THE CORRELATION OF SOIL TYPE, MOISTURE, VARIETY AND RATES OF APPLICATION ON UPTAKE 0F SYSTEMIC INSECTICIDES IN FIELD BEANS Thesis Ior III. Degree OI M. S. MICHIGAN STATE UNIVERSITY Thomas Rosario Castro 1961 LIBRARYI'I Michigan State University , -.. . .uvo- -rv.r‘._‘ ABSTRAC T Th3 CORRELATION Or‘ SOIL TYPE, mOISTURE, VARIETY AND RATES OF AI} LICATIOI'C‘ OI] UI-‘TM‘E OF SYST7mlC INSECTICIDES IN FIELD BEaRS by Thomas Rosario Castro Soil applications of 105 granular Thimet (phorate), (0,0—diethyl S-etnylthiomethyl phOSphorodithioate) and 10$ granular Di-Syston, (0,0-diethyl S-BIethylthio) a ethyl phOSphorodithioate) were evaluated for the effects of soil type, moisture, Variety and rates of application on plant growth and uptaKe of toxicant in field beans. Bioassays to determine effectiveness of the treatments were conducted with the two-Spotted Spider nite, Tetranychus telarius (L.); bean aphid, gpnis fabae Scop.; Mexican bean beetle, Epilachna varivestis mUlS.; and vinegar fly, Drosophila melanogaster Meig. The results of the eXperiments are out- lined as follows: 1) a greenhouse culture of two-egotted Spider mites was found resistant to Thimet when the insecticide was applied in bands in soil at rates up to 12 pounds per acre. 2) Thimet applied in bands in the soil at rates as low as 0.25 pounds per acre was effective in controlling a non-resistant strain of the two-spotted spider mite. 5) Thimet applied at l, a and 4 pounds, and Di-Syston at 2 pounds per acre in bands, were effective against Mexican bean beetle larvae and adults for 44 days after planting. These rates did not control infestations of whiteflies. 4) Better control of Mexican bean beetle larvae was obtained from the Chemical 80 days after seeding than 14 days after seeding. 5) There was no apparent toxic effect on adult Mexican bean beetles, fed on green pods from plants grown in muck, loam and loamy sand soils treated with Thimet at a rate of 2 pounds per acre. 6) Thimet residues from 5 (l pound broadcast) to 5.8 (5 pounds broadcast) p.p.m. were present in bean leaves 15 days after seeding. Thirty-five days after seeding most residues had decreased to l p.p.m. 7) Comparable anounts of Thimet applied to beans in-row with seed or mixed in the soil (broadcast) resulted in more phytotoxicity than when applied in bands 2 inches to the side and 1 inch below the seed. 8) Excessive moisture reduced the effectiveness of systemic application and increased phytotoxicity symptoms. ii THE CORIELATIOK OE SOIL TYIE, mOIfimURE, VAAIETY AND RaTEo OE AIPLICaTION ON UITahE 0“ bYSTEnlC thECTlCIDES 1h FlELD BJANS BY Thomas Rosario Castro A THESIS Submitted to the College of Science and Arts, Michigan State University in partial fulfillment of the requirements for the degree of M"TER OE SCIENCE Department of Entomology 1961 AClxI‘I O I I LET GIVIEI‘TDS My grateful appreciation is extended to Professor Ray hutson, Head, Department of Entomology, for maxing this study possible. I wish to express Special thanns to Dr. Gordon Guyer, under whose direction this investigation was nade. His constant help and enthusiasm never failed me during this research. My grateful appreciation is extended to Mr. Arthur Wells for his invaluable assistance throughout the project. 1 also wisn to thank Dr. R. hoogingarner for his assistance with statistical analysis and in conducting the bioassay tests. my sincere appreciation is also extended to Doctors E.C. Martin, Axel Andersen and J.U. Butcher for their critical reading of the manuscript. I wish to thank messrs. Alfred Borgatti and Robert McClanahan for their nelp and suggestions during this study. iv I. II. III. IV. V. VI. Introduction. rt} "1 - *1 lzLBlJfii Ul‘ Literature Review..... Procedure.... Experiment EXperiment EXperiment Experiment Experiment Experiment Experiment 10.0.0... 11...... V1.00... VIIIOOOOO COIQJfiURj Presentation and Discussion of Results.. Summary..... 00.0.00... Bibliography............................ Table ()1 (I) Nunbers of two-Spotted spider nites (from greenhouse cultures) and bean aphids on leaf dis s, removed fron the (a) first true leaf and (b) top trifoliate leaves of plants grown in three different soils............... Mortality of first instar aexican bean beetle larvae fed on foliage of bean plants grown in three different soils. Ilants were infested with larVae 20 days after planting..................................... Kortality of second and third instar Hexican bean beetle larvae fed on foliage of bean plants grown in three different soils. Larvae placed on plants 55 days after plant- ingo..0.OOOOOIOOOVOOOOOOOOOOOOOOOOOOOOOOOOOOOI nssessnent of feeding injury of hexican bean beetle larvae on bean plants grown in three different soils. Rating of feeding injury: I : scattered feeding; 2 : 1/4-1/3; a : 1/3-1/2; and 4 : nore than 1/2 of leaf corlsu.:ledoooooooooooo00000000.0000000000000000 Evaluation of Mexican bean beetle adult feeding injury on green oean pods removed from plants grown in three different soils. feeding injury rating: 1 a no feeding; 23 - less than 1/4; E = 1/51 to 1/2; and 4 . more than 1/2 of the outer pod surface... Number of mites and aphids on detached leaf discs from the first true and top trifoliate leaves of bean plants grown in treated and untreated soil............................... hortality of first ins ar hexican bean beetle larvae placed on bean plants 52 days after planting; grown in treated and untreated 8011......OOOOOOOOOOOOOOOOOOOOOOO0.0.0.000... Mortality of Lexican bean beetle adults fed on plants grown in treated and untreated soil. Vl Iii {'3 ("i (D m Cr] L0 01 26 28 e o! 'fable IO. 11. I4. 17. mortality of second and third instar Mexican bean beetle larvae fed on bean plants grown in treated and untreated soil. LarVae were placed on plants 44 days after planting....... assessment of feeding injury by nexican bean beetle (on days after planting) on bean plants grown in treated and untreated soil........... Nunber of naturally occurring mites and white- flies on bean plants grown on treated and untreated soils. datings of infestation estimated as nunber of mites and mhiteflies per infested leaf: 1 = less than 20; 2 = 21 to ac; 3 = 41 to 60; and 4 = 61 and aboveOOOOOOIOOOOOOOOOOOOOOOOOOOOOO0.0.0.000... mortality of first instar lexican bean beetle larvae fed on bean plants grown at three soil noisture levels.......................... Assessment of feeding injury of flexican bean beetle larvae on Thimet treated and untreated plants grown at three soil moisture levels.... Mean height and spread (crown width) of the Thinet treated and untreated bean plants grown at three soil moisture levels........... Lortality of first instar hexican bean beetle larVae fed on bean plants treated with three rates of Thimet. Plants were infested 28 days after seeding............................ Populations of the two-Spotted Spider mites (red clover culture) on detached leaf discs from plants treated with three rates of Thimet........................................ Lean nunber of mites per detached leaf disc from the Thimet treated and untreated plants of four different bean varieties.............. MortalityIl) of dros0phila naintained in different dilutions of Thinet in pumpkin Inedia for 24 hat-1118......OOOOOOOOOOOOOOOOOOOOOO vii s4 (3 C 7. (D 34 (-3 CD 37 58 4O 41 45 46 Table l9. ta \ 1 fl 1‘ a 0' H;. H (D (D O te dorta leaf- bean after 2.10rta leaf- DGJIL after .unoun leaf soil ui_;licati onS of -:nribnirl 1 J. tuined on bean for hours, utilizing rleaves removed fron plants 15 days rplanting ity droso Ella main .Jeiiu II 34. A) A ~‘I 49 lity of droso hila nuintained on bean pumpnin ueiia for 2% hours, utilizing leaves removed from plants 35 days ledntin£;00000000OIIOOOOOOOOOOOOOOOOOOO lity of drosophila uaintained on Lean pu;n :diin wediu fo nours, utili7 7ing le .1ves removed from ylunts J5 qifs \Atvs.) pliiétirir 00000000000.00.000000000000000 ‘3 L‘- ‘4‘ 51 L.) the bean plants treated With (EIAiZHGtOOOOO.00.0.0.0... t of toxicunt estinated to be in tis Tunas 'f the viii "~"“1-3- VA‘Vv/jrfi'rr ' “ll‘Ju' VlJUls Insects and insect transmitted diseases are serious problems of the field and snap bean industry in the United States. The nexican bean beetle, figilachna varivestis nuls., is one of the host serious bean pests. It has been present in the southwest for more than a century and agpears to have nigrated fron.mexico, where it was widely distributed. It was first recorded from Alabama in 1920. Records of its Lresence in the central and eastern bean growing regions followed in rapid succession. In LLichigan the bean aphid, (fighis fauae Scogu the six-Spotted leafhotper, nacrosteles fascifrons (Stal.), and the potato leafhopger, Empoasea fabae (harris), are also imyortant bean pests. fne aihids yroduce injury botn by feeding and disease transmission. Nuaerous insecticides have been applied as foliur Sprays or dusts for control of the insect complex on beans. n more recent develofnent nus been the use of systemic insecticides for this furpose. Hay and Keedhan (1957) reported that beans could be grotected fron bean nihid infestations by the application of deaeton directly to seeds or as soil treatnents. Also, Guyer gt a; (1960) retorted satisfactory control of sucaing insects on field and snap beans with soil arplications of thimet (yhorate) and Di-Syston (Bayer 19639). -1- to h o " ' " :1 ll .5 'qacio. '20 T e bdectives or t e rvecti_ t rs re, rted here were: 1) {‘0 03 To study the :ovenent of systenic materials (Thimet and Di-Syston) tnrough the bean plant. To determine by bioassays the iuantity of insecticides in the plant at different stages of growth, when applied at various rates. To eValuate the effects of soil moisture and texture on pnytotoxic symptoms in treated plants. To evaluate the resyonse of different varieties of field beans to treatnents. ’ 'rrn'fit; «’1 r :1 1-1: T“? .LLLIL{.L.L 11:10 [{ALVILH Bennett (1949) defined a systemic insecticide as: "a substance which is absorbed and translocated to the other parts of the plant, thus rendering untreated areas insecti- cidal". The systemic behaviour of chemicals was observed as early as the fifteenth century, when Leonardo da Vinci injected arsenic into a peach tree trunk, killing the pests on the tree and rendering the fruits toxic (U:DA 1960). One of the first Known uses of a system'c insecticide in the United States was the injection of potassium cyanide into a Spanish broom shrub for control of cottony-cushion scale, lcerya purchasi maskell (USDA 1960). One of the most fully explored systemic insecti- cides was sodium fluoroacetate, which was present in a South African plant, uichapetalumjgymosum (Bennett, 1957). David and Gardiner (1951) stated that sodium fluoroacetate was an extremely effective systemic insecticide, when applied to the leaves or roots of beans. However, they indicated that it was not of practical importance because of its high mammalian toxicity. In 1936, hard-harer and loos studied the systemic action of selenium. Ihey observed that wheat growing on seleniferous soils was not injured by aphids. 1n 19b? -5- -4- hurd-harer demonstrated that there was a correlation between the rates of absorption of sulphur and selenium by different plants, and that the sulphur-selenium ratio of the plant increased as the sulphur content of the seleniferous soil was increased. Fulton and mason (1937) were the first worxers to experimentally demonstrate the systemic property of derris. Ihey found that derris when applied to the first two leaves of bean plants reduced the attacx of the nexican bean beetle on the new growth. Extracts from the first and second trifoliate leaves added to water resulted in 100 percent mortality to goldfish. The discovery by ochrader of systehic insecticidal activity in widely separated types of compounds initiated and gave impetus to recent advancements in this field (Bennett 1957). Systemic insecticides have been investi- gated in fruit and forest trees as well as in field and garden crops. in 1950 Metcalf and Uarlson snowed that trunk and foliar applications of octamethyl perpnOSpnoramide and deheton on citrus were effective in controlling the citrus red mite, Panonychus citri (McG.). Jepson at El (1954) studied seasonal influences on the efficiency of foliar Sprays, trunx and soil applications of demeton against citrus red mite and citrus bud mite, nceria sheldoni (Ewing). They indicated that sumaer trunk treatment was the Host effective. -5- haynes gt El (1953) evaluated systemic insecticides for the control of the European pine shoot moth, Rhyacionia buoliana (Schif.), on red pine, and indicated that Thimet as a foliar Spray, soil drench or granular soil application provided significant reductions in the larval populations. In 1959A1shzawi and horris Jr. demonstrated that it was possible to reduce the transmission of Ceratocystis ulmi (Buis.) in elm trees by treating with Chipman 3-6199 at 10, 20 and 50 grams per tree. They observed that the insecticide at 20 grams per tree was also effective in reducing the length of feeding niches of the barn beetle, Scolytus multistriatus (harsh.). Wilcox and howard (1957) conducted tests with Thimet on strawberries and turnips and reported excellent control of aphids and spider mites by band treatment near the plants. In 1958 Guyer gt al, in their investigations of systemic insecticides for control of hessian fly, ihytgphaga destructor (Say) on wheat, reported that Thimet and Bayer 19659 (Di-Syston) were effective in the control of fly larvae on winter wheat. They observed phytotoxicity in Thimet seed treatments in the early stages of plant development. Andres at El (1959) demonstrated that Thimet and Bayer 1903 (Di-Syston) applied as seed or furrow treatments controlled aphids for over a month on cabbage and cauliflower at 1/2 pound toxicant per acre. Applications -6— of Thimet and Bayer 19059 (Di-éyston) on granules gave good control for only 5 days. Reynolds (1957) protected seedlings of alfalfa, cotton and sugar beets by applying systemic insecticides at the time of planting. They applied demeton, Systox thiol isomer and Bayer 19659 (Di-Syston) as coatings on alfalfa seed, and obtained satisfactory results in the control of the spotted alfalfa aphid, Therioaphis maculata (Buck.). 1n radio-active tracer studies with Bayer 19659, demeton and Thimet applied as seed treatments on sugar beets, cotton and alfalfa, these same authors found the highest concentrations of the toxicant in the cotyledons. The insecticide apparently was not translocated in substantial amounts to other parts of the plant. The highest concen- trations were found in the oldest leaves and the lowest in the youngest leaves. Systemic insecticides have been investigated extensively as seed treatments. Chao (1950) protected plants of cotton, peas, beans and nasturtiuns from aphid attack by preplanting seed treatment. Ashdovn and Cordner (1952) protected peas from aphid infestations with diethoxy— thiophoSphoric acid ester of Z-ethyl mercaptoethanol, applied both as a soil and seed treatment. Schradan and demeton were also used successfully for the treatment of peas and beans (David and dardiner 1955). These authors did not observe preferential absorption of water or thiol isomer of -7- demeton by the seed. These findings were in agreement with the conclusion of Atkins (1909), that the membrane of bean seeds becomes semipermeable only after gernination. Chao (1950), however, observed selective absorption of water from a solution of schradan by broad beans. a summary of the many studies associated with soil applications of systemic insecticides indicates that differential absorption of the toxicant by the plant does taxe place in various soil types. Getzin and Chapman (1959) studied the effect of soil type on uptake of systemic insec- ticides and found the highest translocation rates of Thimet and dimethoate in sand; lesser rates in clay loams and lowest in muca. They also studied the binding of the insecticide to soil by measuring leaching rates of radio— active Phosdrin through colunns of twelve soils. They correlated binding of the insecticide with the base-exchange capacity, organic matter, and nitrogen content of the soil, and concluded that organic hatter content was the factor primarily reSponsible for insecticide binding. The translocation of systemic insecticides within the plant is not clearly understood. Bennett (1957) suggested that the process of transloCation would Vary according to the insecticide and the site of absorption. Chao (1950) demonstrated that schradan, when applied as a seed treatnent, tended to accumulate more in the older parts of the plant than in the young leaves. havid and Gardiner (1955) found that the distribution of the thiol isomer of demeton in the plant was fairly uniform after seed treatment. In 1952 Metcalf and march demonstrated that labelled schradan and phOSphoric acid were translocated uniformly throughout lemon seedlings. Metcalf 33 31 (1954) found that movement of the thiono isomer of demeton in lemon seedlings was similar in amount and direction to that of schradan, though it tended to accumulate more in the terminal leaves. Due to the high mammalian toxicity of host systemic insecticides, detoxification is essential in edible crops. however, for the insecticide to be efficient, it should not break down too rapidly. Detoxification has been reported as occurring by actual loss of toxicant from the plant surface or by the breakdown of the toxicant by the plants (Bennett 1957). in 1949 Bennett showed that vapor loss of dimefox and bis (2-fluoroetnoxy) methane occurred from leaves rain, Thomas gt 1 (1955) J * following root absorption. A; demonstrated that the volatile thiol isomer of demeton was translocated to, and given off from the leaves, following root absorption. Guyer 32 a; (1958) found that Thimet residues in wheat seedlings decreased from 4.9 p.p.m., 15 days after planting to below a detectable amount 57 days after planting. hartley and heath (1951) suggested that the breakdown of schradan was by enzynic oxidation. Before the development of systenic insecticides, rotenone, pyretnrins, piperonyl cyclonene, methoxycnlor and other naterials were employed for control of insects on beans. The organophoSphorus insecticides, malathion, parathion, and demeton have been evaluated for control of Dean insects by Brett and nrubaker 1955 and 1958, noore 1950, Ditman and Liley 1958. In 1957 Jay and heedhan systemically protected beans from aphid infestations by applying demeton as a seed treatment and soil application. Guyer gt a; (1960) reported satisfactory control of sucxing insects and a reduction of Mexican bean beetle injury when 1 and 2 pounds of rhimet or Di-Syston were applied per acre. In 1960, Wilcox and Rowland reported good control of two-spotted Spider mites on lima beans for 2 to 5 months by soil appliCations of granular or liquid formulations of Thimet and Di-Syston as seed-furrow treatments. F1 PROCEJUK All experiments in this study were conducted in the Michigan state University llant Science greenhouses at East Lansing, Michigan. Eight-inch clay pots, sterilized greenhouse soil and darx red Kidney beans (Phaseolus vulgaris 1. var. Darx med hidney) were used except for Experiments I and VI, where different soils and bean varieties were sub- stituted. all treatments were randomly arranged in 4 replications. Regular Thimet (phorate) 10p granular was used in all experiments. Di-syston and Ihimet 0.8. both 10p granular were also used in Experiment 11. The rates given throughout this presentation are amount of active toxicant. Insects used for Testing The Mexican bean beetles used for these investi- gations were collected from bean fields in nontcalm County, Michigan. They were then reared in the greenhouse by a modification of the methods described by leterson (1959). The beetles were reared on tender bean plants grown in four-inch clay pots at temperatures from 750 to 850 F. and at relative humidities over 60 percent. A large, wooden framed, galvanized screen cage, measuring 25 x 60 x 50 inches was used for oviposition. cluorescent bulbs were -10- -11- installed over the cage to furnish the light reguired to maintain la to 15 hours photoyeriod. dgg masses were collected on alternate days. The excessive leaf naterial was removed, leaving about 1 sduare-inch-piece of leaf with the egg mass. These egg masses were then placed in a covered Petri dish on a disc of blotting paper, vetted with a saturated solution of sodium chloride. The eggs started to natcn after 5 to 7 days when xept at 700 to 750 F. The larvae, upon hatching, remained clustered on the empty egg shells for about 10 to lo hours. nithin this period the leaf pieces with the young larvae were placed on tender bean foliage. The plants with the young larvae were placed in another wooden framed, galvanized screen cage measuring 15 x 22 x 32 inches. fresh foliage was supplied every 2 days. when the larvae pupated, the yupae were collected in a pint box and placed in the oviposition cage. Bean aphids (aphis fabae bcop.) were collected in august from field beans in oaginaw county, michigan. The cultures were maintained on young bean plants in the green- house. attempts to rear these aphids for an extended period failed. Two different cultures of two-spotted spider mites, Tetranychus telarius (L.) Jere maintained. The culture used for Experiments I and II was from the hichigan State University -12- greenhouse culture. These mites had been subjected to treatments of numerous miticides and organephOSphoric insecticides. The nites used in Experiments V and VI mere collected from a red clover field on the Michigan State University farm, Crop Farm, East Lansing. This mite culture was reared on bean plants in the greenhouse, but was isolated from greenhouse infestations. Vinegar flies, Drosophila melanogaster Meig., were obtained from a calture maintained in laboratory. Methods of Seeding_and llacement of Insecticides In EXperiments I to V1 the insecticide was applied in bands in tne soil. when this method was used an 3-inch clay pot was filled with 5 inches of well compacted soil. A thin band of the insecticide was applied and over this band one inch of soil was added. Five bean seeds were then planted and arranged so that the band of insecticide was 1 inch below and 2 inches to the side of the seeds. The seeds were covered with 1 inch of soil leaving still another 1 inch of Space in the pot for holding water. In the in-row application the insecticide was placed directly with the seed over 0 inches of soil in the clay pot and covered with 1 inch of soil. -15- for the broadcast treatment the insecticide was mixed with the upper l-incn layer of soil around the seed. Bioassay Tests A modifiCation of the floating leaf-disc method as described by Rodriguez (1955) and Eichmeier (1958) was used to test nites. Using this method, leaf discs were excised with a seven-eighth inch diameter cork borer from detached leaves. as the discs were cut, they were fastened to a piece of pencil eraser by a pin and floated in a ietri dish containing a 2 percent sucrose solution. The piece of eraser acted as an ancnor and xept the discs in position. The floating discs were infested with 5 female two-Spotted Spider mites. The Ietri dishes with the infested, floating discs were placed under fluorescent lights and watered regularly. Tests with aphids were conducted in the Same manner using 5 aphids on each leaf disc. The leaf discs were maintained for a period ranging from o to 10 days after which the number of mites and aphids on the discs were counted. The first instar hexican bean beetle larvae tests on bean plants were conducted by placing a piece of leaf containing 50, 8 to 10-hour-old larvae on the foliage. mortality counts were made every 2 days. The second and third instar larvae and adults of hexican bean beetle were placed directly on the bean foliage for feeding and mortality studies. -14- Assessment of the feeding injury by the Mexican bean beetle larvae and adults on bean foliage was rated as follows: 1 - scattered feeding; 2 . 1/4 to 1/3 leaf destroyed; 5 = 1/5 to 1/2 leaf destroyed; 4,: nore than 1/2 leaf destroyed. Experiment I The purpose of the first experiment was to evaluate he uptake of Thimet from widely different soil types. It was begun on July 25, 1900 using the following three soils: 1) much (obtained from the Huck Experi- mental Harm, Clinton 00.). 2) loam (obtained from the Lutz Farm, montcalm 00.). 3) loamy sand (obtained from the hillman Farm, montcalm 00.). Each of the soils received a banded 2-pound Thimet treatment and together with an untreated control, made 6 pots in each of the 4 replications. On August 9 (15 days after planting) bioassays were conducted nith 2 sets of leaf discs excised from the first true leaves of the treated and untreated bean plants, using two-Spotted Spider mites (greenhouse culture) and bean aphids. Counts of mites and aphids were made after a and 9 days reSpectively. On August 15 (21 days after planting) bioassays with 2 more sets of leaf discs -15- from the third and fourth trifoliate leaves of the bean plants were conducted using mites and aphids. In this case counts of mites and aphids were made after a and 10 days respectively. Bioassay tests with first instar hexican bean beetle larvae were conducted from august 19 - 25 on treated and untreated bean plants. mortality counts were made every 2 days. On august 27 (53 days after planting) the plants in the 5rd and 4th replications were infested with 10 second and third instar larvae of the bean beetle. Larval mortality counts were made every 2 days. Feeding injury of ' .V -, ,, -. .V . A 7'. _.+r If) the Mexican bean beetle larvae (as us~esscg aCCori-ng to tho (1 rating described in the methods section. On September 14 (50 days after planting) two green bean pods were picxed from each of the treatments and the controls and placed separately in 1 pint paper boxes along with b newly emerged mexican bean beetle adults. The feeding damage and hortality were evaluated after 5 days. all the plants were removed from the pots on September 21 so the roots could be examined for symptoms of phytotoxicity. xperiment 11 L11 The second experiment was established on august 5 _‘ to correlate various rates 01 Thimet and Di-Syston applications -15- with insect mortality in the aerial plant parts. The insecticide application, eXperimental design and planting methods were the same as those used in Experiment 1. Sterilized greenhouse soil was used with insecticide treat- ments as follows: Treatments Dos:ge per acre Granular Thimet 1 1b. Granular Thimet 2 lbs. Granular Thimet 4 lbs. Granular Thimet C.3. 2 lbs. Granular Di-Syston 2 lbs. Control -_ On August 19 (14 days after plantinq) bioassays were conducted with mites (greenhouse culture) and aphids on leaf discs excised from the first true leaves. After one week, the same tests were run with discs froa the third and fourth trifoliate leaves. Eioassays with first instar larvae of the Mexican bean beetle were conducted on all bean plants, on august 27. The plants were also assessed for phytotoxicity symptoms and stand counts were made. On September 11 (55 days after planting) mortality and feeding tests were carried out with 5 newly eaerged Mexican bean beetle adults on 2 of the a replications. The -17- plants in this experiment were heavily infested with mites and whiteflies, Trialeurodes vaporariorum (Uestwood) as a consequence of normal greenhouse migrations. The bean plants were assessed for the degree of infestation. llants were rated from 1 to a according to the number of mites or whiteflies per infested leaf as follows: 1 - less than 20; 2 = 21-40; 3 = 41-60; 4 = 61 and more. Experiment III The third experiment was set up on august 23, to study the effect of different levels of soil moisture on the uptake of Ihimet. The arrangement of the exgzriment was similar to EXperiments I and II. The soil-moisture levels were designated as high, normal and low and each contained a 2-pound Thimet treatment and an untreated control. The high moisture level was obtained by placing pots in metal trays containing approximately one inch of water which was maintained throughout the experiment. The normal moisture treatments were watered in the regular manner and in the low moisture treatments just enough vater was added for the plants to survive. fifteen days after planting, bioassays were carried out with first instar hexican bean beetle larvae. ht this stage the plants in the low noisture treatments had developed -15- only 2 leaves while the plants in the other treatuents had reached the first and second trifoliate leaf stage. On September 19 a second bioassay test was run again with first instar larvae. Feeding injury, stand and growth were recorded. The normal growth of the bean plants was adversely affected by the nigh and low moisture treatments. E xLe r i ment IV The fourth experiment was established on September a to study the effect of high rates of Ihimet on bean plants and to evaluate the effect of excessive rates of Ihimet on what appeared to be a resistant mite population. Ten percent granular Thimet was applied in soil bands at the rate of a, S and 12 pounds per acre. Records were obtained on stand, growth a.d phytotoxicity. Experiment V This experiment was arranged on September 15 to ascertain the minimum rates of Thimet effective for the control of mites and Mexican bean beetles. The four treat- ments in this eXperinent were 0.25, 0.5, and 1.0 pounds of Thimet (10g granular) per acre and a control. The bean seeds and insecticides were arranged as in the preceding eXperiments. -19- On October lo a bioassay was made with the first instar larvae of Mexican bean beetles in the same manner as in the srevious experiments. Lioassays using the floating leaf disc method were started on October ll and 27 using mites from the red clover field culture. By November 5 the bean plants were heavily infested with mites and whiteflies and were destroyed. Experiment VI On September 2 Experiment VI was arranged to evaluate the uptake of lhimet in four varieties of field beans. The varieties used were hichigan Darx Red hidney, Michigan Yellow Eye, nichelite and nichigan Cranberry. Eacn variety of bean received a l-pound fhimet treatment applied in the same manner as before and a control. On Octooer Zo a bioassay was carried out by the leaf disc method with nites from the red clover culture. Exteriment VII The last exyerinent was begun on December 14 to compare in-row and broalcast treatnents of Thinet, and consisted of 7 treatments. In the in-row agplication, the Thimet was placed with the bean seeds and in the broadcast treatment it was mixed with the upper one inch of soil. The three in-row treatments were 0.5, 1.0 and 2.0 pounds of Thimet per acre, and the broadcast treatments 0.5, l.O and 5.0 pounds per acre. On December 30 (15 days after planting), bioassay tests were conducted with DroSOphila melanogaster heig. on leaf tissue media. The first leaves from replications I and II were removed from the plants. Ten grams of leaf material were collected fron each treatment and placed in a l-pint Mason Jar with 20 grams of distilled water. The leaves were macerated in the water by a blending Osterizer. To the macerated leaf material, 70 grams of pumpkin media were added and mixed thoroughly to give a homogeneous mixture of pumpkin media and leaf matter, which made a dilution of 10 percent leaf matter. Further dilutions of 5, 2.5, 1.25 and 0.625 percent were made with pumpkin media. Thus there were 5 dilutions of leaf mixture for each treat- ment in the eXperiment. Two shell vials were aarxed for each of the different treatment dilutions and small strips of paper towels Mere placed in each vial. Small amounts of the diluted leaf-pumpxin media were Spread on the papers inside the vials according to their reapective dilutions. About 25 to 50 flies were anaesthetised with ether and carefully -31- inserted in each of the vials. The vials here plugged with cotton and naihtained at £00 0. after 3i hours the mortality of flies was recorded for all the vials. a standard test employing pure fhimet dilutions with pumpkin media was also run to establish the 3350 dosage curve. 3350 values for different treatments were estimated by the "Nomograpn Calculations of hose-effect" method of Litchfield and ;ilcoxon and were then compared with the 2350 dosage of the standard Thimet test. These values were then correlated with the amount of Thimet in leaves of the different treat- ments. On January 9 and 19, 1901 two similar bioassay tests were carried out. Leaves from the replications III and IV were used for one test on January 9, and leaves from all tne replications for the test on January 19. The leaf matter concentration in pumpkin media for these tests ranged from 10 to 0.625 percent for the January test and 20 to 1.25 percent for the test on January 19. '4“: Tfl‘yfid (".1, V‘ ‘ ' ’~\ \I ;‘ ,"ITY " W I' ”T1 f“ ”I ' 1"; - ” :' lizuLJLJl‘LJ'Xi ldii run.) thUUQQiUlV Ur :{lbuulid Experiment I Results of bioassays conducted on august 9 and 15 on detached leaf discs with two-Spotted Spider nites (greenhouse culture) and bean aphids are presented in Table 1. After 6 days there were no significant differences between mite populations on the treated and untreated plants. This was in contrast with the findings of fiilcox and Rowland (1960) who reported 2 to a months' control of two—spotted spider mites with Thimet dosages of less than 1 pound per acre applied to the soil. It was postulated that the green- house mite culture used for this assay was resistant to Thimet. There was a significant reduction in numbers of bean aphids on discs from tne first true leaves removed from the Thimet treated plants, grown in loam and loany sand soils. Aphid populations on leaf discs from treated plants grown in muck were not significantly different from those of control plants, in fact, there was a trend toward higher populations on discs from treated plants. Aphid populations on discs from the top trifoliate leaves which were removed from both treated and untreated plants grown in loan and loamy sand soils were significantly lower than those from the plants grown in much. Lonever, there were no significant Table l. hunbers of two~spotted sgider mites (from greenhouse cultures) and bean aphids on leaf discs, removed from tne (a) first true leaf and (0) top trifoliate leaves of plants grown in three different soils. .. i a . .. mean) ) number of nites.or aphids per leaf disc 11 from first from Soil Iounds true leaves top trifoliate of actual 15 days leaves 20 days Thimet per after planting after planting acre Mites Aphids mites aphids duck 2 9 a 55 be 33 a 10 b huck - 22 a 14 ab 23 a 20 0 Loan 2 13 a 2 a 3‘ a 0 a Loan - 25 a 2 be 57 a 4 a Loamy sand 2 19 a 7 a 31 a 1.0 a Loany sand - 22 a 45 c 45 a 5.0 a (i) heans with a cohlon letter are not significantly different fron each other at the 5 percent level (Duncan l95o). (ii) mites and aphids were naintained on the discs for 6 and 9 days respectively. differences in the populations on discs from treated and untreated plants grown in these two soils. The variation in results in this part of Experiment I was thought to be due to the difficulty in maintaining a strong bean aphid culture as well as problems in developing a consistent technique for disc culture. Results of the feeding studies conducted with a) first, and b) second and third instar Mexican bean beetle larvae are summarized in Tables 2 and 5. There was a maximum reduction of only 65 percent of beetle larvae on treated plants grown in muck, in contrast to 100 percent mortality of larvae on plants grown in loam and loamy sand soils. In both tests, however, there was a significant reduction in larval populations 2 and 4 days after infestation on treated plants grown in much. The rating of feeding injury on the leaves is presented in Table 4. All control plants were almost completely destroyed 24 days after infestation. Treated bean plants grown in the loam and loany sand soils were injured the least, and the injury was significantly less than that on the treated plants grown in muck. hexican bean beetle adult feeding damage on the green bean pods was evaluated and the results are given in Table 5. The degree of feeding damage to pods did not 0'— - '90- Table 2. Mortality of first instar Mexican bean beetle larvae fed on foliage of bein plants grown in three different soils. Ilahts were infested with larvae 26 days after planting. ’i Iercent larval nortality‘ ) Iounds of Soil actual Thimet 2 days 4 days per acre after larVal after larval infestation infestation much 2 55 b 65 b muck - 20 c 25 c Loam 2 100 a 100 a Loam - 25 c 53 c Loany sand 2 100 a 100 a Loamy sand - 25 C 50 c (i) means with a comnon letter are not significantly different from each other at the 5 percent level (Duncan 1955). ‘ VI -39.. Table a. mortality of second and third instar hexican bean beetle larvae fed on foliage of bean plants grown in three different soils. Larvae placed on plants 55 days after planting. Percent larval mortality(l) Pounds of Soil actual Ihimet 2 days 4 days per acre after larval after larval infestation infestation muck 2 55 b 55 b Luck - 2 c 12 C Loam 2 57 a 87 a Loan - 7 be 20 C Loamy sand 2 95 d 100 d Loamy sand - 5 C 17 C (i) Means with a common letter are not significantly different from each other at the a percent level (Duncan 1955). “,7 -’ - N Table 4. assessnent of feeding injury of mexican bean beetle larvae on bean plants grown in three different soils. Rating of feeding injury: 1 . scattered feeding; 2 = 1,4- l*3° 3 - 1 5-1/2' and 4 a more than 1’2 of leaf consumed. I 9 / ’ / lounds of Mean feeding Soil actual Thimet (1) per acre injury Loam 2 1.1 c Loam - 3.8 a Loamy sand 2 1.0 c Loamy sand - 4.0 a (i) means with a common letter are not significantly different from each other at the 0 percent level (Duncan 1955). Table 5. CO v kl‘ EValuation of nexican bean beetle adult feeding injury on green bean pods removed from plants grown in three different soils. Feeding injury rating: 1 - no feeding; than 1/2 a a 2 less than 1/4; 5 - l/a to 1/2; and 4 = more of the outer pod surface. Pounds of Mean feeding Soil actual Thimet ( ) per acre injury muck 2 4 a 1'“.le - 4 d Loam 2 4 a Loam - 4 a 0.8 a Loamy sand 2 4 a Loamy sand - (i) means with a common letter are not significantly different from each other at the 5 percent level (Duncan 1955). ‘j. indicate any signifiCant difference among treatments and controls. fhis indiCated that the toxicant, if present in the pods, was in very minute juantities and had no effect” on the feeding of adult nexican bean beetles. There was no indication of phytotoxicity symptoms in any of the soil-insecticide treatment combinations in EXperiment I. Results of this eXperiment indicated that Thimet was less effective in muck than in the loan and loamy sand soils. Getzin and shapnan (1959) reported that Thimet uptake was greater in sand and clay loans than in much. They indicated that organic matter bound the insecticide in the soil. This binding effect of Thimet on much may have influenced the results in Experiment 1. £Xperinent ll The results of the bioassays carried out on nurust 19 and 24 (14 and 19 days after planting) with wo-spotted Spider nites and bean 'phids on excised leaf discs are summarized in Table 0. Mite counts made 6 days after infestation indicated that there were no significant differences in populations of mites on the leaf discs renoved from the treated and control plants. These results give additional support to the hypothesis that the mi es from the greenhouse culture were resistant to Lhimet. Aphid Table 6. Humber of mites and aphids on detached leaf discs fron the first true and top trifoliate leaves of bean plants (1) grown in treated and untreated soil. mean number per disc Pounds of Treatments ingggtiiide First true leaf disc Toplggifglizte per acre mites aphids Aphids Thimet 1 61 a 24 a 4 a Thimet 2 4d a E a 5 a Thimet 4 45 a 2 a 11 a Thimet 2 55 a 7 a 17 a Di-Syston 2 50 a 37 a 12 a Control - 57 a 67 a 18 a (i) Mites and aphids were counted a and 10 days resLectively after infestation. (ii) Means with comnon letter are not significantly differently from each other at the 5 percent level (Duncan 1955). -51- counts were taken 10 days after infestation and in both tests, on discs from the first true leaves and upper tri- foliate leaves, tne djhld populations appeared to be higher in the controls. Differences were not statistically significant, however. The lacx of significant differences between the treatments and controls nay have been due to the variable size of the populations in the different replications. The nortality of first instar Mexican bean beetle larvae was significantly hixher on the treated plants than on the controls (Table 7). all treatnents had 10C percent larVal mortality four days after infestation. Table 8 shows tne results of the nortality studies with Mexican bean beetle adults on oean plants. The 2 and 4-pound applications of Thimet resulted in 100 percent hill of the beetles after 2 days. all other treatnents gave 10o percent beetle mor- tality in 4 days in contrast to no mortality in the control. Results of the assays with second and third instar nexican bean beetle larvae on bean plants are presented in Table 9. The larval aortality on all treated plants was high as early as 2 and 4 days after feeding was initiated. Thimet applications of 2 and 4 pounds per acre gave 100 percent mortality within 2 days. There were no significant differences in mortality among the different treatmen s. assessment of feeding damage by the flexican bean beetle {0 I (3. Table 7. nortality of first instar Mexican bean be;tle larvae placed on bean plants 52 days after plantinr; rro n in treated and untreated soil. Percent larval mortality(l) lounds of Treatments actual insecticide 3 days after 4 days after per acre larval infestation larval infestation Thimet l 87 a 100 a Thimet 2 100 a 100 a Thimet 4 ‘ lOO a 100 a Thimet C.3. 2 87 a 100 a Di-Syston 2 75 a 100 a Control - l4 b 24 b (i) Means with cannon letter do not differ significantly from each other at the 5 percent level (Duncan 1955) Table 8. hortality of genican bean beetle adults fed on plants grown in treated and untreated soil.(l) Percent mexican bean beetle nortality‘ll I’ o und :5 of Treatments actual insecticide 2 days after 5 days after per acre larVal infestation larVal infestation Thimet l 70 a 100 a Thimet 2 10a a lOO a Thinet 4 100 a 100 a Thimet 0.8. 2 O b 100 a Di-Syston 2 u b 100 a Control - O b O b (i) Beetles placed on giant 35 days after planting. (ii) heans with common letter do not differ significantly from each other at the 5 percent level (Duncan 1955). r ~04- fable 9. Mortality of second and third instar Mexican bean beetle larvae fed on bean giants grown in treated and untreated soil. LarVae were placed on plants 43 days after planting. Percent larval nortality(l) lounds of Treatments actual insecticide 2 days after 4 days after per acre larval infestation larval infestation Thinet l 75 a 57 a Thinet 2 ' 100 a 100 a Thimet 4 100 a 100 a Thinet 0.3. 2 7l a 85 a Di-Syston 2 71 a 79 a Control - O b O b (i) means with cannon letter do not differ significantly from each other at the 5 percent level (Duncan l955). larvae and adults (Table l0) indicated that the control plants were nearly destroyed after the tests, with little injury to the treated plants. No significant variations in feeding injuryvere indicated aaong the different insecti- cide treatnents. Variations in natural mite and whitefly infestations in the greenhouse are given in Table ll. Intensive mite infestations were present on plants from both the treataents and control, HOMBVGT, there were significantly more mites on the Di-Syston treatment and control. There Was a relatively high whitefly infestation with no si:nifiCant differences among treatnents and controls. Lith the exception of the a-pound Thimet treatment, the insecticide applications appeared to have no adverse effect on normal gernination and growth of the bean plants. ilants treated with 4 pounds of Thinet per acre showed slight yellowing of leaves during the early period of plant gr 0V3! til 0 EXLCI‘ inent III The results of feeding studies of first instar hexican bean beetle larvae on high, nornal and low soil moisture treatnents are presented in Table l2. Two days after infestation the larval mortality did not vary significantly in any of the treatuents or control. larval Table 10. Assessment of feeding injury by Mexican bean beetle (56 days after glanting) on bean plants grown in treated and untreated soil. m V iounds 0? . Mean of feeding freatnents actual insect101de 'n'u ‘(1) per acre 1‘3 rJ Thimet' 1 1.25 b Thimet 3 1.00 b Thimet 4 1.00 b Thimet 0.8. '2 1.00 b Di-Lystoh 2 1.75 b Control - 4.00 a (1) means with COfllOfl letter do not differ significantly from eacn other aILtnna 5 percent level (Duncan 1955). -Q7_ Table 11. Number of naturally occurring nites and white- flies on bean plants grown on treated and untreated soils. Ratings of infestation estimated as number of nites and whiteflies per infested leaf: 1 = less than 30; 2 = 31 to 40; 5 = 41 to 60; and 4 = 61 and above. hean degree of infestation‘l) 1 ounds of freatnents actual insecticide per acre nites Nhiteflies Thihet 1 2.75 be 1.25 a Thinet 2 2.75 be 1.00 a Thinet 4 2.25 b 1.2 a Thinet 0.5. 2 3.50 b 1.35 a Ji-Syston 2 5.50 ac 1.L5 a Control — 4.00 a 1.75 a (i) means with a codmoh letter do not differ significantly from eacn other at the 5 yer cent level (Duncan 1955). Table 12. Mortality of first instar nexican bean beetle larvae fed on bean slants grown at three soil moisture levels. (i) iercent larval mortality iounds of larval infestations actual _ Moisture levels Thinet per acre 14 days 22 days after planting after glanting (ii) (ii) (ii) (ii) (ii) 2 days 4 days 6 days 2 days 4 days high moisture 2 17.5a 62.5bc 65.00 25.0b 70.0b high moisture - 10.0a 17.5d 22.5d 12.50 20.0d hormal moisture 2 12.53 50.00 95.08 95.0a 1LD.0a Normal moisture - 10.0a 20.0d 25.01 17.5bc 22.55 Low moisture 2 35.0a 100.0d IMD.Oa 110.0a 100.03 Low moisture - 32.5a 75.0b 75.0b 82.5bc 45.00 (1) means with a connon letter do not differ significantly from eacn other at the 5 Lercent level \Duncan 1355). (ii) Days after larVal infestation. mortality was significantly higher in the treatments than in the controls after 4 and a days. rIhe second test, conducted a week after the first, resulted in higher larval mortality within 2 days in the treated plants than in the control plants. Tne delayed effect of the toxicant in the treated bean plants on Mexican bean beetle larvae in the first test may have been due to an insufficient uptake of toxicant in the plant. ht the initiation of the first assay, the plants had completed 7 to 8 days of growth following emergence and may not have taken up enough insecticide from the soil to have an immediate effect on the Mexican bean beetle larvae. Treated plants from low and normal soil moisture levels produced significantly higher Kill than did treated plants grown at the high moisture level. The sig- nificantly higher nortality in the low moisture controls nay have been due to stunted growth and lack of succulent foliage for larval feeding. Assessment of the feeding injury of adult Mexican bean beetles on bean plants is presented in Table 15. All treated plants at the different soil moisture levels had significantly less feeding injury than the plants in the control. The relationships between height and Spread of the plants grown at various soil moisture levels are given in Table 14. The plants in the low moisture level were signifiCantly shorter and had less growth than the plants -4u- Table 13. assessment of feeding injury of Mexican bean beetle larvae on Thimet treated and untreated plants grown at three soil moisture levels. iounds of Mean feeding moisture Levels actual Thimet (i) per acre injury high moisture 3 1.8 bc High moisture - 2.8 d normal moisture 2 1.; ab Normal moisture - 2.3 cd Low moisture N |._J O L) Low moisture - 2.5 cd (i) Means with a comaon letter do not differ significantly from each other at the 5 percent level (Duncan 1955). -41- ‘ Table 14. mean height and spread (crown width) of tne Thimet treated and untreated bean plants grown at three soil moisture levels. iounds of Mean height Mean Spread Moisture levels actual Thimet (.) per acre in inches in inches high moisture 2 15 b 12 b high moisture - 17 a 15 a Normal moisture 2 16 a 17 a Normal moisture - 16 a 16 a Low moisture 2 6 c 10 b Low moisture - 6 c 8 b (i) Means with a common letter do not differ significantly from each other at the 5 percent level (Duncan 1955). -42... in the other moisture levels, with the exception of the plant spread in the high noisture level. In tne high moisture level the treated plants were significantly shorter and had less growth than the controls. factors that may have affected the bioasnhrresults of this eXperiaent were: (a) high temperatures (over 1000 f.) for short periods and (b) excessively high and low soil moisture levels. Experiment IV The bioassar tests with two-spotted s;ider mites (greenhouse culture) showed normal mite reproduction on detached leaf discs, from plants treated with as high as 12 pounds per acre of Thinet soil band treatments. This added additional weight to the hypothesis that the mites from the greenhouse culture were highly resistant to fhinet. Seed germination in all treatments was satisfactory. However, symptoms of phytotoxicity vere observed in all Thimet treat- zue1n.s. EXperiment V Table 15 presents the results of the bioassays conducted With first instar hexican bean beetle larvae on treated and untreated bean plants. The larval aortality Table 15. mortality of first instar nexican bean beetle larvae fed on bean plants treated with three rates of Thimet. Plants were infested 23 days after seeding. (1) Percent larval mortality lounds of Treatments actual l'himet per acre 2 days after infestation Thimet 1.00 100.0 a Thihet 0.50 81.5 ab Thinet 0.25 65.5 b Control - 15.0 c (1) Means with a cannon letter do not differ significantly from each other at the 5 percent level (Duncan 1955). -44- on the treated plants mas significantly higher than the controls. One goind fhimet per acre gave 100 percent hill in 2 days. desults of the bioasSays conducted on October 11 and 37 (80 and a2 days after planting) with two-Spotted Spider mites (red clover culture) gave significant reductions in the nite populations on the discs from plants treated with 1 pound fhimet (Table 15). In the first assay, discs from the 0.25 and 0.5 pound Thinet treatments produced signifiCant reductions in the hite (opulations. Results of these bioassays indicated that Thimet at dosages as low as 0.5 pounds per acre produced mortality of the Mexican bean beetle larvae and two-Spotted Spider mites for 42 days after flanting. Exnerinent VI _L Exteriment VI was designed to study the effects of Thinet in terms of insect aortality and pnytotoxicity to different bean varieties. Uioassay results with two-spotted Spider aites (red clover culture) on excised leaf discs in this exterihent are presented in Taole 17. The leaf discs from tne lhimet treated plants in tne four different bean varieties gave significantly lower mite populations as compared to their controls. There were no significant differences between mite )opulations on leaf discs from fable 15. lopulations of the two-Spotted spider aites (red clover culture) on detached leaf discs from plants treated with three rates of Ihihet. lounds of Treatments actual insecticide mean number of mites after 9 days Leaf discs Leaf discs per acre infested 20 days infested 42 days after planting after planting Thimet 1.0 2.5 a 15.0 b Thimet 0.5 o.0 a 41.0 a Thiaet 0.25 15.0 b 50.0 ab Control -- 0.0 c 47.0 a (i) Jeans having a connon letter 1o not differ significantly fron each other at the 5 percent level (Duncan 1955). -45- Table 17. hean nunber of sites per detached leaf iisc from the lhimet treated and untreated plants of four different bean Varieties. Bounds of Mean nunber of Variety actual Thimet sites 10 day per acre after infestation michigan Dark Red Lidney 1 14.5 * hichigan Darh ded Lidney — 52.5 nicnigan Yellow Eye 1 5.0 x hicnigan Yellow Eye - 55.0 .. a . r x niChelite 1 15.5 Michelite - 61.0 y'. 0 ' v x hichigan branberry l 4.5 nichigan Cranberry - 64.0 3EDiffers from controls at the 5 percent level of significance (L.5.D. test, Snedecor 1957). -137- any of tne treated plants. There was no evidence of phyto- toxicity to any of the Varieties. Enperinent VII Dilutions of 95 percent technical Thimet with puhphin media were bioassayed, using Jrosophila melanogaster neig. as a test animal. The results of this assay are presented in Table 13. The 3D50 of fhimet, as determined by the "honograph Calculations of Jose-effect" method of Litchfield and gilcoxon (1949), was found to be 0.155 p.p.m. (slope of line was 1.84). flesults of the three bioassrys conducted with drosophila, using aacerated leaves in gunphin media, are suhmarized in Tables 19, 20 and 21. The leaves used were removed at different stages in growth of bean plants growing in various soil treataents of Thimet. 3350 values for drosopnila in each treatment of the three assays were deternined as described before in this eXperihent; however, tne EJ5O values were expressed as percent leaf matter concentrations. fable 22 gives the conversion values of gercent leaf hatter concentrations for 2350 into parts per nillion (p.g.a.) of toxicant, comparable to Ihiiet in the leaves. 0n the basis that 3D50 for drosoghila mas 0.155 p.p.i. of Ihinet, it was postulated that all nediah effective doses (3 53) of leaf iatter concentrations had 0.153 p.y.n. _ i M . (i . .. . . . Table lo. hortality ) o1 drosognila maintained in different dilutions of 'fhiaet in punphin media for 24 hours. 1 (ii) “D50 (hedian effective dose) Thinet in p.p.m. . . . ,. Iercent mortality in pumpkin neuia 0.500 100.0 0.230 100.0 0.125 254.0 (111) 0.15:) L’opoqo 0.100 25.0 0.075 15.6 0.005 10.6 (i) flies dead and unable to move counted as affected by Lillilnet 0 (ii) hedian effective dose vas deternined by following the hethod of Litchfield g fiilcoxon (1949). (iii) At 5 percent level ED5F confidence limits were 1.75 and 1.53 and slope of ine 1.84. Table 19. Lortalit leaf-gunpxin uedia f removed from plants 3 of or 24 15 LG QFQSOLH11a naintained on bean hours, utilizing bean leaves days after planting. Percent mortality(l) Y. 00\ rounds of ED50(11’ Tre tne to actual Eercent leaf matter as Slope d “ n o Tninet in media percent of per acre leaf line matter 10 5 2.5 1.25 0.62 In row with seed 2.0 59.7 62.5 21.5 15.9 1.6 4.2 1.95 In row with seed 1.0 91.3 61.8 57.0 6.6 0 5.5 1.96 In row with seed 0.5 66.6 30.7 27.5 6.0 0 6.6 2.46 Broadcast in soil 5.0 100.0 37.5 54.8 20.7 1.9 2.6 2.04 Broadcast 2roadcast in soil 0.5 100.0 54.4 50.1 0 4.2 4.4 1.64 Control - (i) Flies which mere moribund and unable to maln. (ii) an- magnod. 0 determined by litcnfield and nilc xon (1349) '1 -|JU- fable 20. mortality of drosophila nairtained on bcan leaf- yunghin dedia for 24 hours, utilizing bean leaves removed fPOJ giants 25 days after planting. . . i iercent mortality( ) iounds of 3350(11) ' (u ‘ J r e . C. as Slave freatnents actual lelc nt leaf matter h t we Ihimet in nedia I,ercen of per acre leaf Line matter 10 5 2.5 1.25 0.625 In row with seed 2.0 100.0 19.0 52.0 17.8 5.7 5.9 2.63 In row with seed 1.0 24.6 16.1 0 0 0 10.75 1.77 In row With seed 005 $105 0 O O O -- Broadcast in soil 3.0 100.0 50.0 51.5 12.0 2.6 5.45 2.56 Broadcast in soil 1.0 100.0 66.6 0 7.6 6.2 4.2 1.50 Broadcast in soil 0.5 84.6 40.0 20.0 2.9 0 5.25 1.11 (i) Elies which mere moribund and unable to walk. “fl (ii) ED deternined by Litchfield and ”ilcoxon (1949) me nod. -51- 5 Table 21. Mortality of drosohhila aaintained on bean leaf- pumpkin nedia for 24 hours, utilizing bean leaves renoved from plants 55 days after ;1anting. - . i lercent mortality( ) Pounds of 3350(11) Treatuents actual lercent leaf natter as Slope Thinet in hedia percent Of per acre leaf Line matter 2 10 5 2.“ 1.25 In row with seed 2.0 92.5 40.7 26.9 15.9 0 7.0 2.82 In row with seed 1.0 84.1 01.1 5.7 0 O 10.0 1.75 In 1.01” with seed 0.5 96.7 5.1 2.7 0 0 Broadcast in soil 5.0 100.0100.0 71.6 23.0 0 2.5 1.4: Broadcast in soil 1.0 52.0 21.4 0 0 O 10.0 1.49 Troadcast in soil 0.5 75.0 26.6 9.1 0 O 14.2 1.99 (i) flies which mere aoribund and unable to valk. (ii) EDrO deterained by litchfield and wilcOXOn (1949) meenod. “I 1‘ o (u Table 22. amount of toxicant estimated to be in the leaf tissues of the bean plants treated vith soil arplications of fhimet. I.p.m. of toxicant lounds of Treatments actual Days after planting Thimet per acre 15 days 25 days 55 days III-TOW 2.0 5.9 309 2).]- In-row 1.0 4.5 1.4 1.5 In-row 0.5 2.5 - — Broadcast 1.0 5.0 5.6 1.0 Broadcast 0.5 5.4 2.9 1.0 of'fihimet, at the particular concentration. pith this hypothesis, the p.p.m. of Thinet were estimated in leaves for each treatment at particular stage of plant growth. The data in some treatments were heterogeneous, so that the confidence limits included probabilities of high and low values. The 0.5, 1 and 2 pound in-row treatments in the first assay produced highly heterogeneous data. In general, higher amounts of toxicant were estimated to be present in the leaves 15 days after planting, and smaller amounts were found 25 days after planting in all except the 2 pound in-row and 1 pound broadcast treatments of Thimet. These differences may have been due to experimental error. There was a considerable decrease in quantities of toxicant 55 days after planting, with the exception of the 1 pound in-row treatment. It has been determined that Thinet follow- ing uptake in plants is converted into its isomers or metabolites. Some of these metabolites were found to be potentially more effective cholinesterase inhibitors than is pure Thimet (Metcalf et al, 1949). The amounts of toxicant estimated to be present in the leaves, then, could pro- portionally be in much lesser duantities. This experiment also indicated that the amount of toxicant estimated to be in the plants' tissues was not directly proportional to Thimet applied; however, the 2 and 5 pound treatments of Thimet did indicate relatively high amounts. The method enployed in preparin: the leaf and pumpkin media was not completely satisfactory. A fairly high amount of moisture was reauired for a homogeneous mixture, which may have affected the outcome of the bioassays. lhytotoxicity symptoms were observed in the 2 pound in-row and a pound broadcast Thimet treatments, with slight stunt- ing in growth. c YT: : '9 \f ~41 LIIA‘H'J-JLK .1 Soil applications of granular Thinet and Di-Syston were evaluated for (a) phytotoxicity and (b) control of foliage infesting insects and mites on bean plants. Experiments were conducted in the greenhouse vith various rates and methods of insecticide applications, on 5 different soil types, vith 5 soil moisture levels and 4 varieties of field beans. Bioassays were carried out with two—Spotted Spider nite, Tetrahychus telarius (L.), bean aphid,Aphis fabae Scop., mexican bean beetle, fipilachna varivestis Muls. and vinegar fly, hros0phila nelanogaster ‘1 G“ 3-18le The results indicate: 1) a greenhouse nite culture of two-Spotted Spider mites was found to be resistant to soil applications of Thimet as high as 12 pounds per acre. 2) Band applications of Thimet at rates as low as 0.25 pounds per acre were effective in controlling a non- resistant strain of the two-Spotted spider nite. 5) Sand applications of l, 2 and 4 pounds of fhimet, and 2 pounds of Di-Syston per acre were effective against mexican bean beetle larvae and adults for 44 days after planting. These rates did not control infestations of wniteflies. 4) Band applications were less effective for control of Mexican bean beetle larvae at 14 days than they were 20 days after seeding. 5) There was no apparent toxic effect on adult nexican bean beetlesfed on green bean pods from plants grovn in muck, loan and loamy sand soils treated with 2 pounds of Thinet per acre. 6) Thinet residues from 5 (1 pound broadcast) to 5.8 (5 pounds broadcast) p.p.n. were present in bean leaves 15 days after seeding. Thirty-five days after seeding most residues had decreased to l p.p.m. 7) Comparable amounts of Thimet applied to beans in-row with seed, or mixed in the soil (broadcast) resulted in more phytotoxicity than when applied in bands 2 inches to the side and 1 inch below the seed. 8) Excessive noisture reduced the effectiveness of the systemic application and increased phytotoxicity synptons. *1fo 'T'Ix" ;‘ Lj'r -.L..) -..LVJ-.ILIL;1. a 1, n.E. and Dal. E-Jorris, Jr. 1959 Experimental prevention of bark beetle trans- mission of Saratocystis ulni (Buis.) Loreau Lith systemic insecticide Chipnan 3-6199. Jour. Econ. ant. 52: 903-904. Andres, L.a., h.T. Reynolis and T.£. fuxito. 1959 The use of systenic insecticides for control of the cabbage aphid on cabbage and cauliflower. Jour. Econ. Ent. 52: 1045-1050. Ashdown, D. and fi.B. Cordner. 1952 Some effects on insect control and plant response of a systemic insecticide a plied as a Spray, a seed treatment, or a soil treatment. Jour. ficon. Ent. 45: 502-507. litigifls , V” .RoGo 1999 The absorption of water by seeds. Scientific Proceedings, Royal Dublin Society 12: 55-46. Bartlett, 3.8. 1951 a new nethod of testing drosophila and technigue for testing insecticides with this insect. Jour. Econ. Sat. 44: 621. Bennett, 8.2. 1949 lreliminary experinents with systemic insecticides. Ann. appl. Biol., 36: 160-165. 1957 Behaviour of systehic insecticides. Ann. Rev. Ent. 2: 279-296. Brett, c.n. and R.I. Brubaker. 1955 nexican bean beetle control with nalathion compared with eight other materials. Jour. Econ. Ent. 45: 912-915. 1953 rests comparing eight insecticides for control of Mexican bean beetle. Jour. Econ. Ent. 51: 555-554. Chao Sens Tsi. ! t) 1950 lrotection ayainst aphids by seed treatnent. Nature 166: 909-910. David, n.A.L. and 5.0.0. Gardiner. 1951 Investigations on the systemic insecticidal action of sodiun fluoroacetate and the in sphorus compounds of aphis fabae Scop. ann. appl. Biol. 53: 91-110. 1955 The aphicidal action of some systemic insecti- cides applied to seeds. ann. nppl. Biol. 45: 594-514. Ditman, L.P. and 2.0. Kiley. 1958 Effectiveness of several insecticides for control insects on snap beans. Jour. Econ. Ent. 51: 07’ “0:3. Duncan, David B. 1955 multiple range and multiple "F" tests. Bio- metrics 11: 1-42. Eichmeier, Jack and Gordon Guyer. 1955 An evaluation of the rate of reproduction of the two—Spotted spider mite reared on gibberellin- treated bean plants. Jour. Econ. Ent. 55: 661-604. Eulton, R.A. and H.C. mason. 1957 The translocation o 1 0 plants. Jour. ngr. Re:. 55: 905-907. '“J Q; N) (I) H "1 Getzin, 1.1. and R.L. Chapnan. 1959 Effect of soils upon uptake of systemic insec- ticides by plants. Jour. Econ. Ent. 52: 1160- 1165. Guyer, G.E., H.L. Bro n and n. Jells. 1955 nn evaluation of systemic insecticides for control of nessian fly in Michigan. nich. ngr. EXpt. bta. cuart. Bul. 40: 595-502. Guyer, G., a. Hells, A.L. andersen and D. deZeeuw. 1960 nn eValuation of systenic insecticides for control of insects on snap and field beins. Agr. EXpt. Sta. ,uart. Yul. 42: 827-85 P (3 g. k 0 U) Hartley, G.S. and 0.3. heath. 1951 Decomposition of radioactive octamethylpyro- phoSphoramiae in living plants. Kature 157: 513. -5e- Keynes, D.L., G. Guyer and J.W. Butcher. 1958 Use of systemic insecticides for the control of the European Pine Shoot moth infesting Red pine. Mich. Agr. EXpt. Sta. auart. Bul. 41: 259-278. hurd-Larer, n.n. and E.W. P003. 1957 Seleniun absorption by crOp plants as related to their sulphur requirements. dour. agr. Res. 54: 501. 1955 Toxicity of selenium-containing plants to aphids. Science 84: 252. Jepson, L.n., n.J. Jessen and J.0. Complin. 1954 Seasonal weather influence on efficiency of systox applications for control of mites on lenons in southern California. Jour. Econ. Ent. 47: 520-525. Litchfield, J.T. Jr. and E.H. nilcoxon. 1949 a simplified method of evaluating dose-effect experiments. J. Pharn. & Expt. Therap. 96: 99. metcalf, d.L. and 3.5. Carlson. 1950 Testing systenic insecticides against citrus pests. Calif. Citrograph 55: 506, 518. netcalf, 3.1., T.x. Euxuto and 3.3. march. 1957 Plant netabolism of dithio—systox and Thimet. metcalf, R.L. and R.B. March. 1952 Behavior of octamethylpyrophosphoramide in citrus plants. Jour. Econ. Ent. 45: 985-997. Metcalf, 3.1., 3.3. March, T.d. Fukuto and M.G. Maxon. 1954 The behaviour of systox isomers in bean and citrus plants. Jour. Econ. Ent. 47: 1047-1955. Moore, Donald. 1950 Piperonyl cyclonene, pyrethrins and rotenone in dusts to control the hexican bean beetle. Jour. Econ. Ent. 45: 158-190. weterson, Alvah. 1959 Entomological Techniques. Edward Brothers, Inc., nnn Arbor, hichigan. pp. 51—52. Reynolds, K.T., T.R. Fuhuto, R.L. Metcalf and R.3. march. 1957 Seed treatment of field crops with systemic insecticides. Jour. Econ. Ent. 50: 527—559. Rodriguez, J.G. 1955 Detached leaf culture on mite nutrition studies. Jour. Econ. Ent. 46: 715. Snedecor, G.d. 1957 Statistical methods. Iowa State College iress, Ames, Iowa. Thomas, L.D.E., S.h. Bennett and C.P. Lloyd—Jones. 1954 The absorption, breaxdown and systemic behaviour in plants of P-labelled demeton—S. Ann. Appl. Biol. 45: 569-595. May, m.J. and E.h. Needham. 1957 Control of some bean and potato pests using a systemic insecticide applied to the soil and seed. llunthathology 6: 96-105. Jilcox, J. and n.E. howland. 1957 Systemic insecticides applied in low and high- gallonage Sprays on strawberry, lima bean and wise chard. Jour. Econ. Ent. 50: 500-501. 1957 Tests with Thimet on strawberries and turnips. Jour. Econ. Ent. 50: 705—704. 1960 Control of two-Spotted Spider mites on beans with systemic insecticides applied in the soil. Jour. Econ. Ent. 55: 224-227. UOSODOA‘EX. 1960 Systemic chemicals for crop-insect control. n.R.S. Special Report. ARE 22-61. {WW Us “ism .3 f" D- 8"” n