”ital: : fig’k k 52% “Q“ '3 ‘ $37.5 . -:‘-. “a? l W, La“ ’2'w‘ ~51: 21%, .‘ ass. ashafigisxs} r 3‘ ‘t-é}. . n; u- sum.— a ”a ‘fi'Wmi‘zé: . 13$? 5239362. "f, “3} t". {n‘m'y --,,. L, ‘1'». 5 s " E; I .. :55- . Twas aa'érxa s "w. ,_'.3‘:.. ,s 9-: 24:53? imam x1. , ~ ’ 133$~ '3‘; 5 , <- - ,- m- ~::.--:.—_ "WES“: This is to certify that the thesis entitled Observations on the Biology of Meloidogyne nataliei: with Special References to Host’Parasite’fielatiOnShips presented by Carlon Joyce Diamond has been accepted towards fulfillment of the requirements for Master of Science—degree in _F_otomology / * Major professor Dated‘fldfl/ 5/79/ //" 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution 4 .—_‘—.__ ICHIGAN STATE UNIVERSITY LlBRARIES LIBRARY Michigan State University PLACE N RETURN BOX to move this chock“ from your record. TO AVOID FINES Mum on or bdoro duo duo. I DATE DUE DATE DUE DATE DUE l "‘7' l OBSERVATIONS ON THE BIOLOGY OF MELOIDOGYNE NATALIEI: WITH SPECIAL REFERENCES TO HOST PARASITE RELATIONSHIPS BY Carlon Joyce Diamond A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1994 ram (Mi lat rip rug obs Gia wer Alt dev C(- ABSTRACT OBSERVATIONS ON THE BIOLOGY OF MELOIDOGYNE NATALIEI: WITH SPECIAL REFERENCES TO HOST PARASITE RELATIONSHIPS BY Carlon Joyce Diamond The goal of this research was to determine the host range, pathogenicity, histopathology, and describe the life history of Meloidogyne nataliei Golden, Rose & Bird (Michigan Grape Root-Knot Nematode) associated with Vitis labruscana Bailey. Five new hosts were identified (V. berlandieri x riparia cv. 583, V. riparia var. Gloire de MOntpellier, V. rupestris var. du Lot St. George, Parthenocissus quinguefolia, and P. tricuspidata). Pathogenicity was not observed on hosts destructively sampled after 89-182 days. Giant cells were induced in V. labruscana cv. Concord. There were three generations per year in southwestern Michigan. Although each life cycle stage had a specific optimal development temperature in vitro, overall optimal is ca. 9° C (maximum ca. 33° C, base ca. 2.5° C). Copyright by Carlon Joyce Diamond 1994 For Michael and especially my daughter, Dawn iv ACKNOWLEDGMENTS I would like to thank my major professor, Dr. George W. Bird, for his guidance, attention, support, understanding, and encouragement. I would also like to thank my committee member, Dr. Charles P. Cubbage for his assistance and advice, and for the pleasant conversations we had throughout my course of study. Thanks also to my other committee members, Dr. G. Stanley Howell and Dr. James Johnson for their assistance and for guiding my independent studies led by each of them. Thanks also to Ms. Becky Mather for the assistance that she extended to me. Thanks also to Mr. John F. Davenport for all of his assistance with my field work. I also wish to acknowledge Mr. Bryan D. Beverly, my nephew, for helping me to weigh and transplant about 200 grape plants. Thank you. TABLE OF CONTENTS LIST OF TABLES..........................................ix LIST OF FIGURES.........................................xi INTRODUCTION............................ ..... ............1 RESEARCH OBJECTIVES. ............................. ........3 LITERATURE REVIEW................ ..... ...................4 VITIS SPECIES.........................................4 Vitis Roots Morphology and Anatomy..................4 Vitis - Root-knot Interactions.......g..............7 THE GENUS MELOIDOGYNE................................10 Meloidogyne Life Cycle Associated with Hosts.......10 Meloidogyne Morphology ................... ..........11 Meloidogyne Hosts.......... ..... ... ..... ...........12 MELOIDOGYNE NATALIEI.................................14 A New Species......................................14 Morphology.........................................14 Gametogenesis.......... ................... .........16 MATERIALS AND METHODS...................................17 Host Range and Pathogenicity of Meloidogyne nataliej-OOOOO0.000.......OOOOOOOOOOOOOOOOOO0.00.00.0017 Acquisition of Plant Material......................17 Propagation of Plant Material......................19 Containers and Soil................................19 vi Setting Up Experiments.............................21 Terminating and Evaluating Experiments... ....... ...26 Life History of Meloidogyne nataliei...... ....... ....27 Field Population Dynamics..........................27 Ontogeny Under Growth Chamber Conditions...........28 Egg Development Under Incubator Conditions.. ....... 29 Histopathology of Meloidogyne nataliei Associated with Vitis 1abruscana.. ........... ........32 RESULTS ..... ............................................34 Host Range and Pathogenicity of Meloidogyne nataliei................................... ......... .34 Life History of Meloidogyne nataliei ................. 44 Field Population Dynamics............ ............ ..44 Ontogeny Under Growth Chamber Conditions...........54 Egg Development Under Incubator Conditions.. ..... ..55 Histopathology of Meloidogyne nataliei Associated with Vitis 1abruscana...... ........... ....70 DISCUSSION ..... .... ....... . ............................. 74 Host Range and Pathogenicity of Meloidogyne nataliei.............................................74 Life History of Meloidogyne nataliei.................79 Histopathology of Meloidogyne nataliei Associated with Vitis 1abruscana........ ............. 81 CONCLUSION O O O ........... O ........... O O O O OOOOOOOOOOOOO O O O 8 2 Summary ............................ ..................83 APPENDIX A. O O O O O O O C 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 O O O O O O O O O O O .84 Arabidopsis thaliana Research Manuscript.............84 APPENDIX B. 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 O O O O O O O O O O O O O O O O O O O 109 Greenhouse Host Range Studies Manuscript ........... .109 vii LIST OF REFERENCES ....................................119 General References.O......OOOOOOOOOOOOOO0......0.0.0121 viii LIST OF TABLES MATERIALS AND METHODS Table Table Table Table Table Table Table Table 1. 2. 8. RESULTS Table Table Table Table Table 9. 10. 11. 12. 13. Soil nutrients and pH of soil used in all host range and pathogenicity studies..............20 Descriptions of Vitis spp. Meloidogyne nataliei host range studies...........................20 Description Meloidogyne of Parthenocissus tricuspidata nataliei host range study........23 Description Meloidogyne of Parthenocissus quinquefolia nataliei host range study........23 Description of Cissus sp. Meloidogyne nataliei host range studies............ ...... .........23 Description of Tetrastigma sp. Meloidogyne nataliei host range study....................25 Description of Ceanothus sp. Meloidogyne nataliei host range study.............. ...... 25 Description of Citrus sp. Meloidogyne nataliei host range study.............................25 Analysis of variance and regression for Vitis rootstock SBB pathogenicity study............37 Analysis of variance and regression for Vitis rootstock Gloire pathogenicity study.........38 Analysis of variance and regression for Vitis rootstock St. George pathogenicity study.....39 Analysis of variance and regression for Parthenocissus tricuspidata (Boston ivy) pathogenicity study of 2-24-93...............41 Analysis of variance and regression for Parthenocissus quinquefolia (Virginia creeper) ix pathogenicity study of 6-1-93.......... ...... 43 Table 14. Frequencies of plant-parasitic nematodes occurring in 48 soil samples.................50 Table 15. Nutrient content of Concord petiole tissue from a Meloidogyne nataliei infested vineyard at mattawan' MIOOOOOOOIOOOOIOOO......OOOOOOOOOOOSO Table 16. Nutrient content of soil sampled at 0.31 m from a Meloidogyne nataliei infested vineyard at Nattawan' MI..........OOOOOOOOOO00.0.0000000052 Table 17. Nutrient content of soil sampled at 0.92 m from a Meloidogyne nataliei infested vineyard at Mattawan, MI....0.00.00.00.000.0.00000000000052 Table 18. Nutrient content of soil sampled at 1.83 m from a Meloidogyne nataliei infested vineyard at "attawan' MIOOOOOOOOOOOOOOOOO......OOOOOOOCOOSB Table 19. Population density of Meloidogyne nataliei in 100 cm3 soil at 0.31 m, 0.92 m, and 1.83 m sampled from an infested vineyard at Mattawan, MIOOOOOOOOCOO ........... ......OOCOOOOOOOOOOCOSB Table 20. Summary of embryogenesis of eggs of Meloidogyne nataliei from one stage to the next at seven temperatures.0.0.0.000...00.0.00000000000000065 Appendix A Table 1. Mean infection percentages of Criconemella xenoplax and Meloidogyne nataliei on Arabidopsis thaliana with 40 and 100 vermiform and adult nematodes inoculated, respectively...........99 Appendix B Table 1. Plants tested in host range studies of Meloidogyne nataliei........................112 Table 2. Mean number of Meloidogyne nataliei and life stage in root and soil samples..............115 LIST OF FIGURES LITERATURE REVIEW Figure 1. MATERIALS Figure 2. RESULTS Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Concord root section from type location at Mattawan, MI showing massive egg sacs of MeIOidogyne natalieiOOOOOOOO0.00.00.00.00000015 AND METHODS Vitis sp. cuttings in flats for rooting in greenhouse.......O.........OOOOOOOOOOOOOOO0.018 Meloidogyne nataliei population on Vitis rootstock SBB at destructive sampling 157 days after inoculation. Control of zero not shown. Each P,/Pi represents the sum of 5 replications. Pfifinal population Pfidnitial population ..................................35 Meloidogyne nataliei population on new hosts, Vitis rootstocks Gloire and St. George and Parthenocissus tricuspidata (Boston ivy) at destructive sampling 146, 181, and 182 days, respectively, after inoculation. Controls of zero not shown. Pfiiinal population Pfidnitial population...................................36 Meloidogyne nataliei population on Parthenocissus quinquefolia (Virginia creeper) at destructive sampling 89 days after inoculation. Control of zero not shown. Each Pf/Pi represents the sum of 6 replications. Pfifinal population Pfidnitial population.... ....O....Q.O0.00.......OOCOCOOOOOOOCOOOO0.0.042 Population density of Meloidogyne nataliei females in seven root samples [0.5 g], on 23 sampling dates from March 1992 through March 1993..O.........O0.0.....0.0.0.0000000000000045 Population density of Meloidogyne nataliei juveniles in seven soil samples [100 cmfi, on xi Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10. 11. 12. 13. 14. 15. 16. 17. 23 sampling dates from March 1992 through March 1993 ............ 0......0.0.00.00000000000000046 Population density of Meloidogyne nataliei males and juveniles in seven soil samples [100 cm’], on 23 sampling dates from March 1992 through March 1993.00.00.000000......OOOOOOOOCOOOO000......48 Population density of Meloidogyne nataliei males and juveniles in seven root samples [0.5 g], on 23 sampling dates from March 1992 through March 1993....O.........OOOOIOOOOOOOOOOOO0.0.00.00049 Embryogenesis of eggs of Meloidogyne nataliei from the two-cell stage at 2.5°, 4.5°, 9°, 18°, 24°, 27°, and 33° C. The last point on each line represents termination of the 10 day study except at 33° C which represents death of the eggSOOOOOOOOOO.........OOOOOOOOOOCOO0.0.0....56 Embryogenesis of eggs of Meloidogyne nataliei from the two-cell stage to the multi-cell stage at 2.5°, 4.5°, 9°, 18°, 24°, 27°, and 33° c.... ...00.0.0.........OOOOOOOOOO00......0.00.0...58 Embryogenesis of eggs of Meloidogyne nataliei from the multi-cell stage to the gastrula stage at 9°, 18°, 24°, and 27° C. Eggs at 2.5° and 4.5° C are shown although no further development took place during these studies...................59 Embryogenesis of eggs of Meloidogyne nataliei from gastrula stage to the tadpole stage at 9% 18°, 24°, and 27° COOIOOOOOOOOOOOOOO0.0.00.00.60 Embryogenesis of eggs of Meloidogyne nataliei from the tadpole stage to the J1 stage at 9% 18°, and 24° C. There is no information available for this egg change at 27°(L................61 Embryogenesis of eggs of Meloidogyne nataliei development from J1 stage to J2 stage at 9°, 18°, 24°, and 27oCOOOOOOOOOOOOOOOOOOO0.0.00.00000063 Embryogenesis of eggs of Meloidogyne nataliei from J2 to free-living J2 at 9°, 18°, 24°, and 27° C. Free-living J2 were removed from BPI watch glasses upon eclosion..................64 Embryogenesis of eggs of Meloidogyne nataliei incubated at 27° C. Eggs were studied from the given stage. No tadpole egg was available at onset of study...............................66 xii Figure Figure Figure Figure Figure Figure DISCUSSION Figure 24. Figure 25. 18. 19. 20. 21. 22. 23. Embryogenesis of eggs of Meloidogyne nataliei incubated at 9°<:. Eggs were studied from the given stage ......................... .........67 Embryogenesis of eggs of Meloidogyne nataliei incubated at 18° C. Eggs were studied from the given stage..................................68 Embryogenesis of eggs of Meloidogyne nataliei incubated at 24° C. Eggs were studied from the given stage..................................69 A. Control Concord root cross section (x256). B. Giant cell (gc) in cross section, induced by Meloidogyne nataliei, that may be showing extra wall deposits that form on cross-walls between altered cells (x256). C. Cross section of giant cell showing thickened cell wall (x500). D. Same root section as C. indicating giant cell orientation in root cross section (x125).....71 A. and B. Cross sections of parasitized Concord root tissue. Giant cells (gc) parallel to each other, with parenchyma cells in between, induced by the feeding of a single Meloidogyne nataliei female (x256). C. and D. Giant cells in cross section of grape root tissue, induced by a single female, x200 and x500, respectively...72 A. Cross section of Concord root tissue with nematode (n) surrounded by brown cells (b) (x500). B. Longitudinal section of two giant cells (gc) and beginnings of a third (x160). C. Anterior of Meloidogyne nataliei, position indicated in D, at base of three giant cells, position indicated by arrows (x312). D. Same section as B. with position of nematode indicated by arrow (x125).... ...... . ..... ....73 A. Healthy Parthenocissus quinquefolia at onset of host and pathogenicity study in growth chamber. B. Parthenocissus quinquefolia in unhealthy condition prior to termination of study in growth chamber. Most plants are leafless.....................................76 A. Healthy Parthenocissus tricuspidata at onset of host and pathogenicity study in growth chamber. B. Unhealthy Parthenocissus tricuspidata prior to termination of study in growth chamber. Some plants have only desiccated xiii APPENDIX A Figure 1. Figure 2. leaves ....................................... 77 Meloidogyne nataliei juvenile above Arabidopsis thaliana root feeding intracellularly. Dark area of root at nematodes anterior end is due to cell destruction by nematode feeding (x160)......... 0.00.00.........OOOOOOOOOOOO0.00.00.00.00000101 Meloidogyne nataliei juvenile above, not in, Arabidopsis thaliana root feeding intracellularly (x400)............. ...... ...102 xiv INTRODUCTION Grape (Vitis Tournefort spp.) is the most widely planted fruit crop in the world, growing from-temperate to tropical regions. Most vineyards, however, are in the temperate climates (Pearson, 1988). In Michigan (MI) there were 4,453 ha (11,000 acres) of grapevines at bearing age, in 1991 (Anon., 1992). Grape production was 33,636 MT (37,000 tons) of cv. Concord, and 8,182 MT (9,000 tons) of other cultivars (Anon., 1992). Ninety-three percent of the crop was sold for juice, 5% for wine, and 2% for fresh market (Anon., 1992). The crop was valued at $255.5 U.S. per MT or $281 U.S. per ton (Anon., 1992). Michigan ranks fourth in the U.S. in Concord production (8.6%), and fifth in the nation for total grape tonnage produced; behind California, Washington, New York, and Pennsylvania (Anon, 1992). The U.S. ranked seventh in grape acreage worldwide in 1983, following Spain, the Soviet Union, Italy, France, Turkey, and Portugal (Pearson, 1988). Nematodes damage grape plants by feeding on roots, thus reducing root efficiency. The first indication of vine damage is usually observed as reduced vigor and yield with slight yellowing of leaves. Seldom does nematode activity lead to vine death unless there are other stress factors 2 involved. Nematode-infected roots are unable to meet the demands of the shoot system for nutrients and water and are the first to show a nitrogen or water deficiency, especially during peak demand periods. Root-knot nematodes create "sinks" in the root system that channel photosynthates to the infection sites. This is done through the formation of giant cells in which the nematode inserts its stylet and feeds (Wharton, 1986). Giant cells are formed by repeated nuclear mitoses in the absence of cell division, resulting in a large multinucleated cell (Wharton, 1986). It is the giant cell that diverts photosynthates to the nematode feeding site. Meloidogyne nataliei Golden, Rose & Bird (Michigan Grape Root-Knot Nematode) was described in 1981. It is not known if M. nataliei is a pathogen of grape, or if it induces the formation of giant cells. Very little is known about the biology of this nematode. RESEARCH OBJECTIVES The pathogenicity of M. nataliei associated with the ontogeny of Vitis spp. can be determined and described at a field-level and under controlled conditions (growth chamber). The objectives of this research are to: Determine the optimal temperature for egg development and degree days required for egg hatch. Determine the length of life cycle from egg to adult on Vitis labruscana in the growth chamber and field. Determine whether M. nataliei is a pathogen of Vitaceae species. Determine if shoot growth, root growth or both are effected. Determine at what nematode population level most growth suppression, if any, takes place. Observe how and where on plant roots M. nataliei parasitizes Vitis spp. Carry out hosts range studies. Determine root cell responses to M. nataliei feeding and reproduction activities using histopathological techniques. LITERATURE REVIEW VITIS SPECIES Vitis Roots Morphology and Anatomy The vine of Vitis spp. consist of roots, stems, leaves, flowers, and fruit. Roots may be distinguished between germination roots and adventitious roots, according to their origin. Germination roots are produced when grape seeds germinate, producing first, a clear tap-root soon forming lateral roots developing around its neck, and later on, lower down as well. The first lateral roots are primary, the second are secondary, the third are tertiary and so on. Germination roots are of importance primarily in breeding programs. Of more importance are the adventitious roots which are formed on parts of the stem; usually at a node, but may form anywhere on the wood during vegetative propagation. The strongest roots form at nodes (Perold, 1927). Primary roots develop laterals which produce tertiary roots, sometimes called hair roots because they are very thin and fine compared with the primary and secondary roots (Perold, 1927). Tertiary roots, or hair roots, absorb nearly all the nutrients and water which the vine takes up from the soil (Perold, 1927). Hair roots are formed every year on the 5 roots of the vine as soon as winter dormancy has been completed (Perold, 1927). The primary functions of roots are absorption of water and nutrients, storage of reserves, and anchorage (Winkler, et al., 1974). Equally important is that factors, i.e., cytokinins, originating in roots are required for normal inflorescence development (Mullins, 1967). Using grape cuttings, Mullins (1967) observed that the inflorescences failed to develop in the absence of roots. It has also been suggested that gibberellin and cytokinin synthesized by the roots play an important physiological role in controlling the growth of the flower clusters and shoots in addition to controlling the carbohydrate reserves and the photosynthate of newly developing leaves (Niimi, et al., 1978). In every individual root there are four parts: the root cap, subapical meristem, zone of elongation, and the maturation zone. The root cap is a thimble-like mass of cells, usually 2-4 mm long, that protects the subapical meristem, basipetal to the root cap, and aids the root in penetrating the soil (Raven, et al., 1986). The subapical meristem is composed of small (10-20 pm in diameter), many- sided cells with dense cytoplasm and large nuclei (Raven, et al., 1986). Cortex and procambial cylinder are distinguishable close to the root apex (Pratt, 1974). Basipetal to the subapical meristem is the zone of elongation which measures only a few mm in length (Raven, et al., 1986). The cells in this region results in most of the 6 increase in length of the root. Beyond this zone the root does not increase in length. The zone of maturation or absorption zone, is basipetal to the elongation zone and is about 10 cm long (Pratt, 1974). Most of the cells of the primary tissues mature here. The maturation zone may be recognized by the presence of fine root-hairs on it and its yellowish color (Perold, 1927). Root hairs, that are 12-15 pm long, are produced in this region (Pratt, 1974). Root hairs are irregularly distributed. Root hairs and epidermis soon die, and the root is then covered by intercutis (Pratt, 1974). The transportation zone is the rest of the root (recognized by its brown color) and serves for transporting soil solution to the aerial parts of the vine, and for transporting the manufactured food back from there to the root, particularly to the growing points (Perold, 1927). The whole of the maturation zone then existing is converted into a transportation zone in late autumn as the vine enters dormancy and the whole root now turns brown through suberization of its external cell layers (Perold, 1927). The primary vascular cylinder consists of pericycle surrounding alternating bundles of primary xylem and phloem separated by medullary rays (Pratt, 1974). Primary xylem, consisting of vessels alone, differentiates centripetally and primary phloem, consisting of sieve tubes, develops centrifugally (Pratt, 1974). 7 Lateral roots develop where the zone of absorption meets the zone of transportation, a vascular cambium appears and periderm is formed giving the zone of transportation a brown exterior (Pratt, 1974). Vascular cambium arises inside the primary phloem bundles and develops in the pericycle outside of the primary xylem (Pratt, 1974). Secondary xylem consists of large scalariform-pitted vessels, parenchyma, tracheids, and septate lignified fibers (Pratt, 1974). The most important vital processes of the plant take place in the parenchyma cells, and parenchyma cells can at any time again form meristematic cells by cell division (Perold, 1927). Phellogen or cork cambium develops in the pericycle after the vascular cambium is established (Pratt, 1974). By the end of the vegetative season many roots produced during this period have died but the stronger roots are covered with periderm even to their tips (Pratt, 1974). Cambium resumes its division, often as a discontinuous ring, as each dormant period ends exhibiting alternating bands of sieve elements and phloem parenchyma (soft phloem) and of phloem fibers (hard phloem) formed by the cambium toward the outside (Pratt, 1974). New phellogen forms in the secondary phloem and produces periderm which cuts off old phloem and old cork thus restricting food-conducting tissue to the youngest rings of phloem (Pratt, 1974). Vitis - Root-knot Interactions Within the genus Meloidogyne, M. incognita Chitwood (Kofoid & White, 1919) Chitwood, 1949, M. javanica (Treub, 8 1885) Chitwood, 1949, M. hapla Chitwood, 1949, M. arenaria (Neal, 1889) Chitwood, 1949 and other root-knot nematode species are important pathogens of grapevines. Second-stage juveniles emerge from eggs, move through moist soil and penetrate roots. Those most active can travel vertical distances of 30 cm (12 in) in sandy loam soil in three days (McKenry, 1992). Second-stage juveniles of root-knot nematodes are attracted to roots and usually penetrate and enter just behind the subapical meristem. They establish themselves in the vascular tissues, begin feeding, and after two weeks of warm temperatures, females mature into egg-laying adults (McKenry, 1992). The familiar "knot" or "gall" seen on the root surface is the result of stimulation of cellular change in the plant in the immediate vicinity of the feeding site caused by the female nematode development. Internally, this knot results in disruption of conducting tissues. A single knot may be inhabited by one or several adult females and the number of females present influence the size of the gall. If the pearl-colored, sedentary adult female is dissected from the gall, it is barely visible to the unaided eye. Small galls or knots are typically 3 mm (% in) in diameter, but can be larger where there has been a multiple attack (McKenry, 1992). However, they are seldom larger than 12 mm (% in) in diameter (McKenry, 1992). Histopathological techniques are used to determine host cell responses to sedentary endoparasitic nematodes. Through 9 the use of fixation, dehydration, infiltration, embedding, sectioning, and staining, host cells may be studied with the aid of light and electron microscopy. The root-knot nematode life span in grape is from one to several months, with the greatest longevity, but least activity, occurring during winter (McKenry, 1992). A single gall may be maintained by successive females for many years. Male root-knot nematodes do not feed and are sometimes present in low numbers (McKenry, 1992). In California, egg population reaches highest numbers in September when soils of many vineyards are driest. It is presumed that dry soil reduces egg hatch but not egg production with as many as 1,500 eggs produced by a single adult female. Second-stage juveniles are generally two to five times more numerous in soil during fall and winter than in spring and summer (McKenry, 1992). The preponderance of the population is located 15-90 cm (6-36 in) deep beneath the vine row, depending on soil conditions (McKenry, 1992). Meloidogyne spp. are best adapted to coarse-textured soils including sand, loamy sand, and sandy loam (McKenry, 1992). The Meloidogyne spp. host range is vast and includes the roots of many broadleaf weeds and cover crops present in vineyards. 10 THE GENUS MELOIDOGYNE MeloidOgyne Life Cycle Associated with Hosts The life cycle of a root-knot nematode, a sedentary endoparasite, begins with the egg stage that develops a first-stage juvenile within the egg. The second-stage juvenile hatches from the egg and must enter a root to feed. M. nataliei has been observed feeding from root cells while outside the root tissue (Appendix A). Once inside root tissue, second-stage juveniles enlarge to form the sausage- shaped third-stage juveniles of both the pre-male and pre- female that continues as a sausage-shape into the fourth- stage from which vermiform non-feeding males emerge and swollen mature egg-laying females develop (McKenry, 1992). Mature M. nataliei males, removed from egg sacs and inoculated onto Arabidopsis thaliana L., have been observed feeding at root cells (Appendix A). The saccate female produces eggs in a gelatinous matrix, external to the nematode body, that usually develops external to the root surface (McKenry, 1992). In some Meloidogyne spp., intersexes are formed as a result of nutritional deficiencies (Wharton, 1986). Intersexes show a mixture of male and female characteristics (Wharton, 1986). When conditions are favorable, second-stage juveniles of Meloidogyne spp. develop into females, but when under stress most develop into males (Wharton, 1986). These stress conditions include crowding, low host susceptibility and stress in the host plant caused by growth regulators, 11 defoliation, infected roots and high or low temperatures (Triantaphyllou, 1971). Male production in response to stress or crowding may increase genetic variability in a deteriorating environment (Wharton, 1986). Meloidogyne Morphology Female Meloidogyne spp. have a white body, globular to pear shaped with length 295-4250 um, sometimes elongate and with a neck, usually short, sometimes very long (Jepson, 1987). The female stylet is robust with cone usually half the total length with dorsal curvature and distal opening and length 10-25 am having a base with three knobs (Jepson, 1987). The male is vermiform with length about 700-1900 nm and is migratory (Jepson, 1987). The stylet is robust with length 13-30 pm and the cone is straight, usually half the total length, occasionally shorter with an opening posterior to the apex and the base with three knobs (Jepson, 1987). Second-stage juveniles, the infective stage, are migratory with a length of about 250-600 pm the stylet is robust, with a cone about half the total length and a base with three knobs (Jepson, 1987). The third and fourth-stage juveniles are swollen and sedentary within the roots, without a stylet and within the cuticle of second-stage which retains the tail spike (Jepson, 1987). Several species of Meloidogyne are parthenogenetic, obligate or facultative, but the presence or absence of males is not correlated with this characteristic (De Guiran, 12 et al., 1979). Parthenogenetic species usually have a shorter life cycle and are morphologically similar to other parthenogenetic species. They produce large galls or knots with females deeply embedded in root tissue, occur in subtropical to tropical regions and are host generalists (Jepson, 1987). Other species are amphimictic. These have longer life cycles with distinct morphology, and small or no galls are produced. The females are semi-endoparasitic occurring in temperate to subtropical regions and are more host specific than parthenogenetic species (Jepson, 1987). Some species have several generations per year but in others there is only one (De Guiran, et al., 1979). Under controlled conditions at 21°CL five species (M. javanica, M. arenaria, M. hapla, M. incognita, and M. incognita acrita Chitwood, 1949) produced the gelatinous matrix 35-37 days after inoculation, started egg production 37-39 days after inoculation, and had host invasion by the new second-stage juveniles 57-63 days after (Tarjan, 1952). Meloidbgyne Hosts Hosts of M. hapla, M. javanica, M. arenaria and M. incognita include economically important woody hosts and herbaceous hosts. M. hapla has been recorded on more than 550 wild and cultivated plants, including olive, grape, tea, coffee, and pigeon pea (Jepson, 1987). Thirty-two Meloidogyne spp. feed on plants from a single subclass (Jepson, 1987). M. megatyla Baldwin & 13 Sasser, 1979 and M. pini Eisenback, Baojan Yang & Hartman, 1985 are two species that are believed to be confined to the Gymnosperms, Pinus L. spp. (Jepson, 1987). The remaining species are distributed throughout the subclasses of Angiosperms, most species occurring in the recent Rosidae and Asteridae, in the Magnoliaceae fewer Meloidogyne spp. occur in the more primitive Magnoliidae and Hamamelidae (Jepson, 1987). In the Liliidae a large number of species occur, mostly in the climax group Cyperales, in the Commelinidae (Jepson, 1987). Species restricted to the most advanced subclasses are M. enterolobii Yang & Eisenback, 1983, M. incognita wartellei Golden & Birchfield, 1978, M. indica Whitehead, 1968, M. nataliei, and M. tadshikistanica Kirjanova & Ivanova, 1965 restricted to Rosidae and in the Asteridae, M. coffeicola Lordello & Zamith, 1960, and M. decalineata Whitehead, 1968, the coffee species, M. litoraIis Elmiligy, 1968, and M. ovalis Riffle, 1963, the woody species, and finally, M. cruciani Garcia-Martinez, 1982, M. incognita grahami Golden & Slana, 1978, M. kirjanovae Terenteva, 1965, M. megriensis (Poghossian, 1971) Esser, Perry & Taylor, 1976, M. microcephala Cliff & Hirschmann, 1984, and M. sinensis Zhang, 1983, the herbaceous species (Jepson, 1987). l 4 MELOIDOGYNE NATALIEI A New Species Meloidogyne nataliei females were first observed protruding from the root surfaces of Vitis labruscana Bailey cv. Concord when soil and root samples were submitted in 1977 to the Michigan State University Cooperative Extension Service, Nematode Diagnostic Laboratory from a vineyard at Mattawan, MI, USA (Golden, et al., 1981). The roots, bearing no galls or knots, were heavily infested with females surrounded by massive egg sacs containing large numbers of eggs and with male nematodes within the egg sacs (Figure 1). The known distribution of M. nataliei is presently limited to Van Antwerp and Porter Townships in Van Buren County, MI. Morphology Nematode morphology indicated that this was a new species of the genus Meloidogyne Goeldi, 1887 (Golden, et al., 1981). Further investigation revealed that juveniles, extracted from the soil, had a strong stylet averaging about 22 pm in length (Golden, et al., 1981). M. nataliei sexually mature females are large, with a length of 731-1,247 pm, and width of 383-834 pm. They are pearly-white, globular to pear-shaped, and usually with an exceptionally long neck (Golden, et al., 1981). The massive egg sac protrudes posteriorly, containing 50-400 eggs. It is often three or more times the size of the female (Golden, 15 Figure 1. Concord root section from type location at Mattawan, MI showing massive egg sacs of Meloidogyne nataliei. 16 et al., 1981). Males are large and vermiform, with a length of 1,191- 1,757 um, and a midbody width of 33-43 pm. Males have a distinct stylet that ranges from 28.4-29.2 um long (Golden, et al., 1981). Second-stage juveniles are vermiform. They have a body length of 539-641 pm, body width of 22-29 um, a strong stylet guide and a short hyaline tail (Golden, et al., 1981). ‘ M. nataliei eggs are 147-186 um in length and 50-62 am wide (Golden, et al., 1981). As seen with optical microscopy, the egg shell is hyaline without visible markings (Golden, et al., 1981). Gametogenesis Gametogenesis in M. nataliei is by regular meiosis with two maturation divisions that lead to the production of haploid female and male gametes, both with four chromosomes (Triantaphyllou, 1984). Reproduction is exclusively by amphimixis with the chromosomal mechanism of sex determination that could be XX Q—XY d with the male being the heterogametic sex (Triantaphyllou, 1984). The Y chromosome is present in some cleaving eggs and absent in others; apparently revealing the existence of two types of eggs, one giving rise to females and the other to males (Triantaphyllou, 1984). M. nataliei has four very large chromosomes, as compared to the relatively small chromosomes of the other amphimictic or facultatively parthenogenetic species of Meloidogyne (Triantaphyllou, 1984). The behavior of the chromosomes and the morphology of M. nataliei during gametogenesis are more similar to those of the genus Heterodera Schmidt, 1871, than Meloidogyne, and for this reason a re-evaluation of the taxonomic position of this species may be warranted (Triantaphyllou, 1984). MATERIALS AND METHODS Host Range and Pathogenicity of Meloidogyne nataliei Hosts studies were initiated when grapevine plant materials were not available from commercial suppliers making it necessary to collect cuttings for propagation. Other plant species material was obtained from commercial sources. Plants were potted for experiments either in clay or plastic containers. Loamy sand soil was used in each of sixteen different studies all conducted in growth chambers. Plants were inoculated with M. nataliei collected from an infested vineyard. Acquisition of Plant Material Vitis berlandieri Planchon x riparia Michaux cv. 58B Teleki, V. riparia var. Gloire de Montpellier, and V. rupestris Scheele var. du Lot (St. George) cuttings, all rootstocks, were collected from a research plot located at Lawton, MI, and Concord cuttings were collected at a research plot at Michigan State University (MSU) in January, 1992. 17 18 Figure 2. Vitis sp. cuttings in flats for rooting in greenhouse. 19 Cuttings of wild Parthenocissus quinquefolia Planchon were obtained from a woodlot at Mattawan, MI, and from a research vineyard at Fennville, MI. Cuttings of Tetrastigma voinierianum Miguel (chestnut vine) were supplied by MSU Botany Teaching Greenhouse at MSU. Cissus rhombifolia Vahl (grape ivy) stock plants (Table 5), Parthenocissus tricuspidata Planchon (Boston ivy) stock plants, P. quinquefolia (Virginia creeper), Ceanothus sanguineus Pursh (Oregon Tea Tree), and one species not in the Rhamnales but in Sapindales, Citrus sinensis Osbeck (common orange) were obtained commercially. Propagation of Plant Material Cuttings taken from Vitis spp. and P. quinquefolia were placed in flats of steam sterilized loamy sand soil in a greenhouse and placed on top of heating coils ca. 25°-29.4° C (75°-85° F) to root (Figure 2). Cuttings of T. voinierianum, Cissus rhombifolia, and P. tricuspidata were dipped in Dexol® Rooting Powder (active ingredient: Indole-3-Butyric acid 0.3%) and placed either directly into pots or flats of autoclaved, 10432 g/6.5 cm? (23 psi) for 14 h, Baccto® potting soil to root. Ceanothus sanguineus and Citrus sinensis were obtained as rooted plants; no cuttings made. All plant materials were watered as needed. Containers and Soil The rooted cuttings were transplanted from flats into pots. Plants rooted in Baccto® had roots rinsed as free as possible of that soil before transplanting. Vitis spp. 20 Table 1. Soil nutrients and pH of soil used in all host range and pathogenicity studies. Soil pH: 6.8 Percent Bases Phosphorus.. 370 lbs/A Potassium... 133 lbs/A 5 % Calcium.....1200 lbs/A 86 8 Magnesium... 76 lbs/A 9 % ZinCCOOOOOOO Manganese... Copper...... Cation Exchange Capacity: 3 me/lOO 9 Lime Recommendation is 0 lbs/1000 sq. ft. Mechanical Analysis 83.9% sand 9.0% silt 7.1% clay LOAMY SAND Table 2. Descriptions of Vitis spp. Meloidogyne nataliei host range studies. Date Plant Total days Nematode inoculated species of study density 4-14-92 Vitis cv. 588 157 0 500 J2* 1000 J, 1500 J, 2000 J, 5-30-92 Vitis v. Gloire 146 O 750 J, 5-30-92 Vitis v. St.George 181 0 750 J2 *‘VJ2 - second-stage juvenile 21 rootings, P. quinquefolia rootings, and the Citrus sinensis plants were placed into 14-15.3 cm (5.5-6 in with 800 cm3 soil) clay pots. Cissus rhombifolia rootings were transplanted into 7.6 cm (3 in with 160 cm3 soil) clay pots. P. tricuspidata rootings were transplanted into either 7.6 or 15.3 cm (3 or 6 in, 300 or 800 cm3 soil) green plastic pots. T. voinierianum were transplanted into 7.6 cm (3 in with 300 cm? soil) green plastic pots. Ceanothus sanguineus plants were potted into black plastic tubes (400 cm? soil). Container size was based on plant size. Experimental soil used after rooting was a loamy-sand, steam sterilized for two h maintaining 96° C (205° F) for one h (Table 1). The Michigan State University Soil Testing Laboratory (STL) performed the soil analysis (Kenworthy, 1967). Plants were maintained with Hoagland's No. 2 Basal Salts continuously and Peter's® Fertilizer 20-20-20 (100 g/18.92 l) biweekly and watered with tap water or distilled water as required. Setting Up Experiments Inoculum was collected as infected roots from a vineyard at Mattawan, MI. These roots were chopped and placed into 250 ml Erlenmeyer flasks with ca. 100 ml tap water on a gyratory shaker for four days (Bird, 1971). All experiments were inoculated with M. nataliei using a pipet placed above holes made in the soil with a spatula, which were closed afterward, in the root zone near the plant stems, except the first study with P. tricuspidata (Table 5) 22 that had inoculum poured onto the root system from a beaker as the plants were being transplanted. The SBB study was a total of 25 plants, five treatments, and five blocks (Table 2). The Gloire study was six plants in total, two treatments, and three blocks (Table 2). The St. George study was a total of six plants, two treatments, and three blocks (Table 2). There were two studies utilizing P. tricuspidata. The first had two treatments and four blocks for a total of eight plants (Table 3). The second had two treatments and six blocks for a total of 12 plants (Table 3). The first P. guinquefolia study was made up of 25 plants, five treatments, and five blocks (Table 4). The second P. quinquefolia study was 21 plants, three treatments, and seven blocks (Table 4). The third P. quinquefolia study was 18 plants, three treatments, and six blocks (Table 4). The first Cissus rhombifolia study had three treatments and four blocks for a total of 12 plants (Table 5). This experiment met with an accident, mechanical failure of the growth chamber, and was terminated sooner than planned due to plant death (Table 5). The second and third studies with Cissus each had 12 plants, two treatments, and six blocks (Table 5). Two T. voinierianum studies were carried out concurrently in different growth chambers set at approximately the same temperature (Table 6). Both studies 23 Table 3. Description of Parthenocissus tricuspidata Meloidogyne nataliei host range study. Date Plant Total days Nematode inoculated species of study density 2-19-93 P. tricuspidata 182 O 300 eggs + l800 J,* 2-24-93 P. tricuspidata 182 O 200 eggs + 750 J, *J, = second-stage juvenile Table 4. Description of Parthenocissus guinquefolia Meloidogyne nataliei host range study. Date Plant Total days Nematode inoculated species of study density 5-2-92 P. guinquefolia 181 0 (wild) 500 J,* 1000 J, 1500 J, 2000 J, 5-11-93 P. quinquefolia 101 0 (wild) 580 J, 1160 J, 6-1-93 P. guinquefolia 89 0 18 J, 324 J, *J, 8 second-stage juvenile Table 5. Description of Cissus sp. Meloidogyne nataliei host range studies. Date Plant Total days Nematode inoculated species of study density 8-20-92 Cissus sp. 82 0 61 J; 122 J, S-ll-93* Cissus sp. 100 O 180 J, 5-11-93* Cissus sp. 100 0 180 J, *Identical studies in two different growth chambers at ca. 183 C. ‘J, = second-stage juvenile 24 had two treatments and five blocks with 10 plants each (Table 6). Like T. voinierianum, both Ceanothus sanguineus studies were conducted at the same time in different growth chambers at approximately the same temperature (Table 7). Both experiments had two treatments and eight blocks with 16 plants each (Table 7). The Citrus sinensis study was four plants, two treatments and two blocks (Table 8). This study was inoculated a second time because it had been involved in the previously mentioned growth chamber accident (Table 8). All experiments were arranged in randomized complete block design, blocking against initial plant weight. Plants were watered just to leaching after being inoculated and placed in the growth chamber at ca. 18° C (64° F) with 16 h light 8 h dark, 8.7 cal cm‘2 h’l (2,600 foot candles), for the duration of the studies, with one exception. The first study with P. tricuspidata (Table 5) was removed from the growth chamber after approximately two months, and placed in a greenhouse for one month in an attempt to stimulate new shoot and leaf growth. These plants had senesced their leaves due to the low, constant temperatures in the growth chamber. Greenhouse temperatures ranged from 18.3° C (65° F) at night to 32.2° C (90° F) during the day. 25 Table 6. Description of Tetrastigma sp. Meloidogyne nataliei host range study. Date Plant Total days Nematode inoculated species of study density S-l7-93* Tetrastigma sp. 92 0 1000 J; 5-17-93* Tetrastigma sp. 92 0 1000 J, *Identical studies in two different growth chambers at ca. 18°C. Kb - second-stage juvenile Table 7. Descriptions of Ceanothus sp. Meloidogyne nataliei host range study. Date Plant Total days Nematode inoculated species of study density 6-22-93* Ceanothus sp. 61 O 150 J; 6-22-93* Ceanothus sp. 61 0 150 J, 7Identical studies in two different growth chambers at ca. 18°(L KB = second-stage juvenile Table 8. Description of Citrus sp. Meloidogyne nataliei host range study. Date Plant Total days Nematode inoculated species of study density 5-30-92 Citrus sp. 345 0 750 J,* & 12-1-92 & + 300 J, *J, = second-stage juvenile 26 Terminating and Evaluating Experiments When experiments were terminated, whole plant fresh weights were recorded in grams. Leaves and petioles, stems, and roots were then weighed separately. Leaves, petioles and stems were placed in a drying oven for approximately five days, then removed and weighed again. The second experiment with P. tricuspidata (Table 5) grew so well in the growth chamber that the plants had to be pruned after 69 days. The pruned vines were handled as above. Plant weights were statistically evaluated using the analysis of variance and regression tests. Analysis of variance tests were calculated manually. Regression tests were conducted using computer software SYSTATO 2.0. Roots were chopped with a knife on a cutting board, mixed by hand, and 1 9, less if not available, placed in Erlenmeyer flasks with ca. 50 ml ethoxyethyl mercuric chloride 10 pg/ml and streptomycin sulfate 50 ug/ml (EMC), placed on a gyratory shaker for extraction of nematodes (Bird, 1971). Another 0.5 g of roots were cleared with sodium-hypochlorite and stained with acid fuchsin (Byrd, 1972). Soil was processed for nematode extraction using the centrifugal-flotation technique (Jenkins, 1964). All processed roots and extractions were examined microscopically. 27 Life History of Meloidogyne nataliei Field Population Dynamics Soil and root samples were collected biweekly for one year from Spiech Farms at Mattawan, MI, a known infested V. labruscana cv. Concord vineyard. Samples were collected as described by Bird, et al. (1990). Nematodes extracted from soil using Jenkins' (1964) centrifugal-flotation technique, and roots were cleared and stained as described by Byrd (1972). All soil extracted samples and stained root samples were examined microscopically. Soil temperatures at three depths, 0.31 m (12 in), 0.92 m (36 in), and 1.83 m (72 in), were recorded using a hydro- thermograph over a one year period. Additional soil temperature data was supplied by the MSU South West Michigan Research & Extension Center, Benton Harbor, MI. Petioles were sampled for analysis on September 11, 1992 (Hanson, et al., 1986). Soil samples were collected for analysis September 30, 1992 (Hanson, et al., 1987). Petiole tissue analysis and soil analysis from three levels 0.31 m, 0.92 m, and 1.83 m for each vine were submitted to the MSU STL for processing (Kenworthy, 1967). Utilizing seven grapevines for soil samples, all three depths were collected at one vine over a 1.62 ha (four acre) area. Three soil samples from each vine totalling 21 samples were processed for nematode extractions and evaluation (Jenkins, 1964). Degree day development was determined using 2.5°C:(38° F) as the base temperature. Each day this base temperature 28 was subtracted from the average for that day and any remaining degrees accumulated as degree days. Ontogeny Under Growth Chamber Conditions Growth chamber studies were conducted on Concord rootings from cuttings collected in a research vineyard at MSU. Rootings were transplanted from flats to 15.3 cm (6 in) clay pots. Soil used was loamy sand, steam sterilized for two h maintaining 96° C (205° F) for one h (Table 1) . Plants were grown under greenhouse conditions until inoculated. One-year-old Concord grapevines were inoculated with 400 vermiform M. nataliei per pot. Inoculum was collected by placing chopped infected roots, collected from Spiech Farms vineyard, in 250 ml Erlenmeyer flasks with ca. 100 ml tap water on a gyratory shaker for four days (Bird, 1971). Plants were maintained in the growth chamber with Hoagland's No. 2 Basal Salts continuously and Peter's® Fertilizer 20- 20-20 (100 g/18.92 l) biweekly and watered with tap water or distilled water upon demand. Growth chamber temperature was ca. 25° C (77° F) for 67 days then lowered to 18° C (64° F). Growth chamber lighting provided 16 h light 8 h dark, 8.7 cal cm'2 h‘l (2,600 foot candles). One plant was destructively sampled every 7 days and roots examined with a hand lens for swollen females and egg masses. Soil (100 cm’) and roots (0.5 g) were processed as above. One 9 of roots was also placed on a gyratory shaker for nematode extraction (Bird, 1971). Degree day development was determined using 2.5° C as 29 the base temperature. Egg Development Under Incubator Conditions Egg masses were removed from females in Concord root tissue with forceps and placed in a beaker of tap water. These Concord plants had been rooted and grown in the greenhouse until inoculated with M. nataliei vermiform when they were placed in the growth chamber at 18° C. These females and egg masses with eggs had developed at 18° C. The water was brought to a 10% sodium-hypochlorite solution and agitated with a spatula for 4 minutes to denature the gelatinous matrix. The solution with the eggs was poured through a 100 mesh sieve over a 400 mesh sieve, and eggs were rinsed for five minutes with running tap water. The eggs were rinsed with distilled water into another beaker and pipetted into Bureau of Plant Industry (BPI) watch glasses where they were counted and observed for cell number and degree of differentiation microscopically. Eggs at all stages of cell development were utilized. The BPI watch glasses were filled with distilled water, placed one each into Petri dishes, covered, and placed in incubators without light at 33° c (91.4° F), 27° c (80.6 ° F), 24° c (75.2° F), 13° c (64.4° F), and 9° c (48.2° F) to confirm results obtained from the growth chambers and to find optimal development temperature for M. nataliei (Tyler, 1933). To prevent evaporation of water from the BPI glasses at 33° C, distilled water was added to the Petri dishes. BPI glasses incubated at 33° C had an average of 42.2 30 eggs, at 27° C an average of 4.6 eggs, at 24° C an average of 5.3 eggs, at 18° C an average of 6.8 eggs, and at 9° C an average of 29.4 eggs. The 33° and 9° C studies, with the greatest number of eggs per BPI were established first. Due to the difficulty in viewing so many eggs in BPI glasses, the number of eggs per BPI was reduced for the remaining studies. Each treatment was replicated ten times. Treatments were arranged randomly on a tray in the incubators. Each BPI glass was removed from the incubator every ca. 24 h, and, with the aid of a microscope, vermiform M. nataliei were counted and removed. Egg cell development was recorded. BPI glasses had drops of distilled water, kept at the same incubation temperature, added to them to replace any lost through evaporation as needed. Remaining eggs were returned to the incubators until the last eggs had hatched or were determined to be dead. Egg mortality was confirmed utilizing new blue R stain. New blue R was prepared in water to make a 0.05% solution by weight (Shepherd, 1962). New blue R's ability to confirm egg death was verified by staining nematode eggs that had been simmered on a hot plate in water for two min. When cool, the eggs were then stained for one min. The stain solution was drawn off with a pipet. Heat killed eggs were stained mauve or deep purple. Eggs believed to be alive were stained in the same manner and showed no acceptance of stain pigment. After observing egg development at 9° C, additional studies were established to determine the minimum 31 temperature for egg development and for clarification of previous data. Eggs were collected as above, placed in a Petri dish, and two-cell eggs were removed by suction with a pipet and bulb and placed in BPI glasses. Each BPI glass had one two-cell egg per glass at 9° and 4.5°C (40.1° F) and two or three two-cell eggs at 2.5° C i 0.25° (36.05° F). The glasses were filled with distilled water and observed for cell development once every ca. 24 h for ten days. As needed, the watch glasses were refilled with drops of distilled water kept at the same temperature the eggs were incubating. Each treatment was replicated 10 times and arranged randomly on a tray in the incubators. BPI watch glasses were pre-cleaned with sodium-hypochlorite. Degree day development was determined using 2.5° C as the base temperature. Embryogenesis studies terminating after ten days were evaluated using heat units, each degree of temperature above 2.5° C each hour being equal to one heat unit (Tyler, 1933 & Ferris, et al., 1978). Egg development was evaluated by stages. One-cell stage is an un-cleaved egg, that may or may not have been expelled from the female (Bird, 1971). The two-cell stage is two blastomeres containing somatic cells and precursor of gametes (Bird, 1971). The multi-cell stage includes cell cleaving beyond two-cells, T-stage, rhomboid stage, morula, and blastocoele blastula stages (Bird, 1971). Gastrulation starts with the entry of the intestinal precursors followed by the germ line (Bird, 1971). The tadpole stage is the 32 cylindrical embryo, the J1 is a complete, vermiform laying tripled over in the egg, and when the J1 has elongated and molted once in the egg it has become a J2 (Bird, 1971). When the J2 hatches it is said to be free-living (Bird, 1971). Histopathology of Meloidogyne nataliei Associated with Vitis labruscana Root tissue was removed from infected Concord plants that had been inoculated and grown in a growth chamber and from Concord plants that had not been exposed to nematodes to be used as the controls. Grape plants were removed from their pots and the roots were washed free of soil with tap water. Root pieces with females and egg masses were cut from whole roots with a surgical blade. The root pieces were placed in 1.5 ml Eppendorf® micro test tubes for processing. Root tissue was fixed for 24 h in 3% glutaraldehyde and 6.4 pH phosphate buffer solution in a 1:1 mix and two changes of osmium tetroxide with 6.4 phosphate buffer solution at 25% and 50%, and then 100% phosphate buffer all at 0° C, dehydrated with acetone removed and replaced twice for 4 h each at room temperature and a third time for 24 h, refrigerated. Two changes of acetone and Spurr's in a 1:1 mix for four h and 12 h were made before the roots were infiltrated with Spurr's resin, changed once. The Spurr's was prepared by the MSU Center for Electron Optics - 5 9 vinyl cyclohexene dioxide (ERL-4206), 7.4 g Quetol 6510, 26 g nonenyl succinic anhydride (NSA), and 0.4 g 2- 33 dimethylaminoethanol (S-l). Root tissue was placed in a flat mold, each space filled with the resin, tissue oriented, and placed in a desiccator for four h. The molds were then placed in an oven at 65° C (149° F) for 24 h. Upon removal from the oven the mold was cooled for 15 min in a desiccator before the hard blocks were popped out. The blocks were placed in a Petri dish and stored in a desiccator until needed. Hardened blocks were trimmed with a razor blade while being held in place by a chuck attached to a holder sitting on the stage of a dissecting microscope. Sections were cut with glass knives using a SorvallO MT-2 Ultramicrotome. Sections were made in cross section and longitudinally. Sections were cut 4-10 pm thick, lifted with watch maker's forceps and placed on a drop of distilled water on a microscope slide. The slide was placed on a hot plate at 45° C until the water had evaporated in a near dust free area. The sections were stained within a few hours. Sections were stained with toluidine blue 0 - 0.05% toluidine blue 0 solution in benzoate buffer, pH 4.4 (O'Brien, et al., 1964). Sections were stained in toluidine blue 0 solution by placing a drop of solution on the section for 30 sec-2.5 min, rinsed in running water until the resin was free of the stain, dried at room temperature, mounted in Permount, and No. 1 cover slips applied (Feder, et al., 1968). Other sections were stained with acid fuchsin - 1% acid fuchsin in water using the above procedure (Feder, et al., 1968). When over staining occurred, 100% acetone was pipetted over the sections and then water rinsed. All stained sections were examined microscopically. Micrographs were made using a Zeiss III RS Photogenic III 12v60w/Ept. Hw/.10w mercury, v/z phase condenser. RESULTS Host Range and Pathogenicity of Meloidbgyne nataliei All Vitis spp. studied were hosts of M. nataliei. This nematode was able to migrate into the roots, feed, become mature reproducing males; and become females with massive egg sacs, that produced new generations of juveniles. 5BB soil extraction revealed an increase in the juvenile population over the inoculated level, processed roots had numerous juveniles, males and viable eggs, as did stained roots (Figure 3). Gloire soil extraction was negative, no developmental stage found, in each treatment. Root tissues contained juveniles, males, and females with egg masses (Figure 4). St. George soil extraction revealed only dead juveniles but root tissues contained juveniles, males, and females with egg masses (Figure 4). Using the analysis of variance and regression tests for SBB (Table 9), Claire (Table 10), and St. George (Table 11) to evaluate whole plant fresh weight increases, and dry leaves and petioles weights, no statistically significant 34 14' 12‘ . % increase 10‘ [*1 % decrease 500 1000 1500 2000 M. on SBB, Figure 3. Meloidogyne nataliei population on Vitis rootstock 5BB at destructive sampling 157 days after inoculation. Control of zero not shown. Each Pf/Pi represents the sum of 5 replications. Pf=final population Pi=initial population 36 40 ' , I Gloire 3? 35' I St. George H ‘ I Boston ivy ”I, 30 DI "‘ B 0 - fl 94 25 z ‘ ,u 3 20 " Q m . R O a 15 ' é a ' o - I: 10 a - O 5 Replications Figure 4. Meloidogyne nataliei population on new hosts, Vitis rootstocks Claire and St. George and Parthenocissus tricuspidata (Boston ivy) at destructive sampling 146, 181, and 182 days, respectively, after inoculation. Controls of zero not shown. Pfiiinal population Pfidnitial population 37 Table 9. Analysis of variance and regression for Vitis rootstock SBB pathogenicity study. ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT 588 N: 25 Multiple R: .668 Squared multiple R: .446 Source Sum-of-squares DF Mean-square F-ratio P Tmt 4.087 4 1.022 0.424 0.789 Rep 26.947 4 6.0737 2.793 0.062 Error 38.592 16 2.412 REGRESSION LEAF & PETIOLE DRY WEIGHT 588 N: 25 Multiple R: .031 Squared multiple R: .001 Standard error of estimate: 1.739 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 3.103 0.602 0.000 . 5.151 0.000 Population -0.000 0.000 -0.031 1.000 -0.150 0.882 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT 588 Source Sum-of-squares DF Mean-square F-ratio P Regression 0.068 1 0.068 0.022 0.882 Residual 69.558 23 3.024 ANALYSIS OF VARIANCE FRESH WEIGHT 588 N: 25 Multiple R: .740 Squared multiple R: .548 Source Sum-of-squares DF Mean-square F-ratio P Tmt 447.572 4 111.893 0.464 0.761 Rep 4220.130 4 1055.033 4.376 0.014 Error 3857.655 16 241.103 REGRESSION FRESH WEIGHT 588 N: 25 Multiple R: .019 Squared multiple R: .000 Standard error of estimate: 19.249 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 32.742 6.668 0.000 . 4.910 0.000 Population -0.001 0.005 -0.019 1.000 -0.093 0.927 ANALYSIS OF VARIANCE FRESH WEIGHT 5BB Source Sum-of-squares DF Mean-square F-ratio P Regression 3.211 1 3.211 0.009 0.927 Residual 8522.147 23 370.528 38 Table 10. Analysis of variance and regression for Vitis rootstock Gloire pathogenicity study. ANALYSIS OF VARIANCE FRESH WEIGHT GLOIRE N: 6 Multiple R: .733 Squared multiple R: .537 Source Sum-of-squares DF Mean-square F-ratio P Tmt 0.034 1 0.034 0.001 0.978 Rep 84.101 2 42.050 1.161 0.463 Error 72.430 2 36.215 REGRESSION FRESH WEIGHT GLOIRE N: 6 Multiple R: .015 Squared multiple R: .000 Standard error of estimate: 6.256 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 5.860 8.076 0.000 . 0.726 0.508 Population -O.lSO 5.108 -0.015 1.000 -0.029 0.978 ANALYSIS OF VARIANCE FRESH WEIGHT GLOIRE Source Sum-of-squares DF Mean-square F-ratio P Regression 0.034 1 0.034 0.001 0.978 Residual 156.531 4 39.133 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT GLOIRE N: 6 Multiple R: .991 Squared multiple R: .982 Source Sum-of-squares DF Mean-square F-ratio P Tmt 0.031 1 0.031 2.502 0.255 Rep 1.299 2 0.650 52.735 0.019 Error 0.025 2 0.012 REGRESSION LEAF & PETIOLE DRY WEIGHT GLOIRE N: 6 Multiple R: .151 Squared multiple R: .023 Standard error of estimate: 0.575 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 0.487 0.743 0.000 . 0.655 0.548 Population 0.143 0.470 0.151 1.000 0.305 0.775 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT GLOIRE Source Sum-of-squares DF Mean-square F-ratio P Regression 0.031 1 0.031 0.093 0.775 Residual 1.324 4 0.331 39 Table 11. Analysis of variance and regression for Vitis rootstock St. George pathogenicity study. ANALYSIS OF VARIANCE FRESH WEIGHT ST. GEORGE N: 6 Multiple R: .789 Squared multiple R: .623 Source Sum-of-squares DF Mean-square F-ratio P Tmt 5444.491 1 5444.491 1.021 0.419 Rep 12161.325 2 6080.663 1.140 0.467 Error 10664.384 2 5332.192 REGRESSION FRESH WEIGHT ST. GEORGE N: 6 Multiple R: .767 Squared Multiple R: .588 Standard error of estimate: 62.342 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 284.117 101.804 0.000 . 2.791 0.068 Population -52.835 32.696 -0.628 1.000 -1.616 0.181 ANALYSIS OF VARIANCE FRESH WEIGHT ST. GEORGE Source Sum-of-squares DF Mean-square F-ratio P Regression 11166.149 1 11166.149 2.611 0.181 Residual 17104.052 4 4276.013 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT ST. GEORGE N: 6 Multiple R: .877 Squared multiple R: .769 Source Sum-of-squares DF Mean-square F-ratio P Tmt 16.434 1 16.434 1.198 0.388 Rep 74.797 2 37.399 2.726 0.268 Error 27.440 2 13.720 REGRESSION LEAF & PETIOLE DRY WEIGHT ST. GEORGE N: 6 Multiple R: .792 Squared multiple R: .628 Standard error of estimate: 3.324 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 16.398 3.590 0.000 . 4.567 0.010 Population -4.315 1.662 -0.792 1.000 -2.596 0.060 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT ST. GEORGE Source Sum-of-squares DF Mean-square F-ratio P Regression 74.477 1 74.477 6.741 0.060 Residual 44.194 4 11.049 40 differences were found (P < 0.05). Pathogenicity was not determined for these three species. P. guinquefolia and P. tricuspidata are also hosts for M. nataliei. Swollen, sedentary females surrounded by a gelatinous matrix filled with eggs were observed on root tissue of P. tricuspidata. Nematodes recovered by soil extraction methods were juveniles, males, and viable eggs (Figure 4), but only from the second P. tricuspidata experiment (Table 5). Nematodes observed from root tissues were juveniles and females, also from the second experiment (Figure 4). There appears to have been no nematode development in the first experiment (Table 5). There were no statistically significant differences found (Table 12), analyzing data from the second experiment (Table 5), in fresh mass weights, fresh root weights, or dry mass weights using the analysis of variance and regressions tests (P < 0.05). Pathogenicity effects were not detected in P. tricuspidata. Few females and no egg masses were found on root tissues, although soil extraction revealed viable eggs, few juveniles but many males were found from roots and soil of P. quinquefolia (Figure 5). Pathogenicity effects were not detected in P. quinquefolia (Table 13). Using the analysis of variance and regression tests, there were no statistically significant differences found (Table 13) in any of the three experiments on P. quinguefolia between whole plant fresh weights, fresh root 41 Table 12. Analysis of variance and regression for Parthenocissus tricuspidata (Boston ivy) pathogenicity study of 2-24-93. ANALYSIS OF VARIANCE FRESH WEIGHT BOSTON IVY N: 8 Multiple R: .982 Squared multiple R: .965 Source Sum-of-squares DF Mean-square F-ratio P Tmt 634.748 1 634.748 33.708 0.010 Rep 906.575 3 302.192 16.048 0.024 Error 56.492 3 18.831 REGRESSION FRESH WEIGHT BOSTON IVY N: 8 Multiple R: .630 Squared multiple R: .397 Standard error of estimate: 12.669 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 70.258 14.165 0.000 4.960 0.003 Population -17.815 8.959 -0.630 1.000 -1.989 0.094 ANALYSIS OF VARIANCE FRESH WEIGHT BOSTON IVY Source Sum-of-squares DF Mean-square F-ratio P Regression 634.748 1 634.748 3.955 0.094 Residual 963.067 6 160.511 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT BOSTON IVY N: 8 Multiple R: .880 Squared multiple R: .774 Source Sum-of-sguares DF Mean-square F-ratio P Tmt 0.002 1 0.002 0.005 0.950 Rep 3.938 3 1.313 3.423 0.170 Error 1.150 3 0.383 REGRESSION LEAF & PETIOLE DRY WEIGHT BOSTON IVY N: 8 Multiple R: .019 Squared multiple R: .000 Standard error of estimate: 0.921 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 4.435 1.030 0.000 . 4.308 0.005 Population 0.030 0.651 0.019 1.00 0.046 0.965 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT BOSTON IVY Source Sum-of-sguares DF Mean-square F-ratio P Regression 0.002 1 0.002 0.002 0.965 Residual 5.088 6 0.848 nataliei 0 l H .1 .\\\\\\\\\\\\4 I/III/III/III III/IIIIII/IJ U .\\\\\\\\\\\\1 \xxxxxxxxxxxx ///////////// IIII/III/IIIA . _ ,\\\\\\\\\\\\) \\\\\\\\\\\\\ 0.6 ””””””’4 II lIIIII/IIJ ‘ »\\\\\\\\\\\\ \\\\\\\\\\\\\ Ill/IIIIIIIII III/llI/Il/Ia 'FI . »\\\\\\\\\\\\) \\\\\\\\\\\\\ IIIIIII/IIIII, III/IIIIIIIIA a b\\\\\\\\\\\\ \\\\\\\\\\\\\ III/IIIIIIIII II III/IIIIIA .... .\\\\\\\\\\\\‘ \\\\\\\\\\\\\ 04-1 IIIIIIIII/III III/IIIIIIIIJ u ' .\\\\\\\\\\\\‘ \\\\\\\\\\\\\ II/IIIIIIIIII, IIIIIIIIII/l4 H L\\\\\\\\\\\\ \\\\\\\\\\\\\ - III/IIIIIIIII III/IIIIIII/J .... L\\\\\\\\\\\\‘ \\\\\\\\\\\\\ I’ll/IIIIIIII III/IIIIII/I4 > L\\\\\\\\\\\\‘ \\\\\\\\\\\\\ 02‘ ///////////// III/IIIIIIIIA . .\\\\\\\\\\\\‘ \\\\\\\\\\\\\ Ill/IIIIIIIII ////////////4 .\\\\\\\\\\\\‘ \\\\\\\\\\\\\ a .. III/Il/IIIIII III/III/IIIIA .\\\\\\\\\\\\‘ xxsxxxxxxxxxx O Ill/IIIIIIIII IIIIl/Illlllq >\\\\\\\\\\\\‘ \\\\\\\\\\\\\ . 42 1.6“ population 3; . increase I—l 1.4 " °r'| . DI \ fl is: 1.2 population ‘ *I\I\I\I\I\I\I\I\I\I\I‘I\I d. c r . a 8 . >\\\\\\\\\\\\l IIIIIIIIIIIII 1 . 0 '7 r/\/\/\x\/\/\/\/\/\/\/\/\I b\\\\\\\\\\\\‘ p \ \ \ \ \ \ \ \ \ \ \ \ ‘ I I I I I I I I I I I I I b \ \ \ \ \ \ \ \ \ \ \ \ ‘ IIIIIIIIIIIII 18 324 Pi. Figure 5. Meloidogyne nataliei population on Parthenocissus quinquefolia (Virginia creeper) at destructive sampling 89 days after inoculation. Control of zero not shown. Each Pdffi represents the sum of 6 replications. Pfifinal population Pfidnitial population 43 Table 13. Analysis of variance and regression for Parthenocissus guinquefolia (Virginia creeper) pathogenicity study of 6-1-93 0 ANALYSIS OF VARIANCE FRESH WEIGHT VIRGINIA CREEPER N: 18 Multiple R: .519 Squared multiple R: .269 Source Sum-of-squares DF Mean-square F-ratio P Tmt 51.017 2 25.509 0.838 0.461 Rep 61.163 5 12.233 0.402 0.837 Error 304.357 10 30.436 REGRESSION FRESH WEIGHT VIRGINIA CREEPER N: 18 Multiple R: .193 Squared Multiple R: .037 Standard error of estimate: 5.007 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 7.437 3.122 0.000 . 2.382 0.030 Population 1.134 1.445 0.193 1.000 0.785 0.444 ANALYSIS OF VARIANCE FRESH WEIGHT VIRGINIA CREEPER Source Sum-of-squares DF Mean-square F-ratio P Regression 15.436 1 15.436 0.616 0.444 Residual 401.102 16 25.069 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT VIRGINIA CREEPER N: 18 Multiple R: .565 squared multiple R: .319 Source Sum-of-squares DF Mean-square F-ratio P Tmt 0.322 2 0.161 0.044 0.957 Rep 16.777 5 3.355 0.921 0.506 Error 36.440 10 3.644 REGRESSION LEAF & PETIOLE DRY WEIGHT VIRGINIA CREEPER N: 18 Multiple R: .024 Squared multiple R: .001 Standard error of estimate: 1.829 Variable Coefficient Std error Std coef Tolerance T P(2 tail) Constant 5.138 1.140 0.000 . 4.506 0.000 Population 0.050 0.528 0.024 1.00 0.095 0.926 ANALYSIS OF VARIANCE LEAF & PETIOLE DRY WEIGHT VIRGINIA CREEPER Source Sum-of-squares DF Mean-square F-ratio P Regression 2.075 1 2.075 0.650 0.432 Residual 51.047 16 3.190 44 weights or dry leaf, petiole and stem weights (P < 0.05). Cissus rhombifolia, T. voinierianum, Ceanothus sanguineus, and Citrus sinensis, did not show any sign of nematode development within root tissue. Nematodes recovered, usually dead, through soil extraction methods were in lower numbers than the initial population for these four genera of plants. Life History of Meloidogyne nataliei Field Population Dynamics M. nataliei has three generations per year under vineyard conditions in southwestern MI (Figure 6). Sampling of soil and root tissue for M. nataliei began in March, Julian day 78, and ended the following March, Julian day 76. By beginning with Julian day 76, thus becoming Julian day 1, and ending with Julian day 78, now Julian day 366, a complete year may be visualized. The first female population is evident by 80 days, 13 degree days, after the third over-wintering female population (Figure 6). Females from this first generation (Figure 6) lay the eggs that become the second juvenile generation by 100 days or 828 degree days later (Figure 7). The second female generation matures at approximately Julian day 232, 1816 degree days (Figure 6), and produces the third juvenile population by 56 days, or 2742 degree days later (Figure 7). The third female population, over-wintering, is evident about Julian day 332, 2841 degree days (Figure 6), and lays the eggs that are 45 40.. 3 35 .... January 20 March 17 0 o 1 "‘ 30 ~ {3 . November 27 m 25 - o ‘ August 19 20' -.. , \ .2 H 15 .. June 10 U . 4) *o u 10 - a «I g 5 " I"9— females 0 I I I I I I I I I I I I I I I ”i 0 50 100 150 200 250 300 350 400 2846 13 45 02B 1250 2273 2742 3946 2846 Julian. Days (Degree Days) Figure 6. Population density of Meloidogyne nataliei females in seven root samples [0.5 g], on 23 sampling dates from March 1992 through March 1993. 46 40' C91 35_ “—9— juveniles a 30.: October 14 c d June 24 / a 25" January 20 D '3 201 y! a: d 3 15‘ 0 December 23 U . u 10... 0 a. 5-0 a o c D o 0 I I ; 0I0 I I I ° I I I I I I I I 0 50 100 150 200 250 300 350 400 2846 13 45 828 1250 2273 2742 2846 2846 Julian Day (Degree Days) Figure 7. Population density of Meloido ne nataliei juveniles in seven soil samples [100 cm], on 23 sampling dates from March 1992 through March 1993. 47 the over-wintering third juvenile population about 54 days, or 12.3 degree days, later (Figure 7). Egg masses usually contain a mature male nematode. Early stage males, normally in low numbers, reach their highest numbers in the soil at about the same time the first, second, and third juvenile populations reach theirs (Figure 8). Population dynamics of male and juvenile stages of M. nataliei appear in root tissues from Julian day 1 through Julian day 366 (Figure 9). Females are found protruding horizontally on all branches of the mature root system, i.e., primary, secondary, tertiary, etc., and often from the root apex. Other nematode species occurring along with M. nataliei in the vineyard used for the life history study include Hoplolaimus galeatus (Cobb, 1913) Thorne, 1935, Criconemella xenoplax (Raski, 1952) Luc and Raski, 1981, Xiphinema americanum Cobb, 1913, and Pratylenchus neglectus (Rensch, 1924) Filipjev & Schuurmans Stekhoven, 1941. M. nataliei occurs at an absolute frequency of 50% and a relative frequency 19.36% (Table 14). Absolute frequency is how often a species occurs among samples, a measure of distribution uniformity, not abundance, and is expressed as a percentage. absolute frequency = 100 x (number of samples containing a species/ number of samples collected) The relative frequency puts the total frequency on the basis of 100%, the frequency of one species relative to another. 48 30' H —‘3— juveniles '3 25- _'°— males 0 o) . 5 20- O 3 15- 41 o 10- ° 'v-l v-I ,, o I: ° ° . ” 5 . 4 . a . 0' 3! . '5 ' r ‘ 1 ' r ' I 1 I ' I ‘ l 1 1 0 50 100 150 200 250 300 350 400 2846 13 45 828 1250 2273 2742 2846 2846 Jul ian Day (Degree Days) Figure 8. Population density of Meloidogyne nataliei males and juveniles in seven soil samples [100 cm’], on 23 sampling dates from March 1992 through March 1993. 49 1.2" 7 —_9— juveniles 3 1.0“ —0— males 0 O a: D! In 0 DH 0 fl H U 4.: Q I: s' -0.2 ' I ' I l ' I ' I ' I ' I ' j 0 50 100 150 200 250 300 350 400 2846 13 45 828 1250 2273 2742 2846 2846 Jul ian Day (Degree Days) Figure 9. Population density of Meloidogyne nataliei males and juveniles in seven root samples [0.5 g], on 23 sampling dates from March 1992 through March 1993. 50 Table 14. Frequencies of plant-parasitic nematodes occurring in 48 soil samples. Number of samples Absolute Relative containing frequency frequency Nematode species (8) (t) Heplolaimus gaIeatus 23 47.92 18.55 Criconemella xenoplax 46 95.83 37.10 Xiphinema americanum 12 25.00 9.68 Pratylenchus neglectus 19 39.58 15.32 Meloidogyne nataliei 24 50.00 19.36 Total T27 _2sa.33 100.01 Table 15. Nutrient content of Concord petiole tissue from a Meloidogyne nataliei infested vineyard at Mattawan, Actual Optimum nutrient nutrient Nutrient concentrations concentrations N (t) 2.02 .80-1.2 P (8) 0.08 .16-.30 X (a) 1.09 1.5-2.5 Ca (t) 0.61 .5-1.0 Mg (t) 0.11 .25-.40 B (ppm) 18.00 25-50 Zn (ppm) 11.00 30-60 Mn (ppm) 355.00 30-60 Cu (ppm) 10.00 10-50 Fe (ppm) 57.00 20-100 Al (ppm) 113.00 - Mo (ppm) 6.00 - Na (ppm) 245.00 - 51 relative frequency 8 100 X (frequency of species/sum of frequency of all species) In a population trend study of nematodes in 50 V. labruscana vineyards in southwestern MI, Bird, et a1. (1985) found Criconemella DeGrisse & Loof, 1965 sensu Luc & Raski, 1981 spp., X. americanum, PratYlenchus Filipjev, 1936 spp., and H. galeatus absolute frequencies of 96%, 72%, 46%, and 26%, respectively, similar to the results in Table 14. Petiole tissue analysis of the vineyard (Table 15) identified Mn at high levels (355 ppm). The recommended level is 30-60 ppm. The toxic level of Mn for petioles is 1500 ppm (Hanson, 1987). Aluminium was also present at a very high level of 113.0 ppm. This is about 73 ppm higher than acceptable (Hanson, 1987). Nitrogen, K, Ca, Cu, and Fe were all within recommended ranges (Table 15). Phosphorus, Mg, B, and Zn were all at lower than the recommended levels for commercial grape production (Table 15). Soil analysis results indicated low pH ranging from 4.0 to 5.6 in the upper 0.31 m (12 in) of soil (Table 16) , but satisfactory pH at the medium depth 0.92 m (36 in) ranging from 6.0 to 6.8 (Table 17), and at the deep level 1.83 m (72 in) the pH ranged from 5.8 to 8.3 (Table 18). The lime recommendation was 0.6 MT/ha (1.4 tons/A) for the top three inches of soil. Preplant lime recommendations at 0.92 m and 1.83 m would be 0.2 MT/ha (0.5 tons/A) for seven of the sampled sites. The MSU STL recommends that growers consult their County Extension Agent for nitrogen fertilizer recommendations. 52 Table 16. Nutrient content of soil sampled at 0.31 m from a Meloidogyne nataliei infested vineyard at Mattawan, MI. Average of seven samples Sample Sampled Recommended Organic depth nutrient nutrient matter CEC* Lime m Nutrient lb/A amendment pH % me/lOOg index 0.31 P 188.7 35.0l 4.9 1.0' 5.8 66 X 112.4 138.6 Ca 278.7 .0 Mg 39.6 .0 Cu(ppm) 3.9 .0 Fe(ppm) 32.1 .0 Mn(ppm) 7.2I .0 Zn(ppm) 1.7? .0 *Cation Exchange Capacity 1Average of two samples 'Average of five samples Table 17. Nutrient content of soil sampled at 0.92 m from a Meloidogyne nataliei infested vineyard at Mattawan, MI. Average of seven samples Sample Sampled Preplant Organic depth nutrient nutrient matter CEC* Lime m Nutrient lb/A amendment pH % me/lOOg index 0.92 P 57.9 44.3 6.4 0.3 1.5 62.91 X 73.1 177.1 Ca 479.0 .0 Mg 54.9 .0 Cu(ppm) 0.7 .0 Fe(ppm) 16.4 .0 Mn(ppm) 4.1 .0 Zn(ppm) 0.9 .0 *Cationixchange Capacity TAverage of six sample 53 Table 18. Nutrient content of soil sampled at 1.83 m from a Meloidogyne nataliei infested vineyard at Mattawan, MI. Average of seven samples Sample Sampled Preplant Organic depth nutrient nutrient matter CEC* Lime m Nutrient lb/A amendment pH % me/lOOg index 1.83 P 57.6 110.0 6.4 0.3 2.4 72.61 R 72.0 178.0 Ca 795.8 .0 Mg 96.0 .0 Cu(ppm) 0.8 .0 Fe(ppm) 21.6 .0 Mn(ppm) 7.4 .0 Zn(ppm) 1.0 .0 *Cation Exchange Capacity 'Average of five samples Table 19. Population density of Meloidogyne nataliei in 100 cm? soil at 0.31 m, 0.92 m, and 1.83 m sampled from an infested vineyard at Mattawan, MI. Sample Depth m Juveniles Males Females I-l 0.31 2 0 1 I-l 0.92 8 0 0 I-l 1.83 5 0 0 I-2 0.31 5 0 0 I-2 0.92 0 0 0 I-2 1.83 27 0 0 I-3 0.31 0 0 0 I-3 0.92 0 0 0 I-3 1.83 0 0 0 II-l 0.31 l 0 0 II-l 0.92 0 0 0 II-l 1.83 0 0 0 III-1 0.31 0 0 0 III-l 0.92 0 0 0 III-1 1.83 0 0 0 IV-l 0.31 0 0 0 IV-l 0.92 0 0 0 IV-l 1.83 2 0 0 V-l 0.31 0 0 0 V-l 0.92 0 0 0 V-l 1.83 0 0 0 54 Actual numbers from 21 soil samples at all three depths indicate few M. nataliei down to 0.31 m, total of 9, and at about 0.92 m, total of 8 (Table 19). At soil depth about 1.83 m, one sample contained 27 juveniles, and the total for this depth was 34. Only soil was evaluated, no roots, so the female found was dislodged from root tissue. Ontogeny Under Growth Chamber Conditions Swollen, sedentary females were observed 1808 degree days after inoculation. The first 67 days were at 25°C (77°F) and then 18 days at 18° C 1 2° (64° F). The lower temperature was maintained for the duration of the study. Females had produced the beginnings of a gelatinous matrix after 1871 degree days. Thirty-nine egg masses containing ca. 50 eggs that had not undergone the first cell division were observed after 1918 degree days. Four egg masses containing 109 eggs (61-160) per mass were observed after 2051 degree days. Egg development ranged from two-cells to fully differentiated vermiforms. Females had developed secondary masses, attached to, but definitely separate from, the primary mass, each containing fewer than seven eggs that had not undergone the first cell division. On this same plant eleven females were found with egg masSes, each containing a mature male. Eggs began hatching by 3137 degree days. The first sedentary females, 43 were found, were located 7.7 cm (2.5-27 cm) from the stem on primary mature lateral roots. Females were found solitary in position, in 55 pairs, and in clusters of three. Reproducing females were never observed on immature roots. Egg Development Under Incubator Conditions Two-cell egg studies were established at seven temperatures to estimate a minimum temperature and an optimal temperature for egg development. Two-cell eggs cleave at different times at different temperatures (Figure 10). Two-cell eggs at 2.5° C did not cleave during the 240.28 h study (Figure 10). Two-cell eggs at 4.5° C cleaved to a multi-cell stage by 99 h or 223 heat units (Figure 10). At 9° C two-cell eggs cleaved to multi-cell by 133 h, or 899 heat units (Figure 10). Eggs incubated at 18° C and 27° C had two egg stage changes. The first at 18° C was from two-cell to multi-cell by 28 h and the second was from multi-cell to gastrula by 195 h or 439 and 3074 heat units, respectively (Figure 10). Two-cell eggs incubated at 24° C cleaved to multi-cell at 39 h, 853 heat units, and did not cleave again during the remaining 201 h that represented 4374 heat units (Figure 10). The two-cell egg at 27° C cleaved to gastrula, going through the multi-cell stage in less than 24 h, by 100 h, 2485 heat units, and cleaved again at 194 h to the tadpole stage after accumulating an additional 2313 heat units (Figure 10). The two-cell egg at 33° C was the first of the eggs at seven temperatures to cleave (Figure 10). The change from two-cell to multi-cell took place at 22 h, 663 heat units, but no further development had taken place by 120 h, 56 free-living J2 - We.-. 19 c "...... 2.25 c 12- ---O-- 4.5 c —0— 9 c J1 - —v— 24 c —a— 27 c tadpole . —u— 33 c 33.0 C gastrula 1 multi- - cell 2-cell Egg Development Stages ' I I r l U _' I I I l I I 1 0 24 48 72 96 120 144 168 192 216 240 Hours Figure 10. Embryogenesis of eggs of Meloidogyne nataliei from the two-cell stage at 2.5°, 4.5°, 9°, 18°, 24°, 27°, and 33° C. The last point on each line represents termination of the 10 day study except at 33° C which represents death of the eggs. 57 3690 heat units, when it was observed that all the eggs appeared to be filled with air bubbles (Figure 10). At 168 h, 4920 heat units, the stain new blue R was applied, and the mauve to purple coloring of the eggs confirmed that these eggs were dead. The IT’C study was terminated at that time (Figure 10). New blue R was also used on eggs at the other temperatures when the studies were terminated, and all, even those at 2.5° C, were found to be alive. The information revealed from these studies indicate that in vitro, 2.5° C is the approximate temperature at and below which no egg development occurs. These studies also indicate that at approximately IT’C any egg development is halted and death results, a maximum temperature. Two-cell eggs change to multi-cell eggs fastest at 33° C but because that temperature is fatal to embryos and since 2.5° C holds the eggs in suspension indefinitely, the optimal temperature must lie between these extremes. Figure 11 shows that at temperatures where egg development and survival are probable, two-cell eggs change to multi-cell fastest at 18°, then 24° , 27°, 4.5°, and 9° C in descending order. Multi-cell eggs change to gastrula stage fastest at 27°, then 24°, 18°, and 9° C in descending order (Figure 12) . The temperature for the fastest change from gastrula to tadpole is 24° followed by 18°, 9°, and 27° C, in that order (Figure 13). The next egg stage, tadpole to J1 developed the quickest at 9°, then 18° and finally 24° C (Figure 14) . There were no tadpole stage eggs available when the 27° C study {k . - M ///_//////n _///-u UUUUU l [C 7////// 600 ‘ a multi-cell 500 " 400‘ 300 “ . 7/ A é Hours %% m l 7 I 2.25 4.5 9 18 24 27 Temperature [C] 100 ' Figure 12. Embryogenesis of eggs of Meloidogyne nataliei from the multi-cell stage to the gastrula stage at 9°, 18°, 24°, and 27° C. Eggs at 2.5° and 4.5° C are shown although no ftrrther development took place during these studies. ///-uc. ------------- 0000000 222222 HHHHH 61 EH tadpole - J1 253‘ :mm- 150‘ Hours 100‘ 7 2.25 4.5 Temperature [C] I 18 N b Figure 14. Embryogenesis of eggs of Meloidogyne nataliei from the tadpole stage to the J1 stage at 9°, 18°, and 24° C. Tfilere is no information available for this egg change at 27° c. 62 was established, resulting in a missing developmental stage for that incubation temperature (Figure 17). The next egg stage is J1 developing to J2. At 9° C this occurs at 56 h, the fastest, followed by 24° C and 0.33 h later by eggs at 27° C with 18° C taking the longest to change (Figure 15). From the J2 egg emerges a free-living J2 vermiform nematode. This stage passes fastest at 27°, just 56 h, followed by 24°, 9°, and 18° C (Figure 16) . As may be seen at .18°C3 the J2 to free-living J2 stage takes the longest linder these in vitro conditions, a full 568 h which is 8941 heat units or 373 degree days (Figure 16) . There is not a single optimal temperature for all Stages of egg development (Table 20). Each stage has an optimal temperature as indicated by the asterisk in Table 1&5. The total of the indicated hours is 230 which represents a total of 3844 degree days. Within these experimental c“Dnditions, utilizing the three most complete data sets available, 9°, 18°, and 24°C, it seems that 24° C is most optimal for the embryogenesis of M. nataliei eggs when 1lboking at the number of hours (Figure 18, 19, & 20). HOwever, if the heat units are converted to degree days then 9° C is more optimal (Table 20). ////////M u r '96 MW T 4. ............ 0 5 O 5 3 2 2 1 uuuuu F' re 15. Embryogenesis of eggs of Mel ment from J1 sta e to J2 sta e D. ..m . mac v. d2 64 800 j 700‘ B J2/Froe-living J2 600 " 500 1 Hours 400‘ 300 ‘ %m Temperature [C] zoo - —— ‘— 100 " 2.25 4.5 Figure 16. Embryogenesis of eggs of Meloidogyne nataliei from J2 to free-living J2 at 9°, 18°, 24°, and 27° C. Free- living J2 were removed from BPI watch glasses upon eclosion. 65 Table 20. Summary of embryogenesis of eggs of Meloidogyne nataliei from one stage to the next at seven temperatures. Temperature [C] Egg stage 2.25 4.5 9 18 24 27 33 2-cell/ multi-cell - 98.92 139.55 27.90* 58.83 91.06 21.55 multi-cell/ gastrula - - 407.27 195.15 96.00 26.83* - gastrula/tadpole - - 92.01 82.65 36.85* 93.45 - tadpole/J1 - - 26.17* 67.05 210.37 - - J1/J2 - - 56.09* 183.60 155.93 155.60 - J2/free-living - - 195.80 567.67 156.00 55.91* - Totals [h] 916.89 1124.02 713.98 Totals [heat units] 6189.00 17703.32 15529.07 Totals [degree days] 257.88 737.64 647.04 *least number of hours at each egg stage 66 free-living JZ 3 Lw—I 5 J2 I! u l m J” ‘4’ 0 tadpole- E 0 gastrula H 0 3 multi-4 0 cell a 2-cell 8' l-cel I ' I ' I ' I ' I ' I ' I ' I ' I fi I ' I ' 0 48 96 144 192240288 336384432 480 49-44 148.32 247-2 346.08 444.96 0 98.9 197.76 296.64 395.52 494.4 Hours Heat Units Accumulated lf'igure 17. Embryogenesis of eggs of Meloidogyne nataliei incubated at 27° C. Eggs were studied from the given stage. No tadpole egg was available at onset of study. 67 free-living .122 m 8 12 u i U U7 #3 J1 u I: 0 tadpole< E 8' ...gastrula 0 > . 0 multl- o v a cell 3: 2-cell fil l-cellfi.,.,fi,.,.........l 0 48 96 144 192 240 288 336 384 432 13.44 40.32 67.2 94.08 120.96 0 26.88 53.76 80.64 107_52 Hours Heat Units Accumulated Ifigure 18. Embryogenesis of eggs of Meloidogyne nataliei 1ncubated at 9° C. Eggs were studied from the given stage. 68 free-living .12 :[ .12 m 0 u 3 00 J]. u I: tadpole4 0 E D: o gastrula H 0 5 multi-‘ a cell a 2-cell 3 l-cell Ifi j ' I ' I ' I ‘ I ' Tfi I ' I ' I ' I ‘ I ' I ‘ I 0 48 96 144 192 240 288 336 384 432 480 528 576 624 31.53 95.04 158.4 221.76 285.12 348.48 411.84 0 63.36 126.72 190.08 253.44 316.8 380.16 Hours Heat Units Accumulated Figure 19. Embryogenesis of eggs of Meloidogyne nataliei incubated at 18° C. Eggs were studied from the given stage. 69 free-living J2 3 u J2 a! .u m .114 u I 5 tadpole4 : E I: Ogastrula4 PI 0 g multi- n cell a 2-cell In III l'ceIII‘I‘FfiI'I'IT1'I'I'I‘I'l‘ 0 48 96 144 192 240 288 336 384 432 480 528 43.68 131.04 218.4 305.76 393.12 480.48 0 87.36 174.72 262.08 349.44 436.8 Hours Heat Units Accumulated Figure 20. Embryogenesis of eggs of Meloidogyne nataliei incubated at 24° C. Eggs were studied from the given stage. 70 Histopahtology of Meloidogyne nataliei Associated with Vitis labruscana Root tissue cells stained with toluidine blue 0 had blue cell walls when observed under light microscopy. Some sections had nematodes present which also stained blue. Where toluidine blue 0 and acid fuchsin were both utilized, cells appeared red or purple. The host response from Concord to feeding by M. nataliei is the formation of giant cells. These giant cells were found in the vascular tissue. Xylem cells appear to have been utilized but it is not known if xylem vessels were the feeding sites and subsequently the cells used for giant cell formation. Of those observed by light microscopy, the dimensions in longitudinal section are 409.07 pm long and 127.4 um wide (289.9-526.5 um long and 104-150.8 pm wide). Length and width in cross section are 114.83 um x 79.3 pm (114.4 x 79.3 pm). Cell walls of the giant cell are thickened, 5.53 pm as compared to the control cell wall width, 1.3 nm (Figure 21). M. nataliei giant cells are egg shaped in cross section, i.e. oval (Figure 21). In all observations, giant cells lie basipetal to the nematode, in numbers up to 3, appearing to have been induced by a single female (Figure 23). Longitudinally, giant cells are an oblong-circular shape (Figure 23). Giant cells lie parallel to one another with a few, scattered parenchyma cells between them (Figure 22). 71 Figure 21. A. Control Concord root cross section (x256). 8. Giant cell (gc) in cross section, induced by Meloidogyne nataliei, that may be showing extra wall deposits that form on cross—walls between altered cells (x256). C. Cross section of giant cell showing thickened cell wall (x500). D. Same root section as C. indicating giant cell orientation in root cross section (x125). Figure 22. A. and B. Cross sections of parasitized Concord root tissue. Giant cells (gc) parallel to each other, with parenchyma cells in between, induced by the feeding of a single Meloidogyne nataliei female (x256). C. and D. Giant cells in cross section of grape root tissue, induced by a single female, x200 and x500, respectively. 73 Figure 23. A. Cross section of Concord root tissue with nematode (n) surrounded by brown cells (b) (x500). B. Longitudinal section of two giant cells (gc) and beginnings of a third (x160). C. Anterior of Meloidogyne nataliei, position indicated in D., at base of three giant cells, position indicated by arrows (x312). D. Same section as B. with position of nematode indicated by arrow (x125). Plant cells surrounding the anterior portion of the nematode did not stain blue by toluidine blue 0, as did all other cells, but appear to be brown (Figure 23). DISCUSSION Host Range and Pathogenicity of Meloidogyne nataliei It had been observed in the field in years prior to these experiments that M. nataliei becomes sedentary, produces a gelatinous matrix, and begins egg laying as soil and air temperatures begin to decline in October and November. Triantaphyllou (1985) noted that five months after inoculating Concord grape seedlings with M. nataliei and growing the plants in a greenhouse at 22-28° C, only adult females with a few eggs enclosed in a large gelatinous matrix were found, no juvenile stages were in the root tissues. Also, during attempts prior to these studies, to evaluate host ranges at MSU with M. nataliei in the greenhouse with fluctuating temperatures, usually above 24° C (75.2° F), were unsuccessful (Appendix B). For these reasons 18° C 1 2° was selected as a temperature that would encourage nematode development while still allowing the plants to grow. The Vitis spp. and the Parthenocissus Planchon spp. are all hardy in Michigan, USDA Zone 5, but their dormant periods are partly induced by declining temperatures. The low growth chamber temperature maintained throughout these 74 75 studies may have caused problems with senescence, low growth rates and the onset of dormancy that may have masked any pathogenetic effects caused by the nematodes (Figure 24 & 25). M. nataliei did not appear to be a pathogen, a specific cause of disease, with any of the plant species studied. While P. quinquefolia proved to be a host for M. nataliei, its suitability may be questionable. Many more male nematodes were found, both in the soil and roots, than females. The development of excessive numbers of male nematodes is associated with stress conditions including crowding, low host susceptibility and stress in the host plant. Stress in the host plant would be caused by growth regulators, defoliation, infected roots and high or low temperatures. Of these stress conditions, low host susceptibility and/or defoliation may have been the causes. Crowding is not suspect because in the 6-1-93 study, one treatment was 18 vermiform per pot and the other was 324 per pot. 0f the studies that resulted in the identification of a new host, the 6-1-93 study contained the lowest number of nematodes. This same situation of excessive numbers of male nematodes was also found with Gloire. In the one study with Gloire, the male percentage was 52.4% of the population. The Pi was 750 vermiform. These plants were actually defoliated and the stems were desiccated on some of the plants. 76 Figure 24. A. Healthy Parthenocissus quinquefolia at onset of host and pathogenicity study in growth chamber. 8. Parthenocissus quinquefolia in unhealthy condition prior to termination of study in growth chamber. Most plants are leafless. 77 Figure 25. A. Healthy Parthenocissus tricuspidata at onset of host and pathogenicity study in growth chamber. 3. Unhealthy Parthenocissus tricuspidata prior to termination of study in growth chamber. Some plants have only desiccated leaves. 78 In the 5-2-92 study of P. quinquefolia males represented 85% of the population; whereas, in the 5-11-93 study males were 6%, and in the 6-1-93 study males were 86% of the nematode population from soil and roots combined. Compared with 588 and St. George, male nematode percentages were 2% and 4%, respectively. For P. tricuspidata the males were 1.4% of the final population. Cissus L. and Tetrastigma Miguel spp. are both tropical in origin and are grown and utilized as houseplants in the temperate climates. Cissus sp. plants tolerated the growth chamber temperature and grew well. The Tetrastigma sp. went into decline and failed to grow within a few days of exposure to the growth chamber temperature. The leaves became very pale, and it is believed that very little photosynthetic activity took place during the 94 days that these plants remained in the growth chamber. The condition of these plants may have influenced M. nataliei's inability to feed, develop, and reproduce on their roots, although that was not the case with Cissus. There was never any knotting or galling observed on any host plants. Sedentary females of M. nataliei were located on mature roots with fully developed periderm. They were never found on immature roots. Citrus sinensis, a sub-tropical species, grew and developed very well at 18° C in the growth chamber. These plants not only grew well but set and matured fruit. They were still in excellent condition when destructively sampled 79 after 345 days, even though, C. sinensis proved to not be a host of M. nataliei. Life History of Meloidogyne nataliei Although soil pH was low at 0.31 m (4.9), tissue analysis revealed that low pH was not, overall, inhibiting uptake and availability of nutrient elements. One study with grapes indicated that the toxic effects of high Al and Mn concentrations at pH levels below 5.5 greatly affect root growth and development (Himelrick, 1991). Himelrick (1991) tested nine Vitis cv. at high soil pH (6.7) and low soil pH (4.8) for elemental concentrations in foliar tissue. That study established a negative trend in low pH soils manifested by the reduced shoot weight on average by 27% in seven cultivars, reduced root weight on average by 13% in four, and reduced root volume by 21% below values obtained of 6.7 pH soil in six of the nine cultivars tested, one of which was Concord (Himelrick, 1991). The Himelrick (1991) study also revealed that plants grown under low pH contained 416% more Mn and 147% more Al than those grown in high pH soil. Concord tissue concentrations at high soil pH and low soil pH were 406 ppm and 783 ppm of Al and 211 ppm and 752 ppm of Mn, respectively (Himelrick, 1991). Low pH increases available Al in soil which would account for the moderately high (113.0 ppm) Al concentration found in tissue analysis. It has been suggested that high Al concentrations revealed through tissue analysis of sweet 80 cherry (Prunus avium (L.) L.) trees in Michigan may play a significant role in a physiological disfunction, referred to as decline of sweet cherry trees (Melakeberhan, et al., 1993). It was suggested by Melakeberhan, et al., (1993) that raising and maintaining the pH at recommended levels may increase the ability of sweet cherry trees to resist or at least withstand infection by pathogens, including nematodes (Melakeberhan, 1993). The same recommendation might be applied to M. nataliei infested vineyards. In a study on four cv. of Vitis vinifera grown in sand, leaf analysis results from three different sampling dates, July, October, and November, revealed Na concentrations of 0.03-0.14 ppm, 0.08-0.38 ppm, and 0.03-0.14 ppm, respectively (Ehlig, 1960). At these concentration levels, no symptoms of Na toxicity were observed visually (Ehlig, 1960). This indicates that the Na concentration level found in the Mattawan vineyard (245.0 ppm) may be toxic. Leaves in this vineyard were not observed for Na toxicity. Spiech Farms vineyard raises two cultivars of V. labruscana, Concord and Niagara. In this vineyard, M. nataliei is a parasite of both cultivars. Based on the evidence of the 110 complete or partial experiments in water, it is concluded that the maximum temperature for the development of M. nataliei eggs in such culture is near 33° C. For extended periods of time 100% mortality incurred at 33° C. The minimum temperature for development is below 4.5° C. While no development was seen 81 at 2.5° C in 10 days, the eggs were still viable. If a resting stage for M. nataliei exists, it has yet to be identified. The in vitro optimal temperature for complete development is 9° C. Histopathology of Meloidogyne nataliei Associated with Vitis labruscana Giant cell formation begins within hours of infection for many Meloidogyne spp. The length of time the grape roots were exposed to parasitism was not tracked in this study. For the first 48 to 60 h, the stimulative effect of the nematode on the surrounding cells, plerome layer, is to retard differentiation (Christie, 1936). At the end of that period these undifferentiated cells begin to enlarge and division of the nucleus may take place. Original cell walls disappear and contents of adjoining cells coalesce to form the beginning of a giant cell. As many as eight cells may coalesce to form the giant cell. Giant cell wall thickening is characteristic of cellular alterations induced by Meloidogyne spp. Three types of structures have been recognized in the boundary formations, related to size of tubules and vesicular objects (Huang, et al., 1969). Feeding tubes have been identified as well as enlarged and sometimes multinuclei of various shapes and sizes. These objects were not observed in this study. Electron microscopy studies will be initiated at MSU to identify and confirm the presence of feeding tubes. The brown cells seen surrounding the nematode in Figure 23 may be due to an accumulation of phenolic compounds around the infection site. The accumulation is thought to be a resistance response by the host to the nematode. CONCLUSION 1. Five previously unknown hosts for M. nataliei were found, Vitis rootstocks SBB, Claire, and St. George, and P. quinquefolia, and P. tricuspidata. 2. M. nataliei has three generations in the field. The first or over-wintering generation appears as mature females about Julian day 332 or 2841 degree days. A second generation with mature females appears about Julian day 76 or 13 degree days. A third generation has mature females in place on root tissue about Julian day 232 or 1816 degree days. 3. In the growth chamber, M. nataliei develops from free-living vermiform, to sexually mature adults, to egg laying females, and a new generation of free-living vermiform after 3137 degree days, at ca. 18°(L 4. Embryogenesis occurs in vitro optimally at 9° C. Each egg development stage has an optimal temperature. Maximum and minimum temperatures in vitro are near 33° and 2.5° C, respectively. 5. M. nataliei induces giant cell formation in Concord. Several giant cells may be formed by one female. The giant cells are oriented parallel to each other. 82 83 Summary Further research is needed on The Michigan Grape Root Knot Nematode. In vitro studies testing temperatures below 2.5° C for embryogenesis and female egg laying would be valuable. I observed, non-experimentally, that M. nataliei continues to lay eggs at 4.0°CL Such temperatures do not kill eggs. Studies testing egg development at different temperatures and different stages might more closely mimic field conditions. APPENDICES APPENDIX A Arabidopsis Thaliana Research Manuscript Culturing of Criconemella Xenoplax and Meloidogyne Nataliei on Arabidopsis Thaliana C.J. Diamond‘, H. Melakeberhan‘, and G.W. Birdl Received for publication ‘ Department of Entomology, Michigan State University, East Lansing, Michigan 48824. 84 85 Annual crop losses from plant-parasitic nematodes have been estimated at 12.3% of the world's major crops, equivalent to 100 billion United States (US) dollars (24). The loss is 14.6% in developing countries and 8.8% in developed countries (24). Longer growing seasons and more optimum environmental conditions are among the reasons for higher overall yield losses in more developing countries than in developed countries. Nematode damage symptoms are often subtle and difficult to distinguish from other disease symptoms of parasitic and nonparasitic nature (19). Based on their feeding behavior, plant-parasitic nematodes can generally be grouped as ectoparasites, migratory endoparasites or sedentary endoparasites (8). Ectoparasites feed on plant parts from the outside by inserting their stylets into plant cells (21). Endoparasitic nematodes enter plant tissues completely or with a large portion of their body and are divided into two groups: sessile endoparasites that remain immobile after entry into plant tissues and migratory endoparasites that move from one site to another while feeding within plant tissues (8). Examples are Criconemella xenoplax (Raski) Luc & Raski, Heterodera glycines Ichinohe and Pratylenchus penetrans (Cobb, 1917) Filipjev & Schuurmans-Stekhoven, 1941, respectively. Symptoms of plant-parasitic nematode damage may be considered at the whole plant levels. Meloidogyne Goeldi spp., are migratory and sessile endoparasites, in addition to a decline in fruit production, cause plants to 86 be stunted in heavy infestations and plants can exhibit symptoms of nutritional deficiency and produce galls or knots (3). At the cellular level, Meloidogyne spp. must be able to initiate and maintain syncytia, also known as giant cells (3). Our ability to control plant-parasitic nematodes, in part, depends on understanding the mechanisms of host- parasite interactions. In nematology, progress has been slow mainly due to the complexity of this interaction and the specific demands of ectoparasites and endoparasites during in-vitro culture (25). Culturing plant-parasitic nematodes is more difficult because they are obligate parasites, and more expensive than the culture of fungi or bacteria because of the need to use host-plant tissue (14). The need for an accessible model system to help understand the plant- pathogen interaction is accepted and recent developments in Arabidopsis thaliana L. research, such as RFLP maps of Arabidopsis Thal (7), the amenability of Arabidopsis with Agrobacterium tumefaciens (27) and A. rhizogenes (28), offer encouraging hopes (25). A. thaliana is a small weed in the Cruciferae family and has been a convenient subject for studies in classical genetics since the 19405 (11). While the exact origin of the plant is not known, it is adapted to temperature ranges of 4-27°C (15). It takes four-six weeks to complete its' life cycle and at maturity, depending on nutritional factors, plants may grow up to 30 or 40 cm (17). Genetic studies have 87 established that A. thaliana has haploid chromosome number of 5 (13). Arabidopsis, so far, has the smallest genome among the higher plants with a haploid nuclear genome size of roughly 70,000 kilobase (kb) pairs compared to 1,600,000 kb pairs of tobacco (17). The inflorescence may be self fertilizing or cross-fertilized in the laboratory with each silique, i.e. fruit, containing 30-60 seeds at maturity making it possible to collect well over 10,000 seeds within months from an individual plant that may be grown to maturity in an area of 1 cm2 (17) . The use of A. thaliana as a host will provide a new model system for the molecular genetic analysis of the plant-parasitic nematode interaction (25). The significance of this small DNA content for molecular genetics is that a genomic library of Arabidopsis chromosomal fragments is easy to make, and simple and economical to screen, also, a very small amount of dispersed repetitive DNA and a high percentage of single copy sequences (15). The ability to identify specific genes involved in the plant-parasitic nematode interaction may lead to the development of tolerant and resistant plant varieties. In-vivo studies for successful infection and development on A. thaliana have been established for several economically important cyst- forming nematodes, H. schachtii, H. trifolii, H. cajani and the root-knot nematodes M. incognita (Kofoid & White, 1919) Chitwood, 1949 and M. arenaria (Neal, 1889) Chitwood, 1949 as well as P. penetrans, a migratory endoparasitic nematode 88 (25). There is a need to use A. thaliana as a model host in understanding the biology of other plant-parasitic nematodes. Pome and stone fruits and grapes are among the important crops in Michigan (MI). MI is one of the nation's major pome and stone fruit producing states with total production of 494.4 million kilograms of apples, tart cherries, sweet cherries, peaches, grapes, pears, and prunes and plums valued at 139,268,000 US dollars in 1990 (2). MI ranks third in the nation in sweet cherry production, 61,235 kilograms, and sixth in peach production, 20,411.? kilograms (2). MI's sweet cherry industry was valued at 6,913,000 US dollars and its peach industry was valued at 9,447,000 US dollars in 1990 (2). C. xenoplax is among the important plant-parasitic nematodes of stone fruit trees (6). In South Carolina, Criconemella is the only genus of nematode that has been determined to be a part of the symptom complex known as "peach tree short life" (6). criconemella spp. increase peach tree susceptibility to bacterial canker and reduce tree growth and productivity (6). In MI, bacterial canker is a component in the symptom complex known as "stone fruit decline" that affects almost all of the sweet cherry orchards located there (16). Extensive surveying of sweet cherry orchards located in the northwest and southwest regions of MI showed the C. xenoplax and P. penetrans, with P. penetrans being highest in frequency followed by C. xenoplax, along with Meloidogyne spp., Paratylenchus spp., 89 and Xiphinema Cobb, 1913 spp. have a field association with bacterial canker (16). Grape (Vitis Tournefort spp.) is the most widely planted fruit crop in the world (20). The USA grape crop for 1990 was 4.95 million kilograms (2). MI production in that same year was 41,730.5 kilograms from 4452 ha valued at 13,372,000 US dollars (2). Meloidogyne nataliei Golden, Rose & Bird, 1981 is a parasite of grape that may result in reduced plant vigor and decreased yield (10). Currently, little is known about M. nataliei but another Meloidogyne spp., M. hapla Chitwood, 1949, parasitizes grape plants causing decline in vigor and greater susceptibility to stress, which may ultimately lead to yield loss, but seldom kills the vines (22). C. xenoplax, P. penetrans and M. hapla were identified in a study for population trends and vertical distribution of plant- parasitic nematodes in 50 MI vineyards with C. xenoplax found in 48 of the 50, P. penetrans was found in less than 25 and M. hapla in 30 of the 50 sites (5). The objective of this study is to establish culture conditions for successful infection and development of C. xenoplax and M. nataliei on A. thaliana and to study the host-parasite interaction of these organisms under monoxenic conditions to be followed by cellular and sub-cellular studies in the future. 90 MATERIALS AND METHODS To initiate this study it was thought to be desirable to have a readily available supply of sterile nematodes. Nematodes were cultured on root explants of carrot; of grapes and host-parasite interactions studied on Arabidopsis. Four experiments were completed. All experiments were carried out under sterile conditions. Sources of materials Arabidopsis thaliana seeds 'Columbia' and Daichin agar were provided by Dr. C.E. Novitski (Central Michigan University) and 'WT' seeds by Dr. C.R. Somerville (Michigan State University). C010 Q30 Agar was obtained from Thomas/Scientific Apparatus. Salt mixtures for medium and other components were obtained from Sigma. Square phage typing grid dishes were obtained from Fisher. 'Chantenay' and 'Danvers' carrot seeds were purchased commercially. Microscope photographs were provided by Dr. D. Roberts (Michigan State University). Negative development and photograph enlargement equipment were provided by Dr. K. Klomparens (Michigan State University). Preparation of medium Growth medium was autoclaved at 120° C for 20 minutes. Petri dishes were used for root explant seed germination and culturing of nematodes on root explants and carrot discs. Phage plates were used for Arabidopsis because they are square and do not roll when put on an angle. Phage plates containing Arabidopsis seeds and plants were wiped with 91 Kimwipes wetted with 95% ethanol and wrapped with Parafilm before being placed in an enclosed, clear plastic container that had been soaked for 15 minutes in 30% (w/v) sodium hypochlorite, drained but not rinsed, before being put in a growth chamber. Phage plates were wiped with ethanol again before Parafilm was removed for inoculating nematodes. All medium plates were stored in a refrigerator at 4-6° C until needed. Sterilization of seed and other plant material Carrot, grape, and Arabidopsis seeds were surface sterilized, after imbibing water for 1 h, by first being placed on and folded in Whatman filter paper N21, 5.5 cm in diameter to prevent the seed floating free, immersed in 70% ethanol and agitated to remove air bubbles for 2 minutes, drained and immersed in 0.05% (w/v) sodium hypochlorite (with 0.5% SDS added for Arabidopsis) for 15 minutes and then drained and rinsed in sterile distilled water for 5 minutes two times as outlined by Balcells (provided by MOGEN International). The seed packets were placed in Petri dishes and after the filter paper had dried, seeds left over after sowing were kept there. Carrot shoots and the tips were removed and the carrots soaked in tap water for five minutes to loosen soil particles, washed in tap water and soap to help remove remaining soil particles, and soaked in 95% ethanol (18). The carrots were flame-sterilized and the epidermal tissue was removed with a flame-sterilized vegetable peeler and 92 then cut into 2-3 cm discs (18). Grape roots were rinsed with tap water to remove soil particles and immersed in 0.05% (w/v) sodium hypochlorite for 20 minutes and rinsed in sterile distilled water. Sterilization of nematodes Both C. xenoplax and M. nataliei were surface sterilized in mercuric chloride (15 g/ml) for 3-15 minutes and rinsed three times in sterile distilled water for five minutes (29). A 400 mesh screen cleansed with full strength commercial bleach was used for retaining the nematodes. Inoculating with nematodes A Pipetman, with a tip cleaned with 70% ethanol and rinsed in sterile water, was used for inoculating the nematodes onto plant media. Establishing root explants and carrot discs for nematode cultures To establish and maintain a steady source of sterile C. xenoplax, two susceptible genotypes of carrot (Daucus carota L. cv. 'Chantenay' and 'Danvers') were used for root explants. Sterile seeds were placed on seed germination medium, four plates of each cultivar, prepared with Bacto- Agar (23). The Petri dishes were then placed in an incubator without light at 25° C to germinate. Carrot discs, were also prepared to maintain sterile cultures of C. xenoplax. The discs were placed in Petri dishes containing Bacto-Agar and Gamborg's B-S medium and placed in a dark incubator at 25°C:(9). The fresh carrots 93 were obtained from the supermarket. The inoculation process was repeated one time. Grape root explants were produced from Vitis labrusca L. cv. Concord roots, to maintain sterile cultures of M. nataliei. Sterile grape root sections were out 2-3 cm in length and placed in Petri dishes on Gamborg's B-5 medium prepared with Bacto-Agar (9). These dishes were also placed in a dark incubator at 25° C. The Concord roots were obtained from an established vineyard at Mattawan, MI. The sterile Concord seed were placed on the seed germination medium in Petri plates and incubated in the dark at 25°CL All Petri plates used for establishing root explants and carrot discs were sealed with Parafilm. Inoculation for establishing sterile nematode cultures Adults and juveniles of C. xenoplax were collected from greenhouse cultures at Michigan State University that are maintained on hairy vetch and were extracted by centrifugation-flotation (12). Adult males and juveniles of M. nataliei were collected from roots from a vineyard at Mattawan, MI, and extracted by shaking in ethoxyethyl mercuric chloride (10 pg/ml) and streptomycin sulfate (50 ug/ml) solution (3). Carrot discs were inoculated by placing 100 juveniles and adults in 0.1 m1 sterile water on the agar surface in the vicinity of the discs. Petri plates were wrapped with Parafilm and placed in a dark incubator at 25°(L 94 Arabidopsis growth medium Medium was prepared with Knop solution, 1% sucrose and 0.8% Daichin agar as per the Sijmons' (1991) protocol (25). This medium was poured into square phage typing grid plates (ca. 20 ml/plate) and placed at a 60° angle to cool. The medium collected in the bottom 25 mm of the plates at the same angle. Using the basic Sijmons' (1991) formula but with a nonspecific agar labeled C010 Q30 Agar and 1 g ampicillin, medium was prepared again. This was poured into phage plates (ca. 30 ml/plate) and allowed to cool laying flat. The third time medium was prepared using Sijmons' (1991) formula but with purified agar and 1 g ampicillin. The medium was poured into phage plates (ca. 20 ml/plate) and allowed to cool laying flat. The addition of ampicillin to the growth medium was due to the many incidence of loosing plates to contamination. Daichin agar medium using the original formula from Sijmons (1991) was prepared again and was poured into phage plates (ca. 20 ml/plate) and allowed to cool laying flat. Seeding of Arabidopsis Two ecotypes of Arabidopsis were used, 'Columbia' and 'WT'. The original set of Daichin agar plates were seeded first by placing four seeds within the medium at 2 mm depth. The seeds were placed in the middle of the plates. Four seeds per plate were sown on the nonspecific agar using forceps to place the seeds on the medium surface 30 mm from the bottom edge of the phage plates and the purified 95 agar, and the second set of Daichin agar plates were sown three seeds per plate with the seeds on the medium surface 60 mm from the bottom edge (Figure 1). All were then laid flat for the seeds to germinate. All seeds were put in place with flame-sterilized forceps that were re-flamed between the seeding of each plate. All seeding took place in a Laminar flow hood using sterile technique (1). Phage plates were put in a growth chamber at 25° C with a 16 h light and 8 h dark cycle for the seeds to germinate. Inoculation of Arabidopsis with nematodes Phage plates were removed from the growth chamber when Arabidopsis plants were ten-days-old. C. xenoplax and M. nataliei were inoculated, at 40 and 100 vermiform juveniles and adults, respectively, onto the medium surface, in separate plates, around the area that the plant emerged from the medium and directly above the roots. This procedure was repeated three times. For the first experiment, four of the twelve angled Daichin plates were inoculated, two with each genus of nematode, onto ten-day-old Arabidopsis plants. For the second experiment, six Daichin plates with angled medium and four plates with the nonspecific agar medium were inoculated. Half of each type of medium was inoculated with each genus of nematode. For the third experiment, three angled Daichin and two plates of nonspecific agar were inoculated with M. nataliei while two angled Daichin plates 96 and two nonspecific agar plates were inoculated with C. xenoplax. For the final experiment, five plates of purified agar were inoculated with each genus of nematode. In the second, third and fourth experiments, plates that had been poured and cooled flat remained flat for 24 h after inoculation, after which time they were placed at a 35° angle for plant growth. The Daichin plates that contained medium on an angle were put back at that angle immediately following inoculation and returned to the growth chamber. Observation of host-parasite interactions Observation for root infection began 16 to 24 h after inoculation. Plates were inverted and observed on a dissecting microscope at a magnification of 20X until infection was noted at which time observations were made on a compound scope at a magnification of 100x. Photographs were made of root infections with a Polaroid camera, using black and white, positive-negative film, mounted on a Nikon AFX-IIA compound microscope at the MSU Plant Diagnostic Laboratory. Photographs were taken at magnifications of 160x and 400x. The negatives were developed and enlarged contrast enhanced photographs were made at the MSU Electron Microscopy Laboratory. RESULTS Preparation of Arabidopsis growing medium The first batch of Daichin agar medium was allowed to cool to 18° C before it was poured. The medium became lumpy 97 and was sometimes difficult to see through with the microscope. A. thaliana plants grown on the nonspecific agar medium were small, etiolated, and appeared to have altered geotropism although they received the same light-dark cycle as the other plates. Plant leaves were in the medium and the roots were on the surface and above it. These plants failed to grow after 15 days and died within 25 days after germination. Sterilization of Arabidopsis seed and seeding Sterilization of Arabidopsis seeds seemed very effective. When contamination of a plate took place after seeding it was not noted in the vicinity of the seed itself. On Daichin agar medium, A. thaliana ecotype 'Columbia' had 45% germination. Ecotype 'WT' had 65% on Daichin and purified agar and 45% on the nonspecific agar. Both ecotypes of A. thaliana germinated in 3-4 days in a growth chamber at 25’Ciand produced plants ready for inoculation ten days after germination. Sterilization of Nematodes Surface sterilization of both genera of nematodes produced high mortality at first. Time in the mercuric chloride was initially 15 minutes but was reduced at each subsequent attempt as follows; 7 minutes, 5 minutes and 3 minutes. Mortality was reduced by 78% going to 7-3 minutes. Establishing root explants for nematode cultures Establishing sterile cultures of nematodes in root explants was attempted once but not achieved. Carrot seed 98 plates became contaminated within 24 h and were discarded. The contamination was directly at the seed-medium contact site. Grape root plates showed contamination within four days and were discarded. Sterile cultures of nematodes on carrot discs was attempted two times but not achieved. Both genera of nematode died within 48 h of inoculation. Observation of host-parasite interactions C. xenoplax migrated into the root zone 18-24 h after inoculation. This nematode was observed moving within the root zone, sometimes directly along side a root and moving from one root to another. Stylet thrusting was never observed in any of the experiments. No molting, mating or egg production was observed. C. xenoplax would remain in the root zone until death, or cessation of movement. Percentage of C. xenoplax moving into root zone was always low (Table 1) . M. nataliei invaded the root zone 18-24 h after inoculation. This nematode was observed moving within the root zone and at the root feeding. Stylet thrusting and metacarpal pumping were observed for as long as 1.5 h and was still in progress when observation was terminated. At one observation of infection that was less than 24 h old, a juvenile was observed feeding on a root 195 pm (194.5-195.5 pm, SD 0.5 pm) basipetal to the root tip. Cell damage was clearly visible and began 162 pm (161.5-162.5 pm, SD 1.0 pm) basipetal to the root and extended 161.4 pm 99 Table 1. Mean infection percentages of Criconemella xenoplax and Meloidogyne nataliei on Arabidopsis thaliana with 40 and 100 vermiform and adult nematodes inoculated, respectively. Experiment Number C. xenoplax M. nataliei I 0% 65% II 12% 22% III 4% 10% IV 0% 6.3% I 100 (159.3-162.5 um, SD 0.5 pm) (Figure 1). The root width at the feeding site was 91 um (SD 0.0 pm). M. nataliei feeds on Arabidopsis intracellularly (Figure 2). At another observation of infection the juvenile nematode was feeding at a root hair. The root hair was 468 pm (468-471.3 pm, SD 0.0 pm) in length and 15 um (13-16.3 an, SD 0.5 pm) wide. This particular root hair was 357 um (357 pm, SD 0.0 pm) basipetal to the root tip. A male M. nataliei was observed feeding at several roots and feeding sites, exhibiting migratory behavior. When M. nataliei was feeding on primary and lateral roots, their stylets were inserted into the vascular cylinder. All juveniles exhibited migratory behavior. No juvenile swelling or female swelling was seen. DISCUSSION Attempts to establish sterile cultures of nematodes on root explants was abandoned in this experiment because of the ease of extracting and sterilizing the nematodes and the many problems encountered with contamination. Growth medium for the host-parasite interaction was prepared, using Daichin agar, poured and cooled on a 60° angle. The medium was contained in the bottom 25 mm of the phage plates and was thick and lumpy because it had cooled too much before pouring. This did not present a problem when using a microscope to observe but attempts to take photographs were hampered because this medium presented observation problems. A second batch of medium was prepared Figure 1. Meloidogyne nataliei juvenile above Arabidopsis thaliana root feeding intracellularly. Dark area of root at nematodes anterior end is due to cell destruction by nematode feeding (x160). 102 Figure 2. Meloidogyne nataliei juvenile above, not in, Arabidopsis thaliana root feeding intracellularly (x400) . 103 using a nonspecific agar. The nonspecific agar medium was poured and cooled flat but was too thick. After gelling this medium was cloudy and that combined with the thickness also prevented adequate photographs of parasite-host interactions. Plants grown in this medium were small, chlorotic, and etiolated with altered geotropism that failed to grow and died within 25 days of germinating. The purified agar medium was prepared to provide a clear, thin medium for photography. In the first of these phage plates that were sown, the seed failed to germinate. The final growth medium was prepared with Daichin agar per the Sijmons' (1991) protocol. When the seeds in these plates failed to germinate another group of Arabidopsis seeds were sterilized and sown. For the third and fourth experiments the Arabidopsis seeds were placed on the growth medium 60 mm from the bottom edge of the phage plates. This allowed a greater distance for the roots to grow before reaching the bottom edge of the plate where visibility became obscured by the bend at the edge of the phage plate. The greater distance was 55 mm as compared to 17-27 mm that was available with previously seeded plates. The nematodes tended to move toward root tips for infection. The longest observations of parasite-host interactions were made on three plants, in the same phage plate, all with stunted roots. The roots were stunted before the nematodes were inoculated onto them and the reason for the stunting was not determined but these roots remained 104 within 15 mm of the hypocotyl base until this experiment was terminated after 23 days. Nematode surface sterilization time in mercuric chloride was reduced from 15 minutes to three. This was done because of high mortality with the nematodes at the longer time. Three minutes was used for the fourth experiment. While many more nematodes survived, the plates they were inoculated onto became severely contaminated with quick growing fungi and bacteria within five days and had to be discarded. The five minute time gave the best results, that is, low mortality without contamination. M. nataliei was not observed feeding on epidermal cells, breaking the walls or entering the cortex. Nematodes seen at a location could be found within the same area for 2-3 days but no sedentary positioning was seen. Meloidogyne spp. cause a most obvious morphological response to infection by the host, galling or knotting, that starts relatively rapidly, i.e. sometimes several hours after invasion (3). Calling is not essential for nematode development and growth and can take place in plants in which the nematode does not become established (3). Gall or knot formations were not seen with M. nataliei on A. thaliana. Four plates with infection taking place were removed from the 25° C growth chamber after eight days, the plates were contaminated but not discarded as was the normal policy, and placed in a growth chamber for 20 days at 15° C. At 15° and 25°CL the nematodes accumulated 5 and 15 degree-days (DD- 105 base 10° C) per day, respectively, and no development changes in the nematodes were seen. Temperature is not, however, the only factor influencing developmental rate of nematodes, in a study done by Godfrey and Oliveira (1932) cowpea and pineapple plants were grown side by side in a greenhouse, yet under identical conditions, development to egg-laying took 35 days in pineapple and only 19 days in cowpea for H. marioni (Cornu) (26). Nutrition plays a role in the rate of development and M. nataliei may not find A. thaliana a suitable host for development which remains to be determined in future studies. 106 Literature Cited 1. 2. 10. 11. Agrios, G.N.. 1988. Elan; Pathology. Third Edition. Academic, New York and London. pp. 276-277. Anon. (1991). Michigan Agricultural Statistics. Michigan Department of Agriculture, Lansing, MI. pp. 4, 32-35. Bird, A.F. 1974. Plant response to root-knot nematode. Anggal Review of Phytopathology. 12:69-85. Bird, G.W. 1971. Influence of incubation solution on the rate of recovery of Pratylenchus brachyurus from cotton roots. J. Nematol. 3:378-385. Bird, G.W. & D.C. Ramsdell. 1985. Population trends and vertical distribution of plant-parasitic nematodes associated with Vitis labrusca L. in Michigan. J. Nematol. 17(2):100-107. Brittain, J.A. & R.W. Miller. 1976. Managing Beach Tree Short Life In South Carolina. Clemson University and USDA Extension Services Clemson, S.C. pp. 3-4. Chang, D., J.L. Bowman, A.W. Dejohn, L.S. Lander & E.M. Meyerowitz. 1988. Restriction fragment length polymorphism linkage map for Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA, 85:6856-6860. Dropkin, V.H. 1989. Introduction to Plant Nematology Second Edition. John Wiley & Sons, Toronto. p. 219. Gamborg, O.L., T. Murashige, T.A. Thorpe, & I.K. Vasil. 1976. Plant tissue culture media. In-Vitro. 12:473-478. Golden, A.M., L.M. Rose & G.W. Bird. 1981. Description of Meloidogyne nataliei n. sp. (nematoda: Meloidogynidae) from grape (Vitis labrusca) in Michigan, with SEM observations. J. Nematol. 13(3):393-400. Griffing, B. & R.L. Scholl. 1991. Qualitative and quantitative genetic studies of Arabidopsis thaliana. Genetics Society of America. pp. 605-609. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 107 Jenkins, W.R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter. 48:692. Laibach, F. 1907. Zur Frage nach der Individualitat der Chromosomen im Pflanzenreich. Beih. Bot. Zentralbl. 1 Abt. 22:191-210. Mai, W.F., R.M. Riedel & S.C. Rabatin. 1988. Introduction. Conference on Nematode Culturing. p. 3. Marton, L. & J. Browse. 1991. Facile transformation of Arabidopsis. Plant Cell Reports. 10:235-239. Melakeberhan, H., A.L. Jones, P. Sobiczewski, & G.W. Bird. 1993. Factors associated with the decline of sweet cherry trees in Michigan: nematodes, bacterial canker, nutrition, soil pH, and winter injury. Plant Disease. 77(3):266-271. Meyerowitz, E.M. 1987. Arabidopsis thaliana. Annual Review of Genetics. 21:93-111. Moody, E.H., B.F. Lownsbery & J.M. Ahmmed. 1973. Culture of the root-lesion nematode Pratylenchus vulnus on carrot disks. J. Nematol. 5:225-226. Nickle, W.R. 1984. Plan; and Insect Nematodes. Marcel Dekker, Inc., N.Y. p. 7. Pearson, R.C. & A.C. Goheen. 1988. Compendipm pf Grape Diseases. APS Press, St. Paul, MN. p. 1. Poinar, 6.0. 1983. The Natural History of Nematodes. Prentice-Hall, Englewood Cliffs, N.J. pp. 122,128. Raski, D.J. 1988. p. 55. in R.C. Pearson & A.C. Goheen, eds. Nematode parasites of grape. Compendium of Grape Diseases. APS Press, St. Paul, MN. Riedel, R.M. 1985. Plant Nematology Laboratory Manual. pp. 159-162. Sasser, J.N. & D.W. Freckman. 1987. A World Perspective on Nematology: The Role of the Society. pp. 7-14, in J.A. Veech & D.W. Dickson, eds. Vistas op e atolo ° Commemoration of The Twent - t Anniversary of The Society of Nematologists. Society of Nematologists, Hyattsville, MD. Sijmons, P.C., F.M.W. Grundler, M. von Mende, P.R.Burrows & U. Wyss. 1991. Arabidopsis thaliana as a new model host for plant-parasitic nematodes. Plant 26. 27. 28. 29. 108 Journal. 1(2):245-254. Tyler, J. 1933. Development of the root-knot nematode as affected by temperature. Hilgardia. 7(10):391-415. Valvekens, D., M. Van Montagu & M. Van Lijsebettens. 1988. Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA, 85:5536-5540. Van Sluys, M.A., J. Tempe & N. Federoff. 1987. Studies on the introduction and mobility of the maize activator element in Arabidopsis thaliana and Daucus carota. EMBO Journal. 6:3881-3889. Westcott III, S.W. 1988. In-vitro Culture of Criconemella xenoplax. J. Nematol. 20:664 (Abstr). APPENDIX B Greenhouse Host Range Studies Manuscript Greenhouse Host Range Studies of Michigan Grape Root-Knot Nematode Meloidogyne nataliei (Nematoda: Meloidogynidae) on 22 Plant Species C.J. Diamond‘, G.W. Bird‘, and J.F. Davenport‘ Received for publication ‘ Department of Entomology, Michigan State University, East Lansing, Michigan 48824. 109 110 Abstract: Meloidogyne nataliei is a recently discovered parasite on grape root. Twenty-two plant species, representing a diversity of monocotyledon, dicotyledon, annual, biennial, and perennial plants, were used for host- range studies. Our greenhouse data showed that, unlike other nematodes of this genus, M. nataliei has a narrow host-range. No development and reproduction took place on 21 of the plant species used indicating that, except for grape, they were not hosts for the Michigan Grape Root-Knot Nematode. Field observations of M. nataliei indicate that older, woody roots may be preferred for feeding sites. More information on the biology of this nematode is needed for more detailed information from host range studies. Key wopd : host-range, Meloidogyne nataliei, grape. 111 One of the most widespread pests limiting world agricultural productivity is the root-knot nematode Meloidogyne Goeldi, 1887 species. Nearly all of the plants that account for the majority of the world's food supply are susceptible to these species. Worldwide average annual yield loss of major crops due to plant-parasitic nematode damage is estimated to be 12.3% (6). Of that 12.3% loss, 5% is due to Meloidogyne spp. (5). In developing countries, root-knot nematode infection is so widespread and common that galled roots are considered "normal" (5). With the discovery of a new Meloidogyne spp. in 1977; Meloidogyne nataliei Golden, Rose & Bird, 1981; steps were taken to determine the host range. M. nataliei was found parasitizing grape (Vitis labruscana Bailey cv. Concord) in a vineyard at Mattawan, Michigan (MI). Greenhouse host range studies were carried out on 22 plant species. The objective of this study was to determine if plant species other than V. labruscana could also be hosts for M. nataliei, that is, not only provide a nutrition site but one for penetration, growth, development and reproduction also. Materials and Methods Twenty-two plant species, ranging from oat to watermelon, were selected for hosts determination (Table 1). These represented a diversity of monocotyledon, dicotyledon, annual, biennial, and perennial plants, some are cultivated and others are weed species. VFF.IT 112 Table 1. Plants tested in host range studies of Meloidogyne nataliei. Plant tested Common name & Duration of stud Y cultivar (days) ; Vitis labruscana grape, Concord 62 5 Daucus carota carrot, 60 ' Gold Pak Elite : Pyrus malus apple, Empire 79 France avium cherry, 79 ' Montmorency/Mahaleb ' Prunus persica peach, 79 I . Cresthaven/Halford ;.As are as officinalis asparagus, * 65 l : Echinochloa crusgalli barnyard grass, * 65 ; Amaranthus sp. pigweed, * 51 I Avena sativa oat, Ausauble 62 I_Trifolium vulgaris red clover, * 60 Zea mays corn, Pioneer 3901 62 Cucumis sativus cucumber, 62 Northrup King Glycine max soybean, Hark 63 Gossypium sp. cotton, Delphine 16 64 Citrullus vulgaris watermelon, 63 Charleston Gray Capsicum annuum pepper, 65 California Wonder Lycopersicon tomato, Rutgers 51 esculentum Beta vulgaris sugar beet, * 51 Arachis hypogaea peanut, Florunner 51 Solanum tuberosum potato, Superior 35 Fragaria x ananassa strawberry, Red Chief 50 *cultivar unknown or not applicable 113 Egg masses of M. nataliei were obtained from grape roots at type location, Mattawan, MI. Eggs were removed from the gelatinous matrix using the NaOCl-extraction method (2). Seedlings of each species were planted in 3.05 decimeter clay pots, 1 seedling per pot, 6 replications of each, filled with 2500 cm? sandy loam soil which had been steam sterilized for 4 h. Soil was inoculated with 3,000 eggs/plant for penetration and reproduction studies using holes made with a metal spatula into the soil next to the seedling (nematode eggs not mixed into the soil). After inoculation, plants were placed in the greenhouse arranged in a randomized complete-block design. Plants were maintained at 24° 1 3° C. Supplemental light for a 19 h day length was provided by fluorescent lamps. The number of days for sampling after inoculation varied (Table 1). Separate assaying tests for roots and soil were performed to determine penetration and reproduction by nematodes. Roots were washed free of soil, cut into 1-2 cm lengths and a l g random sample was taken and stained with acid fuchsin (4). Nematodes within the roots were counted with the aid of a dissecting microscope. Total numbers of nematodes in the roots were recorded and individuals were assigned to one of three growth stages: vermiform juveniles that showed no evidence of swelling, males with well developed spicules, and swollen sedentary females. 114 Soil was assayed by mixing and then removing 100 cm3 which was used for nematode extraction using centrifugation- flotation (3). Total numbers of nematodes in the soil were recorded as in the roots. Results Other than on grape, the known host for M. nataliei, penetration of the root by M. nataliei was observed on cotton, cherry, peach, and peanut (Table 2). For a plant species to qualify as a host, the nematode needs to be able to not only penetrate the root but also develop through further molts to a mature stage and reproduce. No development and reproduction took place indicating that except for grape the plant species used were not hosts for the Michigan Grape Root-Knot Nematode. Very few vermiform juveniles were recovered from the soil (Table 2). The number of eggs that hatched is not known. Discussion Meloidogyne spp. typically differ from most other sedentary endoparasites by having extensive host ranges; parasitizing monocotyledons, dicotyledons and woody and herbaceous plants (5). Our greenhouse data show that, unlike other nematodes of this genus, M. nataliei has a narrow host range. Of the plants used in this study, grape remains the only known host for M. nataliei. 115 Table 2. Mean number of Meloidogyne nataliei and life stage in root and soil samples. fl Host Common Name Mean # in Soil Mean I in Roots l cotton 0 2J grape 1M 24J carrot 2J 0 apple 2J O cherry 0 1J peach 0 5M peanut 0 2J “ strawberry lJ 0 “ = J= uven le M=male Plants from Table 1 not listed had no nematodes in soil or roots. No females were found. 116 The plant species in this study were inoculated with eggs that had been removed from their gelatinous matrix. Hatching of Meloidogyne eggs is usually spontaneous and readily occurs without any stimulus from plant roots (5). When the soil was assayed for nematodes only vermiforms were considered. A count of unhatched eggs may have been indicative of the low numbers of vermiforms found in the soil and roots. Egg production has been observed to commence in the field, at infested vineyards, when soil temperatures begin to decline in October (personal observation). Further, most M. nataliei activity seems to take place during the winter months in MI (personal observation). Greenhouse temperatures may have been too high for egg hatch, penetration, growth and development of this nematode. No adult females were found in the root tissue of the 22 plant species indicating that development from the juvenile stage did not take place and that reproduction was not achieved. In general, once the juveniles of the genus Meloidogyne hatch they infect nearby roots of susceptible plants to continue their life cycles. Research in the genus Meloidogyne has shown that infective second-stage juveniles do not find roots by random movement but are attracted to plants in response to stimuli emanating from roots (5). Thus far, it is not known if the second-juvenile is the infective stage in M. nataliei. Actively elongating roots have been found to be more attractive to juveniles of other 117 Meloidogyne spp. than roots in which extension had slowed (7). It is now suspected that M. nataliei is attracted to older, woody roots in grape. Because of the swollen female size, they may be readily observed on roots unaided by magnification in the field but as yet have only been observed on older, woody roots while the younger roots remain uninfected. I Second-stage juveniles undergo several morphological changes following penetration of host tissue that are associated with parasitism in other Meloidogyne species. All three esophageal glands and the metacorpus enlarge during the early stages of parasitism along with many other changes such as granules in the subventral glands, etc. (1). The juveniles found within the roots of the cotton, cherry, peach, and peanut were not observed for these changes. Extensive research is needed on the biology of M. nataliei, including temperature requirements for egg hatch, penetration, development and reproduction, and time required to complete a full life cycle. Because field observations indicate that the Michigan Grape Root-Knot Nematode uses the older, woody roots of grape for reproduction, this experiment may have been terminated too soon. Future host .range studies may have different results if the inoculated ‘plants and nematodes are given more time to develop at lower temperatures. 118 Literature Cited Bird, A.F. 1968. Changes associated with parasitism in nematodes. III. Ultrastructure of the egg shell, larval cuticle, and contents of the subventral esophageal glands in Meloidogyne javanica, with some observations on hatching. J. Parasitol. 54:475-489. Hussey, R. S. and K. R. Barker. 1973. A comparison of methods of collecting inocula for Meloidogyne spp., including a new technique. Plant Dis. Rep. 57:1025- 1028. Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Dis. Rep. 48:692. McBeth, C. W., A. L. Taylor, and A. L. Smith. 1941. Note on staining nematodes in root tissue. Proc. Helminthol. Soc. Wash. 8:26. Sasser, J. N. and C. C. Carter. 1985. An Advanced Treatise on Meloidogyne Volume I: Biology and Control. North Carolina State University Graphics. 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Ramsdell. 1985 Population trends and vertical distribution of plant-parasitic nematodes associated with Vitis labrusca L. in Michigan. J. Nematol. 17(2):100—107. Bird, G.W. & F. Warner. 1990. Detecting and Avoiding Nematode Problems. MSU CES Bulletin E-2199, East Lansing, MI. p. 3. Byrd, D.W., Jr., T. Kirkpatrick, & K.R. Barker. 1983. An improved technique for clearing and staining plant tissue for detection of nematodes. J. Nematol. 15:142-143. Christie, J.R. 1936. The development of root-knot nematode galls. Phytopathology 26(1):1-22. De Guiran, G. & M. Ritter. 1979. Life Cycle of Meloidogyne Species and Factors Influencing Their Development. pp. 173- 191 in F. Lamberti & C.E. Taylor, eds. Roo - n Nema (Mgigigpgypg Spegies) Systematics, piglogy and Qonppgl. Academic, New York and London. Ehlig, C.F. 1960. Effects of salinity on four varieties of table grapes grown in sand culture. in J.R. Magness, ed. Proc. Am. Soc. Hort. Sci. 76:323-331. Feder, N. & T.P. O'Brien. 1968. Plant microtechnique: some principles and new methods. Amer. J. Bot. 55(1):123-142. Ferris, H., H.S. Du Vernay, & R.H. Small. 1978. Development of a soil-temperature data base on Meloidogyne arenaria for a simulation model. J. Nematol. 10(1):39-42. 119 120 Gahan, P.B. 1984. Piant Histochemistry and Cytochemistry an lhgrgdugpigp. Academic, London. pp. 20-21, 193-194. Golden, A.M., L.M. Rose & G.W. Bird. 1981. Description of Meloidogyne nataliei n. sp. (Nematoda: Meloidogynidae) from grape (Vitis labrusca) in Michigan, with SEM observations. J. Nematol. 13(3):393-400. Hanson, E. 6 J. Hull. 1986. Plant Tissue Analysis for Determining Fertilizer Needs of Michigan Fruit Crops. Extension Bulletin E-449 (Revised) March 1986. CES, MSU. Hanson, E. & C. Kesner. 1987. Fertilizing Fruit Crops. Extension Bulletin E-852 Major Revision December 1987. CES, MSU. Himelrick, D.C. 1991. Growth and nutritional responses of nine grape cultivars to low soil pH. HortScience 26(3):269- 271. Huang, C.S. & A.R. Maggenti. Wall modifications in developing giant cells of Vicia faba and Cucumis sativus induced by root knot nematode, Meloidogyne javanica. Phytopathology 59:931-937. Jenkins, W.R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48:692. Jepson, S.B. 1987. Identification of Root-Knop Nemgpgdgg (Meioigogype species). C'A-B International, United Kingdom. pp. 2-4, 14. Kenworthy, A.L. 1967. Plant analysis and interpretation of analysis for horticultural crops. pp. 59-92 in Soil Testing gpd Plant Analysis. Soil Sci. Soc. Am. Publ. 2. McKenry, M.V. 1992. Nematodes. pp. 279-293 in D.L. Flaherty, L.P. Christensen, W.T. Lanini, J.J. Marois, P.A. Phillips & L.T. Wilson, eds. §pppp_Pg§t Management Seggpd_£dipigp. University of California Division of Agriculture and Natural Resources. Melakeberhan, H., A.L. Jones, P. Sobiczewski, & G.W. Bird. 1993. 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