THE PWNTJJ’ARASHIC mmmwues ASSOCIATED WETH CULTEVf‘TEEI BLUEBERWES EN MXCMGAN _—__ THESIS J L [b A R Y Michigan Stag University ABSTRACT THE PLANT-PARASITIC NEMATODES ASSOCIATED WITH CULTIVATED BLUEBERRIES IN MICHIGAN by James Peter Tjepkema Two hundred and seventy soil samples from 30 blueberry farms were processed to determine the frequency of occurrence of plant-parasitic nematodes in Michigan cultivated blue- berries. Soil from beneath blueberry bushes with shoestring and necrotic ringspot virus symptoms was processed to learn if any nematode was associated with the spread of these viruses. Studies on the vertical distribution and seasonal variation of pOpulations of nematodes were conducted to evaluate the sampling procedures. Attempts were made to rear several species of nematodes in the greenhouse on blueberries. Fourteen species or genera of plant-parasitic nematodes occurred in soil samples from blueberry fields but only Pratylenchus crenatus (Loof) and Trichodorus christiei (Allen) occurred frequently. Xiphinema americanum (Cobb) was associ— ated with bushes which had necrotic ringspot virus symptoms. The low number of nematodes in samples was partly accounted for by a seasonal reduction in nematode pOpulations and was not due to the practice of sampling only the top 6 inches of soil. 2. christiei and Tetylenchus Joctus (Thorne) increased on blueberries in the greenhouse; 6 other species failed to increase. THE PLANT-PARASITIC NEMATODES ASSOCIATED WITH CULTIVATED BLUEBERRIES IN MICHIGAN By James Peter Tjepkema A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1966 ACKNOWLEDGMENTS I wish to thank the Michigan Blueberry Grower's Association and their research director, Mr. John Nelson, who suggested this project and employed me during the summers when I worked on it. I also wish to thank my committee members: Dr. Gordon Guyer, chairman, Entomology Department; Dr. A. J. Howitt of the Department of Entomology; Dr. Erwin Barnes of the Department of Botany and Plant Pathology; and Dr. J. A. Knierim, my Major Professor. I am eSpecially thankful for the guidance and support Dr. Knierim provided. I am indebted to Dr. Paul Wooley for taking the photographs in this thesis and also serving on my committee. I am also indebted to Dr. James Bath who gave me advice. I am especially grateful for help in identification of nematodes given by Mrs. Natalie Knobloch. I am also grateful to Drs. M. w. Allen, S. A. Sher, and W. R. Jenkins who gave opinions on the identity of several species of nematodes. ii TABLE OF CONTENTS INTRODUCTION LITERATURE REVIEW GENERAL PROCEDURES Soil Sampling Procedures Nematode Extraction Procedures . The Christiei-Perry Technique The Inverted Flask Technique The Paper Cup Baerman Funnel The Sugar Flotation Technique Discussion of Nematode Extraction Techniques Procedures for Identifying and Counting Nematodes THE GENERAL SURVEY Procedures Results and Discussion. SURVEY FOR NEMATODES ASSOCIATED WITH NECROTIC RINGSPOT AND SHOESTRING VIRUSES . Procedures. . . . . . . . Results and Discussion. SEASONAL VARIATION IN NUMBERS OF NEMATODES Procedures. Results and Discussion. THE VERTICAL DISTRIBUTION OF NEMATODES Procedures. Results and Discussion. THE GREENHOUSE STUDY Procedures. Results and Discussion. iii l7 l7 l7 23 23 23 29 29 29 32 32 32 A LIST OF PLANT PARASITIC NEMATODES ASSOCIATED WITH CULTIVATED BLUEBERRIES IN MICHIGAN. SUMMARY AND CONCLUSIONS. LITERATURE CITED iv Page 36 Al A4 LIST OF TABLES Incidence of several genera and species of nematodes on blueberry farms from 3 regions in Michigan Incidence of several genera and species of nematodes in 3 varieties of Michigan blueberries . . . . . . . . Incidence of several genera and Species of nematodes near blueberries with necrotic ringspot virus (NRSV) or shoestring virus (SSV) symptoms . . . The occurrence of several genera and species of nematodes on blueberry farms where necrotic ringspot virus symptoms appeared The occurrence of several genera and species of nematodes on blueberry farms where shoestring virus symptoms appeared. The increase of a 3 month period of nematodes on potted blueberries. Page 15 15 19 21 22 35 Figure LIST OF FIGURES Seasonal variation in numbers of Trichodorus christiei (Allen) on the Jones blueberry farm . . . Seasonal variation in numbers of Trichodorus christiei (Allen) on the Hartman blueberry farm . . Seasonal variation in numbers of Pratylenchus crenatus (Loof) on the Hartman blueberry farm Seasonal variation in numbers of Xiphinema americanum (Cobb) on the Pollard blueberry farm Distribution of several nematodes between the first 6 inches of soil and the second 6 inches of soil on 3 farms vi Page 24 25 26 27 3O Plate II. III. IV. LIST OF PLATES Filter cup and body of the paper cup Baerman funnel . . Adapter and stopper with handle Inverted flask apparatus ready for operation. . . . . Inverted flask apparatus in operation vii Page INTRODUCTION Michigan shares leadership with New Jersey in the production of cultivated blueberries. There are 9000 acres of blueberries under cultivation in Michigan. Most of the acreage is in Berrien, Van Buren, Allegan, Ottawa, and Muskegon counties. These counties occur along the southern half of the eastern shore of Lake Michigan. Little is known about the effect of nematodes on the production of blueberries. Nematodes may influence blueberry production by [l] spreading plant viruses, and [2] acting as plant pathogens. Since information on either of these two possibilities is inadequate, the following studies were undertaken: [l] a general nema- tode survey, [2] a survey of nematodes asSociated with plants with necrotic ringspot and shoestring virus symptoms, [3] a study on seasonal variation in numbers of nematodes, [A] a study on the vertical distribution of nematodes, and [5] a study on the increase of nematodes in potted blueberry plants. The purpose of the general survey was to determine which nematodes are associated with blueberries and the frequency of occurrence of these nematodes. The survey of fields which exhibited virus symptoms was designed to l learn whether there were any particular nematodes associat— ed with these virus symptoms. Studies on seasonal varia- tion in numbers and vertical distribution of nematodes were included to help evaluate the accuracy of the survey data. Research on the increase of nematodes on potted plants was included to give some indication of the ability of nematodes found in the surveys to act as parasites of blueberries. LITERATURE REVIEW There are only a few papers which deal with nematodes associated with blueberries in Michigan. Knierim (9) listed several genera of nematodes found in soil samples from Michigan blueberry fields. Xiphinema americanum (Cobb)from Michigan was used to transmit necrotic ringspot virus (NRSV) from blueberries to cucumbers (5), Attempts to use nematodes to transmit shoestring virus of blueberries were frustrated by difficulties in rearing nematodes, (12). A number of surveys for nematodes in blueberry fields have been conducted in eastern United States. Blueberries were included in surveys of nematodes associated with crops in both New Jersey (7) and Massachusetts (18). The occur— rence of nematodes associated with blueberries in five states was recorded in a paper titled "Distribution of Stylet-Bearing Nematodes in the Northeastern United States" (ll). In another paper the occurrence and relative abun— dance of some nematodes from blueberries in four eastern States were presented (4). Information from these surveys will be presented under the sectionwlisting plant parasitic nematodes associated with Michigan blueberries. Race and Hutchinson reported that blueberry roots are unattractive to Pratylenchus penetrans (Cobb) (13). This statement was based on observations of the behavior of §;_ penetrans in relation to roots of blueberries and other plants grown in petri dishes. Hutchinson et al. (6) ob— served large numbers of Trichodorus christiei (Allen) associated with stunted blueberry cuttings. Zuckerman innoculated blueberry plants grown in pots with nematodes and observed nematodes feeding on the roots of blueberry seedlings grown in agar (l6, 17). He found that Trichodorus christiei, Hemicycliophora similis (Thorne) and Tetylenchus Joctus (Thorne) reproduced in pots and feed on the roots grown in agar. Stubby roots were associated with the feeding of T; christiei and terminal root galls were associated with H; similis feeding. There were no symptoms associated with Tetylenchus Joctus feeding. Root growth of cuttings set into soil innoculated with T;_christiei and with H; similis was significantly reduced. Root growth of cuttings set into soil innoculated with Tetylenchus Joctus was not significantly reduced. Cuttings innoculated with T; christiei after root growth had started showed a variable amount of root reduction. Lister et al. (10) presented evidence that NRSV is similar to tobacco ringspot virus which is known to be transmitted by Xiphinema americanum. Also, they reported that X. americanum was associated with NRSV in several different geographical locations. GENERAL PROCEDURES Soil Sampling Procedures Soil samples were taken within 2 ft. of the bases of blueberry bushes with a soil sampling tube. Four or more probes were taken with the sampling tube to accumulate one half pint of soil in each sample. Only the top 6 inches of soil were sampled except in the vertical distribution study where 12 inch probes were taken and divided into two 6 inch sections. Samples were processed shortly after they were collected or were refrigerated at 40 to 50° F. and kept in plastic bags to prevent them from losing moisture. Nematode Extraction Procedures The following methods were used to extract nematodes from soil: [1] the Christie-Perry technique (3), [2] a modification of the inverted flask technique (1) combined with a paper cup Baermann funnel, and [3] Jenkins' modi- fication of the sugar flotation technique (8). The Christie—Perry Technique.-—A soil sample was mixed throughly with water in a bucket. Then the mixture was allowed to stand for a few seconds so that heavy particles in the soil could settle to the bottom of the bucket. The particles which remained suspended in the water were de- canted through a 25-mesh screen into a second bucket. The washed particles remaining in the first bucket were washed two additional times by the process just described. This procedure seperated the nematodes in the sample from heavy particles such as sand which remained in the first bucket and from large particles, such as leaves and sticks, which were caught on the 25—mesh screen. The contents of the second bucket were stirred, allowed to stand for a few seconds, and decanted onto a 325—mesh screen. Particles which remained in the bucket were washed by the process Just described two additional times. Some heavy particles remained in the bucket. Nema— todes were caught on the 325—mesh screen along with other small organic and mineral particles. Very small particles such as silt and clay passed through the 325-mesh screen. The nematodes were seperated from the residue on the 325—mesh screen by means of a Baermann funnel. The nema~ todes and residue on the screen were washed into a 250 ml beaker. A piece of muslin cloth was fasted over the mouth of the beaker and the beaker was inverted and partly sub- merged in a water filled funnel. The nematodes seperated themselves from the residue by wiggling through the cloth; they settled into a test tube which was fastened to the bottom of the funnel with a piece of rubber tubing. The extraction was completed when the test tube was removed after four days. The Inverted Flask Technique.--A 2000 ml flask was filled with water and one half pint of soil which had been screened through one quarter inch mesh hardware cloth. An adapter fitted with a rubber stopper (Plate II) was attached to the mouth of the flask. The flask was inverted several times to suspend the nematodes in the water. Then, the flask was placed upside—down in a ring stand above another 2000 ml water filled flask with the adapter submerged in the water in the neck of the lower flask (Plate III). The stopper was removed while submerged with a wire designed for this purpose. After 2 to 3 minutes, when the larger particles had settled into the lower flask, the stopper was replaced. The contents remaining in the upper flask were poured onto a 325—mesh screen; nematodes and small particles were caught on the screen and washed into a paper cup Baermann funnel. The particles which had settled into the lower flask were processed by repeating the extraction tech— nique just described two additional times to remove nematodes which may not have been recovered. The Paper Cup Baermann Funnel.--The body of the funnel (Plate I) was constructed from a 12 oz. paraffin coated, conical paper cup. The tip of the cup was cut off to make a hole one quarter inch diameter. A short glass tube was cemented to tip of the cup with paraffin and connected to a test tube with a short length of rubber tubing. Plate I.-—Filter cup and body of paper cup Baerman funnel. Plate II.—-Adapter and stopper with handle. .coflpmnmmo CH mzpmnmmdm .cofipmnodo pom momma mapmanQm gmmam pmpnm>2Hnu.>H mpmam gmmHM vmppo>CHlu.HHH mumam 10 A filter cup (Plate I) was designed to fit inside the body of the funnel. It was constructed from the upper part of two conical paper cups and a piece of muslin cloth. The cloth was cemented between the two telescoped pieces of paper cup with paraffin. The cloth formed the bottom of the filter cup. The diameter of the bottom was 2/3's the dia- meter of the top of the paper cup. The filter cup was partly submerged in water inside the body of the funnel; then the exract from the inverted flask technique was placed in the filter cup. The nema- todes separated themselves from the small particles by wiggling through the cloth and settled to the bottom of the test tube. They were collected after four days by re— moving the test tube. The Sugar Flotation Technique.——Nematodes and small particles were exracted from 50 cc of soil by the sieving- decanting process described in the Christie—Perry technique. The nematodes and particles were washed into two 50 ml tubes and spun for 3 to 4 min. at full speed in a clinical centri- fuge. The nematodes were retained in the sediment in the bottom of the tube and the supernatant was discarded. The sediment was mixed with a solution containing one pound of sugar per liter of water. The tubes were centrifuged again at full speed for l min. The supernatant, which then con- tained the nematodes, was poured onto a 325—mesh screen, rinsed with water, and washed into a test tube. Nematodes 11 which did not remain in suspension during centrifugation were extracted by centrifuging a second time after sus— pending them again in the sugar solution. Discussion of Nematode Extraction Techniques Each of the three extraction techniques described above was used for one season of sampling. Since the efficiency of the three techniques was unknown, the re- cords of nematodes extracted during one season could not be accurately compared with the records of any other season. However, exact comparison between nematodes extracted in different seasons was not essential to this study. The advantage of the changes from one extraction technique to another was in conservation of time. The inverted flask technique combined with the paper cup Baermann funnel which was used in 1964 was faster than the Christie—Perry technique which was used in 1963, and the sugar flotation technique which was used in 1965 was faster than the other two techniques. Procedures for Identifying and Counting Nematodes To count and identify nematodes to genus a small counting-chamber was scanned at a magnification of 100 diameters with a compound microscope by manipulating the microscope's mechanical stage. All nematodes which were known, or suspected, to be parasitic on higher plants were recorded with a few exceptions. Aphelenchoides spp., Aphelenchus spp., and Tylenchus spp., though present in 12 some samples were not recorded. These genera are small and hard to identify and contain species which may not be para- sites of higher plants. Most of the species determinations were made from specimens mounted in glycerin, although tem- porary water mounts were used on occasion. The nematodes which were mounted in glycerin were killed with heat, fixed in F. A. A., and processed by Seinhorst's method (15). THE GENERAL SURVEY Procedures From July through August 1964, soil samples were taken from 10 farms in each of three regions; (1) Grand Junction in Allegan county, (2) Holland in Ottawa county, and (3) Fruitport in Muskegon county. There were three sample sites on each farm; one site for each of the blue- berry varieties: Rubel, Jersey, and Stanley. On a few occasions when only two of the three varieties could be found on one farm, the other variety was sampled on a nearby farm. At each site three one cup samples of soil were taken within a block of 100 bushes. Each sample was com— posed of soil cores which were taken from the bases of several bushes. Nematodes were extracted by the inverted flask method combined with the paper cup Baermann funnel, and were identified and counted. Results and Discussion Pratylenchus spp. and Trichodorus christiei (Allen) occurred on 2/3's and 1/3 of the farms sampled respectively; other genera of nematodes were found infrequently (Table l). Three-fourths of the Pratylenchus spp. were P;_crenatus; the remaining were P; penetrans (Cobb). Pratylenchus l3 14 spp. and E; christiei were found in samples from all three regions and all three varieties that were sampled (Tables 1 and 2). The occurrence of various genera of nematodes in soil samples from Michigan blueberry fields which were processed by Knierim (9) corresponded roughly to the occurrence of the same genera of nematodes reported here. Pratylenchus spp. were also the most common nematodes found in samples examined by Knierim. More direct comparison between Knierims findings and the findings in this study was not possible because samples on which Knierim's report was based were collected in an irregular manner by growers and agricul- tural agents for various purposes. In the eastern United States Tetylenchus sp. and Hemicycliophors spp. are the most common nematodes from blueberry fields; Pratylenchus spp. is relatively uncommon there (7, ll, 18). Tetylenchus sp. and Hemicycligphora spp. also occur in wild blueberries in the east (6). Wild blueberries were not sampled extensively in Michigan; how— ever, they are not generally found near commerical blue— berry fields here. This suggests that the principle nema- todes found in eastern blueberry fields may have come from wild blueberries and that this may not be the case here. The largest number of nematodes per cup of soil re— corded in the survey was 43. Several hundred nematodes per cup of soil have been recorded from blueberries in New 15 TABLE l.——Incidence of several genera and species of nema- todes on blueberry farms from 3 regions in Michigan. Number of Locations per Genera of Nematode** Region Total Number of Locations* P T X H Ty Pa L Grand Junction 10 7 3 2 l l 2 Holland 10 6 Fruitport 10 6 2 0 l l 0 Total 3O 19 9 2 2 2 2 l *Nine samples per location. **Key to the genera and Species at bottom of this page. TABLE 2.--Incidence of several genera and species of nema- todes in 3 varieties of Michigan blueberries. Number of Locations per Total Number Genera of Nematode** Region of Locations* P T X H Ty Pa L Jersey 30 ll 3 l Rubel 3O 7 6 Stanley 30 12 2 *Three samples per location. **Key to genera and species below. Key to genus and species of nematodes: A -- Atylenchus decalineatus (Cobb) C —- Criconemoides Sp. H -— Hemicycliophora spp. Ho —- Hoplolaimus galeatus (Cobb) L -- Longidorus Sp. P -— Pratylenchus crenatus (Loff) and P. penetrans (Cobb) Pa -— Paratylenchus sp. T -- Trichodorus christiei (Allen) Ty -- Tylenchorhynchus claytoni (Steiner) X -- Xiphinema americanum (Cobb) 16 Jersey (7). Either the season when fields were sampled or a more efficient extraction technique may account for larger numbers recorded there. (The effect of season will be discussed in another section). SURVEY FOR NEMATODES ASSOCIATED WITH NECROTIC RINGSPOT AND SHOESTRING VIRUSES Procedures Soil samples were taken from the bases of bushes which had symptoms of necrotic ringspot (NRSV) or shoe— string virus (SSV) on several farms during the summers of 1963, 1964, and 1965. Approximately equal numbers of samples were taken from plants with each virus symptom each year. A sample consisted of soil from be— neath a single bush. Soil samples were extracted on the first, second, and third summers by the Christie- Perry technique, the inverted flask technique combined with the paper cup Baermann funnel, and the sugar flota— tion technique, reSpectively; the extracted nematodes were identified and counted. v Results and Discussion Xiphinems americanum (Cobb) was associated with NRSV symptoms on 8 to 12 farms; the same nematode was not associated with SSV symptoms of 14 farms. Other nema- todes Were associated about as often with NRSV as they 17 18 were with SSV. Pratylenchus Spp. and Trichodorus christiei (Allen) were common near bushes with either SSV or NRSV symptoms (Table 3). The frequent association of §;_americanum with NRSV symptoms indicates that it may be the primary vector of NRSV. Pratylenchus spp., primarily represented by P; crenatus according to the general survey, was as frequently associated with NRSV as was §L_americanum, but it was also common in fields with SSV symptoms and in the general survey. Therefore, its prevalence near NRSV symptoms may only re— flect its overall prevalence in blueberries. The repeated association of Pratylenchus spp. with SSV symptoms and T; christiei with both SSV and NRSV symptoms may be explained the same way. However, X. americanum seems to occur in- frequently in blueberries, except in those with NRSV symptoms, since it was not found in any fields with SSV symptoms and was found on only 2 of 30 farms in the general survey. §;_americanum may have originally acquired NRSV from weeds and spread it to blueberries or it may have originally acquired NRSV from diseased cultivated blue— berries and spread it to other blueberry plants. If X; americanum originally acquired NRSV from weeds, it could have become a vector of the virus in blueberries when blue— berries were planted where the nematodes and weeds infected with NRSV occurred. If §;_americanum originally acquired NRSV from cultivated blueberries, it could have become the TABLE 3.——Incidence of several genera and species of nema— todes near blueberries with necrotic ringspot virus (NRSV) or shoestring virus (SSV) symptoms. Number of Locations Per Type of Virus Total Number Genera or Species of Nematodes* Symptom 0f Locations P T X H Ty C C Ho L Pa NRSV l2 8 6 8 2 0 3 0 2 l l SSV 14 A A 0 3 2 2 2 l l l * Key to genera and species of nematodes on p. 15. PO vector of NRSV in blueberries when both blueberries with the virus and healthy blueberries were planted where the nematode occurred. Since X; americanum was found on only 2 of 30 farms in the general survey, it may have occurred infrequently where blueberries were planted. If_X; americanum origi- nally acquired NRSV from cultivated blueberries and the nematode occurred infrequently where blueberries were planted, it would very rarely become a vector of NRSV unless many of the planted blueberries were virus in— fected. If X;_americanum originally acquired NRSV from weeds and Spread it to blueberries it could have become a vector of the virus in blueberries even though only healthy blueberries were planted where the nematode occurred. Cases of X;_americanum spreading NRSV in blueberries would not be extremely rare even if the nematode occurred infrequently where blueberries were planted if weeds with NRSV were always present. However, nothing is known about the weed hosts of NRSV since no attempts to find weed hosts of NRSV have been reported. 21 TABLE 4.——The occurrence of several genera and species of nematodes on blueberry farms where necrotic ringspot virus symptoms appeared. Number Number Genera and Species of of of Nematodes Recovered * Farm Year—"Sites Samples X” P T H C Ho L Pa Benkie 63 2 2 + — — — — + + — AA #3 53 3 5 - + + — — — — — 64 1 3 — + - _ _ _ - _. AA #9 65 l l + + — — — — — Pollard 63 1 l + + + - — + - + 64 1 many** + + — — — — - + 65 l 1.. + + - — + — - — Foretsch 63 2 2 — + + — — _ _ _ 64 2 2 + + — _ _ _ _ _ Hartman 63 1 1 — _ _ _ _ _ _ _ Woods 63 l 1 — _ _ _ _ _ _ _ 64 l 3 — + + — _ _ _ _ Johnson 63 1 1 _ + _ _ _ _ _ 64 l 3 — — — _ _ _ _ _ Decker 64 2 2 + + — _ _ _ _ _ Krohn 65 l l + - - — + — - _ N. Lake 65 l 1 + + + _ _ _ _ _ Bush 65 1 1 + — + — + _ _ _ * Key to genera and Species of nematodes on p. 15. ** Sampled through out the year for study on seasonal variation in numbers. 22 TABLE 5.--The occurrence of several genera and species of nematodes on blueberry farms where shoestring virus symptoms appeared. Number Number Genera and Species of of of Nematodes Recovered * Farm Year Sites Samples P T H Ty A C Pa L Ho Isreal 63 3 5 — - _ _ _ _ _ _ _ 64 1 1 — — — - - .. .. _ _ 65 1 1 - - — — - - - _ - Wiepert 63 3 5 — — + _ _ _ - _ _ Hartman 63 2 3 + — _ + _ _ + + + 64 1 1 + + - — — — - - _ 65 1 1 + + — - — — _ _ Depree 63 3 6 + — — — — - _ _ _ 64 l l — - — — - _ - _ _ 65 l 1 — - - - + — - — — Elhart 63 1 2 — _ + - _ _ _ - _ 64 l 1 — — - + - - - - - Behm 63 2 2 — — — — — - _ _ _ Peterson 63 l 1 — — — _ _ _ _ _ _ G. Kiel 63 1 1 - + — - - - _ _ - R. Kiel 63 l 1 + — — — _ _ - - _ Aussicer 63 l l — + — — — _ _ _ _ 64 1 1 + - — - - — — - - Lippenga 63 1 1 — _ _ _ _ _ - _ _ 64 l l — - — — _ _ _ _ _ Diepen» 63 1 1 — — — _ - _ _ _ _ horst 64 l 1 - — - _ _ _ - - _ Patzloff 64 2 2 — — — — _ _ _ _ - Fischer 65 1 1 — + + _ + + _ _ _ *Key to genera and species on p. 15. SEASONAL VARIATION IN NUMBERS OF NEMATODES Procedures Samples from each of 3 farms near Grand Junction were taken at 2 week intervals in the summer and at 1 month intervals in the winter, except in January and February when the ground was frozen and could not be sampled. Three samples were taken on each sampling date from each site (the area beneath a single blueberry bush). Nematodes were extracted from the samples by the inverted flask combined with the paper cup Baermann funnel and were identified and counted. Results and Discussion Pratylenchus crenatus (Loof) (Figure 3) and Xiphinema americanum (Cobb) (Figure 4) at Hartman's and Pollard's farms, respectively, and Trichodorus christiei (Allen) (Figures 1 and 2) at Hartman's and Jones' farms were highest in numbers in the fall or early winter and lowest in numbers in the summer. T; christiei showed a strong population reduction from late July until the middle of September. This reduction occurred at a time when there was little rain fall and at the time when the blueberry 23 Number of Nematodes 24 140 "’ + S an 81” 6V a on ’ _. @1522; ELSE... / 8o 1 ’ ’ .1. ///’o," .0 .1 I /. .0 .. / :‘M<%/”‘W/I * point based on 2 samples A I A L L A l I l l I 1 June July Aug. Sep. Oct. Nov. Dec. Jan. Figure 3.--Seasonal variation in numbers of Pratylenchus crenatus (Loff) on the Hartman blueberry farm. Number of Nematodes 25 +standard deviation * point based on 50_ ;;Z mean ‘_ 2 samples ___25 -standard deviation ** point based on 1 20+ lOm 1 May June July Aug. Sep. Oct. Nov. Dec. Jan. Figure 4.--Seasonal variation in numbers of Xiphinems americanum (Cobb) the Pollard blueberry farm. Number of Nematodes 360.. 330-. 300-. 270-. 240-- 210" 180» 150* l20~ 901 601 30w 26 +standard deviation mean —standard deviation * point based on 2 samples // L ' -1 __. Pi..- ___.-’/ - May June JdIy’fA g. Sep. dct. Nov. Dec. Figure l.—-Seasonal variation in numbers of Trichodorus christiei (Allen) on the Jones blueberry farm. Jan, Numbers of Nematodes 300 280 260 240 220 2OO 180 160 140 l2O 100 80 60 40 2O 27 +standard deviation / ///; mean . } —standard deviation l“‘ A I \\\\\\\ ‘\\‘\;\‘\\\\:\\ --“flf‘n-u-u-..“ "‘ g / / , I“ “'"' 'v . '— "-‘I‘ Juhe VJilly Aug{" ep. act. Nov. Deb. Jan. Figure 2.——Seasonal variation in numbers of Trichodorus christiei (Allen) on the Hartman blueberry farm. 28 fruit matured. The peak number of nematodes recorded for P;_crenatus and X;_americanum were 81 per half pint of soil in late September and 41 per half pint of soil in early October respectively. ‘2; christiei had a peak of 200 per half pint of soil in the middle of November at one location and 209 per half pint of soil in late December at the other location. Although the sampling procedure could not be followed in January and February because the ground was frozen, some chunks of frozen soil were extracted but only a few nematodes were recovered. On March 16 nor— mal sampling was resumed and the average number of nema- todes per half pint of soil was 92.2; christiei at Jones', 94 T; christiei at Hartman's, ll X;_americanum at Pollard's, and 77 P;_crenatus at Hartman's. The seasonal variation in numbers of nematodes suggests that the low number of nematodes found in the general survey may be partly accounted for by seasonal factors and may not be characteristic of plant parasitic nematodes in blueberry fields in Michigan throughout the year. The reduction in numbers of T; christiei which was found to occur in the summer, which is the time when most of the samples were taken, indicates that T;_christiei may be even more wide spread than was reported. The probabi- lity of recovering T; christiei would have been greater if the samples had not been taken in the season when its population was low. THE VERTICAL DISTRIBUTION OF NEMATODES Procedures The procedures used in this study are the same as those used in the study of seasonal variation in numbers except that the soil was sampled to a depth of 12 inches instead of 6 inches. The samples were taken at 2 week intervals from August 28 to November 10. To determine vertical distribution, the 12-inch core of soil was evaluated in two 6 inch sections. Results and Discussion Pratylenchus crenatus (Loof) and Trichodorus chris- tiei (Allen) at Hartman's farm, 2; christiei at Jones' farm, and Xiphinema americanum (Cobb) at Pollard's farm were as prevalent or more prevalent at the first 6 inches of soil as they were in the second 6 inches of soil (Figure 5). Soil samples from the top 6 inches of soil contained numbers of these nematodes that at least equaled the numbers in a sample of the same size taken from the top 12 inches of soil. Therefore, the procedure used throughout this investigation of sampling only the top 6 inches of soil rather than sampling to a greater 29 Number of Nematodes 30 50 -. 50-7 '7‘ 40-' , \ 40* I 30 -> \ 30—)- \ \ 20" \ 20'+ e I I ”—3' \ lOJ. \ lei T f -’ .— -- .. rfl/ j T Sept. Oct. N6v. sépt. Oct. N03? Xiphinema americanum (Cobb) on Trichodorus christiei (Allen) on Pollard's farm. Jones' farm. 0 0 first 6 inches ._ _. second 6 inches 100.. ' 1501 804 //////’ 120.. 60 "‘ /° 90 .L 40-4 ////////// 60W 20-» 30-) . I.‘ \ \ l, v " ’ .— .. _ _ _ _.l ‘0 ’. i i i-D #— I T9 Sept. Oct. Nov. Sept. Oct, Nov. Pratylenchus crenatus (Loof) on Trichodorus christiei (Allen) on Hartman's farm. Hartman'sIfarm. Figure 5.--Distribution of several nematodes between the first 6 inches of soil and the second 6 inches of soil on 3 blueberry farms. 31 depth, probably does not account for the small number of nematodes that were usually recovered. However, the re- covery of small numbers of nematodes was partly accounted for by a seasonal reduction in nematode populations. In- formation on nematodes found at a depth greater than 12 inches would be desirable but it is likely that most nema- todes that are associated with blueberries can be found within the top 12 inches of soil. Blueberries are shallow rooted and the distribution of nematodes associated with them would be expected to coincide with the distribution of their roots. THE GREENHOUSE STUDY Procedures Thirty to 150 nematodes of several Species were intro- duced into pots containing young blueberry plants to study their ability to survive and increase on blueberry roots. The nematodes were extracted by the sugar flotation techni- que from soil collected from blueberry fields; no attempt was made to separate them from small numbers of unidenti— fied plant parasitic nematodes and other soil microflora and microfauna which were found in the soil extract. Two or three pots were used for each species of nematodes; three pots which were not treated with nematodes were kept as controls. Usually a pot was treated with one species, however, in two cases where two species were prevalent in one soil sample both species were added to the same pot. The plants used in the study were rooted cuttings, variety Jersey, which were taken from cutting beds in the fall and kept in cold storage for a month, then potted in one quart cans on soil made up of 50% peat and 50% potting soil. The plants lost their leaves in cold storage but developed new growth shortly after they were potted. Three months after the nematodes were added to the pots, five 50 32 33 cc samples from each pot were processed using the sugar flotation extraction technique to determine the degree of increase or survival of the nematodes. Results and Discussion Nematodes of all the species which were introduced into the pots were recovered after 3 months. Only Tetylenchus Joctus (Thorne) and Trichodorus christiei (Allen) increased in numbers. There were a few more Xiphinema americanum (Cobb) extracted than were added, however, extraction of the controls indicated that there may have been some XL_americanum in the pots before any nematodes were added. The slight increase in numbers of X;_americanum may be due to survival but not increase of added nematodes, plus the survival of nematodes that were there before X;_americanum was added. There were also a small number Of.2; christiei extracted from the control pots. The number of g;_christiei extracted from the treated pots was so much larger than the number of those in the control pots and the number of those that were origi— nally added to the treated pots that the increase can not be explained the same way the small increase of X;_americanum was eXplained. The increase in T; christiei and Tetylenchus Joctus is taken as evidence that they established themselves as parasites of blueberries. The failure of the other Species to increase may or may not indicate that they are not 34 parasites of blueberries. The conditions of this study may have prevented nematodes that can increase on blueberries in the field from increasing in the greenhouse. The in— crease Of.2; christiei and Tetylenchus Joctus supports similar findings of Zuckerman (l6, l7). Greenhouse studies using blueberry plants as potential hosts for the nematodes which failed to increase in this study have not been previously reported. Hemigycliophora similis (Thorne), which was not included in this study because sufficient numbers of it could not be found, has been shown to in- crease on blueberries in the greenhouse by Zuckerman (l7). 35 .Umcfienopop was mHonucoo ca canon manocoamumnm mo mucow onp mace: o N H.o m mm m .Qm mmUHoEocooaao cam mma x H and wma m Sandcaposm mEoGHQQHx o x m amm oma m mspm0n manocmfiaume .Ucm : x ma mama mm m fimfipmahno mSLOUOSOHHB ”pom comm on coups mums mmaomdm 03p scans 2H mpmoa so: x m.o mm OHH m mumpuocmm mscocmampmpm o ,x m.o mm mm m .Qm mszoqwmonocoaww so: .x n.o mm om m mapmcono manocoampmpm o x m.o am AHA m .am manocuwwpooaflmm : x w mum mma m Hofipmfipno mSAOBOQOHpe Honpcoo Eopm msucoz m popu¢ pooo< mpom omuomnpxm mmmmpocH vopompuxm .oz can: no .02 opoumEmz .oz cam: .02 2mm: .mmfinnmnmSHn coupon so mmUOpMEm: uo voanmm zucoE m m mm>o mmmmhonfi oneul.m mqm¢e A LIST OF PLANT PARASITIC NEMATODES ASSOCIATED WITH CULTIVATED BLUEBERRIES IN MICHIGAN Atylenchus decalineatus (Cobb) This nematode has been found on a few blueberry farms in Michigan as well as in blueberry fields in New Jersey (7) and Massachusetts (18). It is found in cranberry bogs as well as blueberries fields (11). The high water table necessary for these two crops favors A;_decalineatus since it is known to have affinity for wet soil (2). Another nematode which has an affinity for west soil is Dolichodorus heterocephalus (Cobb) which has been reported from blue- berries in New Jersey but not in Michigan. Criconemoides Sp. This genus, of which some specimens from Michigan were identified as g;_sphaerocephalus (Taylor) has been found only occasionally in blueberries here and in New Jersey (7). This may have been due to the inability of Criconemoides sp. to wiggle through the filter in Baerman funnels which were used to extract it. When the sugar flotation technique of extraction was used Criconemoides sp. was found in samples from 5 of 9 farms. 36 37 Helicotylenchus sp. In both Michigan and New Jersey (7) Helicotylenchus sp. has been recovered a few times from blueberries. Hemicycliophora Spp. Nematodes from this genus were occasionally found in Michigan blueberry fields; most of those that were deter- mined to species were H; similis (Thorne) although H; vaccinium (Reed and Jenkins) was identified once. Hemicyc- liophora spp., including g; gracilis, (Thorne) H; similis, and H; uniformis (Thorne) were among the most common nema— todes found on blueberry farms in the eastern United States and were also common in various species of wild blueberries and cranberries in those states (17, 6). Zuckerman found galls and reduced root growth associated with the parasitism of blueberry cuttings by g, similis (17). Hoplolaimus galeatus (Cobb) H;_galeatus was recovered from a few blueberry fields in Michigan and has been reported from blueberry fields in New Jersey (7). Longidorus sp. In Michigan a few specimens of this genus have been recovered from blueberry fields. Paratylenchus Sp. Nematodes of this genus were recorded a few times from blueberry fields both in Michigan and eastern United States (11). 38. Pratylenchus spp. Members of this genus were the most common nematodes that were found in Michigan blueberries. About 3/4's of those that were identified were P; crenatus (Loof); the rest were P;_penetrans (Cobb). Pratylenchus spp. were in— frequently recovered from blueberry fields in New Jersey (7) and Massachusetts (18). HutChinson and Race rated blueberries as a poor host for P;_penetnans (13). Both P;_penetrans and P;_crenatus failed to become established as parasites of blueberries in the greenhouse. Rotylenchus sp. This nematode was not found by the author but has been recorded from Michigan blueberries by Knierim (9) and was found in blueberries in New Jersey and Maryland (11). Tetylenchus Joctus (Thorne) T; JoctuS‘was recovered once by the author while collecting nematodes for the greenhouse study. The only other record of this genus in Michigan blueberries is from a soil sample sent to Knierim by a grower. In New Jersey and Massachusetts, 2; Joctus is the most common nematode from blueberry farms and is also common in wild blueberries and cranberries (7, l7). Greenhouse pot studies described in this thesis and studies made by Zuckerman (17) indicate 39 that it is a parasite of blueberries. Zuckerman's tests, however, indicate that it may not be destructive to blue- berries. Trichodorus christiei (Allen) The only Species of Trichodorus that was identified from Michigan blueberries is T; christiei. Because of difficulties in determining species in this genus there is a possibility that other species were over looked. .2; christiei was found to be wide spread in Michigan but was apparently not so wide spread in New Jersey (7) and Massachusetts (18). Zuckerman's research (16) and research reported herezfndicates that T;_christiei is a parasite of blueberries. Zuckerman also has shown that E; christiei is associated with reduced growth of blueberry cuttings and with a stubby root condition in blueberries. Tylenchorynchus claytoni (Steiner) T; claytoni was found infrequently in Michigan blue- berry fields. g;_claytoni and Tylenchorynchus sp. have been reported from cultivated blueberries in several eastern states (11). Xiphinema americanum (Cobb) X;_americanum has been implicated as the vector of necrotic ringspot virus of blueberries by the research of Griffin et a1. (5) and Lister et al. (10). It was found in several fields in which symptoms of necrotic ringspot 40 virus were present. It was not recovered from several fields in which shoestring virus symptoms were evident. Generally it was found infrequently in blueberries in both Michigan and the eastern United States (18, 11). SUMMARY AND CONCLUSIONS Plant parasitic nematodes from 11 genera were found in Michigan blueberry fields. Only the genus Pratylenchus, represented primarily by P;_crenatus (Loof) and Trichodorus christei (Allen) were found frequently. They were found on three blueberry varieties and in three blueberry regions. Study on the relation of E; crenatus and T; christiei to blueberry production is merited on the basis of their wide spread occurrence. Their effect on blueberry production could be important due to such frequent occurrence. The procedure of sampling the top 6 inches of 8011 proved to be suitable, at least for P;_crenatus, 2, chris- tiei, and Xiphinema americanum (Cobb). There were as many or more of these nematodes in the top 6 inches of soil as there were in the second 6 inches of soil. The numbers of E;_christiei and to a lesser extent P; crenatus and X; americanum were lowest in the summer. The probability of recovering these nematodes was reduced by sampling during the summer. X; americanum was found frequently in fields with necrotic ringSpot virus (NRSV) symptoms and was found in- 41 42 frequently in other fields. It is likely that XL_americanum is a vector of NRSV becausq [1] it is frequently associated with NRSV symptoms, [2] it has been used to transmit NRSV to cucumbers (5), and [3] it can transmit tobacco rings- pot virus which is closely related to NRSV (10). There was no particular nematode associated with bushes showing shoe— string virus (SSV) symptoms. However, 2; christiei Should be considered as a possible vector of SSV since it is common in Michigan blueberry fields and since members of the genus Trichodorus can transmit other plant viruses(l4). X;_americanum may have spread NRSV from weeds that had the virus to blueberries. If this is the case infesta- tions of NRSV in blueberries could start even if NRSV was eliminated from existing blueberries and only NRSV free blueberries were planted. Since there is not information available on the weed hosts of NRSV, it would be desire— able to determine if any weeds in areas where blueberries are grown harbor NRSV. T; christiei and Tetylenchus lgctus (Thorne) increased in numbers on potted blueberry plants in the greenhouse over a three month period; species which did not increase were P;_crenatus, P;_penetrans (Cobb) Tylenchorhynchus claytoni (Steiner) Helicotylenchus sp., Criconemoides sp., and Xiphinema americanum. The nematodes that did not increase may not be parasites of blueberries or they may have been prevented from increasing by abnormal environmental condi- tions imposed on them in the greenhouse. 43 The ability of T; christiei and Tetylenchus joctus to increase on blueberries confirms similar findings by Zuckerman (16, 17). These greenhouse tests support the recommendation that work should be done on the relation of I; christiei to blueberry production even though T;_christiei did not consistently reduce growth of young blueberry plants in a test conducted by Zuckerman (16). Although Zuckerman's test suggests that, except in cutting beds, 2, christiei may not damage blueberries, more extensive proof of this is needed due to the wide occurrence of T; christiei in Michigan blueberry fields and its ability to increase on blueberry roots. lO. LITERATURE CITED Chapman, R. A. 1958. An evaluation of methods for determining the number of nematodes in the soil. Plant Disease Reptr. 42:1351. Chitwood, B. G. and A. C. Tarjan. 1957. A rede- scription of Atylenchus decalineatus Cobb, 1913 (Nematode: Tylenchinae). Proc. Helminth. Soc. Wash. 24:48-52. Christie, J. R. and V. G. Perry. 1951. Removing nema— todes from the soil. Proc. Helminth, Soc. Wash. 18:106—108. _ Goheen, A. C. and A. J. Braun. 1955. Some parasitic' nematodes associated with blueberry roots. Plant Disease Reptr. 39:908. Griffin, G. D., J. E. Huguelet, and J. W. Nelson. 1963. Xiphinema americanum as a vector of necrotic ring- spot virus of blueberries. Plant Disease Reptr. 47:703. Hutchinson, M. T., J. P. Reed, and S. R. Race. 1960. Nematodes stunt blueberry plants. New Jersey Agr. 42:12-13. Hutchinson, M. T., J. P. Reed, H. T. Streu, A. A. DiEdwardo, and P. H. Schroeder. 1961. Distribution of Stylet—Bearing Nematodes in the Northeastern United States. New Jersey EXpt. Sta. Bull. 796:1-62. Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reptr. 49:692. Knierim, J. A. 1963. Nematodes assoicated with crop plants in Michigan. Quarterly Bull. 46:254—262. Lister, R. M., L. C. Raniere, and E. H. Varney. 1963. Relationships of viruses associated with ringspot diseases of blueberry. Phytopathology 53:1031-1035. 44 ll. 12. l3. l4. 15. 16. 17. 18. 45 Mai, W. F., H. W. Crittenden, and W. R. Jenkins. 1960. Distribution of stylet-bearing nematodes in the northeastern United States. New Jersey Agr. EXpt. Sta. Bull. 795:1—32. Progress report of research attempted on funds granted to J. E. Huguelet by the Michigan Blueberry Grower's Association. Race, S. R., and M. T. Hutchinson. 1959. Susceptibi— lity of various plants to Pratylenchus penetrans as determined by behavior of the nematode, lieson formation, and root growth. (Abstr.) Phytopathology 49:525. Raski, D. J., and Wm. B. Hewitt. 1963. Plant-parasitic nematodes as vectors of plant viruses. Phytopathology 53:39-47. Seinhorst, J. W., 1959. A rapid method for transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4:67—69. Zuckerman, B. M. 1962. Parasitism and pathogenesis of the cultivated high bush blueberry by stubby root nematodes. Phytopathology 52:1017—1019. Zuckerman, B. M. 1964. Studies of two nematode species associated with roots of the cultivated highbush blueberry. Plant Disease Reptr. 48:107- 171. Zuckerman, B. M., and J. W. Coughlin. 1960. Nema- todes associated with some crop plants in Massachu- setts. Mass. Agr. EXpt. Sta. Bull. 521:1—18. “ HIGAN STATE UNIVERSITY LIBRAR III II III III IIIIIIIII IESI