ur—r- SYUBEES QM FHE §URWVAL OF TYLENCHED HEMAYQDES MSGCEAFED WETH GIGAMC SGD Thesis for “is Dug?“ 0? M. 5. meme“! 3mg mmm Robert Akpam Ram. 1968 ‘.h“-‘A ‘4 -A “m-‘y;’ THESIS _'_—--.--: -§""O LIB RA M hfiCngan Staifi Univ "ersizy f ¢v~—— 0 t; I} h ABSTRACT STUDIES ON THE SURVIVAL OF TYLENCHID NEMATODES ASSOCIATED WITH ORGANIC SOD by Robert Akpan Itam Fourteen genera of plant nematodes were observed for occurrence and vertical distribution in Merion grass grown on organic soil. The effect of sod heating on 6 of these was studied at 3 ranges of tempera- ture namely: 60-104°F for 37 hours; 60-1140F for 50 hours; 68-1170F for 36 hours. Comparisons of extraction techniques and nematode occurrence in organic and mineral sod were also studied. Marion Kentucky bluegrass sod grown on mineral soil contained larger numbers of genera and individual tylenchid nematodes than in sod raised on organic soil. Heat accumulation in merion sod stacks to 115°F or higher signified the maturation within the stacks of conditions lethal to most of the associated tylenchid nematodes, including Aphelenchoides sp., Aphelenchus sp., Ditylenchus sp., and xylenchus sp. Populations of Pratylenchus and pre-adult Paratylenchus survived merion sod heating beyond 115°F. These findings indicate that practically all tylenchid nematodes shipped in organic merion sod would survive the heat development in the sod stacks during the first day of shipment. As the stack period ex- ceeded 24 hours and the temperature rose above 104°F, the effect of "sod-heating" would become increasingly harmful to both the nematodes Robert Akpan Itam and the grass. A more precise knowledge of nematode species involved and heat development within sod stacks is needed before the necessity of control measures can be assessed. STUDIES ON THE SURVIVAL OF TYLENCHID NEMATODES ASSOCIATED WITH ORGANIC SOD By Robert Akpan Itam A THESIS Submitted to , Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1968 ACKNOWLEDGEMENTS my thanks are due to the Michigan State Department of Agriculture for suggesting this project; and to Dr. G. E. Guyer, Chairman of the Department of Entomology, Michigan State University, for providing the opportunity, facilities, and support for the research. Special thanks are also due to the Proprietor and Staff of the Halmich Sod Nurseries, East Lansing, Michigan, for supplying the sod samples utilized in the investigation. I am indebted also to all the Professors on my Thesis Committee for their interest and close supervision. My gratitude goes to Dr. E. C. Martin, Chairman of the Committee, for many helpful hints, and for the photographs; and to Dr. J. B. Beard, who made available all relevant information on turf science, research, and industry; to Dr. J. A. Knierim, my Major Professor, for guidance and technical assistance; and to both Dr. J. Bath and Dr. P. H. WOoley, for useful comments on the purview of the investigation, and for information on the principles of scientific rhetoric. I also owe my gratitude to Mrs. Natalie Knobloch, Technician in Nematology, for help in the identification of specimens; to Mr. John King, for his preliminary data on sod-heating; and to Dr. D. L. Haynes and Mr. W. G. Ruesink, for help in the statistical analysis of results. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . 3 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . 8 EXPERIMENTS AND RESULTS . . . . . . . . . . . . . . . . . . . . 19 A. Preliminary Temperature Tests . . . . . . . . . . . . . . 19 B. Sod Heating Tests . . . . . . . . . . . . . . . . . . . . 21 C. Vertical Distribution Studies . . . . . . . . . . . . . . 37 D. Occurrence in Organic Sod Strips . . . . . . . . . . . . 37 DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . 43 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 47 iii Table 4A. 5A. 6A. 10. 11. 12. LIST OF TABLES An Evaluation of Baermann Funnel Extraction of Muck-Sod Nematodes A Combination of Funnel Filtration with the Centrifugal Flotation Mathod, as a Means of Separating the Nematodes from Plant Matter . Effect of Heat on the Survival of Muck-Sod Nematodes . . . . . . . . . . . . . . . . . . . Effect of Sod-Heating on the Survival of Mbck— Sod Nematodes . . . . . . . . . . . . . . . . Analysis of the Data of Table 4 . Effect of Sod-Heating on the Survival of Muck- Sod Nematodes . . . . . Analysis of the Data of Table 5 . Effect of Sod-Heating on the Survival of Muck— Sod Nematodes . . . Analysis of the Data of Table 6 . Distribution of Tylenchorhynchus sp. in 0.5 m1 Subsamples of a Mass Inoculum of Turfgrass Nematodes . . . Effect of Sod-Heating on the Survival of Tylenchorhynchus sp. . . . . . . . . A Check on the Effectiveness of the Recovery of Tylenchorhynchus sp. from the Funnel Filters . Vertical Distribution of Stylet-Bearing Nematodes in Organic-Sod Soil Occurrence of Stylet-Bearing Nematodes in Organic Sod . An Estimate of Stylet—Bearing Nematodes Associated with Mineral Sod . iv Page 14 15 20 26 28 29 31 32 34 35 36 38 39 41 42 LIST OF FIGURES Figure 1. Plastic Cylinders as Used for the Dynamic Filtration Technique . . . . . . . 2. Four—inch Diameter Cores and Standard Dimension Sod Strip . . . . 3. Heating Oven and Thermostatic Controls Page 13 23 23 INTRODUCTION The importance of the Turfgrass industry to American economy, as measured in terms of maintenance expenditures, has been estimated recently at $4.3 billion per annum (Nutter, 1965). An estimate of the farm value on a national scale has not been made; but the total acreage under commercial sod production for the year 1965 was 48,000, and 95,000 for 1967 (Beard, 1967). The corresponding 1966 figures for the State of Michigan were: $149,133,200 for the annual maintenance cost (Beard & Hoglund, 1966), with 19,546 acres and 22,000 acres under cultivation in 1966 and 1967 respectively. The farm value to Michigan sod growers in 1966 was $26,200,000 (Beard, 1967). These figures are the highest for any single State in the United States. Sixty-five percent of Michigan's sod in 1966 was raised on organic soils and 35 percent on mineral soils; the total value of the industry, including growing, shipping, and laying, being $73,360,000 (Beard, 1967). The industry is currently ranked as the fastest growing industry, and the fifth leading agricultural industry, in this State (Beard, 1967). The foregoing statistical evaluation of the American turf in- dustry, impressive though it is, does not reveal the probable role of sod in the dissemination of harmful nematodes that destroy our food and ornamental crops. Several species of pathogenic nematodes are already known to possess host ranges that include most of the turfgrass species grown on mineral soils. For example the stubby-root nematode, l 2 Trichodorus christei Allen has over 100 potential host plants, including grains, legumes, vegetables, ornamentals, and turfgrasses (Rhode Efiuilr’ 1957; Coursen gt_gl:, 1958). It is therefore presumable that mineral sod is involved in the dispersal of these destructive nematodes. Such a role cannot, however, be readily inferred for organic sod, owing to the lack of information on the identity and biology of the nematodes that are supported by sodgrasses produced on organic soils. Moreover, for organic as well as mineral sod, it is yet to be experimentally established that associated sod nematodes survive the metabolic heat which develops within the stacks of harvested sod rolls. The present studies are therefore an attempt to elucidate some of these unknowns as they relate to organic sod. The objectives were twofold: (i) to identify some of the tylenchid nematodes associated with muck sod; and (ii) to determine, by means of laboratory experiments, which of these nematodes would likely survive the heat build-up in sod- stacks. Certain parallel studies were also conducted with mineral sod samples, for purposes of comparison. In addition, a study of the ver- tical distribution of muck sod nematodes was also carried out to help in establishing the validity of the results and conclusions. LITERATURE REVIEW Existing knowledge of the occurrence and biology of turf nema- todes pertains particularly to nematodes associated with turfgrasses grown on mineral soils. Similar information on nematodes associated with turf grown on highly organic soils, like muck, is not available. In relation to nematode occurrence in mineral sod, Goodey (1933) studied gall formation on the leaves of fine bentgrass, Agrostis tenuis Sibth., by the seed—gall nematode, Anuguina graminophila. In the United States, the first published work, which documented the parasitic association of nematodes with turfgrass, appeared in 1951. Tarjan g£_§l: (1951) investigated the parasitic involvement of Panagrolaimus rigidus and Eucephalobus oxyuroides in the yellow tuft disease of bentgrass, Agrostis sp. Eleven years later, Rhoades (1962) listed 21 genera and species of nematodes known to be pests of turf in the United States; but Taylor's (1962) critical analysis of the situation showed that valid experimental evidence for pathogenicity existed for only seven of these. A tentative estimate of the figures today indi- cates that more than twice as many parasites and pathogens have been established. The newly added pathogenic species include: Hypsoperine graminis, (Dickerson, 1966; Minton gt_gl,, 1967); Meloidogyne incognita acrita, (Gaskin, 1965; Hodges_e£”al., 1963); Pratylenchus scribneri, (Minton, 1965); Panagrolaimus sp., (Pepper, 1965); Heterodera leuceilyma, (Perry, 1965); Belonolaimus longicaudatus, (Thoades, 1962); Trichodorus 3 4 christei, (Rhoades, 1962); Trichodorus proximus, (Rhoades, 1965); and Ditylenchus radicicola, (Smithson_e£_§l:, 1963). Rhoades' (1962) list of turfgrass nematodes shows that parasitic mineral sod nematodes belong to two taxonomic orders: Tylenchida and Dorylaimida; the greater majority of them being tylenchids. Couch's (1962) account of turf diseases caused by nematodes shows that each turfgrass species is usually associated with more than one of these worms. For instance, Kentucky bluegrass (Poa pratensis L.), the species selected for the present investigation, is associated with no less than seventeen parasitic tylenchids. Five of these have been proven to be pathogens: -- viz., three species of Helicotylenchus which cause the 'summer dormancy' disease of Kentucky bluegrass,_H. digonicus,_§. microlobus, and_H. pumilus (Perry, 1959); an unidentified species of Panagrolaimus which has been connected with the 'melting-out' disease of the bluegrass (Pepper, 1965); and the grass root-knot nematode, Meloidogyne incognita acrita Chitwood (Gaskin, 1965). Other parasitic species which have been associated with this turfgrass include, Tylenchorhynchus maximus Allen, T, nudus Allen, T, dubius Butschli, Helicotylenchus platyurus Perry, Pratylenpratensis (de Man) Filipjev, Heterodera trifolii Goffart, Xiphinema americanum Cobb, Hoplolaimus coronatus Cobb, and Meloidogyne hapla Chitwood, (Perry, 1959); Trichodorus christei Allen, (Rhode 35 21., 1957); Paratylenchus projectus Jenkins, (Coursen g£_§13, 1958b); and a species of Aphelenchus (Pepper, 1965). The ecological life of plant parasitic nematodes, as recently reviewed by Wallace (1963), is under the influence of a complex inter- play of various environmental factors: -- including, the biotic, 5 physical, and chemical factors of the soil environment; the host—plant factor; cropping practice and history; the original native vegetation of the soil; climatic factors; and factors due to the inherent specific differences of the nematodes themselves. For this reason, Wallace points out, it is usually difficult to relate cause and effect in many field and laboratory studies of the ecology and biology of plant nema- todes. Heterodera schachtii, for example, is absent from the very acid peaty soils in the Fen district of England (Petherbridge & Jones, 1944). Heterodera rostochiensis, on the other hand, is particularly abundant and active in these same areas of the Fen district (Wallace, 1960). Some authors find no relationship between soil type and nematode occurrence; while others suggest that there is an association between soil type and the distribution of some nematode species (wallace, 1963). For example, Caveness (1957) studied the nematodes associated with sugar beet production in some northwest and north central States of the U.S.A. He found no evidence for an association between any genus or species of parasitic nematodes and any particular soil type. Sasser (1954), on the other hand, found that infestations of Meloidogyne incognita,_M. incognita acrita, and M. hapla were more severe in the sandy loam soils than in the heavy clay soils of Eastern Maryland. Laboratory studies of the temperature relations of plant nema- todes have also been shown to present similar complications, because the host plant itself is affected by the temperature treatment. For example, Krusberg (1959) found that the optimum temperature for repro- duction of Tylenchorhynchus claytoni was 21 to 27°C on wheat and 29 to to 35°C on tobacco. Blake (1962) showed that reproduction of Ditylenchus dipsaci in oats was greater at 8°C than at 15°C. 6 Other factors which have been shown to influence the temperature relations of plant parasitic nematodes are those introduced by the nematodes themselves; -- e.g. developmental stage, age, and physiological state. Thus, Ditylenchus dipsaci is more resistant to low temperatures (Bosher & MCKeen, 1954) and to high temperatures (Courtney & Latta, 1934) when the preadult stage —- i.e. the fourth stage larva -— is in the dry quiescent state. Sherman (1934) and Cairns (1953) also showed that this stage is more resistant to heat than other stages of the nematode. Similarly, Rhoades and Linford (1961) found that the preadult stage of Paratylenchus projectus could survive sudden exposure to low temperatures (about -19°C) better than other stages. The thermal death curves of different plant nematodes also ex- hibit a wide variation between species of the same genus (Wallace, 1963). Apart from factors stemming from intrinsic differences between species, much of this variation has been traced to one essential factor: dif- ferences in the heat penetrability of the medium or tissue in which the tested nematode is enclosed (Staniland, 1950; Blake, 1961). This varia- tion notwithstanding, Wallace (1963) tentatively concludes that plant nematodes are probably killed instantly at 52°C (129°F), and that long exposures to temperatures above 40°C (104°F) may prove lethal to the nematodes. A recent exemplification of this conclusion by Heald and Wells (1967) showed that an infestation of Criconemoides sp., Hypsoperine sp., and Tylenchorhynchus sp. in cores of bermudagrass turf could be completely eradicated by hot water treatment at 55°C (131°F) for 15 minutes. In contrast with this, the optimum temperatures for popula- tion increase of five nematode species maintained on Tall Fescue host fall 10 or more degrees below the lethal temperature range of 104 to 7 129°F, as follows: Hoplolaimus tylenchiformis, 77 to 84°F; Helicoty- lenchus mannus, 70 to 94°F; Tylenchorhynchus claytoni, 70 to 94°F; Trichodorus christei, 63 to 94°F; and Paratylenchus projectus, 63 to 77°F (McGlohon 3551., 1962). There are at present no published observations on the subject of sod-heating per se, nor on its effects on any of the associated macroorganisms. Unconfirmed observations, however, seem to indicate that the lethal temperature to the turfgrass lies between 104°F and 115°F, (King and Beard's preliminary data, 1967). Sod farmers claim the first 24 hours after harvesting to be safe from harmful heating in the stack but the safe period probably varies widely, depending on the size of the sod-stack, the temperature and relative humidity of the atmosphere, and the metabolic rate of the microflora in the thatch and roo 1: zones . MATERIALS AND METHODS Sampling Procedures Two forms of sod samples were employed in these investigations: i) freshly cut commercial sod strips of standard dimensions, 52 in. by 24 in. by l in., ii) 6-in. deep cores cut with a 4-in. diameter corer. Yates and Finney (1942) recommended the use of the 4-in. diameter corer as best for soil animals, since it maintains a relatively uniform sampling efficiency at low as well as at high animal population den— sities. Both types of samples were obtained from healthy merion Kentucky bluegrass turf farms within the State of Michigan. Five farms, maintained on muck soils, served as sources for the samples. One of these, the Halmich Sod Nurseries in East Lansing, provided all the samples for the stack-heating tests. Samples used for the preliminary temperature tests were obtained from the Green Acres Turf farm, Mason. The three remaining farms which provided samples for the survey inves- tigations were Baldwin Sod Farms, Stockbridge, Emerald Valley Nurseries, Gregory and Reding's Sod Farms, Livonia. For a comparative study of nematode occurrence in mineral sod, samples were obtained from two other sources —- viz., Hiram F. Godwin & Son mineral sod farms, South Lyon; and the Horticultural garden at Michigan State University, East Lansing. Also, samples from the Battle Creek Golf Course, taken ex- clusively from the putting greens, supplied the inoculum of Tylenchor- hynchus sp. used in one of the heating tests. 8 9 Samplings were made throughout most of Spring, Summer, and Autumn of 1967. The collection of samples was, as a rule, done in the evenings, when the average soil temperature, measured at a depth of) one inch, varied from 44°F in May to 71°F in mid-summer, and down again to 43°F in October. Samples were collected in polythene bags; and were preserved in a cold chamber at 40°F when it was not possible to utilize them immediately. However, all samples were usually put to use within 48 hours after collection; otherwise they were discarded. The pattern of collecting samples from the field was as follows: The sod strips were picked up from the field at random. The 6-inch deep cores were cut from various casually selected spots on the field, simply by walking across the farm diagonally and removing cores at in- tervals along the line. Similar methods of sampling were used by Tunstall and Matthews (1961) who estimated the level of occurrence of red bollworm eggs (Diparopsis castanea) by taking two diagonal traverses the field and counting the eggs at regular intervals. A more systematic pattern of sampling was adopted in taking core samples from the harvested strips of sod. In the preliminary temperature tests, five adjacent rows of sod cores were sequentially removed from the entire surface area of a piece of sod strip. Thus, the results of each row served as a check on those of contiguous rows. For the sod-heating tests, two adjoining pairs of cores were drawn from the centre of each experimental piece of sod; one of each pair of cores serving as the control. Each experiment was repeated four times in the preliminary tests, and eight times in the sod-heating tests. lO Extraction of the Nematodes (i) The Christie-Perry Technique -- Nematodes present in sod samples used for the preliminary tempera- ture tests were recovered by a modification of Christie-Perry's (1951) technique. Two operations were involved: wet sieving, and funnel fil- tration. Each core of sod was teased and washed into a plastic bucket of about 2.5 gallons in capacity. A jet of cold water, directed from a sprinkler, ensured a thorough rinsing of the grass shoots and roots which, after squeezing to expel the water, were discarded. Large clods of soil were broken, and more water added to fill about half the capac- ity of the bucket. The mixture was then thoroughly stirred; allowed to stand for about 30 seconds, and decanted through a 25-mesh sieve into a second bucket. In this way, sand and other heavy particles were re- tained in the bucket, while the lighter and larger organic debris were caught on the sieve. The filtrate in the second bucket was also stirred thoroughly, allowed to stand for 30 seconds, and then decanted through a 325-mesh sieve. The 325-mesh sieve, together with the residues, was set aside, and the whole screening process repeated, be- ginning with the 25-mesh sieve. Three such repetitions, making a total of four screening Operations for each core of sod, were carried out. At the start of each repeat operation, the residues on the 25-mesh sieve were returned to the first bucket, half a bucket of water added, and the operation carried through to the 325-mesh screen stage. The total amount of residues on the 325-mesh sieve was then rinsed with water to eliminate all fine and silty matter. 11 The separation of nematodes from unwanted components of the residues was achieved by the funnel dynamic filtration technique. The residues were washed into a filtering apparatus made from a short length of thin walled metal tubing three inches in diameter and having a piece of muslin cloth fastened to the bottom with a rubber band. The filtering can, together with its contents, was then placed in a 6-inch Baermann funnel, and the latter filled with water so that only the lower part of the filtering can was submerged. The nematodes left the residues, filtered through the cloth, and fell to the bottom of the funnel to collect in a small test-tube attached to the stem of the funnel. The filtration process was continued for four days. (ii) Centrifugal-Flotation Technique —- Jenkin's (1964) modification of the centrifugal-flotation technique, in combination with Christie-Perry's filtering technique, was used in extracting nematodes present in sod cores employed for the sod—heating tests. This procedure included a sieving operation ex- actly like that already described in the preceding section; differential flotation of the nematodes by the use of a solution of specific gravity greater than that of the nematodes and funnel filtration of the pro- ducts of flotation, as a means of separating the nematodes from other matter of similar specific gravity. This last step was carried out in a manner similar to that already described under the Christie-Perry technique. Differential flotation of the nematodes was achieved as follows: The products of the sieving operation were first shared equally between four lOO-ml centrifuge tubes. Equal amounts of water were then added 12 to the tubes to nearly fill them; after which, the tubes with their contents were centrifuged for five minutes at full speed. This threw down all solid matter, leaving a clear supernatant liquid in each tube. The liquid was carefully poured off and discarded, and a solution of sucrose sugar (1 lb. per litre of water) was added in its place. After thoroughly mixing the sugar solution and sediment the tubes were again centrifuged at full speed for 30 seconds. The supernatant suspension, containing the nematodes as well as other matter of similar density, was then filtered through a 325-mesh sieve. The residues were thoroughly rinsed with water to wash away the sugar, and then transferred to the muslin cloth filterr The nematodes wriggled through the cloth, leaving the debris behind, and collected below in a small test—tube connected to the stem of the funnel. Mbst of the nematodes were recovered within the first day but filtration was not terminated until after four days. Evaluation of the Extraction Methods The low numbers of tylenchid nematodes extracted from the sod cores, using Christie and Perry's modification of Baermann's technique (Table 3), made necessary a critical examination of the effectiveness of this extraction method. Christie-Perry's funnel residues were pro- cessed for unrecovered nematodes by the centrifugal-flotation method of Jenkins. Nematode counts showed that the degree of efficiency of the Funnel method varied with different genera (Table 1). The more active nematodes e.g. Aphelenchoides sp. and Aphelenchus sp., filtered through more successfully than sluggish ones such as Paratylenchus sp. and Ditylenchus sp. .osvwccomu GOwumuuaflm owEmC%w ecu wow wow: mm muoucfiaxo owummHmII.H .wwm l4 mm N a NH O o 2 3582538 mm H m e o o e m>um~ muoeououom 3 «2 m2 Se 93 o 2 3583.3 S 2 «m a S 3 o 3 35:32.: mm mm m em NH NH o mmwwosocmfimnam N .uuxm .csm .w>< HmuoH m ummH N umoH H ummH um>ummno mumcmw uomuuxm a“ .w>< amacsm mmavwwmm floccsm cw mmUOumaoz mo .02 mercumamc mOmlxosa mo coHuomuuxo amass“ camaummm mo cofiumsfim>o qumN mumeoumumm o o o o o o o N o N o o o o o o o o moaoamNNNooNNmm o o o o o o N m o o o m o o o o o o manoamNsNNn o o o o o o o o o o o o s 0N o o o 0N manocmNmsa< o o o o o o N m o o o m N a m N o N mmeNoaUamNmea< .ms< NsNoN >N NNN NN N .w>< Nance >N NNN NN N .w>¢ NmNoN >N NNN NN N «Magoo .mue we ”NooN .N .uaya .mue «N mecca .N .usus mNouNaoo mom «0 ouoo you momoumaoz mo Nonasz mooOumBo: oOmeosa mo Hm>H>H3m onu so use: no uoomwmnl.m mqmo wcwumomu|.m .wNm .awuum oOm cowmcoENm oumocmum one mouoo HouoEmNo sochusomll.N .wNm 24 temperatures comparable to that in the second set of tests. Other procedural details remained the same as for the first set of tests. In the fourth set of tests, the effect of sod-heating on the survival of an introduced population of Tylenchorhynchus sp. was studied. A mass inoculum, containing the selected nematode as well as others was obtained from bentgrass sod by Christie-Perry's funnel ex- traction method. Eight 0.5 ml aliquots of the mass inoculum were measured out by a method of volumetric subsampling and the average number of_Tylenchorhynchus sp. per aliquot determined (Table 7). A 5-ml inoculum per core of sod was then measured out by the same method of subsampling. The estimated number of Tylenchorhynchus sp. contained in each 5-ml measure was obtained by multiplying the average count per 0.5 ml aliquot tenfold. A.magnetic stirrer was used to maintain uni- form dispersion of the stock suspension throughout the subsampling process. From each of the four experimental strips of sod, four contig- uous cores were drawn -- one as the control and three for the treat- ments. The control core was processed immediately to determine the kinds and numbers of indigenous nematodes present. Each of the three experimental cores was mass inoculated with about 1500 specimens of Tylenchorhynchus sp., this being the estimated number contained in each 5-ml inoculum. One of these three cores was processed immediately after inoculation so as to give an estimate of the expected yield of ,Tylenchorhynchus sp. (Treatment 1); another was subjected to heating in the sod-stack, (Treatment 3); and the third core was enclosed in a plastic bag and set aside at 77°F for the duration of the stack heating operation, (Treatment 2). These last two treated cores were processed 25 for nematodes soon after treatment; the former expressing the effect of sod-heating on the nematodes and the latter indicating the influence of the new environment on them. The heating in the sod-stack lasted for 50 hours and increased from 76°F to 114°F. Results of the Sod-heating Tests - The results of the sod—heating tests showed, on analysis, that only Tylenchus sp. suffered a significant reduction in population numbers under the heat treatment of 60 to 104°F in 37 hours (Tables 4, 4A). Aphelenchoides sp., Aphelenchus sp., and Ditylenchus sp. were definitely not adversely affected by this treatment. Nbr were Para- tylenchus sp. and Pratylenchus sp., despite their low numbers in the different samplings. When heated from 60 to 114°F in 50 hours, Tylenchus sp., Aphelenchoides sp., Aphelenchus sp., and Ditylenchus sp., in that order, suffered significant numerical losses. Paratylenchus sp. and Praty- lenchus sp. were still unaffected by this degree of treatment (Tables 5, 5A). Pratylenchus sp. also survived the more intensive heat treatment of 68 to 117°F in 36 hours and although scanty, the data for Paraty- lenchus sp. indicated a similar survival ability for this nematode under the same conditions (Tables 6, 6A). As regards the inoculation tests with Tylenchorhynchus sp., the results indicated a conclusive lethal effect of the tested level of sod-heating on this nematode (Table 8). The data also bore out the re- sults obtained with indigenous sod nematodes under a similar heat treatment (Table 5). However, when the indispensable losses in numbers of Tylenchorhynchus sp. were taken into account, the significance of 26 N q N N mm Cu mm mm manoamamH a m o N e m e e maaucmNsumum m oN ON 0 m o o o maaocmNsumumN «a as NN ow as mmN oN mNN msaocoNsuNn A.mua va N s e o a NN oN w masoamNmaa< moqu . 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