v—o 9“ —‘M—QW"— v-— —— -.-_, -— -‘- ...-- ... '3’ u: . A STUDY OF A MICHIGAN ISOLATE o ‘ 0F .MELOIDOGYNE HAPLA Thesis for the Degree of M. S. M‘lCHiGAN STATE UNIVERSITY JAMES K. BRODY, JR. 1972 .. _ . p o . _ . . o . o ._ . .. .. . .. . - ... .. .. 1 . . .. .. o. . . .. .. .. . . . . . 1.. .. . ... .... . . . .. .....,zuw.4.”"... _ . . . . ., .. . .....t .9 .. ... ,. .. .y, .E ...?ANr-a' _ . . ... .... .l..,.ogr.vw....u - _ .0. . ,. . .. . . .. o i . r. . _ . . .. . . p _ . . a .. a. . u . . . .rA. . .... . . g ...D. . . or, Ow .. . o ......_P .vHo.a.-o.”.:nd.no\O/;rl ... o .. o ... . if? . .. . ....r..-..’..2. ...l...L¢H;..o.!or / ., 1,. .o. ...-D. '1... 70¢... . . _ v. _ Iv; ....I..’c...¢v _. . . .. ..D Q u-‘1(wt‘ . .1 . 7. .oth,¢'3 1.1. .. .oh.~0>.1..l1_..flr 0.. .I."-1.O:vl“ _ . ’ . 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LIBRARY Michigan State -. v ad — g BINEING BY nuns & suus' BUUK anm m LIBRARY BINDERS ‘L SPRMGPOFI. IMCIIGA] ‘w. 5% 3-3:» OCT25‘2004 ()5. 06 84: ABSTRACT A STUDY OF A MICHIGAN ISOLATE OF MELOIDOGYNE HAPLA BY James K. Brody, Jr. Various cropping sequences influenced the popu— lations of Meloidogyne haplg Chitwood 1949 in field microplot studies. Populations of g, haplg on celery increased during the growing season. Low populations were noted on carrots, and populations declined in microplots maintained in onions, sweetcorn, or fallow. Greenhouse studies supported the field study. Onions appeared more effective in decreasing g, haplg associated macrodamage to a subsequent crop of carrots than were other cropping sequences studied in the greenhouse. Successive crops of radishes had a trap cropping effect, reducing g, haplg_populations. Host crops affected the generation time of g, haplg. Generation times for g, £22l2.°f 45, 56, and 72 days were found on carrots, celery, and onions, respectively. Sweetcorn appeared to be a nonhost. One generation failed to be completed on radishes. James K. Brody, Jr. Meloidogyne hapla penetrated carrot seedlings within 24 hours. Penetration occurred adjacent to the root cap, and the second stage larvae were oriented with their anterior ends towards the distal terminis of the root. Survival of g, hapla was studied at temperatures of 7 and ~14 C, and at ambient room temperature. Greater survival occurred at 7 C than at ambient room temperature or -14 C. Eggs of g, hapla appeared to survive better than the larvae at -14 C. After three days at that temperature, however, neither stage appeared to survive. Bioassays were found to be an important tool since indicator plants often showed galling in soil where no g, hapla larvae were found. A STUDY OF A MICHIGAN ISOLATE OF MELOIDOGYNE HAPLA By D“! .' '- James K.UBrody, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1972 ACKNOWLEDGMENTS Special thanks go to Dr. Charles Laughlin who offered guidance and encouragement throughout this study. To Drs. E. C. Martin, James Bath, and Melvyn Lacy, I extend my appreciation for serving on my guidance com- mittee. Appreciation is also extended to John Davenport and Mark Otto for their manual labors. ii TABLE OF CONTENTS Page LIST OF TABLES O O O O O O O O O O O O 0 iv LIST OF FIGURES. . . . . . . . . . . . . v INTRODUCTION 0 O O O O C O C O O O O O O 1 LITERATURE REVIEW . . . . . . . . . . . . 2 MATERIALS AND METHODS. . . . . . . . . . . 7 Cropping Study . . . . . . . . . . . . 8 Generation Time Study . . . . . . . . . . ll Penetration Study . . . . . . . . . . . 12 Cold Survival Study. . . . . . . . . . . 13 RESULTS AND DISCUSSION . . . . . . . . . . 14 Cropping Study . . . . . . . . . . . . 14 Generation Time Study . . . . . . . . . . 25 Penetration Study . . . . . . . . . . . 29 Cold Survival Study. . . . . . . . . . . 32 SUMMARY AND CONCLUSIONS . . . . . . . . . . 38 LITERATURE CITED . . . . . . . . . . . . 41 iii LIST OF TABLES Influence of cropping sequences on M. hapla populations in field microplots . . . . . Effect of cropping sequences on the number of galls caused by M. hapla on 0.5 gm of roots of tomato indicator plants in the 1972 season. . . . . . . . . . . . Effect of cropping sequences on the number of M. hapla second stage larvae in 100 cc of sail un er greenhouse conditions. . . . Effects of cropping sequences on macro- symptoms associated with M. hapla damage on carrots . . . .. . . . . . . . . Development of M, hapla and galling on four different crops . . . . . . . . . . Penetration of 'Nantes' carrot seedlings following inoculation with 50 M. hag a larvae . . . . . . . . . . . . . Influence of temperature on survival of M. hapla larvae maintained at ambient room temperature, 7, and -14 C for two, six, and eight weeks . . . . . . . . . . Influence of temperature on survival of M. ha la eggs and larvae as indicated by gal 1ng on 0.5 gm of roots of tomato plants grown in soil previously maintained at ambient room temperature, 7, and -14 C for two, six, and eight weeks . . . . . . . . . . Influence of exposure time at -14 C on the survival of M. hapla eggs and larvae in soil as indicated —by galling on 0.5 gm of tomato plants grown in soil previously maintained at -14 C for 0-3 days . . . . . . . . iv Page 15 17 19 23 26 30 33 34 36 LI ST OF FIGURES Figure Page 1. Microplot cropping diagram showing crops grown in individual microplots in the 1971 and 1972 growing seasons . . . . . . . 10 2. Effect of various carrot rotations on M. ha la populations as shown by roots of four weeE old tomato indicator plants . . . . 16 3. Effect of cropping sequences on producing carrots free of damage caused by M. hapla . 21 4. Carrots showing galling, forking of the taproot, and a hairy root condition caused by M, hapla . . . . . . . . . 24 INTRODUCTION Northern root-knot nematodes (Meloidogyne Mgplg Chitwood 1949) are commonly found in Michigan, as well as other states, where numerous agricultural crops serve as host plants. Northern root-knot nematodes are the only nematodes known to cause economic losses to growers of car- rots in Michigan, which ranks third in the nation in carrot production (19). Nationally, losses to carrots attributed to nematodes are estimated to be 20% annually (27). The objectives of this study were: (1) to study the effect of cropping sequences upon population develop- ment of a Michigan isolate of M. Mgpia; (2) to determine the generation time of this isolate of M. Mgplg on various host crops; (3) to study the rate of larval penetration by M. 32213; (4) to investigate the effects of soil temperature upon the survival of M. 23213. Hopefully, such knowledge will contribute to a better understanding of the northern root-knot nematode in Michigan and assist in the develop- ment of improved control methods. LITERATURE REVIEW The genus Meloidogyne Goeldi 1887, commonly called the root-knot nematode, was first described as a plant parasite in 1855 when Berkeley (4) described root ”ex- crescences" on the roots of cucumbers in England. In the United States, May (17) described the symptoms caused by root-knot nematodes on violets in 1888. The first studies of root-knot nematodes in the United States were carried out by Neal (21), Atkinson (1), and Stone and Smith (29). Root-knot nematodes were considered to be of one species until 1949 when Chitwood (9) reclassified Heterodera marioni (Cornu 1879) Goodey 1932 into five species of Meloidogyne, including M. Mgpl§_Chitwood 1949, the northern root-knot nematode. Due to numerous name changes, it is difficult to ascertain which species of root-knot nematodes investigators were discussing prior to 1949. The symptoms identifiable on above ground plant parts of plants infected with root-knot nematodes are quite general and are attributed to a disruption of the vascular system in the roots. Dwarfing, low yield, wilting, premature death, and poor bloom are common expressions. Below ground symptoms of root-knot nematode infected plants are generally more pronounced than are the above ground symptoms. Galling, stunting, necrosis and proliferation are common external symptoms on roots. Profuse lateral root formation is associated with galls caused by the northern root-knot nematode. Smith and Mai (26) describe lateral root formation in onion (Allium 3323 L.) roots. The same phenomenon is described by Sasser (24) on peanuts (Arachis hypogaea L.), soybeans (Glycines m3§ (L.) Merr.), and tomatoes (Lycopersicon esculentum Mill.). Whereas not all plants exhibit external symptoms, the formation of giant cells within the roots is a universal internal symptom (14). Christie (10) states that giant cell formation and gall formation may be a means by which a plant attempts to isolate the nematode. Dropkin and Nelson (12) found, however, that good nematode growth and fecundity were directly associated with giant cells of good quality. Galls with no giant cells, small giant cells, and highly vocuolated giant cells produced slower growing nematodes with a reduced fecundity. The second stage larva of M. 33213, the stage which emerges from the egg, is the only infective stage. Once established in the root near the root tip with the head in the stele, the second stage larva becomes 4 sedentary. The larva molts three times within the root, and after each molt the nematode becomes more saccate. After the last molt, the nematode is considered an adult (14). Meloidogyne 33233 has a wide host range as do most root-knot nematodes. Host plants include numerous agri- cultural and horticultural plants as well as many weeds (24, 31). CrOps grown on organic soils and which are known to be susceptible to M. hapla include carrots (Daucus carota L.), onions, and celery (3pium graveolens L.). Wilson (36) found that after four or five years of con- tinuous carrots in muck soil, 95-98% of the carrots were deformed by M. 33233. Onions have been described as a good host crop for M. M3pl3_by Lewis et a1. (16). Olthof et a1. (22) estimates a 10% loss in muck grown onions in Ontario due to the northern root-knot nematode. Wilson (35, 36) reported that onions were not good hosts for M. 33213 and that M. 33233 populations decreased on onions. The generation time of M. hapla is variable. Smith and Mai (26) found that egg masses were produced on onions in about 36 days after infection. Using tomatoes as host plants, Bird (5) found that egg laying occurred 29 days after infection. Tyler (32), working with an un- identified species of root-knot nematode inoculated on tomatoes, found that egg laying occurred in 19-27 days and second stage larvae were present in 29-39 days. In addition, Tyler showed that root-knot nematode generation time is temperature dependent. Penetration of roots by M. 33233 second stage larvae has been found to be quite variable. Bird and Wallace (6) found that 2.9% of a high population (60,000) of M. 33233 second stage larvae entered the roots of a tomato plant within 48 hours. Kinlock and Allen (15), also using tomato plants as hosts, found that after ten days the amount of starting inoculum which had penetrated the host roots ranged from 65.3% of an initial larval count of 125 to 47.3% of an initial larval count of 1,000. Working with M. 33233 on onions, Smith and Mai (26) found penetration to occur within 24 hours. They found the second stage larvae within the roots initially oriented with their anterior end towards the distal end of the root. Later, the larvae reoriented so their anterior ends were oriented towards the proximal end of the root. In addition, these investigators found that more than one second stage larva may enter the root through a single site. M. 33233 appears to be able to survive adverse cold conditions since it is a recurring problem in Michigan. Daulton and Nusbaum (11) found that eggs of M. 33233 survived under field conditions with temperatures ranging down to O C. Bergeson (3) reported that eggs of the northern root-knot nematode survived better at 0 C than did the second stage larvae. Sayre (25b) found that M. hapla populations under winter field conditions in Ontario declined to 1/4 of the original population, but then remained constant. MATERIALS AND METHODS Inoculum for this study came from a Michigan isolate of Meloidogyne M3233thich had been maintained on 1 greenhouse grown carrots. A portion of the isolate was 1 mixed with steam sterilized potting soil (5 parts loam, 1 part sand, and 1 part peat) and planted to 'Fireball' tomatoes or pascal celery. The above procedure was repeated as necessary to maintain and increase inoculum. To facilitate locating nematodes and early gall formation, roots were stained with acid fuchsin in a 1:1 solution of glacial acetic acid and 95% ethanol, a modi- fication of the McBryde method (18). The roots were stained for 24 hours and then cleared for 24 hours in chloral hydrate. After the above procedure had been performed, nematodes within the cleared roots appeared red or pink. Larvae of M. M3pl3'were recovered from soil by either a modified Baermann funnel technique (2) or the centrifugation-flotation method (20). Soil samples of 100 cc were processed in both cases. Samples from the field were obtained to a depth of 20 cm using a 3 cm diameter soil sampler. Sufficient cores were obtained from each sampling site to constitute 500 cc of soil. Soil was placed in plastic freezer bags and stored at 7 C until processed. Nematodes in processed samples were counted in a syracuse watch glass under the disecting microscope at 40 X. To determine soil infectivity, bioassays were conducted using one week old tomato seedlings which were transplanted into 7.6 cm plastic pots containing the soil under consideration. After four weeks of growth, the tomato roots were stained as previously described, and the galls 0.5 gm of roots were counted. Cropping Study To study the effects of cropping sequences on M. 33233 populations, field microplots were established in Houghton muck at the Michigan State University Organic Soils Farm. The microplots consisted of 25 bottomless, galvanized steel garbage cans of 75 liter capacity. The cans were placed in the soil, five cans across and five cans deep on 3 m centers, to within 5 cm of the upper lip. During the spring and summer of 1971, the following crops were grown in the microplots, five microplots per crop: 'Golden Beauty' sweetcorn (£33_m3yg_L.), 'Yellow Globe' onions, 'Nantes' carrots, and pascel celery. In addition, five microplots were maintained clean fallow. Prior to planting, each of the microplots was treated with captan fungicide at the rate of 6.7 kg active per hectare and then inoculated with the below ground contents of three 7.6 cm pots of M. 33333 inoculum, about 30,000 larvae. The inoculum was incorporated into the top 0.3 m of soil within the microplots. Soil populations were determined three times during the growing season using the modified Baermann funnel technique. During the spring and summer of 1972, the same crops were grown, but were rotated as shown in Fig. 1. In this manner all possible two year cropping sequences were obtained. Bioassays were conducted with the microplot soil three times during the growing season as were larval counts by the modified Baermann funnel technique. In conjunction with the field microplot study, a greenhouse study was undertaken to determine the effects of various cropping sequences on M. M3233_populations. Three parts potting soil was mixed with one part M. 33233 infested soil and placed in 90 clay pots of 20 cm diameter. The following cropping sequences were investigated: carrots followed by cabbage (Brassica oleracea var. capitata L.); carrot followed by rye (Secale cereale L.); carrot followed by onion; carrot followed by one, two, and three crops of radishes (Raphanus sativus L.); carrot followed by one and two crops of carrots; carrot followed by fallow. Larval numbers were determined by the 10 1972 SWEETCORN ONION CARROT CELE RY O O O O O O O O O O O O O O O O FALLOW O SWEETCORN O ONION O CARROT 1 9 7 1 O CELERY O FALLOW FIG. 1. Microplot cropping diagram showing crops grown in individual microplots in the 1971 and 1972 growing seasons. ll centrifugal-flotation method before planting and after harvesting. Crops were allowed to grow for the approximate number of days which are needed in commercial production (after 78 days in the case of rye). Carrots were planted in the pots of soil which had contained rye, onion, cabbage, and the third crop of radishes and allowed to grow for about 70 days. The number of carrots with galls, split tap roots, and/or a hairy root condition served to indicate the influence of the preceding cropping sequences on M. 33233 populations. In addition, the carrot roots of the second and third carrot crops in the carrot, carrot, carrot rotation were similarly evaluated. Generation Time Study To determine the generation time of M. M3pl3_on various crops, 'Golden Beauty' sweetcorn, 'Nantes' carrots, 'Yellow Globe' onions, pascal celery, and 'Champion' radishes were grown in the greenhouse in 20 cm clay pots containing sand which had been fumigated with methyl bromide. The fumigated soil was aired for four days prior to planting. Immediately before planting, the pots were inoculated with infected roots from the stock culture by incorporating the infected roots into the sand in the pots. After the pots were inoculated, seeds of the previously mentioned crOps were planted. 12 At approximately 14 day intervals, two pots of each crop were examined. The soil was processed by the centrifugal-flotation method. Roots were stained and the amount of galling, the development of the galls, and the development of the nematodes noted. Penetration 35332 A 24 hour penetration study of M. M3pl3_on fNantes' carrot seedlings was conducted using the method described by Chapman and Eason (8). Seeds were treated in 1% Chlorox for 15 minutes, rinsed in distilled water, and allowed to germinate on dampened.Miracloth in a covered petri dish at room temperature. Two day old seedlings were transferred to syracuse watch glasses, two seedlings per glass, which had discs of Miracloth covering the bottoms. The seedling roots were covered with a second disc of Miracloth in such a manner that the seedling roots were sandwiched between discs of Miracloth and the cotyledons and stems were above the sandwich. Each watch glass was then inoculated with 50 M. 33233 larvae, which were no more than 24 hours old, in 2 ml of water, covered, and incubated at 21 C in the dark for various lengths of time. Every two hours a dish GMDRegistered.trademark, The Chlorox Company, P. O. Box 24305, Oakland, California 94623. Registered trademark, Chicopee Mills, Inc., 1450 Broad- way, New York, N. Y. 10018. 13 was removed from the incubator and the roots stained. The occurance of penetration and the orientation of the nematodes were noted. 9333 Survival 32331 Cold survival studies were conducted to determine whether eggs or larvae of M. 33233 have the greatest over- wintering potential in Michigan. 500 cc quantities of I M. 33233 infested soil were sealed in plastic freezer bags and placed at either ambient room temperature, 7 C, or -14 C. At the end of two, six, and eight weeks, four bags of soil from each temperature treatment were sampled. Larvae were extracted by the centrifugal-flotation method and the remaining soil in each sample was planted with a tomato indicator plant. In addition to the above pro- cedure, four bags of soil were removed daily for ten days from the -14 C treatment and the soil was processed as described aboVe. RESULTS AND DISCUSS ION Croppi 3 Study The cropping study conducted in the field micro— plots showed that cropping sequences can distinctly affect the northern root-knot nematode populations in the soil (Table 1; Fig. 2). Both in 1971 and in 1972, celery caused M. 33233 populations to increase greatly during the growing season. In 1971 the M. 33233 population in the celery microplots went from an average of 40 larvae in 100 cc of soil on May 18 to an average of 576 larvae in 100 cc of soil on September 14. With one exception, the M. 33233 population in the celery microplots also in- creased in 1972, though not as dramatically as in 1971. It is interesting to note that on the May 18 and July 20 sampling dates in 1972, only the microplots which had been in celery during the 1971 season had a detectable M. 33233 larval population in the soil. Onions, fallow, and sweetcorn suppressed M. 33233 populations, particularly when rotated with each other (Table 2). Rotations of these crops resulted in low mean populations of larvae and low mean number of 14 15 .connou ahma cw «wanes: uonouoaa o» ccomuouuou cannon ~hma cw «Hones: paddouowz N .Huou no 00 ooa a“ ou>uua cocoa ucooou How and muonesz H o o o o o o voa vN vu cm 0 a o a on o o o o o o o o o o .094 an o «n o o o o o o o o o a o o o 0 ea c o o o o o o o hash ON a o o o o 0 av o o o c an o o o o av o o o a we a o o an: ad oumo Hm mu an m H am he NH n N mm ca ma a n vn as vH a v mm on ma oa m Nuonouowz soaaah uuoHoo uouuuu acaao auou mono condom «had o o a o c was mow mac New ova «N a vN m o o o o o o o o o o o .umom «a o o o o o o o o o o o o o o o o o o o o o o o o o mash ma 0 a ma 0 vs um vw vn cw «n ov vN mm o vu on «n «n v» av o o o a gun as: ma ouuo mu vn mm mm Hm ow ma ma 5H ma ma vu ma ~H Ha oH m o h m m v n a H uonouowx soaaah mucuao uouuuo acaco :uoo mono sownom anaa .nuoHQOHUAE panda ad acouuuanaoa damn: .fi no neocosvom unannouo mo oocosaucn .H manta 16 .cuoouwmsm .Aoav “coflco .Amv uponumo .Amv “mumamo .Abv 130Hamm cmmao .Amv "muouumo mcH3OHH0m mmono H hmucmHm woumowccfl oumEou UHo xmms HDOM mo muoou an csonm mm mcowumasmom mamas .2 co mGOAumuou uouumo msoflum> mo poommm .m .me Ill-alt]; 17 TABLE 2. Effect of cropping sequences on the number of galls caused by M. hapla on 0.5 gm of roots of tomato indicator plants in the 1972 season. 1971 Crop 1972 Crop 18 May 20 July 29 Aug. Onion Celery 0 2 133 Celery Celery 86 25 108 Carrot Celery 57 28 92 Corn Celery 0 17 71 Fallow Celery 4 0 38 Onion Carrot 2 0 0 Celery Carrot 79 5 32 Carrot Carrot l4 2 1? Corn Carrot 0 3 0 Fallow Carrot 1 0 0 Onion Onion 6 0 0 Celery Onion 62 13 17 Carrot Onion 20 0 6 Corn Onion 2 0 0 Fallow Onion 2 0 0 Onion Corn 0 0 0 Celery Corn 46 4 0 Carrot Corn 10 0 0 Corn Corn 0 0 0 Fallow Corn 1 0 0 Onion Fallow 0 0 0 Celery Fallow 40 31 23 Carrot Fallow 9 5 0 Corn Fallow 0 0 0 Fallow Fallow 1 0 0 l8 galls on tomato indicator plants. M. 33233 populations in the onion micr0plots decreased to the same extent as popu- lations in the sweetcorn and fallow microplots. Of the crOps studied, carrots were intermediate in their ability to support the northern root-knot nematode. M. hapla populations in the carrot microplots were much less than in the celery microplots, but greater than in the sweetcorn, onion, or fallow microplots. Meloidogyne M3233 populations in the carrot microplots were less at the end of the 1972 season than at the end of the 1971 season. The greenhouse cropping study tended to support the findings of the field microplot study. Cabbage greatly increased the number of M. 33233 larvae in the soil (Table 3). In the case of radishes, each successive crOp decreased the M. 33233 population. Successive crops of carrots increased populations of M. 33233 while rye and fallow tended to suppress M. 33233 populations. Onions maintained a small population of M. 33233. When considering damage done to carrots planted after various cropping sequences, onions preceeding carrots gave apparent suppression of root-knot nematode symptoms on the carrots (Fig. 3). Continuous carrots resulted in very few carrots showing no symptoms of root- knot nematode damage. TABLE 3. Effect of cropping sequences on the number of M. hapla second stage larvae in 100 cc of soil under greenhouse conditions. Crop Rotation1 M. hapla Population2 Cabbage Onion Rye Fallow Radish Radish after radish Radish after radish after radish Carrot Carrot after carrot 93.6 a 13.6 7.2 5.6 14.4 27.2 bc bc be be be be 1 One crop of carrots preceeded all rotations shown. 2 Numbers are means of ten replications. indicate Duncans new multiple range groupings which do not differ significantly at the 5% level. The small letters 20 FIG. 3. Effect of cropping sequences on producing carrots free of damage caused by M. hapla. l Cropping sequences, most recent crop listed first: (1) carrot, carrot; (2) carrot, carrot, carrot; (3) carrot, three successive radish crops, carrot; (4) carrot, rye, carrot; (5) carrot, onion, carrot; (6) carrot, cabbage, carrot. 21 700 651* 60" 550 50“ 45* 404)- p I 5 1 35.. 300 25+» 200 whommfiu om0<2