to the lesion nematode (Pratylenchus scribneri) and the stubby root nematode (Trichodorus christiei). As many as three- to fivefold increases in concentration are required to give similar levels of kill of the latter two nematodes as the former two. Propargyl bromide is about equally toxic to all four nematodes and considerably more toxic than 1,3-D, EDB, and DBCP. Cysts and larvae of the sugar-beet nematode are killed by lower dosages of 1,3-D than of EDB or DBCP. Two new chemicals, the organic phosphate nematocide, Nellite (phenyl jV.yVl-dimethyl phosphorodiamidate), and SD 7727 (2,4-dichlorophenyl methanesulfonate), are reported to have a high degree of selectivity for root- knot nematodes. Both are effective in controlling root-knot nematodes in soils at concentrations below 5 ppm, but neither is effective against numerous other plant-parasitic nematode species tested. SD 7727 is particularly interest- ing in that its mode of action appears to be inhibition of infection rather than direct toxicity. The general trend is toward development of selective nematocides. The reasons for such selectivity by certain compounds are little known and offer an interesting area for research. DEVELOPMENT OF RESISTANCE TO NEMATOCIDES The potential development of resistance of nematodes to the present nema- tocides is of continual concern to nematologists. One hears reports that nema- todes in a given field are more difficult to control with a certain nematocide than are nematodes in comparable fields, but no experimental evidence exists demonstrating that nematodes have actually developed resistance to the commonly used nematocides. That nematodes have not developed resistance is not too surprising considering the relatively low selection pressure to which most nematode populations are exposed. Most fields are treated only once a year, and nematocides have been in general use in most areas only since 1950. Just a few fields have been treated annually since 1943. In addition, many fields are row-treated, or treated with minimal dosages, so that the population that persists is not likely to consist merely of resistant individuals, if such

CONTROL BY CHEMICALS 155 exist. Well-planned laboratory and field tests are needed to determine whether some of the important plant-parasitic nematodes can develop resistance to nematocides. FUTURE RESEARCH NEEDS Despite the outstanding progress in control of plant-parasitic nematodes by chemicals, additional research is needed. Very little is known concerning the mode of action of the commercial nematocides. Although such information would probably not increase the use of these compounds, it might help in the development of improved materials and in understanding the susceptibility of nematode species and their stages of development to nematocides. The fate of volatile chemicals applied to the soil is not well known. Such materials spread in all directions-up, down, and laterally. Do they break down physically, microbiologically, or chemically? Do the intact materials leave the rhizosphere by leaching or by gaseous diffusion? Some of the new chemicals are quite specific. Are they directly specific for certain nematode species or indirectly through the host plant, such as by repellent action? Can nematodes become resistant to nematocides? How do nematocides move in water? Many field observations have been made, but carefully planned, well-controlled tests are needed. Such informa- tion would be especially helpful in the application of chemicals in irrigation water, either by flood or sprinkler. How do nematocides move in plants? It is agreed that a systemic material applied to the foliage of plants and translocated downward to control nema- todes in the roots is desirable. Do nematocides follow patterns that have been worked out for insecticides and herbicides? Is it possible for a nematocide to act in this manner? Does the chemical need to be toxic to the nematode when it reaches the root, or can it be merely repellent, or perhaps change the metabo- lism of the plant, making it immune? Techniques for evaluating these actions are desirable. Cheap, accurate, dependable application equipment, both for liquids and granules, is needed. In many areas, the development of nematocide usage has been delayed by lack of suitable equipment. Improved equipment for injecting volatile chemicals into the root zone of established plants, without injuring the root system, is desirable. Probe or spike injection, high-velocity liquid injection (Spitnik), and water injection under pressure are examples of methods that are under trial or that have been suggested.

156 BASIC PRINCIPLES OF CONTROL BIBLIOGRAPHY Allen, M. W., and D. J. Raski. 1950. The effect of soil type on the dispersion of soil fumigants. Phytopathology 40:1043-1053. Carter, W. 1943. A promising new soil amendment and disinfectant. Science 97(2521): 383-384. Castro, C. E. 1964. The rapid oxidation of iron (II) porphyrins by alkyl halides. A pos- sible mode of intoxication of organisms by alkyl halides. J. Amer. Chem. Soc. 86:2310-2311. Chitwood, B. G. 1952. Nematocidal action of halogenated hydrocarbons. Advan. Chem. Ser. 7:91-99. Christie, J. R. 1945. Some preliminary tests to determine the efficacy of certain sub- stances when used as soil fumigants to control the root-knot nematode, Heterodera marioni (Cornu) Goodey. Proc. Helminthol. Soc. Wash. 12(1):14-19. Good, J. M., and A. L. Taylor. 1965. Chemical control of plant-parasitic nematodes. U.S. Department of Agriculture Handbook No. 286. Goring, C. A. I. 1957. Factors influencing diffusion and nematode control by soil fumi- gants. The Dow Chemical Company ACD. Information Bulletin No. 110. Goring, C. A. I., and C. R. Youngson. 1957. Factors influencing nematode control by ethylene dibromide in soil. Soil Sci. 83(5):377-389. Ichikawa, S. T. 1956. Nematode control versus application depths of Nemagon. Phyto- pathology 46:637. (Abstract) Lear, B., and D. J. Raski. 1962. Survival of root-knot nematodes in excised grape roots in moist soil fumigated with ethylene dibromide. Phytopathology 52:1309-1310. Moje, W. 1960. The chemistry and nematocidal activity of organic halides. Advan. Pest Contr. Res. 3:181-217. Rohde, R. A. 1960. Acetylcholinesterase in plant-parasitic nematodes and an anticholin- esterase from asparagus. Proc. Helminthol. Soc. Wash. 27:121-123. Stromberg, L., L. W. Carter, J. R. Stockton, and G. A. Paxman. 1965. Placement of fumigant affects root knot control. California Farmer, February 6, 1965, p. 20.

CHAPTER 14 Evaluation and Selection of Control Measures A nematode disease problem can usually be solved in a number of different ways. The research nematologist, agricultural specialist, and farmer are faced with the evaluation of the disease problem. From the various control methods available, they must select the method that is the most effective, most economi- cal, or both. The method selected will depend on the nematode species, the host plant, the environmental situation, the cash value of the crop, and the relative cost of available control methods. INTEGRATION OF CONTROL MEASURES In most cases, practical control of a nematode disease involves integration of several diverse control measures. Some nematode diseases can be prevented merely by using nematode-free seed or vegetative propagating materials. For example, the disease caused by the wheat nematode (Anguina tritici) is pre- vented by planting clean seed in land that has not been planted to wheat for at least one year. The garlic disease caused by the stem nematode (Ditylen- chus dipsacf) can be prevented by planting nematode-free garlic cloves in clean soil. The disease of banana caused by the burrowing nematode (Ra- dopholus similis) can be controlled in banana plantations by eradicating the nematode from the rhizomes used to propagate bananas and planting this seed in soil that is free of the nematode. New citrus orchards can be kept free of either the citrus nematode (Tylenchulus semipenetrans) or the burrowing nematode by using trees produced in clean nursery soils and planting in orchard sites free of these nematodes. 157

158 BASIC PRINCIPLES OF CONTROL The application of a preplan! nematocide is often all that is required to control a nematode disease effectively. For example, diseases of a number of annual crops caused by the root-knot nematodes (Meloidogyne spp.) can be controlled by preplant soil fumigation. Several procedures, however, are necessary to control nematode diseases on a number of perennial and annual crops propagated from vegetative materials such as roots, bulbs, corms, rhizomes, slips, and transplants. The propagating stock and soil must be rela- tively free of nematodes. For perennials, such as tree and vine crops, the com- bination of nursery propagation of nematode-free plants and preplant soil fumigation is required when the soil is infested. In The Netherlands, the pro- duction of bulbs requires the use of treated bulbs in fields that have had care- fully controlled rotations. In addition, the field must be surveyed prior to planting and must be free of the golden nematode (Heterodera rostochiensis) to prevent contamination of the bulbs by infested soil at harvest. Control of root-knot damage to sweet potatoes involves at least three phases of the pro- duction: first, the selection of seed roots that are either free of nematodes or freed of nematodes by hot-water or dry-heat treatments; next, the placing of the seed roots into beds of sand or coarse-textured soil that either is nematode- free or, if infested, is preplant fumigated, preferably under tarps, or steamed to eliminate the nematodes; and, finally, the transplanting of the clean "slips" into nematode-free soil or preplant-fumigated soil. With many nematode problems, there is a tendency to rely too heavily on only one method of control. More research is needed on utilization and inte- gration of nematode control measures. EFFECTIVENESS AND ECONOMICS The ideal way to control a nematode disease is to eradicate the nematode, or nematodes, involved. This has been accomplished only in very limited areas such as greenhouses or propagating beds. With the soil fumigants in use today, it is not feasible to eradicate nematodes that are distributed throughout the soil mass to a depth of 12 to 15 feet, such as root-knot nematodes on grape or burrowing nematode on citrus. In such cases, preplanting fumigation is di- rected at reducing the nematode population density to well below the tolerance level of young transplanted trees and vines. However, eradication of nema- todes with present-day preplant soil fumigants is more likely for those nema- todes distributed in the surface soil, such as the potato rot nematode (Dity- lenchus destructor). Large sums of money are spent for nematocides to limit spread and pos- sibly achieve eradication of incipient infestations of introduced nematode pests that cause severe crop damage and are difficult to control by other means.

EVALUATION AND SELECTION OF CONTROL MEASURES 159 Such control involves high nematocide dosage rates, gas-proof tarps, and re- peated treatment. Funds expended for this purpose are justified because of the potential crop loss if noninfested acreage becomes infested. The actual cost of restoring the infested acreage to a productive level is not involved. On a more restricted basis, the same cost-effectiveness criteria can be ap- plied to nurseries. With the recent advent of quarantine laws requiring close inspection of nursery plants moving in inter- and intrastate commerce, nema- tode problems have become much more acute for nurserymen. In some cases, nematocide at a cost in excess of $200 per acre is applied to nursery soils; the expenditure is justified, since noninfected nursery plants can generally move freely in the trade. The use of nematocides for possible eradication of nematodes in nurseries has received considerable attention. New chemicals or combinations of chemi- cals for testing are continually available. New application equipment, such as mechanized tarp layers, has been developed. Research to determine which pre- plant nematocide to use and how to use it most effectively should be inte- grated with studies on the use of postplant nematocides designed to keep nematode populations below economic levels on living plants. Further studies on the detection of nematodes at very low population densities also are needed. This would assist in quarantine work and in the evaluation of the ef- fectiveness of control practices. In some cases, treating soil with high dosages of nematocides before plant- ing perennial crops is justified purely on the basis of increased plant growth and production. An initial cost of $250 per acre for preplant treatment of citrus soil with 1,3-D (1,3-dichloropropene) can be less than the increased value of fruit in the first three years of production. The investment of $250 per acre for soil fumigation is not unreasonable for a crop such as citrus, for which the investment in land and trees may exceed $4,000 per acre. The effectiveness of preplant soil fumigation of soils planted to perennial crops has been judged on the basis of how closely nematode control approxi- mates eradication. This reflected the long-term nature of the crop, the inability to rotate, and the lack of an effective nematocide that could be used for postplant treatment. The development of DBCP (l,2-dibromo-3-chloropropane), which is effective in controlling a limited number of nematode species around the roots of living plants, changes this situation somewhat and points the way for additional research. A critical need remains for nematocides to use in postplant treatments to kill endoparasitic nematodes in the roots of perennial crops. Such chemicals would further reduce the necessity of striving for eradi- cation, with its high cost in preplant treatments for perennial crops. After soil application, such a chemical would enter roots from the soil, or, after foliar application, it would move from the foliage to the roots. Various com- binations of nematocide treatments could then be utilized to control all kinds of nematodes that parasitize plants.

160 BASIC PRINCIPLES OF CONTROL The economic return from soil fumigation is an important consideration. A compilation of reports from the United States compares crop yields on plots treated with nematocides with yields on untreated plots. In 853 comparisons involving seven crops, an average increase in yield of 87 percent resulted from nematocide application. Yield increases for crops for which more than 25 comparisons were available are shown in Table 1. Although the evidence does not support the conclusion that differences were solely the result of nematode control, it is evident that nematocides have a favorable influence on the yield of several crops in many parts of the United States. While these data provide strong circumstantial evidence that nematodes severely reduce crop yields, they indicate little about the economic returns from fumigation. An increase of 100 percent in the yield of sugar beets may not result in a net profit to the grower, whereas an increase of 13 percent in tobacco may be profitable. In California, yields of sugar beets are increased consistently when soil infested with the sugar-beet nematode (Heterodera schachtii) is fumigated with nematocides containing 1,3-D; but if yields are increased from 9 to 18 tons per acre, no profit is returned to the grower, be- cause 18 tons per acre are required to pay production costs. Thus, although fumigation is successful from the standpoint of nematode control and in- creased sugar-beet yields, it is generally not considered economically successful and is not recommended. In other states, such as Colorado and Utah, soil fumigation consistently results in profits to sugar-beet growers and is recom- mended. In California, soil fumigation for root-knot nematode control on sugar beets in the San Joaquin Valley is recommended, because the nematode drastically reduces the tonnage of roots produced and also affects the quality of the root and, consequently, the ease with which sugar is extracted. As another example, the average yield of cotton in the United States is about one bale per acre. Yet, in Arizona and California, soils producing two to three bales per acre respond to soil fumigation. When the soil is sandy loam and root-knot nematodes are prevalent, soil fumigation consistently improves yields. Nematodes are the primary limiting factor to plant growth in these TABLE 1 Yield Increase Following Soil Application of a Nematocide Crop Plant Increase (%) Lima bean 35 Cotton 91 Soybean 126 Sugar beet 175 Tobacco 13 Tomato 73

EVALUATION AND SELECTION OF CONTROL MEASURES 161 fields, since other crop inputs, including soil moisture, are rigidly controlled. On farms with large acreages, an increased return of $50 per acre is important. Shortly after the introduction of such successful nematocides as 1,3-D and EDB (ethylene dibromide), it was suggested that the increase in crop value compared with the cost of fumigation should be a ratio of 4 to 1. Actually, no generalized practical formula is applicable: every nematode-crop inter- action must be analyzed with respect to its individual geographic and economic circumstances. Data accumulated over a 20-year period on nematode prob- lems and soil-fumigation results on a certain crop in a specific geographic location provide much better criteria for making fumigation recommendations than do arbitrary figures on returns per dollar invested in nematocides. Root crops such as carrots, sweet potatoes, table beets, and white potatoes are markedly reduced in quality and in market acceptance by diseases caused by root-knot nematodes, even though the crop yield may not be materially affected. Cash returns on a crop of sweet potatoes are increased as much as $300 per acre from an expenditure of only $30 per acre for soil fumigation. This results from an increase in the pack-out of higher grades without any significant increase in total production. In Georgia, control of the lesion nematode (Pratylenchus brachyurus) on peanuts by soil fumigation increases the yield of high-grade peanuts. Postplant treatment of Valencia and navel oranges with DBCP significantly increases the percent of larger fruit, thereby increasing profits. At present, quality of crop produce as related to nematode control is usually considered only in terms of economics. However, with increased knowl- edge concerning the influence of nematodes on the nutrient status and physi- ology of host plants, it will be important to explore the effects of nematodes on factors such as fiber length and strength in cotton, flavor and vitamin con- tent in edible annual and perennial crops, and nutrient content of forage crops for livestock. Soil fumigation may adversely affect the growth and quality of some plants. Bromine-sensitive crops, such as onion, carnation, and citrus, are sometimes stunted by preplant soil fumigation with nematocides containing bromine. In some cases, this may be sensitivity to the nematocide itself rather than to bromine. These points need additional research for clarification. LEVEL OF AGRICULTURAL DEVELOPMENT RECOGNITION OF A PROBLEM In some areas of the United States, and in many of the underdeveloped coun- tries of the world, there are two primary obstacles to effective nematode

162 BASIC PRINCIPLES OF CONTROL control. The first, and perhaps the most important, is lack of recognition that plant-parasitic nematodes are seriously limiting the potential yield of crops. The main reason for this is the shortage of agriculturists at all levels who recognize and understand nematode diseases. This situation is changing as nematologists increase in numbers and more agriculturists receive training in nematode control. The second obstacle is primarily economic and represents a lack of capital to invest in equipment and nematocides. Cultural control methods and resistant varieties should be used where feasible, since, for many nematode diseases, nematocides are too expensive. In some countries, import duties and marketing costs raise the price of fumigants to levels that are pro- hibitive for most uses. Under such circumstances, it is not surprising that soil fumigation is not a widespread practice in all agricultural areas of the world. GROWTH OF SOIL FUMIGATION IN THE UNITED STATES The increase of soil fumigation in the United States is impressive. Since its infancy in 1943, it has developed into an established agricultural practice. According to the U.S. Bureau of Census, 1963 Census of Manufacturers, pro- duction of soil fumigants in 1958 amounted to 25,446,000 pounds, as com- pared with 61,356,000 pounds in 1963. Under intensive cultural conditions, large quantities of methyl bromide are used for the control of weeds, nema- todes, and soil insects. Large quantities are applied to tobacco seedbeds in southeastern states. In California, one of the oldest soil treatments is a mixture of methyl bromide and chloropicrin, which is applied, before planting, on a large proportion of the strawberry acreage at the rate of 300 pounds per acre, costing approximately $270. An estimated 10 million pounds of EDB are used annually in the United States for soil fumigation. EDB and 1,3-D are used extensively in tobacco fields of the southeast. In the pineapple fields of Hawaii, 1,3-D is the principal preplant fumigant that is used. In the cotton fields of Arizona and California, 1,3-D is also widely used as a preplant fumigant. Use of DBCP as a soil fumi- gant has increased rapidly; in 1962,1,545,000 pounds; in 1964, 5,314,000 pounds; and in 1966, 8,722,000 pounds were produced. Although the use of nematocides is expanding, only a fraction of the world's soils that could benefit from nematocide applications are presently treated. SPECIFIC METHODS OF EVALUATING CONTROL MEASURES NEMATODE CONTROL In previous sections, control measures were considered primarily from the standpoints of effectiveness in killing nematodes and of cost. The technical

EVALUATION AND SELECTION OF CONTROL MEASURES 163 problems involved in actually evaluating the relative effectiveness of the control measures were not emphasized. With regard to nematode control, questions arise as to when, how, and where to sample fields for detection of crop-damaging nematodes. Detailed studies on sampling methods were made in England and The Netherlands on the cyst nematodes, Heterodera schachtii and Heterodera rostochiensis. Tech- niques that give consistent and statistically sound results were developed. With these nematodes, the level of infestation in the top 1 or 2 feet of soil is the primary concern. Reliable estimates of soil populations to depths of 8 to 10 feet or more are needed for many nematode species, particularly for those that are parasitic on deep-rooted perennials. Several reliable techniques are available for extracting nematodes from soil and plant samples. New procedures for collecting samples and obtaining information on the number of samples required for reliable data are needed. The most urgently needed information includes dependable procedures for making predictions on potential crop losses, based on the kinds and numbers of plant-pathogenic nematodes recovered from soil and root samples. On the basis of this information, recommendations can be made concerning whether or not soil fumigation of a particular area of land would be profitable. Such predictions cannot be made now even for fields infested with the most eco- nomically important species in the United States, namely the root-knot nema- todes. The cropping history of the land is presently the important source of information concerning possible infestation with the root-knot nematodes and with certain other species. ECONOMIC EVALUATION Efficient techniques are available for determining the value of nematode con- trol. These evaluations require detailed data on yield, quality, date of maturity, and other factors that might influence the value of the crop. The cost and effectiveness of alternate control methods also must be considered. The im- pact of nematode control measures on other cultural operations, such as rota- tion, must be taken into account. A nematode control measure, at first judged too expensive, may in practice prove economical if multiple benefits result, such as control of weeds and fungi, thereby eliminating weeding costs and in- suring a uniform stand and maturity. Nematode control may also aid in more efficient use of fertilizer and water. Although an important factor in the over- all economics of crop production, the economics of nematode control has yet to receive attention from the agricultural economists.

PART IV RESEARCH NEEDS