TAXONOMY AND BIOLOGY OF Verutus volvingentis N. GEN. N, SP. (TYLENCHIDA-NEMATA) BY ROBERT PAUL ESSER A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1980 ACKNOWLEDGEMENTS The author is deeply grateful to the chairmen of his supervisory committee. Dr. V, G. Perry and Dr. A. C. Tarjan, who have given considerable time and immeasurable assistance during the term of this study. Gratitude is also expressed to Dr. R. A. Dunn and Dr. D. F. Rothwell for invaluable assistance and encourage- ment, while serving as members of my supervisory committee. A special debt of thanks must also go to Dr. G. C. Smart, Mr. A. L. Taylor, and Dr. K. R. Langdon for sugges- tions and assistance pertinent to this study. Thanks are also given to Mr. W. W. Smith who origi- nally found the new nematode, and provided much data and material essential to expediting the objectives of this study. I am also deeply grateful to Agricultural Commissioner Doyle Conner, and H. L. Jones, director of the Division of Plant Industry, for encouragement and very generous vouch- safement in this endeavor. Finally, I am very appreciative for considerable en- couragement and forebearance from my wife Hannelore in this term of trial. TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES V LIST OF FIGURES vi ABSTRACT X INTRODUCTION . ^ 1 HISTORY 2 SECTION I TAXONOMY AND SYSTEMATICS 3 Taxonomic Position of the New Genus 3 Taxonomy of the New Genus 17 Anatomy 36 SECTION II REPRODUCTIVE DEVELOPMENT 45 Early Development 45 Male Development 47 Female Development 50 SECTION III HOST-PARASITE RELATIONSHIPS 54 Methods 54 Behavior Studies 56 Page SECTION IV HOST PLANT INVESTIGATIONS 7 0 Host Plant 70 Host Habitat 70 Host Testing 72 Host Symptoms 7 6 Pathogenicity 78 Distribution of Verutus volvingentis in Florida 83 SECTION V BIOLOGICAL CONTROL INTERACTIONS 87 LITERATURE CITED 90 BIBLIOGRAPHY 95 BIOGRAPHICAL SKETCH 100 LIST OF TABLES Table Page 1. Comparative distinguishing characteristics of females in genera of Heteroderidae 7 2. Comparative distinguishing characteristics of males contained in the Heteroderidae 8 3. Male development 49 4. Number of fully developed eggs detected in 123 mature females 66 5. Survival stage and numbers of nematodes recovered before and after a longivity test ... 67 6. Larval hatch from eggs kept in fallow soil 3 years 68 7. Phytoparasitic nematodes found in soil associated with roots of buttonweed 73 8. Verutus volvingentis host testing results .... 74 9. Effect of Verutus volvingentis on foliage and seed pod production of inoculated plants 81 10. Nematode population density in treated and untreated soil 81 11. Areas in Florida sampled for Verutus volvingentis 84 12. Plants examined for the new nematode in the Florida survey 85 LIST OF FIGURES Figure Page 1. Meloidogyne sp. shov/ing spheroid shape, and terminal vulva of a mature female 5 2 . Meloidodera f loridensis 5 3. Verutus n. gen. female in root tissue with a single deposited egg 5 4. Cryphodera eucalypti 10 5 . Hypsoperine graminus 10 6. Heteroderidae male tail types 11 7. A comparison of female appearances in three subfamilies. A) Verutinae, B) Nacobbinae, C) Rotylenchulinae 12 8. First-stage larva of Verutus volvingentis n. gen. , n. sp 13 9. A comparison of first and second-stage larval esophageal glands in the Heteroderidae 15 10 . Water agar en face method 19 11. Verutus volvingentis n. gen. n. sp. Mature female 21 12. Verutus volvingentis n. gen. n. sp., female body shapes 22 13. Vestigial larval tail tip on posterior area of a mature female 23 14. En face presentation of a mature female 23 15. Mature male of Verutus volvingentis n. gen. n. sp 25 16. Verutus volvingentis male en face viev^; 2 8 17. Female tail area showing lateral field irregularities 28 vi Figure Page 18. Telorhabdions of a mature female posterior view 28 19. Posterior region of mature females 28 20. Anus (left) and rectum of a mature female ... 28 21. Excreta exuded from the anus of a mature female 29 22. Vulva and vulva muscles of a mature female ... 29 23. Lateral views of an unprolapsed (left) and prolapsed (right) vagina of mature females ... 30 24. Uterus of a mature female 31 25. Male tail showing tubus 31 26. Male tail in a ventral view 34 27. Larval tail shapes 34 28. Ova 37 29. Anterior portion of third-stage larval male . . 37 30. Rectal musculature 40 31. Anterior nervous system in a first-stage larva 40 32. Ventral nerve cord in the area of the genital primordia 42 33. Nerves in the tail of a first-stage larva ... 42 34. Crystalline layer 43 35. Gubernaculum 46 36. Larvae forced from egg by applying cover slip pressure 46 37. Spicular primordia cells in the cloacal area of an early second-stage male 46 38. Male reproductive system development 48 39. Early third-stage female gonad 51 vii Figure Page 40. Vaginal development of a late third- stage female 51 41. Virgin female with gonad development complete 51 42. Final female ecdysis 52 43. Mature female 52 44. Anatomy of the female reproductive system from vulva to uteri 52 45. A root map 58 46. Mode of root entry by larvae 58 47. Larval head in contact with middle lamella ... 59 48. A mature female in a shallow, hand-cut epidermal section 59 49. Tissue discoloration 60 50. Large lesion occupied by 4 larvae 62 51. Attack sites 63 52. Spheroid bodies attached to anterior region of a mature female 65 53. Appearance of a single spheroid body 65 54. Exudates on the anterior end of Meloidodera f loridensis 55 55. Female containing 7 eggs 65 56. Diodia virginiana in flower 71 57. A mat of buttonweed mixed with herbaceous plants 71 58. Paynes Prairie, Gainesville, Florida 72 59. Young female in root, showing large dark lesion at feeding site 77 60. Appearance of inoculated and uninoculated plants at the conclusion of the pathogenicity trial 80 viii Figure Page 61. A comparison of leaves from inoculated and uninoculated plants 82 62. A comparison of seeds from inoculated and uninoculated plants 82 63. Biological control interactions 88 64. Sporangium of Rhizophidium sp. attached to an egg 8 8 Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TAXONOMY AND BIOLOGY OF Verutus volvingentis N. GEN. N. SP. (TYLENCHIDArNEMATA) By ROBERT PAUL ESSER JUNE, 19 80 Chairman: Armen C. Tar j an Major Department: Entomology and Nematology A new subfamily Verutinae is proposed. Females dif- fer from all other subfamilies in the Heteroderidae in pos- sessing a sausage shaped body with an uncommonly large sub- equatorial vulva. The genus and species named Verutus volvingentis is described. Larval ecdysis was not noted v/ithin the egg. Eggs are not deposited in a gelatinous matrix. Anatomical features include a phasmid that appears only on tail of cast cuticles of the first stage larvae. A previously un- described muscle, the "median dilator vulvae," was named. A description of the rectal musculature and nervous system is given. A detailed account of male and female develop- ment is presented. Male development is completed in 6-15 days; female development took 17 days. Larvae entered the root by pushing through the middle lamella between 2 epidermal cells. Tissue discoloration occurred 3-4 days after feeding. Nuclei of invaded cells enlarged and exu- date production by the host was incited. Larvae and eggs survived at least 3 years in the absence of food. Eggs are the dominant survival stage. The nematode is widely distributed in Florida in moist habitats. Testing of selected economic crops as hosts proved negative. Host plants inoculated with a minimal number of nematodes died in 13y months. Control plants were m.aintained in a healthy vigorous condition. Catenaria anguillulae killed males but not larvae or eggs in biological control tests. INTRODUCTION The family Heteroderidae contains a large number of highly pathogenic species included in 17 genera, 14 of which have been erected since 1956. Pathogenicity has not been proved for the following genera: Atalodera Wouts & Sher, 1971; Meloidoderita Poghossian, 1966; Meloidodorella Khan, 1972; Meloinema Choi & Geraert, 1973; Punctodera Mulvey & Stone, 1976; Sarisodera Wouts & Sher, 1971; Sherodera Wouts, 1973; and Thecavermiculatus Robbins, 1978, The principal objectives of this research were to establish the systematic position of the new genus of nema- todes described in this work and to investigate the patho- genic potential of the new taxon. Secondary objectives included: host testing, life cycle and developmental studies, longevity, host-parasite relationships and anatomical studies. HISTORY In March, 1969, Mr. Wayne W. Smith, "Agricultural Products Specialist," with the Florida Department of Agri- culture submitted 14 samples from a field near Apopka, Florida, for regulatory analysis. Four of the samples were infested with larvae that resembled Heterodera sp. A search of the sample material for Heterodera cysts revealed females that did not fit the generic concept of any known nematode phytoparasitic genus described at that time. In May, 1969, the site from which the samples origi- nated was surveyed in an attempt to isolate and identify the host plant of the undescribed nematode. The host was found to be buttonweed (Diodia virginiana L.) and was sub- sequently infected with the nematode in greenhouse culture. SECTION I TAXONOMY AND SYSTEMATICS Taxonomic Position of the New Genus Verutinae n. subf. Diagnosis: Heteroderidae (Filipjev, 1934) Skarbilovitch , 1947. Female: Mature female saccate, sausage to renif orm-shaped (Fig. 12) , vulva uncommonly large, subequatorial in posi- tion, vulval lips strongly protuberant (Fig. 11), ovaries reflexed, anus subterminal, cyst stage absent, body striae present, phasraid obscure, strong sexual dimorphism present. Male: (Fig. 15) Body vermiform, caudal alae absent, tail rounded flatly to truncate, body untwisted, one testis present. Type genus Verutus n. gen. (from the Latin "armed with a dart" ) . Affinities with the Family Heteroderidae Females . Table 1 shows comparative female distin- guishing characteristics of genera contained in the sub- families of Heteroderidae. Verutus n. gen. differs from all other members of the Heteroderinae in lacking a cyst stage and a terminal vulva. It differs from all members of the Zvtaloderinae , and Meloidogyninae in lacking a terminal or subterminal vulva, and a spheroid body (Fig. 1) . Verutus is most closely re- lated to Meloidoderinae, one member of which, Meloidodera floridensis Chitwood, Hannon, & Esser, 1956, possesses a subequatorial vulva (Fig. 2) and a spheroid or subspheroid body shape. Verutus eggs are deposited as they mature (Fig. 3) , and not retained in large numbers within the fe- male body as in females of Meloidoderinae (Fig. 4) . The body is completely annulated in the subfamilies Meloidoderi- nae, Meloidogyninae, and Verutinae n. subfam. Members of Ataloderinae and Heteroderinae possess an irregular body pattern, or lack body markings. In some members of these 2 subfamilies, annulation may be present on the cervical area or about the vulva, but not on the body. The vulva is widely separated from the anus in the Verutinae (Fig. 11) and in the genus Meloidodera (Fig. 2) in the Meloidoderinae. In all other subfamilies the vulva is located in the peri- neal area or near the anus (Fig. 4, Table 1). In some mem- bers of Ataloderinae, Heteroderinae, and Meloidoderinae the vulva is situated on a papule (Fig. 5) . Neither Meloido- derinae or Verutinae are so equipped. A labial disc (Fig. 14) is described only in Ataloderinae, Meloidoderinae, and Verutinae. An attempt was made to utilize the presence of a gelatinous matrix, or a sub-crystalline layer (Fig. 34) in the diagnosis. This was not possible due to lack of data concerning these criteria in many species descriptions. Genera of the Heteroderidae were also compared to the new Figure 3. Verutus n. gen. female in root tissue with a single deposited egg. Figure 1. Meloidogyne sp., showing spheroid shape and terminal vulva of a mature female. Figure 2. Meloidodera f loridensis showing spheroid shape and equatorial vulva position. genus on the basis of the measurement (length/greatest body width) alpha. This was found to be infeasible due to omitted data and differences in the measurement criteria used by some authors. Some measure the total body length, others exclude the neck and head from the measurement (Mulvey & Stone, 1976). A key to the subfamilies of Heteroderidae based on mature females is as follows. Key to subfamilies of the Heteroderidae 1. Female forms a cyst Heteroderinae Female does not form a cyst 2 2. Annulation absent or limited only to cervical or vulva area Ataloderinae Annulation present on body (may be sparse) 3 3. Body sausage or reniform-shaped vulva uncommonly large Verutinae n. gen. Body ovoid or pear-shaped, vulva not uncommonly large 4 4. Eggs retained in large num.bers in female body, labial disc present Meloidoderinae Eggs not retained in body in large numbers (exception Meloidoderita) , labial disc present Meloidogyninae Males . Table 2 compares selected male characteris- tics of genera included in the subfamilies of the Heteroderidae. It can be seen that few definitive differ- ences exist between males. Only the subfamilies Ataloder- inae and Verutinae contain genera without a twist in the male body (Fig. 6-C) . Males of both subfamilies also pos- sess a truncate tail terminus, similar spicules and guber- naculum, and a tubus (Fig. 6-A,B) . Stylet and body length Table 1. Comparative distinguishing characteristics of females in genera of Heteroderidae. Sub- family Vulva Vulva Egg Galls Cyst Cuticle Anus on a Lip Reten- ^ on Egg ^,^ Vulva Genus Stage Markings Gap Papule Disc tion HPR Host Sac C L Position 1/70 no head & vulva striae very close yes yes yes 7 7 7 ? terminal 1/89 no none „ „ " " " " " " " " " 1/121 " head & vulva striae " " no " ■ semi- endo 7 no yes 2/118 yes pattern close " no " „ „ no " no „ „ 2/13 " „ „ „ „ no & yes " " " " yes yes " 2/99 " pattern & punc- tations It tt no " " endo yes " 7 2/102 " „ „ " " " " " semi- endo no " " " 2/71 " lace- like very close " " " ? ? 7 7 terminal recessed 3/63 no striae not close " yes " semi- endo no no yes terminal 3/16 " " " far ■ apart " " " " " " " mid-body 3/95 " M „ not close " " " ? ? 7 7 terminal 4/41 " " " very close yes " no endo yes yes yes " " 4/56 4/17 4/92 5/130 yes semi- endo no & no no endo yes striae close no sparse striae far apart yes no semi- rare no yes mid-body endo , *HPR = Host parasite relationship, Semi-endo=semi-endoparasite, Endo=endoparasite. **CL = Crystalline layer. Legend Subfamily & Genus l=Ataloderinae; 70=Atalodera, 89=Sherodera, 121=Thecavermiculatus 2=Heteroderinae; 118=Globodera, 13=Heterodera, 99=Meloidodorella, 10 2=Punctodera 71=Sarisodera 3=Meloidoderinae; 63=Cryphodera , 16=Meloidodera, 95=Zeylandodera 4=Meloidogyninae; 41=Hypsoperine , 56=Meloidoderita , 17=Meloidogyne, 9 2=Meloinema 5=Verutinae; 130=Verutus Egg filled cyst develops. Golden, 1976; Andrews et al, 1977. ;2 P? ^ a: t-' tS) ro 0 fD K O rD ra 0 o O w o fj 0 LJ- H- 0 o 3 H- 3 a. & X! n a 0 0 0 0 ^ O 0 ^ -Q Ui f1 z n. CJ K fi) r-^- M C; ra 3 ti ^ f-i O H- O > UJ (H rT M P rr OJ n >-i cr n 01 --1 3 0 0 H' 0 3- c a 3 C C 3 CJ 3 C 0 3 3 0 1 H- Qj fi- H-13 :j ^ a ri! (^ & in ^. 3 M 3 f- ' H- M i 1 m rr H- o 0 ti Uj '.I, 1) m O '. ii ^1 ^ O y (1) H* \-i sa |ra a 3 = = = o --J O 1-i I I ^ M- K* I— ' CO Qj £. Ln o cr O m '<' fi) H- n r:5 () q rt-l:) ;t' fu ro I-! Q) m n- fD H- rt < fl) (D ^ 0 a- u. p- (i) en h rt H- P- Oi 3 pj uq fD C • H- cn ^ H- 3 iXj O l^- OJ li pj o rt (1) ^-i H- 01 rt H- n U3 -^ Oi l/l rt ON rr rt fD o f3 o i-( measurements also overlap in both subfamilies. Except for the striated gubernaculum of the genus Sherodera it would be difficult to differentiate between males of Ataloderidae and Verutinae. The only other genus with a truncate tail terminus and tubus is Sarisodera in the subfamily Hetero- derinae. The Sarisodera male can be separated from males of Ataloderinae and Verutinae by its long stylet (38-46 ym) . Meloidoderita possesses a sharp conoid tail and is the only male in the Heteroderidae with caudal alae. All other genera, not included in the aforementioned, have rounded or bluntly conoid tails (Fig. 6-C) V7ith or without a twist. The only small males have been described in Meloidoderita (350-432 ym) , and Meloidodera (457-505 ym) . Males in the genus Meloinema stand apart from all other males in posses- sing a distinct subacutely conoid tail (Fig. 6-D) . Larvae. The larvae of the new genus (Fig. 8) close- ly resemble larvae in the subfamilies Atalodorinae , Meloidoderinae, and all of the larvae in the genera of Heteroderinae (except Meloidodorella, which has a short stylet [11-16 ym and reduced telorhabdions]) . Larvae of the Meloidogyninae differ from the new genus, for the most part, in having a small stylet, small telorhabdions, and fine body striae. The principal character peculiar to first- stage larvae of the new genus is the absence of a detect- able phasmid. 10 Figure 4. Cryphodera eucalypti (after Colbran) , showing egg retention in the mature female, and separation of the vulva and anus. Figure 5. Hypsoperine graminus Sledge and Golden"^^ 1954 . A mature female showing a vulva situated on a papule. 11 Figure 6. Heteroderidae male tail types; A,B - Truncate with tubus. A - Sherodera (redrawn from Wouts & Sher) , B - Verutus , C - Rounded twisted type. Meloidodera (after Hopper), D - Blunt conoid. Meloinema (redrawn from Choi & Geraert) . Comparisons were made of esophageal gland structures in the 15 genera of the Heteroderidae to determine if the glands could be used in generic or subfamily diagnosis. Esophageal glands with two lobes, the anterior lobe over- lapping the posterior lobe, were found in the Verutinae, Meloidoderinae , and Heteroderinae (Fig. 9 A,D,F,H,J). Considerable variation in esophageal gland structure was noted in the 27 species descriptions examined in 4 genera of the Heteroderinae (Fig. 9 F-K) . Esophageal glands in 29 species of Meloidogynidae all consisted of a single lobe. Meloinema differed from all other genera in having an extremely long esophagus (300 ym) . However, in the description the esophagus length range was listed at 125- 130 ym, which contradicts the illustration. The genera 12 Figure 7. A comparison of female appearances in three subfamilies. A) Verutinae, B) Nacobbinae, C) Rotylenchulinae, 13 14 Sherodera and Zelandodera, Wouts , 1973 are not included in Figure 9 since neither genus is represented by an illustra- tion of the larval esophageal glands in the literature. The Verutinae also differ from the Ataloderinae and Meloidogyninae in possessing two esophageal gland lobes. Affinities with the Subfamily Rotylenchulinae , Husain & Khan, 1967 The females of Verutus (Fig. 7-A) closely resemble females in Rotylenchulinae (Fig. 7-C) . Verutus females differ in the absence of a well-defined tail tip, a dorsal gland orifice that originates less than one stylet length from the base of the telorhabdions , possession of a large muscular uterus, and, principally, by the absence of a vermiform, vulvate juvenile femiale stage. Verutinae males differ markedly in general appearance from Rotylenchulinae males which have a tapering conoid tail, are usually less than 500 pm long, and assume a C- shaped body position. Rotylenchulinae males possess an elongate, truncate cephalic framework in contrast to the slightly convex, shallow cephalic framework of Verutinae males . Affinities with the Subfamily Nacobbinae, Chitv/ood & Chitwood, 193 7 Nacobbinae females differ from females of Verutinae in body shape (Fig. 7-B) , position of a posteriorly sit- uated vulva, and in having a single gonad. Nacobbinae 15 ABC Figure 9. A comparison of first and second- stage larval esophageal glands in the Heteroderidae. A) Verutinae; Verutus . B,C) Ataloderinae; B) Atalodcra , C) Thecavermiculatus D,E) Meloidoderinae; D) Meloidodera, E^ Cryphodera Colbran, 1966 . F,K) Heteroderinae; F) Sarisodera, G) Meloidodorella, H) Punctodera, I, J) Globodera or Heterodera . K) Heterodera. L-0 Meloidogynidae ; L) Meloidoderi ta , Ml Meloidogyne , N) Hypsoperine , Ol Meloinema . (Esophageal glands are directly proportional to the size of the metacorpus shown.) 16 females form prominent root galls while Verutinae females do not. Nacobbinae males possess conoid tails and caudal alae, both of which are absent in Verutinae males. Nacobbinae larvae differ from Verutinae larvae in having a bluntly rounded tail tip. Discussion. The new subfamily Verutinae does not fit within the concepts of the subfamilies in the family Heteroderinae , or the subfamilies, Nacobbinae or Rotylen- chulinae. It is therefore proposed as a new subfamily. Subfamilies proposed by Husain, 1976, but not included in the analysis are Meloineminae and Meloidoderellinae . The position of the Verutinae in the Animal Kingdom is shown in the following scheme: Kingdom-Animalia Subkingdom-Metazoa Branch- En terozoa Division- Bilateria Section-Pseudocoelomata Phylum-Nemata (Rudolphi , 180 8) , Cobb, 1919 Class-Secernentia (von Linstow, 190 5) Chitwood, 19 5 8 Order-Tylenchida Thorne,1949 Suborder-Tylenchina Pearse,1942 Super family- He terodero idea (Fi lip jev, 193 4) Golden, 19 71 Family-Heteroderidae (Filipjev ,1934) Skarbilovitch , 1947 17 Subfamily-Verutinae Genus -Verutus Species- vol vingentis In the above scheme categories above Phylum are based on Storer and Usinger, 1957. Classifications below Section are based on schemes proposed by Golden, 1971, Wouts , 1972, Andrassy, 1976, Husain, 1976, and Stone, 1977. Taxonomy of the New Genus Methods; Measurement Preparation Specimens to be measured were placed in water within a "Zut" ring on a glass microscope slide (Esser, 1973-b) , and a cover slip placed on the zut. The nematodes ceased moving in 3 to 5 min. , and measurements were taken according to the method proposed by Esser, 1971. While the nematodes are in the quiescent state one has 20 to 30 min. to make observations and measurements before deterioration, swelling and/or shrinkage occur. Permanent Fixation When specimens on slides are to be fixed permanently the cover slip is removed and 2 or 3 drops of 2% formalin are added to the exposed zut well. The specimens are then transferred to a BPI watch glass for permanent fixation in lactophenol (Esser, 1973-a) . En face Preparation A new method was devised to study en face prepara- tions. Live immobile females or males, or freshly killed nematodes in 2% formalin were used as subjects. Procedure . A 12 X 12 X 3 mm square of 1.7% water agar is cut very evenly with a razor blade (Fig. 10-A) and placed on a microscope slide. A 3- to 4-mm piece is pre- cisely cut from the square (Fig. 10-A) and laid with the outer face down (Fig. 10-B) . Nematodes are placed on the upper side of the cut piece with the longitudinal axis of the head parallel v/ith the outer edge of the cut piece (Fig. 10-B) . The cut piece is then placed back into the same position it occupied in Fig. 10-A, then gently pushed into its original position against the parent block (Fig. 10-C) . A small (4-mm) drop of water is applied to a 15-mm cover slip that is then placed waterside down over the cut line (Fig. 10-C) . A drop of immersion oil is applied to the center of the cover slip at the junction of the cut pieces. When the body of the nematode is properly aligned the en face appears as in Fig. 10-D . If the en face is off-center or below the field of focus, the cover slip is removed, the cut piece placed backside down, and the speci- mens reoriented. Water must be added to the cover slip each time it is placed on the agar block. It takes 10 to 15 min. to prepare an en face ready for viewing using this technique. Locating the en face is rather easy since it lies within the cut line. 19 Figure 10. Water agar en face method; A) 12 X 12 X 3 mm square of water agar with a 3- to 4-mm piece cut off, B) Specimens aligned on outer edge of the inner face of the cut piece, C) Re-alignment of the separated agar pieces, with cover slip in place, D) Closeup of en face in junction line of re-aligned agar. 20 External Cuticle Preparation Lateral lines and phasmids were not well-defined in live or fixed specimens. Several stains were tested to bring out the lateral lines including: methyl blue, acid fuchsin, merthiolate, iodine, chlorazol black-E, and pro- pionic carmine, none of which enhanced cuticular incisures or phasmids. Lateral incisures were brought out clearly by making squash mounts. Cut or uncut females, males, and larvae were placed in a 4-mm drop of water, and a 18-mm cover slip applied. A needle point was pressed against the cover slip until the body contents gushed out. Examination for the phasmid was made of each squash mount specimen. Nervous System Preparation Chlorazol black-E in lactophenol was used with the 4-min. fixation method (Esser, 1973-a). Verutus n. gen. Diagnosis: Verutinae, with characters of the subfamily. Mature female (Fig. 11) : Body swollen, reniform or sausage- shaped (Fig. 12). Cephalic framework moderately sclerotized, lips striated, set off, amphids obscure, oral disc hexagonal (Fig. 14). Body striated, lateral lines irregular, some- times indistinct. Crystalline layer present (Fig. 34). Stylet tylenchoid, dorsal gland orifice near telorhabdion base. Uncommonly large protuberant post-equatorial vulva. 21 22 Figure 12. Verutus volvingentis n. gen. n, female body shapes. sp, 23 Figure 13. Vestigial larval tail tip on posterior area of a mature female. Figure 14. En face view of a mature female. Gonads didelphic and amphidelphic. Ovaries reflexed. Anus subterminal forming small depression (Fig. 13). Tail vestigial (Fig. 13) or absent. Female. (Table 1) Females differ from all other fe- males in the Heteroderidae in possessing a reniform or sausage-shaped body, with an uncommonly large vulva in a post equatorial position with strongly protuberant lips. Esophagus typically tylenchoid, procorpus moderately expan- ded, metacorpus moderate in size. Isthmus narrower than procorpus, esophageal gland a single lobe moderately over- lapping the intestine. Deirids and phasmids not observed. 24 Male. (Fig. 15) Body vermiform, monodelphic, lips striated not set off, oral disc circular (Fig. 16), amphid- ial openings elliptical on lateral lips. Body untwisted. Spicules and gubernaculum tylenchoid. Caudal alae absent, tail terminus angular truncate (Fig. 6-B) . Phasmids or deirids not detected. Verutus volvingentis n. sp. Female. (35 specimens) Total length = 662.7 (500- 930) ym; width = 141.4 (94-207) ym; tail = 10.2 (3.9-15.7) ym; esophagus = 188.3 (150-290) ym; a = 4.7 (3.0-6.5); c = 69,4 (34-155); total stylet length = 26.1 (23.5-29.4) ym; vulva % = 67,5 (50-75); excretory pore 139.8 (122-183) ym. Female holotype. Total length = 540 ym; width = 118 ym.; tail = 15 ym; esophagus = 148 ym; a = 4.6; b = 3.6; c = 36; stylet = 27.2 ym; vulva % = 71.2; excretory pore = 114 ym from anterior end. Female description. (Fig. 11) Body pearly white, reniform or sausage-shaped (Fig. 12) , anterior part of body sometimes twisted upward lying in a different plane than the posterior swollen portion. Head and neck occasionally reflexed across the posterior body. Six equidistant lips surround a hexagon-shaped oral disc (Fig. 14) . Amphid apertures or lip papillae not observed. Lips set off, com- prised of 2 annules. Cuticle 9-10 ym thick, evenly stria- ted, striae about 2.5 ym apart. The occurrence and volv = vulva, ingen = remarkable size, ^5^ Pigure 15. Mature male of Verutus volvingenti n. gen- n. 26 appearance of lateral lines are variable: lines may proceed for a short distance beyond anus and fade out, or appear as midline cuticular interruptions or irregularities extending slightly past the vulva area. They appear as 1 or 2 lines of irregular blocks in the cervical area. In the tail area they appear as a mass of irregularities in the tail tip area with sometimes a wide separation (5 ym) of the striae (Fig. 17) . In a few females lateral lines were not observed. Stylet tylenchoid, telorhabdions (Fig. 11, 18) , 4-5 wide by 1-2 ym long, directed posteriad. Prorhabdions 14 ym. Two stylets with tips protruding from the body measured 25.5 and 25.6 ym, respectively. The dorsal gland orifice appears 7-11 ym posterior to the telorhabdion base. A moderately swollen procorpus narrows prior to the well- developed metacorpus. A clearly defined metacorpal valve is present. A short narrow isthmus leads from the meta- corpus followed by a single distinct esophageal gland lay- ing ventrally over the intestine. The intestine extends from beneath the mid-area of the esophageal gland to the rectal intestinal valve. The sclerotized portion of the rectum is 12 ym long in a lateral view. The rectum dilates anteriorly extending 30 ym beyond the sclerotized portion (Fig. 19) as a finely sclerotized tube (15 ym long) which joins the intestine. The oval anus (Fig. 20) lies in a depression (Fig. 19) . Tail is usually absent, occasionally vestigial (Fig. 19) . Differences in orientation of the body 27 do not permit an accurate tail annule count, or anal body diaraeter measurement. One female was noted (Fig. 21) v/ith a granular mass over the anus, assumed to be excreta. The excretory pore lies at the level of the esophageal gland 134.4 (113-183) ijm from the oral opening. The nerve ring appears as a mass of tissue surrounding the isthmus. Gonad amphidelphic, anterior branch 136-147 ym long, posterior branch 117-130 \m long. The vulva appears as a transverse slit (Fig. 22) about 62 m v/ide. In some fe- males the vulva lips protrude markedly. The vulva striae do not form a distinctive pattern, but surround the vulva rather uniformly (Fig. 22) . In some females prolapse of the vaginal walls causes the vulva to widen, and the vaginal lining prolapses externally (Fig. 23). Wide muscle bands, the dilator vulvae appear at either end of the vulva under- lying the cuticle (Fig. 22) . Vulva epitygma were not ob- served. The vagina extends 42 to 70.5 ym into the body where it joins the well-developed vagina uterina (58-70 ym; Fig. 11) . A constriction appears at the junction of the vagina uterina and the uterus. The uterus is a large mus- cular sac (70 X 35 ym) that joins directly with the sper- matheca (Fig. 24) . It comprises 4 to 5 rows of large cells with a furrow in the center for expansion. The spermatheca is a roughly circular thick walled chamber 35 to 40 ym in diameter. The oviduct, a thickened area comprised of small cells, lies between the spermatheca and the maturation zone of the ovaries. The ovary is reflexed at the spermatheca. Figure 16. Verutus volvingentis male en face view. Figure 17. Female tail area showing lateral field irregularities . Figure 18. Telorhabdions of a mature female posterior view. Figure 20. Anus (left) and rectum of a mature female. Figure 19. Posterior region of mature females, showing vestigial larval tail tips (T) , and rectum (R) . 29 '3Qy Figure 21. Excreta exuded Figure 22. Vulva and vulva from the anus of a mature muscles of a mature female. V= female. vulva lips, S=striae surround- ing vulva, D=dilator vulvae muscles, M-median dilator vulvae muscles (striae cutaway to show underlying muscles) . and 1 or 2 times in the maturation zone area. The cap cell and germinal zone cells are rarely delineated in live or fixed specimens. Males . (24 specimens, Fig. 15) Body length = 830.8 (650-1020) ym; width = 28.9 (25.5-35.5) ym; tail = 9.5 (5-12.7) urn; esophagus = 153.5 (122-188) \m; a = 28.7 (24.3-32.8) ym; b = 5.4 (4.5-6.6) ym; c = 99.2 (59.1-178.6) ym; total stylet length = 25.3 (21.5-27.4) ym; dorsal gland orifice = 4.6 (2-6.8) ym behind the base of the telorhabdions; 30 W^UUUUULUUUUUU/ Figure 23. Lateral views of an unprolapsod (left) and prolapsed (right) vagina of mature females P-prolapsed vaginal tissue. spicules = 40.4 (36.2-46.6) ym; gubernaculura = 16.2 (14.7- 18.6) ym. Allotype. Total body length 790 ym; width = 25 ym; tail = 6 ym; esophagus = 140 ym; a = 31.6; b = 5.6; c = 131.7; total stylet length = 22 ym; dorsal gland orifice = 6 ym; spicules = 40 ym; gubernaculum = 15 ym. Male description. Body vermiform, untwisted (Fig. 15) , 6 equidistant lips (Fig. 16) surround a circular oral disc that stands out clearly in profile. Crescent-shaped amphids appear indistinctly on posterior margin of lateral 31 Figure 24. Uterus of a mature female 1) spermatheca, 2) uterus, 3) egg. Figure 25. Male tail showing tubus (arrow) . 32 lips. Labium moderately sclerotized, comprising 4 to 7 labial annules, counting from first reduced annule at onset of cephalic sclerotization. Labium rounded, not set off, papillae not observed. Body striae about 2 um wide, some- times ending irregularly at the terminus (Fig. 15) . Four unareolated lateral fields present, extending from region of corpus to cloacal area. Phasmid not observed. Excre- tory pore lying in posterior esophageal gland area, 103- 146 ym from oral disc (mean = 124 ym) . Hemizonid 4 ym long, located just posterior to excretory pore. Stylet typi- cally tylenchoid. Telorhabdions sloping posteriorly. Cheilorhabdions extending through lip annules 2 to 4 . Esophagus comprising a moderately swollen procorpus that dilates just prior to oval, distinct metacorpus containing a valve slightly smaller than that of female. A narrow isthmus precedes a single ventral esophageal gland with single nucleus. Cardia not observed. Nerve ring appear- ing as an irregular band of tissue overlapping isthmus, and extending past the esophageal gland about 1/3 of its length (Fig. 15) . Intestine overlapping about 1/2 of esophageal gland and extending uniformly to cloaca. Tail bluntly hemispherical to truncate; tail terminus annu- lated. Anal lips in form of tubus (Fig. 25) . Caudal alae absent. Spicules equal and slightly arcuate when seen in lateral view. Capitulum moderately swollen, followed by slight constriction, and moderately swollen calomus . Lamina wide at the center tapering at both 33 extremities. Sclerotized piece arising at junction of lamina and calomus and projecting along ventral wall of calomus. The gubemaculum with teeth on lateral sides of cuneus seen in ventral view when cuneus is situated between spicules (Fig. 26) . Male gonaduct originating from ventral face of the cloaca. Narrow vas deferens (Fig. 15) about 90 ym long is followed by rather long seminal vesicle, usu- ally filled with sperm. Germinal and growth zones sometimes indistinguishable. Testes have been observed in which en- tire tube was filled with sperm and an observable germinal and growth zones were not present. Cephalids and deirids not observed. Larval description. (49 first-stage larvae; Fig. 8) Length = 492 (430-540) ym; width = 18.5 (16-20.2) ym; tail = 53.6 (46-64) ym; esophagus = 163 (132-190) ym; a = 26.5 (22- 30); b = 3.2 (2.6-3.7); c = 9.1 (6.7-10.5); anal body diam- eter = 4.5 (4.C-5.2) ym; stylet = 23.1 (21.5-24.5) ym; dorsal gland orifice 6.4 (4-8.9) ym posterior to telorhabdions ; excretory pore 93.8 (79-103) ym from oral disc. Body vermiform, labium rounded, cephalic framework consisting of 16, C-shaped sclerotized pieces lying 4 ym below oral disc. Head bearing 6 annules. Four lateral incisures beginning as single line, 45 ym posterior to oral disc forming 4 lines at median procorpus. The 4 lines resolve into a single line just posterior to anus (30 ym from the tail tip) . Width of lateral incisures at mid-body is 5-7 ym. Excretory pore located in mid-isthmus 34 Figure 26. Male tail in a ventral viev;, showing toothed cuneus of gubernaculum (arrow) Figure 27. Larval tail shapes 35 area. Hemizonid located 1 annule anterior to excretory pore. Stylet well-developed, prorhabdion 10.8-12 ym long. Rounded telorhabdions usually laying in an even plane, occa- sionally sloping posteriorly. Procorpus (38 ym long by 6-7 ym wide) , moderately swollen, narrowing just prior to the well-developed metacorpus (15 yin long by 12 ym wide). Metacorpus valve a wide oval shape. Isthmus narrow, 25 ym long. Esophageal glands about 35 ym long, lying on ventral side of body. Posterior lobe sometimes filled with coarse granules (digestive fluid) . Coarse granules also appearing in anterior end of anterior lobe, in isthmus, and in a large vesicle in posterior part of metacorpus (Fig. 8). A single large nucleus present in the posterior esophageal gland lobe. Anterior esophageal gland lobe not strongly set-off. It is delineated by a weak line of demarcation on posterior lobe, and has a very large nucleus surrounded by a large clear area (Fig. 8) . Esophageal glands overlapping intestine by about 1/2 of their length, extending as a straight tube to undilated rectum. Anus oval, 1 to 2 ym wide. Nerve ring appearing either as group of nerve cells (Fig. 31) , or as fine band of tissue surrounding isthmus (Fig. 8) . Genital primordia appearing about 160 ym ante- rior to tail tip (Fig. 8). Tail conoid (Fig. 27), with 26-29 annules. Tail tip usually awl-shaped. Hyaline area of tail 25.4 (23.5-31.3) ym long. Deirids, phasmids and cardia not observed. 36 Ova. (Fig. 28) Eggs broadly oval 50 X 100 ym. No markings observed on shell. An en-utero egg was 52 X 103 ym. Third-stage larvae. (10 specimens) Length = 572 (500-677) ym; width = 31.3 (27.4-34.3) ym; tail = 11.1 (9.8-13.7) ym; a = 19.5 (17.3-22.6); c = 54.5 (46.7-63.2); dorsal gland orifice = 3.9 (2.9-6.8) ym; excretory pore = 115.9 (109-122) ym. Body slightly swollen, tail rounded, head and esoph- agus similar to that of first-stage larvae (Fig. 29) . Type specimens. Holotype collected May, 19 69 by Wayne W. Smith. Collection number B-5018; Allotype same data as holotype. Type slides in Bureau of Nematology nema- tode collection, Division of Plant Industry, Florida De- partment of Agriculture. Type habitat. Soil about roots, and roots of Diodia virginiana growing near bodies of water. Type locality. Irrigation ditch bank bordering Hwy 50, 4 miles west of Hwy 27 near Clermont, Florida. (Orig- inal site now commercially developed. Anatomy Lateral Incisures These structures are very difficult to see in live or fixed specimens even when various stains were used. It was possible to see them, however, by squeezing out the body contents and examining the lateral sides of the integument 37 l^.-.j!>J-'i-^'^-^ i;^iJiU-U.:iJ Figure 23. Ova. Top to bottom: 2-cell, 3-cell, 4-cell, 5-cell, tadpole stage, first-stage larva . Figure 29. Anterior portion of third- stage larval male. using an oil immersion objective. This procedure was not necessary for males and females. Phasmid. Over a hundred each of males, females, and first-stage larvae were examined for phasmids with negative results in both ventral and lateral views. The phasmid and its lining were only detected on the cast integument of first-stage larvae early in the first molt. The phasmid was located between the 14th and 18th annule from the tail tip. When its location was known, fixed and living first- stage larvae were examined to see if the phasmid was detect- able; in no case was it observed. Muscles Vulva. The dilator vulvae musculature are well devel- oped in broad bands in mature females, extending from vagin- al epithelium to a ventrolateral insertion in the hydodermis (Fig. 22) . A band of muscle also attaches to vagina on either side of median part of vulva, herein called "median dilator vulvae" (Fig. 22) . Rectal muscles. Rectal musculature was observed in a third-stage female (Fig. 30) . The H-shaped muscle surrounds the rectum or rectal intestinal valve. The depressor ani extends into the dorsal hypodermis, and the dilator ani is inserted in ventral hypodermis. A sarcoplasm band nucleus as described by Chitwood & Chitwood, 1937 was not observed. 39 Procorpus and Metacorpus In third-stage larvae the procorpus is short and stout while metacorpus is a well-developed, wide oval. The esophagus of mature females is very similar to that of third-stage larvae. Geraert, 1978, found that the metacor- pus enlarges in saccate females (Heterodera carotae Jones, 1950) , as was the case in V, volvingentis . In males the esophagus is shorter, more slender and the metacorpus is smaller and more elongate. Nervous System In males and females stained with chlorazol black-E the circura-esophageal commissure appears as a flat band of tissue that surrounds the posterior part of isthmus (Fig. 11, 15) and proceeds posteriorly a short distance past the ante- rior part of the basal bulb as 2 ventral ganglion. Anterior and posterior nerve cords were not seen. In first-stage larvae stained with chlorazol black-E, the circum-esophageal commissure appears looped around the isthmus, either as a flat band of tissue (Fig. 8) or as an accumulation of nerve cells (Fig. 31) . The ventral ganglion proceeds posteriorly a short distance, branching dorsally and ventrally. The dorsal nerve arises from the dorsal portion of the ventral ganglion, and becomes indistinguishable a short distance posterior to the esophageal gland. The ventral nerve arises from the ventral portion of the ventral gnaglion, and pro- ceeds as a chain of ganglia (92 in 1 specimen) in the 40 Eigure 30. Rectal muscvi.lature . A) Rectal-intestinal valve area: (1) sarcoplasm, (2) depressor ani, (3) dilator ani, T4) rectal intestinal valve. B) Rectal area: (5) mid-rectum. D) Dorsal side. V) Ventral side. -ff^ Figure 31. Anterior nervous system in a first-stage larva, A) Anterior ventral nerve cord; b) Circum-oral commissure; C) Ventral nerve; D) Dorsal nerve; E) Hemizonad 41 hypodermis. The ganglial chain forms rectal commissures that surround the rectum with 3 dorsal and 5 ventral ganglia (Fig. 33) . A dorsal rectal ganglion (Fig. 33) is present where rectal commissures rejoin post-rectally in the dorsal position. Three ganglia are present in the medial caudal nerve (Fig. 33) . Anteriorly, a large ganglion arises from the dorsal portion of the nerve ring, and one from the ven- tral side (Fig. 31) . The 2 nerve cords extend around either side of the metacorpus forming a small mass of nerve cells just anterior to the metacorpus. Dorsal and ventral nerves proceed from this ganglion to sclerotized area of the labium. Cephalic nerves appear as elongate, spindle-shaped proc- esses. Lateral and papillary nerves were not detected. Crystalline Layer Brown et al, 1971, reported the subcrystalline layer is a complex of long-chain fatty acids. It was hypothesized that sugar exudates from the integument of Heterodera spp. are converted to long chain fatty acids by soil fungi there- by producing the crystalline layer. A crystalline layer was observed on the integument of about 10% of the females of the new genus. This layer assumes the form of the striae and other designs and modifications present in the parent integument (Fig. 34-B,C) . The subcrystalline layer is usu- ally fragmented and sloughs off the female body (Fig. 34-A, D). 42 Figure 32. Ventral nerve cord (arrow) in the area of the genital priir.ordia. Figure 33. Nerves in the tail of a first-stage larva. A ventral ganalion; b) rectal commissure; C) dorsal rectal ganglion; D) dorsal viev/; E) medial caudal nerve; L) lateral view. 43 Figure 34. Crystalline layer. A) Separation of the layer from the anterior end; B,C) Layer at mid-body; D) Layer fragmenting from tail area. 44 Gubernaculum A gubernaculum was isolated from the surrounding tissue for observation of the dorsal and ventral faces (Fig. 35) . The dorsal face is longer, measuring 16 ym, and shows serrated margins on the cuneus (Fig. 35-D) . The ventral face is shorter (11 ym) , ventrally grooved, and serrations were not observed in the focal plane (Fig. 35-V) . SECTION II REPRODUCTIVE DEVELOPMENT Early Development The female deposits a naked undivided egg in the environment (gelatinous matrix absent) . Four-hundred eggs in lots of 50 were examined under the oil immersion lens to determine if a molt occurred in the egg as described for Heterodera rostochiensis Wollen- weber by Hagemeyer, 19 51, and in Meloidogyne sp. by Christie and Cobb, 1941. In no case was evidence of ecdysis present. After examination, the larvae were expelled from the eggs (Fig. 36) by exerting a gentle pressure with a fine needle tip on the cover slip. None of the larvae ex- pelled from the 400 eggs showed evidence of ecdysis. It is concluded based on these data that a molt does not occur in the egg. First-stage larvae possess binucleate genital primor- dia with posterior and anterior cap cells (Fig. 32, 38-A) . Shortly after the first molt, determination of sex is pos- sible by examination of the rectal area. If spicular pri- mordia cells are present (Fig. 37) , a male is developing. Absence of spicular primordia cells indicate a female is developing. 45 4 b '\ Xh. (^ Figure 35. Gubernaculum (top is anterior) . V) ventral face (4 ym wide by 11 ym long) ; D) dorsal face (5 um wide by 16 m long); l=cuneus , 2= corpus, 3=crura. Figure 36 . Larvae forced from egg by applying cover slip pressure. Figure 37. Spicular primordia cells in the cloacal area of an early second-stage male. Phasmid is shown (arrow) on first-stage exuviae. 47 Male Developinent Shortly after the first molt, and before the first- stage integument is cast off, the genital primordiura begins to divide and proliferate posteriorly (Fig. 38 B,C) . The body widens, and the testes join the spicular primordia shortly before, and after, the first molted integument is lost (Fig. 38-D) . Sperm cells are large and angular at this stage. Following the second molt, little development is evident in the testes and spicular primordia. The esoph- agus is not clearly differentiated at this stage of devel- opment. The gubernaculum is the anlage of sclerotization, followed by the lamina of the spicules. The calomus and capitulum are the last to become sclerotized. Development proceeds to completion after the second exuviae is cast. In 2 cases observed, the male left the third-stage exuviae embedded in the root. Empty exuviae are not uncommon in in- fected roots. Table 3 shov/s the length of time required for development of males. Male development from penetration of the first-stage larva until a fully developed male was ob- served took place in a minimum of 6 days and a maximum of 15 days in roots growing in water agar. Three first-stage larvae that entered a root about the same time all molted to the second-stage in 48 hours. Twenty-four hours later, all 3 molted to the third-stage. Three days later the final molt occurred for all 3 males within 9-^ hours. Total aver- age time required was 10 days and 20 hours. Other periods 48 Figure 38. Male reproduc- tive system development: A) genital primordium; B) Genital primordium, 4-cell stage; C) Genital primor- dium, early third-stage male; D) Reproductive system in late third- stage male. l=spicular primordia. 2=vas deferens. 49 of male development observed were: a 15-day cycle; a 10-day, 4-hour cycle (Table 3); and a 6-day cycle. Table 3. Male Development Time (hours) Activity Width (um) 0 penetration 2^1 32^ 48 feeding 11 11 " " 72 ecdysis (first) 81 M M 96 " " 105 feeding 122 " " 145 shedding integument 168 176y 192^ feeding 11 11 ecdysis (second) 28.2 32.9 32.9 42.3 37.6 37.6 37.6 42.3 49.3 49.3 47.0 51.7 42.3 19 5 Third-stage larva emerged from 4 2.3 exuviae which rem.ained in root. 197 Migration along root (length 37.6 700 ym) , stylet 24 ym. 202 Migration to a nev; root, no 38.6 physical change. 212 no change 3 8.6 236 ecdysis (third & fourth) 38.6 24 4 development complete 33.3 Total time = 10 days, 4 hours. It is shown in Table 3 that the width of the feeding larva increases with time until 1762" hours have elapsed when a maximum width of 51.7 ym is attained. After the second ecdysis the width decreases until the male is fully devel- oped with a width of 33.3 ym. Feeding has ceased at this time and it is postulated that the decrease in width is due to energy expended during ecdysis and migration. 50 Female Development Shortly after the first molt the body swells and the genital primordium proliferates anteriorly, and posteriorly (Fig. 38-A,B) . The cells in the center bulge toward the body wall forming the vaginal primordium, after which the anterior and posterior branches elongate and develop (Fig. 39). The vagina first appears as a large opening with very large vaginal primordia cells on either side (Fig. 40). After the second molt the body swells and the gonad com- pletes its development (Fig. 41) . The reproductive system is complete when the third-stage exuviae is cast (Fig. 42) . Mature females (Fig. 11,43) are usually swollen more than virgin females, possess convoluted ovaries, and contain sperm in the spermatheca (Fig. 43) . The vagina uterina was very narrow in a few females (Fig. 44-C) . In older females the vagina uterina is well-developed, with thick folds capa- ble of containing several eggs. In several females a severely prolapsed vagina was noted (Fig. 23-right) . Female Life History Only 1 female developed to maturity in life history tests. The onset of ecdysis was never observed. Vulva development was seen 4 days after root penetration by the first-stage larva. The ovaries were defined 7 days after penetration, a fully developed female was evident 17 days after penetration. Seventeen eggs were deposited on the 51 .^,m'^m&mm Figure 39. Early third-stage female gonad. Figure 40. Vaginal development of a late third-stage female . Figure 41. Virgin female with gonad development complete. 52 Fiqure 44. Anatomy of the female reproductive system from vulva to uteri: A=Egg in uterus; B=uterus cells; C=Narrow vagina uterina; D= Vagina; and E=:Vaqinal muscles. Figure 42. Final female ecdy Figure 43. Mature female, 53 same day development was considered complete. Males were not observed near the female prior to oviposition. Conclusions Critical examination failed to reveal ecdysis in the egg. Early development proceeded as known in most phy to- parasitic nematodes. The distinct spicular primordia that appeared early in male development was not noted in other similar studies (Chitwood & Buhrer, 19 46; Christie and Cobb, 1941; Hirschmann & Triantaphyllou, 1971; and Raski, 1950). Another unique feature of male development occurred when the male abandoned the third-stage larval integument, leaving it embedded in the root following final ecdysis. Male growth measured by body widths during develop- ment has not been reported in other developmental reports examined by the author. A loss in width of 18 ym was shown from a maximum of 52 ym. Time for male development varied from 6 to 15 days. The unique feature of female development was the occurrence of huge vaginal primordia cells. SECTION III HOST-PARASITE RELATIONSHIPS Methods A variety of devices and ideas were tested to study the host-parasite relationships and life history of the new nematode species, all but one of which were unsuccess- ful. Macro-Observation Boxes Wood chambers were patterned after rearing chambers used by Dean, 1929, and by Minton, 1962. The box is 23-cm square with a 2.3-cm chamber enclosed by glass and removable wood sides. Buttonweed plants, well established in white sand in the chamber, were inoculated with groups of 100 larvae at the site of healthy root flushes under the glass. Development of the nematode either failed to occur or took place in areas away from visible root sites. This method was abandoned after a number of failures. In the next trial plastic boxes, 17.5 cm long by 9 . 3 cm wide by 3 cm deep, were filled with either white, or with black volcanic sand, planted with buttonweed and then inoculated with 100 larvae of the new species. In this system the activities of the nematodes were obscured by the substrate. The macro- observation boxes were used to observe nematodes on roots growing in soil. Limitations of magnification and depth 54 55 of focus severely handicapped close observation by this method. Micro-Observation Units Trials were conducted utilizing small plastic boxes of various sizes, and plastic petri dishes containing a poured 4-miTi layer of water agar. Success was assured using the following procedure: A 4-ram layer of 1% sterile v;ater agar is poured into a 9 . 2-cm plastic petri dish lid. A 5-mm ring of water agar is removed from the outside peri- meter of the agar ring after hardening. A 5-mm glass rod is heated over a glass flam.e until slightly red, then used to burn a hole into the side of the closed petri dish. The burn area should be sanded so the dish can be easily sepa- rated. A stem cutting of buttonweed with 1 or 2 small leaves is inserted through the hole and into the agar with the leaves external to the dish. When primary roots emerge and grow into the agar, a 5-mm well is cut into the agar 1-cm lateral to a primary root. Twenty-five first-stage larvae, and 5 raature males in a small drop of sterile water were inoculated into the agar well. A root map was drawn (Fig. 45) wiien larvae made contact with the root. Each larvae that situated itself at a particular site on the root was assigned an alphabetical letter which was placed at the approximate site on the root map. For each observation, the date, time, dish number, and larva letter was recorded. Ob- servations and measurements were made using the oil 56 immersion lens by placing a small drop of water on a cover slip, which was inverted and placed over the root site where larvae were attached. Basic data taken when possible included: time elapsed from the inoculation to the time larvae penetrated the root, time elapsed between penetration of the larva to the appearance of root discoloration, if and when a larva left its feeding site, body width measurements, and ecdysis observations. Behavior Studies Eighteen plates containing plants in agar were inocu- lated. Life history activities were observed in 5 plates; the remainder were abandoned due to plant death, visibility problems, or severe bacterial contamination. As soon as the water in the inoculation well dried the males and larvae migrated into the agar. Male Behavior Males migrated at random in the agar. A proclivity to the root by males was not noted. Several males were seen with lips in contact with the epidermis of a primary root. In no case was stylet movement or metacorpus valve pulsation noted in such contacts. One male lay quiescent very close to a primary root the duration of the trial. Most males migrated slowly through the agar after which they became quiescent. 57 Larval Behavior A total of 49 larvae of 450 inoculated was observed penetrating roots. Data were taken until they departed, ceased activity, or completed development. Root penetration. Larvae migrated to the root follow- ing pathways peculiar to most nematodes in agar (Wallace, 1964). One group of 4 larvae reached the root in 19, 23, 30, and 36 min. , respectively, following inoculation. Sty- let movement was initiated about 4 min. after lip contact with the root. Stylet thrusts were recorded at 92 per min., and 112 per min. by 2 larvae shortly after contact with the root. Klinkenberg, 1963, recorded 69 thrusts per min. for Pratylenchus crenatus Loof, 1960. Pressure was exerted on the epidermal surface by thrusts of the nematode head and stylet. The head slid over the cell surface as it thrust until the stylet tip was over the middle lamella between 2 epidermal cells (Fig. 46-A) . At this point the metacorpus valve moved intermittently indicating digestive enzymes were extruded into the attack site (Fig. 47) . The lamella between the 2 cells separated and the nematode slipped laterally into the opening (Fig. 46-B) . After penetration, the larvae migrated obliquely 1-3 cells and 1-3 cells deep (Fig. 46-C) . One root with a 100 ym diameter was penetrated 61 ym laterally and 50 ym deep. Feeding. Once the larvae were situated in the root, feeding began immediately. In very small roots, feeding 58 V ^ Figure 45. A root map charting the progress of each nematode that occupied a feeding site in one of the inoculated petri dishes. Each letter represents a larva at a feeding site. Figure 46. Mode of root entry by larvae. A=larva with head cen- tered on middle lamella between two cells. B=Penetration. C=Feeding site. 59 ^^^'\k'r'^ ^ ''^" >^^ Vx /.- ■^w^ v^^^^-.;- 1 "^. Figure 47. Larval head in contact with middle lamella. Note small area of discoloration in front of oral aperture. Figure 48. A mature female in a shallow, hand-cut epidermal section. occurred in the cortex or pericycle. One larva fed in a cortical cell occupied by another larva feeding in the peri- cycle. Feeding sites were rather shallow (1-4 epidermal cells deep) in mature roots. A very shallow hand cut longi- tudinal section (Fig. 48) underneath a feeding female rare- ly cuts the female. Tissue discoloration. Yellowing of the tissue (Fig. 49-A) appeared initially 3, 4, and 4^ hours following pene- tration. In some cases (Fig. 49-B) the discoloration was confined to the cell wall. Larvae were also noted with the stylet inserted in the cell wall (Fig. 49-B). Nuclei of 60 Figure 49. Tissue discoloration. A) Heavy stipipled area indicates yellov; discoloration in cells 42" hours after larval entry. B) Stippled area indicates yellov7ing in cell walls. Note difference in nuclei size between healthy and attacked cell. 61 cells with discolored walls were consistently enlarged (11-13 ym) in comparison to healthy cells containing nuclei (5-7 ym, Fig. 49-B) . Feeding migration. A few larvae touched the root and departed without entry. Eleven larvae entered the root, fed, and after a few hours or several days departed. Some of the departing nematodes took up a new feeding position on the same root or entered a different root and resumed feeding. Some larvae were never seen again after leaving a feeding site. Entry of an epidermal site predisposes the site for entry of searching larvae. Nine larvae were seen feeding together in a single, large longitudinal lesion (Fig. 50) . Such lesions are usually abandoned by feeding larvae. One assumes the excess of enzymes and metabolites in such a large open lesion renders the site unfavorable for the development of the nematode. Attack sites. Larvae have been detected feeding at root tips (Fig. 51-A) , root scales (Fig. 51-B) , along feeder roots, and rhizomes, singly, or in groups (Fig. SI- CD). In mature rhizomes, females are commonly seen either singly or in groups (Fig. 51-E) . Females can almost always be found at the junction of secondary roots emerging from the rhizome (Fig. 51-F) . Larvae and third-stage males have also been observed on chlorophyll-bearing stem tissue at the soil line. Larvae and mature females have been de- tected in root leaf scales just below chlorophyll-bearing aerial leaf scales (Fig. 51-B) . Many females resembling 62 Figure 50. Large lesion occupied by 4 larvae. 63 Figure 51. Attack sites. A) Larvae feeding at root tip; B) Larvae feeding on root-leaf scale; C) Larvae feeding on feeder root; D) Large group of larvae in rhizome; E) Group of mature females in mature rhizome; F) Females at secon- dary root juncture. 64 small white sausages lie appressed to large rhizome pieces (Fig. 51-E) . Host exudates. About 5% of the females examined pos- sessed an accumulation of spheroid objects around the cervi- cal region (Fig. 52) . The exudates were closely associated with the integument but did not adhere as do the cement bodies adhering to the integument of cyst nematodes de- scribed by Shepherd and Clark, 1978. The cement bodies are depicted as brown hardened exudates originating from the integument and grossly resemble the spheroid bodies of V. volvinqentis. Exudates of the new species differ in ap- pearing to have a crystalline composition (Fig. 53) . The spheroid bodies appear to be exudates originating from the host in response to feeding activities of the new genus. Exudates have also been noted in Meloidodera flori- densis (Fig. 54) , Oviposition and fecundity. Eggs are deposited naked in the substrate. Five to 25 eggs usually lie inside the ventral space formed by the body coil, or they are scattered about the female body near the vulva. One large egg mass contained 18 5 eggs in various states of development. Stan- dard soil washing procedures usually wash the eggs from the female so the number of eggs deposited is difficult to as- certain. To determine the number of eggs contained in ma- ture females, 123 female specimens were examined (Table 4). 65 ''M& <<^ ^-1^ ^•'S/^ Figure 52. Spheroid bodies attached to anterior region of a mature female. Figure 53. Appearance of a single spheroid body . Figure 55. Female containing 7 eggs, Figure 54. Exudates on the anterior end of Meloidodera f loridensis . 66 Table 4. Number of fully developed eggs detected in 123 mature females. Egg number Females 0 47 1 28 2 22 3 8 4 12 5 5 6 0 7 1 Most females contained 1 or 2 eggs with a maximum of 7 eggs in 1 female (Fig. 55) . The largest egg measured was 128 X 70.5 ym, inside a female. Longevity. All plants which had been inoculated with V. volvingentis for pathogenicity trials had died by September, 1973. Five 1.9 X 20.3 cm soil plugs were re- moved from each of 7 pots and the nematode population counted (Table 5) . Most pots contained large numb)ers of eggs. The pots were maintained in the greenhouse in a fal- low condition until October, 1976, when a sample similar to the soil sample in September, 1973, was taken and the nema- tode population counted (Table 5) . No females survived the longevity test while male and larval survival was minimal. It appears feasible that eggs are the prime survival stage in this species. Egg viability. A 15-cm clay pot in which buttonweed was root-bound, but nematode free, was saturated with sterile water. The pot was then placed in a beaker and an addition- al 100 ml of sterile water added. The effluent was 67 Table 5. Survival stage and numbers of nematodes recovered before and after a longevity test. Pot Fema les Mai es Larvae Ecfq 3 Sept. Oct. Sept. Oct. Sept. Oct. Sept. Oct. 1973 1976 1973 1976 1973 1976 1973 19 7 6 1 2 0 0 0 18 6 552 257 2 0 0 18 0 8 0 1282 5 3 1 0 0 0 0 0 198 6 4 0 0 1 0 3 0 41 287 5 0 0 0 0 0 0 1 2 6 1 0 1 0 6 0 811 2 7 0 0 0 2 8 6 336 129 Total 4 0 20 2 43 12 3221 688 Mean .57 0 2.85 .28 6.1 1.7 460 98.2 collected, and added back to the pot. The collection and addition procedure was repeated 9 times, after which the final effluent was filtered and the leachate placed in an Erlenmeyer flask in the refrigerator. To test egg viability, 10 eggs from 3-year-old fal- lowed soil were placed in a drop of sterile water in each of 4 dishes. Ten drops of stock leachate was added to the water containing eggs in each of the 4 dishes. Four simi- lar dishes containing eggs in sterile water but without leachate served as controls. Results of the test are shown in Table 6. Table 6. Larval hatch from eggs kept in fallow soil 3 years. Larvae emerged Examination Sterile water Sterile water date and leachate only Dish no. Dish no, 1-2-3-4 1-2-3-4 10/26/76 0-0-0-0 0-0-0-0 10/27/76 0-0-0-0 0-0-0-0 10/28/76 1-0-2-0 0-0-0-0 10/29/76 0-0-1-0 0-0-0-0 11/ 1/76 1-0-0-0 0-0-0-0 11/ 4/76 0-0-1-0 0-0-0-0 11/ 8/76 1-3-4-2 0-0-0-0 Total 3-3-8-2 0-0-0-0 Results. Larvae em.erged from eggs only to which leachate was added. Eggs placed in sterile water failed to hatch. The test demonstrated that eggs can survive 3 years in fallow soil. Attempts to inoculate buttonweed with lar- vae hatched from 3-year-old eggs from fallow soil were 69 unsuccessful. Failure is attributed to low inoculum levels and reduced nematode viability. Conclusions Larvae entered roots by penetrating the middle lamiel- la bet^veen 2 epidermal cells. Tissue discoloration became evident 3-4 hours after entry of the nematode into the root. A number of larvae abandoned the site after actively feed- ing. Nuclei in invaded cells were distinctly larger than nuclei in cells not entered by the nematode. Host exudates were noticeably extruded at attack sites and these exudates adhered to the cervical area of female feeding at the site. In longevity tests, eggs and larvae survived 3 years in the absence of a host. Results indicate that ova are the survival staae of this nematode. SECTION IV HOST PLANT INVESTIGATIONS Host Plant Diodia virqiniana L. (buttonweed) in the family Rubi- aceae is a perennial of no known economic importance. Buttonweed (Fig. 56) is comprised of smooth, weedy stems bearing lanceolate leaves. The plant creeps across the soil as it grows forming a dense mat v/hen abundant (Fig. 57) . It usually is found in a mixture of herbaceous plants peculiar to vegetation growing near bodies of water. The vegetative form is found from Florida West to Texas, and North to New England and Missouri (Small, 1933; Rickett, 1967) . Host Habitat Buttonweed is found growing on moist soil adjacent to bodies of vjater such as lakes, ponds, water-bearing ditches, swamps, and prairies (Fig. 58). Maximum growth appears from 1 to 40 m.eters from the water's edae. As soil becomes less moist and elevation increases, buttonweed decreases until none are found . When collecting buttonweed a body of water will almost always be in sight. Norton, 1978, lists 15 genera of phytoparasitic nem.a- todes comprising 30 species in aquatic habitats. Eight genera and 2 species listed by Norton were found associated 70 71 Figure 56. Diodia virginiana in flower. (A Susan Anthony dollar Ts in the background for comparison. ) Figure 57. A mat of buttonweed mixed with herbaceous plants. 72 Figure 58. Paynes Prairie, Gainesville, Florida. A prime habitat for buttonweed and Verutus volvingentis . with buttonweed in an aquatic habitat. Ten genera and 23 species of phytoparasitic nematodes not included in Norton's list were detected (Table 7) . HosL Testing A primary consideration, when a previously undescribed phytoparasite is encountered, is the determination of its potential as a parasite of economic crop plants. In the original site where the new genus was collected corn and soybean plantings were established adjacent to buttonweed plants. The 2 aforementioned plants, in addition to 19 other plants, were placed in soil infested \/ith the new genus. Results of the host testing are shov/n in Table 8. 73 Table 7. Phytoparasitic nematodes found in soil associated with roots of buttonweed. Phytoparasite Occurrence Belonolaimus sp, infrequent Cacopaurus sp . moderate Criconema sp. infrequent Criconemoides curvatum Raski, 1952 frequent mutabile Taylor, 1936 infrequent " " xenoplax Raski, 1952 Dolichodorus he teroc'eph a lus Cobb, 1914 moderate Helicotylenchus crenicauda Sher, 1966 frequent dihystora (Cobb, 189 3) infrequent Sher, 1961 " " erythrinae (Zimmermann, " " ~ 19 04) Golden, 1961 " " longicaudatus Sher, j.9 6 5 paxilli'~Yuen, 19 6 4 Hemicriconemoides wessoni Chitv/ood & Birchfield, 1957 " " Hemicycliophora zuckermani Erze ski, 19 6 5 Heterodera sp. Meloidogyne arenaria (Neal, 1889) Chitwood, 19 4 9 Pratylenchus brachyurus (Godfrey, 1929) Filipjev and Schuurmans Stek- hoven, 19 41 Trichodorus christiei 7>.llen, 19 57 frequent proximus " " " " infrequent Trophoncmr. arenarium (Raski, 19 56) Raski, m^oderate 19 57 Tylenchorhynchus irregularis VJu, 19 6 9 Xiphinema americanum Cobb, 1913 infrequent 74 m 4J cH to C •H to ■P 4J to O Xi c CD tn •H > O > m d Vi > w H H PI 'X3 1 1 1 w 1 W K W C 03 c^j n 1 O 1 1 1 ^ iH X ^-q Oi H X W W '/; "^ -^ y. o> H UD •=3' X n ro 0 •H 0 1 1 O 1 1 o o 0 o 1 C M-l 0 CO ^ iH 1-1 ^ CM ro XJ (J hq X! M J W 1 1 1 1 1 1 i 1 0) CN CM T— 1 rH n rvi CM o 4J cn (C -P Pm E O o O o o o o O o O O O 1 O o o o o O O O (U O yo S PC ^ -p (U m 0) o S -p (NroTOr\i'^'cofN«*corsicO'^cooo(N tn & c X! O !h (S iH (U U nH m , — . O •H >i ^-^ 5 C 0 > C ,-^ >i o T-f -rH tr> Sh ^ M a- ^ 4-1 rH --- d Gi -p -H -- cri 0 m (U !h C •H M-l c a j:; O 4-1 O CO 0 U xi fd ^ o Jh fC' CQ ^- iH (1) C> HH Vi — -tJ E E — C • Sh M-l N Oi - f3 -- -p •r-l O fd K fd Q) ^— G.i >i G ;>- 0 >H 4-1 4-1 (1) P, a E • rH 4J .13 U CL— - O p.— 4-1 ^ <^ — ' OJ 0 4J I 3 u 0 c — 0 -- ri.'. C"" t/: r, fd >. CJ ^ U rd T^ -^ • X 0 G j:: -P fd Sh (U Q) n ,q U CO QJ C fd fd ^ u o O U C !H .-. 0 .C x;' •H C) CD .Q rd iH !h -H CO U E O P XI • t-. 4-1 a- 1) E — • 0 CU ^ fd -H fd E « ra iji fd Sh ■J] -H O K S — -Q O Sh O • O O ""^ Q-i :i! 4-J i-q ^Q r:i • o u . g (D S TD r-l > CO ^ ^ O 0 — — — — E t^ 4-1 --UP Tl '"-' — • ^-^ r-\ , — 4J e 3 i-i • • |3 •-^ •-^ -r4 i:; •H i-T a) s u yt m • • >J i-:i 4-1 • CO T3 fd (D to to W (U E -- ,-0 -H !h h^ ^ c fd . t-q c Sh iH W ■H - w c ^ ^ U rH Sh U5 OJ 4-1 h^ GJ id ^-' C P 13 -H — • — rci OJ E 3 fd rH fd U) 'iJ o (L) Sh , — . to CO h^ C 4-1 G p 4-1 E ■rH r^ >i . •H 0 C o CU • --H *"' ^1 c fd 0 c-H p Sh m 1-q rH M-; !h s:; .•3 h^ W U5 h^ :3 • =3 0 tH w fd u fd o o ■H 0 •I-! -P :i tn h^ ID r— > (U y. (d Cr. U2 fd N 4-1 rj Cij fo 0! £ • > rs G Jh p p 0) x; ,-H • 4J fd C ^-' m c ■H -; CU-rl 4-1 fd ^^ M o Sh c fd :^ c Qa fd u fd 0.1 S^ '^ in 4-1 0 0) fd Lc; CO a 0 w 4-1 > 0 W 4-1 Xt •H • 00 '.4 rO 5 trl U SI cy (-2, u ■H Sh ,Q 4- 'V ^ M o D"' ^ rr! W rO fd g •H 0-, fd m ""0 P -H rd fd i^ rH 4-1 C O -r4 n ;:? en fT CO G) c D r- •0 > •H m 4J m 13 C •H Cfi !h OJ -H p •H Sh r-l fd rH oi 7 rd y~l n ■H > (d rH :3 in fd c r. u (T. (U 0 •H o 'u to Sh fd rd en W 0) g P 4-1 -H >- (Ti •H illj iH 4-1 G 0 ■H RJ c; E Vi 11) d '^", 1 U -H O CO ■H > o ^ O W rH Sh -c; 0 fC T^ jj p o p tn >i w S2 rrj o "■• fd ■H 0) o i2 fd e S-l (U .c 0 p fd -rH iH o , p I>1 0 ■H -C _r:; 4-1 ■H P Q) .id of no economic im- portance. Larvae were not detected in any of the other host plants examined. Results of this host test and because this nematode has not been reported in numerous surveys of economic crop plants indicate the nematode is doubtful as a threat to economic crops, and vei'y likely has a limited host rjtnge. In the original site, the infested buttonvveed was found growing into plantings of both corn and soybeans, neither of which proved to be hosts in field samples or host tests. Since the nematode habitat includes irrigation 76 ditch banks serving as borders for numerous crops, the nema- tode has ample opportunity to infest a variety of crops, should they prove susceptible. Host Symptoms Aboveground symptoms of nematode injury v/ere not noted in any site examined. Symptom.s were noted only in inoculated population maintainence boxes, inoculated pots, and in plants grown in water agar. Generalized aboveground symptom.s include stunting, chlorosis, seed pod reduction, and death. Healthy green stoloiis and leaves gradually turn chlorotic until the entire plant is a pale yellow-gree^n color. Plants begin to decline and die 6 months after inoculation until no living plants remain. In the final stages of decline, germinating seeds produce small plants with 2-4 ].eaves that rarely assume a healthy g-ecn color. Such plants rarely survive very long, and their roots are almost always infected with fem.ales and larvae of V. volvingentis . Root sym.ptoms are expressed as shallow epidermal lesions of a pale yellow to brov/n or dark brown color. Lesions comprise 3-15 cells and are present at feeding sites. Lesions enlarge and darKen with time (Fig. 59). In a few cases, epidermal swelling in the form of a small rounded protuberance appeared at the feeding site. In very small roots lesions sometimes encircle the root resulting in a necrotic constriction that causes blocl'ing of 77 Figure 59. Young female in root, showing large dark lesion at feeding site. conductive elements in the root and subsequent detachment of the root at the lesion site. Feeder roots of buttonweed are very succulent and de- tach quite easily. Thus, it is not practical to compare root systems of infected and nematode- free plants under ex- perimental conditions. Pathogenicity Twenty cuttings of buttonweed were taken from plants grown in steamed soil and transplanted to steamed soil in 20-cm clay pots. Four months later all foliage extending outside the pot perimeters were clipped off, and 25 larvae of V. volvingentis were inoculated into each of 10 pots. Ten pots were left as uninoculated checks. Pots were ar- ranged in a randomized block design using "Tippets Random Number Table" (Le Clerg, Leonard & Clark, 1966). Pathogenicity was evaluated by clipping foliage ex- tending beyond the pot perimeter, and calculating the dry weights. Clippings were dried in a heat chamber for 3 weeks at 57 °C. The total number of seed pods produced were coun- ted on each plant just prior to taking perimeter clippings. Eight 20 cm deep by 2 cm wide soil plugs were removed from each pot to evaluate the nematode population. Soil from the 8 plugs was thoroughly mixed and 150 cc subsamples 79 were removed from each of the mixtures. Samples were proc- essed using the sugar-centrifuge technique. Results Table 9 shows the severe effect of the nematodes on the plant after 1 year. Thirteen days after final evalua- tion data were taken, 4 chlorotic, severely stunted plants still survived the initial inoculation (Fig. 60-A) . Leaves on the surviving plants were very chlorotic (Fig, 61) and seed production was reduced considerably (Fig. 62) . Inocu- lated plants yielded 2,9 gram.s of seed pods, while check plants yielded 15.7 grans. Seeds sprouting in infested soil in pots inoculated with the nematode were attacked at a very early stage of developm.ent and rarely produced more than 1 or 2 yellowed leaves before dying. Within 60 days of the final leaf evaluation, the 4 surviving inoculated plants were dead. All control plants were in a healthy state of growth. Table 10 shows the developmental stage and numbers of individuals in each stage in the 150 cc of soil examined from each replication. This table indicated that the egg is the survival stage of the nematode. Discussion The severe parasitisiri by the nematode resulted in eventual death of all inoculated plants. This occurred under greenhouse conditions when the plants were maintained Figure 60. Appearance of inoculated (A) and uninoculated (B) plants at the conclusion of the pathogejiicity trial. 81 Table 9. Effect of Verutus volvingentis on foliage and seed pod production of inoculated plants. Mean dry weight Days after of pot clippings Mean number of seed inoculation per plant in grams pods per plant untreated treated untreate _d treated 95 11.54 11.40 320.2 324.0 217 4.9 2.6* 0 0 398 24.0 7.2** 512.0 168.3 2 inoculated plants died. 5 inoculated plants died. Table 10. Nematode population density in treated and untreated soil. Days after Mean numbers of eggs or nema- Uninoculated inoculation todes from inoculated plants plants eggs larvae males females 406 1144 26 7 3.0 0 504 728 9 2 0.4 0 82 o^a)lHUMi£.^ - -""^- -■li'rniiii , j i i Figure 62. A comparison of seeds from inoculated (left) and uninoculated (right) . Figure 61. Comparison of leaves from inoculated (top) , and uninoculated plants (bottom) . several months past their normal annual cycle. Symptoms produced in the greenhouse were never observed in the natu- ral habitat. It is believed that the nematode population is in ecological balance with the environment a.nd is not large enough to severely damage its host when buttonweed dies out after about 6 months of growth, following the first frost. During the next 6 months enough nematode eggs and seeds of buttonweed survive to establish a well balanced association between the host and its parasite. In the greenhouse there is no intervening period to retard nematode population 83 growth. As a consequence the parasite increases unchecked, severely damaging or killing its host. Factors in the nat- ural habitat such as biological control agents and population-limiting physical changes are also absent from the controlled environment of the greenhouse. Distribution of V. volvingentis in Florida A total of 266 soil sam,ples, and an almost correspon- ding number of root samples, were examined from collection sites in 13 Florida counties (Table 11) . Ninety-eight samples were positive and 168 samples were negative. Plants examined in the survey are shov/n in Table 12. The nematode was detected in soil from 13 hosts in the survey, but in roots of buttonweed only. It is doubtful if the 12 plants surveyed are true hosts of the new nematode since most of them were growing close to buttonweed. The nematode v/as detected in Alachua, Lake, Orange, and Sumter counties. The largest population of the nema- tode occurred in Alachua county within Paynes prairie (Fig. 57) , which is a relatively undisturbed, natural, ecological unit in Florida. The prairie supports large plantings of buttonweed. Samples taken at the waterline or from high dry sites in the prairie are almost always free of the nem- atode and buttonweed. About 90% of the negative sample results originate from plants other than buttonweed in 84 Table 11. Areas in Florida samples for Verutus volvingentis , County Site SairiDle results Positive Negative Alachua Gainesville-Terwilliger school Nev/nans lake Paynes prairie Earleton Baker roadside Bradford " " Broward Pompano Beach Gadsden Chattahoocliee Lake Clermont Grand Island Howey-in-the-Hills Leesburg Minneola Marion Ocala Orange Plymouth* Zellwood Putnam Florahome* Seminole Sanford Sumter Oxford Rutland Tarrytown Union roadside Volusia Barberville 3 9 1 2 65 29 0 3 0 1 0 1 0 1 0 1 27 91 0 5 0 1 0 2 1 0 0 1 0 1 0 2 0 6 0 3 0 5 0 1 1 1 0 1 0 1 Buttonv/eed was not sam.pled at this site. Table 12. Plants examined for Verutus volvingentis in the Florida Survey. Scientific name Vernacular name Alternanthera so. Anciropogon glomoratus (Wa]t)B.S.P. Baccaris halinifo] ia~L. Bacopa caroliniana (Walt) G.L. Robinson Bidens laevis ( L . ) B . S . P . Brassica oleraceae L. (acephala group) Cassia obtusitolia L. Cephalarithus oc. ^Tden ::alas L. Circium sp. Cuphea cartitagensis (Jacq) Macbride Cyperus odoratus L. Diodia teres Walt " " vj rginiana L. Eupatorium sp. Geranium carolinianum L. Clottidium vesicarium (Jacq) Mohr Glycines iiiax (L.) Merr FjYpericurn iriutilum L. Juncus effu; L. Lachnanthes caroliniana (Lam.) Dandy Linaria sp. Ludwigia arcuata Walt leptocarpa (Nutt.) Hara Mikania scandens (L.) Willd. Nelumbo lut^a (Willd.) Pers. Paronychia' baldwinii Fenzl Faspalum urvillei Steud . Polygonum hydropiperoides Michx. " persicaria l' " punctatum Ell. Ptili.mnium c:a_£ill,-i_ceuiri (Michx.) Raf. RJiexia mariana L. Sabal palmetE^ (Walt.) Todd ex. Sohult & Schult f. Salix sp. "Scirpus sp. Scoparia dulcis L. StenotaTjhrum secundi Typha sp. Zantedeschia sp. Zea mavs L. (Walt, ) 0. Kuntze bushy beardgrass saltbush water hyssop beggars tick collards coffee-weed buttonbush thistle poor Joe buttonweed cranebill bladderpod soybean dwarf St. Johns wort soft rush redroot toadflax false -loosestrife climbing hempweed American lotus vasey grass smartweed heartweed water smartv;eed mock bishop weed meadow beauty cabbage pali:otto willovJ bulrush sweet broom St. Augustine gras cattail calla lily negative positive negative positive negative positive negative positive positive negative positive negative positive negative positive negative positive negative positive negative positive 86 higher, less moist areas, or from areas in water or at the watei-'s edge. Conclusions V. volvingenti.s occurs in Florida in moist habitats. Paynes Prairie contains large populations of the nematode and its host. The host range appears to be very limited. None of the economic host plants tested were susceptible to the nematode. Three months following a low level inocula- tion of the nematode on its host, little or no effect was evident. Severe symptoms occurred 7 months after inocula- tion. Fourteen months after inoculation all inoculated plants were dead. All uninoculated plants in the test were in a healthy vigorous condition. It is believed that death of the inoculated plants was a result of growing an annual plant in the greehouse, devoid of natural inimical ecologi- cal factors present in its natural habitat, and past its time of normal growth. SECTION V BIOLOGICAL CONTROL INTEPACTIONS Males and larvae were exposed to zoospores of Cate- naria ang-uillulae Sorokin. Within 10 min., zoospores were attached to the cephalic region of larvae (Fig. 63-A), and males (Fig. 63-B) , and to the male cloacal area (Fig. 6.3-C) . Development of the fungus was only completed in males. Zoospores were released from an infected male 78 hours after the initial infection. Tv/o-hundred eggs were exposed to a culture of zoo- spores to test the susceptibility of ova to the fungus. Zoospores became attached to 25% of the eggs in culture but no thalli developed subsequently. Eggs were also exposed to a culture of an aquatic Phycomycete, Rhizophidium sp., a member of the Chytridiales , known to attack eggs of invertebrates. Sporangia of the fungus was observed attached to a number of eggs (Fig. 64); however, further development of the fungus was not noted. A natural population of the nematode v/as found infes- ted with endospores of Pasteuria ramosa Metchnikoff (Fig. 63-D) . A single larvae hatching fromi an egg in the popula- tion was also noted with endospores attached. 88 Figure 63. Biological control interactions. Catenaria anguillulae zoospores attached to the cephalic region of a larva (A) , a male (B) , male cloacal region (C) . Endospores of Pas teuria ramosa on a larva (D) . y '^^^-^m Figure 64. Sporangium of Rhizophidium sp. attached to an egg. 89 Females were not noted in biological control inter- actions. Biological control agents attached to ova but failed to penetrate the shell and complete development. LITERATURE CITED Andrassy, I. 1976. Evolution as the basis for the system- atization of nematodes. Pittman, London. pp. 288. Andrews, S.W., L. R. Krusberg, and A. M. Golden. 1977. The host range of a Meloidoderita species from Maryland. J. Nematol. 9:261-262 (Abstr.). Brown, G. , R. K. Callow, C. D. Green, F. G. W. Jones, J. 11. Rayner, A. M. Shepherd, and T. D. Williams. 1971. The structure, composition and origin of the sub-crystalline layer in some species of the genus Heterodera. Nematologica 17:591-599. Chitwood, B. G. 1958. The classification of plant para- sitic nemas and related forms. XV th Int. Congr. Zool. Paper 28:1-2. Chitwood, B. G. and E. M. Buhrer. 1946. The life history of the golden nematode of potatoes, Heterodera rosto- chiensis Wollenweber, under Long Island, New York, con- ditions. Phytopathology 36:180-189. Chitwood, B. G. and M. B. Chitwood. 19 37. An introduction to Nematology, Section 1, Anatomy. (Rev. ed, , 19 50) . Monumental Printing Co., Baltim.ore, Md . pp. 213. Chitwood, B. G., C. I. Hannon, and R. P. Esser. 19 56. A new nematode genus Meloidodera linking the genera Heterodera and Meloidogyne. Phytopathology 46:264-266. Choi, Y. E,, and E. Geraert. 19 73. Description of Meloi- nema kerongense N.G. Nsp. (Nematoda :Meloidogynidae) from Korea. Nematologica 19:334-341. Christie, J. R. and G. S. Cobb. 1941. Notes on the life history of the root-knot nematode Heterodera marioni. Proc, Helrainthol. Soc. Wash. 8:23-26. Cobb, N. A. 1919. The orders and classes of nemas. Contrib. Sci. Nematol. 8:213-216. 90 91 Colbran, R. C. 1966. Studies of plant and soil nematodes. 12. 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Scanning electron micro- graphs of the anterior region of Tylenchoidea (Tylen- chida :Nematoda) . J. Nematol. 7:69-83. Siddiqi, M. A. 1971. Structure of the esophagus in the classification of the superfamily Tylenchoidea (Nema- toda) . Indian J. Nematol. 1:25-43. Sokal, R. P. and P. H. A. Sneath. 1963. Principals of numerical taxonomy. W. H. Freeman & Co., London, pp. 359. Sparrow, F. K. 196 0. Aquatic phycomiycetes . Univ. Mich. Press, Ann Arbor, Mich. pp. 1187. Steiner, G. 1925. The problem of host selection and host specialization of certain plant-infesting nomas and its application in the study of nemic pests. Phytopathol- ogy 15:499-534. Stone, A. R. 1972. Heterodera pallida n. sp. (Nematoda: Heteroderidae) , a second species of potato cyst nematode. Nematologica 18:591-606. Thorne, G. 1961. Principles of nematology. McGraw-Hill Book Co., Inc., New York. pp. 553. 99 Thorne, G. and M. W. Allen. 1944. Macobbus dorsalis , nov. gen. , nov. spec. (NematodarTylenchidae) producing galls on the roots of alfileria, Erodium cicutarium (L.) L'Her. Proc . Helminthol. Soc. Wash. 11:27-31. Tyler, J. 1938. Egg output of the root-knot nematode. Proc. Helminthol. Soc. Wash. 5:49-54. Wouts , W. M. 1973. A revision of the family Heteroderidae (Nematoda:Tylenchoidea) . II. The subfamily Meloiderinae . Nematologica 19:218-235. Wouts, W. M. and B. Weisher. 1973. The authority of the subfamily name Heteroderinae . Nematologica 19: 563-565. Yuen, P. H. 1964. The female gonad in the subfamily Hoplolaiminae with a note on the spermatheca of Tylen- chorhynchus . Nematologica 10:570-580. Yuen, P. H. 1971. Electron microscope studies on Aphelen- choides blastophthorus (Nematoda) . I. The female genital tube. Nematologica 17:1-12. BIOGRAPHICAL SKETCH Robert Paul Esser was born November 8, 1924, in Hackensack, New Jersey. He graduated from Hillsborough High School in Tampa, Florida, served 4 years in the United States Coast Guard during World War II, and worked as a watchmaker prior to entering the University of Florida in 1951. He graduated from the University of Florida in 1955 with a B.S.in Agriculture. In 1955 he was employed by the "State Plant Board of Florida" (now "Florida Department of Agriculture and Consumer Services "). In 1955 he joined the Nematology Department as a Laboratory Technician. In 1956 he was appointed Nematologist . From 1956 to 1980 he has published 70 articles dealing with nematodes. He has lectured in Invertebrate Zoology and Soil Microbiology every year for the past 15 years. He was co-editor of the "Nema- tology Newsletter" for 3 years, Associate Editor of the "Society of Nematologists " (SON) for 2 years, and served on the Executive Committee of SON for 3 years. He was also chairman of the SON Data Retrieval Committee, and organized and chaired the first SON regulatory colloquium at Ottawa, Canada. He served 2 years on the survey committee of the "In-cersociety Consortium for Plant Protection" and is pre- sently on a special data retrieval analysis committee of the same organization. He is a member of FNF, ESN, SON, 100 101 OTAN, Soil and Crop Sci. Soc. Fla., and the Helminthol. Soc. Wash, He is married to Hannelore Esser, and has 3 daughters, Lorelei, Victoria, and Robin. I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. e.(' ', CHairme Ar](Qeji/C. Tar jan, Chaxrman Professor, Entomology-Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. VerrtonG. Per^ Professor, Entomology-Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Robert A. Diinn Assistant Professor Entomology-Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pre- sentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. )onald F. Rothwell' Professor, Soil Science This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. June, 1980 ^cp^k J ■ cv), Deafi,/ College of Agriculture Dean, Graduate School UNIVERSITY OF FLORIDA 3 1262 08553 5234