APPROVAL SHEE'l' Title of Thesis: Nematodes associated with roses and the root injury caused by Meloi~ogyne hapla Chitwood 1949, Xiphinema diversieaudatum (Micoletzey 1927) Thorne 1939, and ---..,.........____...__ Helicoty~nchus nannu~ Steiner 1945. Name of Candidate; Ronald Allan Davis }laster of Science, 1959 Thesis and Abstract Appraved: ~ ----- w. R. Je~ Assistant Professor Department of Botaey Date approved~ / ~ J !f :!!.. NEMATODES As.SOCIA.TED WITH ROOES lL\lD THE ROOT I NJURY CAUSED BY ME!,<):pxDYNE HAPIA CHI'lWOOD 1949, XIBIINEt1A. ~ICAUDATUM (MICOIETZKY 1927) THORNE 1939, AND HELICOTYIENCHUS NANNUS STEINER 19L~5. --- by Ronald Allan Davis ,,, Thesis submit ted to the Faculty of the Graduate School of the University of Maryland in partial fulfillment of the requi rements for the degree of Master of Science 1959 LIBRARY iNIVERSITY OF /Y/ARYLANO COLLEGE PARK. MO. The author wishes to express his since1~ appreciation to Dr. Wo R • • Jenkins for his guidance throughout the course of this work and for his suggestions and advice in the preparation of this manuscript. Gratitude is also expressed to the American Rose Society for providing financ.ial assistance in partial support of these investigations. Acknowledgment is expressed to Dr. B. A. Rohde for his maey helpful suggestions in the preparation of this manuscript, and to rr...ernbers of the Botaey Department, University of Maryland, who contributed advice and aid throughout this work. ii TABJE OF COHTENTS • • • • • • • • • • • • • • • 0 • • • • • • • • • TI~TRODUCTION • • • •. • • • • • • • • ~ . . . . . . . . . . . . MAT:&.1UA.IS JU© ME:l'HODS • • • • • • • • • • • • • • • Survey • • • • • • • • • • • • • • • • • • • • • • • • • • • Source of material for cytological study o. ~ • • • • • • • ... Preparation of slides RESULTS AND DISCUSSION • • • • • 9 • • • • • • • • • • •• • •••• • • • • • • • Sul"'Vey • • 0 • e e • e e O • ~ e e ~ e e O • 9 e e • • • • • • • • • • • • • Effects of Meloido~ne hapla ••• • • • • • • • • • • • • • Effects of Xiphin~ ~~icaudatum • • • • • • • • • • • • Effects of He~~~~lenchus n.annus •• • • • • • • • • • • • iii Page i i 1 4 4 4 5 6 6 8 21 28 SUMMA.RY •• • • •• 0 • • • • • • • • . • • • • • • • • • • • • • 31 LITERATURE CITED • • • • 0 0 0 • • • • • • • • • • • • • • • • • 33 Table 1 LIST OF TABIES AND FIGURES Nematodes associated with rose roots in 61 samples collected from 27 states ••••••• ., •••••• Cl • • • iv Page 7 Figure· Pa ge 1 ~ multiflora root system heavi.:cy· infected with 2 J 4 5 6 7 and 8 9 10 11 Meloidogyne hapla •• • • • • • • • • • • • e • • • • • • • Longitudinal section through a Rosa multiflora root tip gall infected with Meloidowne hapla showing a swollen female and several giant cells. • • • • • • • • • • • •• • Cross section of a root-knot gall on Rosa multiflora showing a Meloidogrne hapla female and an accumulated egg mass near the surface of the root •• o ••••• • • Cross section through a galled root of~ multiflora • • Cross section of an uninoculated Rosa multiflora root approxi.amte]y the same age as those roots illustrated in Figures J and 1+. • • • • • • • • • • • • • • • • • • • Cross sect.ion through a Rosa multiflora root gall incited by Meloidogyne hapla showing a group of 4 giant cells clustered about the anteri.or end of the nematode. 0 longitudinal serial sections t hrough a~ multi.flora root gall infected with Meloidogyne hapla ill.ustrating giant cell development in differentiated tissues • • •• • Longitudinal section through~ nrultiflora root tip showing a Meloidogyne hapla larva , imbedded in the region of elongation, a.nd a developing giant cello • ., • L:>ngi tudinal section through ~ nrul tiflora root tip infected with Meloidogyne ~ showing many nuclei aggregated in the center of a young giant cell • •• • • Cross section through a~ multiflora root tip gall infected with Meloidogne hapla. showing a giant cell nucleus which appears to be partitioned off into sectors, each containing a nucleolus ••••••••• • •• 0 • • 12 12 1J 1J 14 14 16 16 17 Figure 12 13 15 16 17 Cross section through a~ multiflora root gall infected with Meloidogyne hapla illustrating a donut shaped giant cell nucleus in which nucleoli can be observed around the ring • • •••••••••• •.••• Cross section through a Rosa multiflora root gall inf'ected with Meloidogyn~pla illustrating t he disintegratior1 of nuclear membranes of nuclei in a giant cell. • • • • • • • • o • • • • • • • • • • • • • • I.ongitudinal section through a~ multiflora root gall inf'ected with Meloi~~wne hapla illustrating the short, reticulate, :xylem elements which have formed from :xylem parenchyma •••• ., • • ••••• • • • • • • L::mgitudinal section through a Rosa multi:t'lora root gaJJ. infected with Meloido~ne hapia. showing inter- ruption of vessels by gi ant cells.• ••••• • • • • • Cross section of an older Rosa nrultiflora root attacked by Meloidogyne hapla. • • • •••••• • • • • • • • • • Cross section of uninf'ected Rosa multi:t'lor-~ root of approximately the same age a.sinfected root illustrated in Figure 16. • • • • • • • • • • • • • • •. • • • • • • 18 Cross section of Rosa multiflora. root showing 12 or more Meloidogyne hapla~les and egg masses imbedded in the V Page 17 18 18 19 19 20 root • • • •• • •• • • • • • •. 0 ••• •. • •••• o 20 19 20 21 22 23 24 Longitudinal section through a rose root gall produced by Xiphi.nema. diversicau.datum., • •• ., ••• • • • • • • • Cross section through an uninocuJ.ated rose root illustrating normal development ••• ., • •• • • • • • • Cross section through a rose root gall incited by Xiphinema diversicaudatum illustrating injury to cortical tissue at a single infection site •••• o • • o Cross sectional view of Xi.phinema diversicaudaturn injury site.• • • • o • ., • • •• ., o •• • • • • • • • Cross section through a rose root ini'ested with Xiphinema. diversicaudatum illustrating giant cells which have developed as the result of nematode feeding • • o • • wngitudinal section through a rose root infested with Xiphinema diversicaudaturn illustrating deep staining, spherictl structures adhering to the walls of a cortical cell ••••••• • ••• o • ••••• • o • • • • • • • 23 23 24 24 25 25 vi Figure Page 25 wngitudinal section through a rose root infested with Xiphinema diversicaudatum illustrating dark staining, spherical structures clustered in the lumen of a cortical cell ••• 0 0 ••• • •••• •. •...... 26 26 27 wngitudinal section through an uninoculated rose root tip illustrating norm.al development ••••• • • • • • • Longitudinal section through a rose root tip gall incited by Y..iphinema diversicaudatum. • •• • • • • • • 26 27 28 Cross sectional view of uninocul;;i.ted ~ multlflora root showing noi,nal development •••• • ••• 8 •• o • 29 29 Cross section of Rosa mu.ltiflora root attacked by Helicotylenchus nannus •••••••• ~ • • • • • • • • JO Cross section of Rosa multiflora root parasitized by Helicotylenchus n~s illustrating affected cortical --------cells beneath the epider.miso •••• • • • • • • • • • • 29 JO INTRODUCTION Few papers have been published concerning the cytological and histological effects of plant parasitic nematodes on their hosts. Most of this type of work has been done on the root-knot disease. Christie (2) described the development of root-knot nematode incited galls on tomato seedlings, reporting that these nematodes caused hypertrophy of cortical, peric-y-clic, and endodermal cells, hyperplasia of the pericycle, formation of Jcy"lem elements from parenchyma surrounding giant cells, and retardation of meristematic activity in the root tip. He also reported on the development and morphology of giant cells (large, multinucleate cells resulting from a stimulatory effect of nematode feeding). Krusberg and Neilsen (8) observed similar cytological responses in their. work with Meloidogyne incognita acrita Chitwood 1949 infections of Porto Rico variety of sweet potato. other investigators worked primariJy on the cytology and morphology of giant cells and giant cell nuclei. According to Tischler (from Christie, 2), division of giant cell nuclei was by nonual mitosis in early stages of giant cell development, but later divisions occurred by amitosis and by fragmentation. However, Nemec (from Christie, 2) felt that divisions by amitosis and .fragmentation as reported by Tischler were actually stages of nuclear coalescence. Linford (9) described the method by which root-lmot nematodes feed on giant cells and noted that substances were extruded from the szylet during feeding. Kostoff and Kendall (7), working with galled roots of Nicotiana 1 2 hybrids, reported that secretions by the nematode increased cell wall permeability causing exosmosis and resulting in an accumulation of food in the region of invasion. Consequently, growth of plant tissues i n these regions was accelerated and was expressed morphologically as swellings or galls on the roots. I n 1942, Alstatt (1) tested the sus- ceptibility of several strains and varieties of rose stocks, including ~ mu.1tiflora Thunb. to a root-knot nematode. Of 1.3 different under- stocks , on]y one was found resistant. cyle (10) and ¥iassey (12) indicate t hat root-knot nematodes cause a serious disease of rose. Reynolds (15) found that in Meloidogyne incognita (Kofoid and White 1919) Chitwood 1949 infections of~. nntl.tiflora seedlings, the nematode entered t he root and stimulated giant cell development ; but galls occurred on]y rarely and were sometimes found on the end of long roots as a result of the pene- tration of maey larvae. Martin (11) reported t!.• hapla as producing small, hard, galls on rose ro()ts in Rhodesia and l'zy"asaland. t1• hapla was reported by Van Der Linde (22) to infest a rose thornless understock. Two genera of ectoparasitic nematodes have been associated with root gall formation. Van Gundy (23) reported that galls induced on rough lemon roots by Hemicycliophora arenaria Raski 1958 were due to a hyperpl.astic response of the cortical tissue. Sch:mdler (18) demonstrated that galling of rose roots was caused by Xiphinema diversicaudatum (Micoletzky 1927) Thorne 1939, but he did not investigate their cytological effects. In a survey of greenhouse roses , Schindler (17) found Xiphinema and Pratylenchus to be the most widely distributed genera and to occur more frequently than ar~r other nematodes . Other genera found were: Criconem- oides, Paratylenchus, Helicotylenchus, Hemicycliophora, Belonolainius, Trichodorus, T,ylenchus, .Aphelenchoides, Psilenchus, and Meloid.ogyne . Sher (21) described the pathogenicity of Pratylenchus vulnus Allen and Jensen 1951 on rose, reporting that rose plants infested with this species were stunted and chlorotic and the root systems were necrotic with few feeder roots. other nematodes which have been found associ- ated with rose are Pratylenchus pratensis (de Man 1880) Filipjev 1936 ----., - --- J (J, '.1.4·), P. penetrans (11.i-) Sher and Allen 1953 P. scribneri steim)r 1943 - - (1J), and Ditylenchus dipsaci (Kuhn 1857) F'ilipjev 19,36 (5). 'Ihis pre[Sent study was initiated to determine the occurrence and d i s tribution of nematodes associated with roses grown outdoors. In addition the cytological and histological effects of Meloidogyne hapla Chitwood 1949, Xiphinema diversicaudatum (Micoletzky 1927) 'Ihorne 1939, and Hel~cot;rlenchus nannus steiner 1945 on rose roots was determinedo MATERIALS AND METHODS Survey.--Soil samples were examined during the period June to October, 1958 to deterrnine the occurrence and distribution of plant- parasitic nematodes associated with roses grown outdoors. letters were sent to members of the .American Rose Society in Z? states and Washil"gton, D. C. requesting soil and root srunples and including instructions for the proper collection of these samples. One pint of soil from each of 61 samples received was processed in a Seinhorst extraction apparatus (20). The water-soj_l suspension thus obtained was passed through 100 and 270 mesh screens, the residue washed off with a slow stream of water, 2nd the resultant suspension further separated and concentrated through use of a modified Baermann funnel apparatus. Nematodes and water were drawn off into a. syracuse dish after 15-20 hours and heat relaxed in an oven held. at 48°c. .Nematodes were then mounted in warm FAA and identified. Source of material for cytological study. --To obtain galls produced by Meloid~~ !.;§i.pl.a Chitwood 19l.J-9, two ~ mul.tiflora 'Ihunb. seedlings were planted in 4-inch-diameter clay pots contai ning soil infested with this species. Root-knot galls caused by ~ · hapla were also obtained from natu- ral.J.y infected multiflo:r.a seedlings from a commercial nursery. Galls produced by Xiphinema diversicaudaturn (Micoletzl,;y 192'?) Thorne 19J9 were obtained from rose, variety Better Times, which had been inocu- lated with specimens of t his species. Inoculation with Helicotylenchus ~~ Steiner 1945 1rras made with specimens obtained from populations maintained in the greenhouse on tanato. 4 5 Extraction and concentration of nematodes was accomplished by the method previously described. The nematode-water suspension thus obtained was brought to a knm,m volume and .3, 1cc. aliquots were pi- petted off, placed in individual syracuse di shes, and counted. The total number of nematodes present was then approximated. Four-week oJd Ii• multiflora cuttings which had been rooted in sand were placed in steamed clay pots half full of steamed soil • .AJ.iquots of the nematode suspension (2,150 spec:iW:ms per pot) were then pipetted directly onto the roots and the pots filled with soil. All plants in these experiments were held at a night temperature of 65-70°F. Roots of inoculated and check plants were washed free of soil and compared under a stereoscopic microscope to determine external symptoms of injury. Preparation of slides.--Several por tions of roots from plants inoculat ed with !i• hael a, ! • diversicaudatum, and H. n'3.nnus and from check pJ;J.nts were fixed in FAA, dehydr ated by the tertiary butyl alcohol method (6), infiltrated with paraffin, and irnbeddede Sections were cut at 15 micron thicknesses with a microtome and fixed to slides wi th Ha.upt •s adhesive. Sect.ions were then stained with safranin and fast green and mounted in balsam according to standard methods. RESULTS AND DISCUSSION Survey.--The number of soil and root samples received from each state were as follows: STATE NO. OF SA.l1PLES STATE NO. OF SAl"lPLES Cal:i.fornia 1 New Jersey 4 Georgia 1 New York 5 Illinois 2 North Carolina 2 Indiana 2 North Dakot~ 1 Iowa L1. Ohio 1 }~ansas 2 Pennsylvania 4 Kentuclcy- 2 Rhode Island 1 Louisi~.na 1 South Carolina 1 NaI'IJland 8 Tennessee 2 Michigan 1 Texas 1 Minnesota 1 Utah 1 Hissouri 1 Virginia 7 Nebraska 1 West Virginia 2 l ew Hampshire 1 Washington, D. c. 1 All 61 samples contained knovm and possible plant-parasitic nematodes. Their occurrence is given in Table 1. Host commonly found were the genera Xiphinema, occurrine 42 times; Pratylench~, 41 times; Helicotylenchus, 37 times; T,ylenchorhynchus, 29 times; and T.Ylenchus, 28 times. These are substantially the same results 6 Table 1. Nematode~ c,s sociated with rose roots in 61 samples collected from 27 states Genus Number Genus Number Genus Number and of and oi' and of Species Occurrences Species Occurrences Species Occurrences Aphelenchoides spp. 8 L<)ngidorus sp. 1 Rotylenchus robustus ,.., .:> A. tenuicaudatus 1 Meloidogyne haplE. 7 Trichodorus spp. 6 Aphelenchus spp. 8 Belonolai.rnus gracilis 1 Neotylenchidae 20 'Iylenchorh..ynchus SPo 1 .... T. brevidens 8 Criconemoides SPPo 4 Paratylenchus spp. 7 T. claytoni 1J Po dianthus '< T. cylindricus 1 _,I Ditylenchus SPo 1 P. projectus 8 T. dubius 4 Gottholdsteineria buxophila 1 T. nudus 1 Pratylenchus T. parvus 1 Helicotylemchus penetrans 26 rnulticinctus 1 P. pratensis 7 'Iylenchus spp. 28 H. nannus J4 P. scribneri 1 P. thornei 1 Xiphinerna americanum 41 Hem.icycliophora spp. 8 P. vulnus 6 Xo krugi 1 Hoplolaimus tylenchifonnis 6 Psilenchus sppo 4 --.J as those reported by Schindler (17). The most commonly found species was Xiphinen~ americanum Cobb 1913, occurring in 41 samples. Helico- ty1ench~ nannus steiner :1.91.J-5, t.he next most common species, occurred 8 J4 times; Pratylenchus penetrans (Cobb 1917) Chitwood and Otelia 1952, occurred 26 times; and 'Iylenchorh;ynchus clay~ Steiner 1937, occurred 13 times. Ma.qy of these genera and species are known pathogens on other hosts and it is therefore probable t hat they may also cause diseases of roses. Effects of Meloidogyne hapla.. --A root system heavily infected with H. hapla is shown in Fig. 1. Sectioned and stained galls showed the presence of individuals of this nematode within the cortex, stele , and root tip (Figs. 2,J,4,6,9,18). For comparison, cross sections of uninocu.la.ted roots are illustrated in Figs. 5 and 17. Vascular tissue was apparently the preferred feeding site since most nematodes were ob- served lying with their anterior ends imbedded witi1in this tissue with t heir bodies extending into the cortex (Figs. 2,3,4 ). Females depositoo. their eggs near the surface of the root (Figs. J,18). In some infected root tips, the presence of the parasite apparently suppressed mitotic activity i.n the apical meristem and growth was retarded (Fig. 2). Hyperplasia of the cortex and vascular parenchyma (Fig. 4) and giant cell formation were observed to accompar.w all infections. Giant cells formed around the anterior end of the nematode (Figs. 2,J,4,6, 9) and were gener- ally located in the vascular tissue or root tip. These cells were less often obse:rved in the cortex, a development similar to that described by Christie (2)o Giant cell formation in differentiated tissues began with the enclosing of a group of vascular parenchyma cells by a thick wall. Walls of the enclosed celJ.s then disintegrated, protoplasmic contents coalesced, and a giant cell resulted (Figs. 7,8) . This type of giant eel]_ development has not been previously reported in the literature and may be specific for this host. 9 In undifferentiated tissues of the root tip, giant cells developed in the same manner as reported by Christie (2); i.e., from the dissolu- t ion or cell walls with a subsequent dePosition of a thick wall around the coalesced cytoplasm and nuclei. As illustrated in Fig. 9, a giant cell of this type can be seen developing in the region of elongation. LYsis of procarribial cells was evident. The cytopla i,m appeared granula2· in all giant cells; however, the cytoplasm stained red in old giant cells; gray in young or developing giant cells. This reaction was also observed by Krusberg and Neilsen working with !i• incognita acrita infections of Porto Rico variety of sweet potato (8). It is possible that the differ- ential staining reaction is due to a chemical change which occurs in the giant cells as they grow older. Nuclei of giant cells varied in nwnber depending upon the region in which they were located and their age. 'Ihey varied in appearance even between adjacent giant cel ls. Nucleoli in all giant cells were as large, or larger than normal nuclei and stained deeply. In root tips, young giant cells contained J0-40 nuclei aggregated in the center of the cell (Fig. 10), while within the stele the nuclei were less numerous and usu- ally much larger (Figs . 11,12,1.3). The number of nuclei in a single giant cell may depend upon the number of cells which go into its forma- tion. If this were the case, it would explain the increased number of nuclei in root tip giant cells since ma.ey procambial cells are involved 10 in their formation. Also , nuclei of older giant cells may have coalesced or disintegrated, thus reducing the number of nuclei observed. Nuclei in some giant cells appeared to be partitioned off with a nucleo- lus in each cavity (Fig. 11), ·while in other giant cells they were donut shaped and nucleoli were arranged around this ring (Figo 12). As re- ported by Krusl::>erg and Neilsen (8), and Christie (2), nuclear membranes of nuclei in young giant cells were distinct (Fig. 10), while nuclear membranes of nuclei in some older giant ceD.s were indistinct and aP- peared to be disintegrating (Fig. 13). In some infections the nuclei of cortical cells surrounding the nematode body and the nuclei of those ceD.s around the new]y formed giant cells were increased approximate]y 2-3 times in size (Figs. 4,9) . As reported by other investigators (2,8), ~lem elements appeared to form from ~lem parenchyma around some giant ceD.s . 'IhesE~ cells had no definite arrangement or shape and were short, reticulate elements (Fig. 14). I.oneitudinal sections through galled roots revealed that giant cells in the vascu1ar cylinder interrupted the continuity of some vessels and other ,rascuJ..ar tissues (Fig. 15). The interruption of these stelar tissues undoubtedly would have an effect on translocati.on of water and nutrients through the roots and could account for much of the injury resulting from root-knot infection. When roots in which a considerable amount of secondary growth had occurred were attacked, giant cells formed but were sma11er and less numerous than giant cells formed in younger roots. In these cases, the anterior end of the nen~tode became imbedded in the stele and the posterior portion in the periderrn. Peridermal tissues pro- liferated around the body of the nematode and a layer of cork was formed around the anterior end of the nematode (Fig. 16). Figure 17 is a photo- micrograph of a cros s section of a non-infected root of the same age. When multiple infection of single primary roots occurred, roots became rough and increased great]y in diameter. As many as 12 females a.nd egg masses i.mbedded in a single cross section were observed (Fig. 18). large cavities occurred in the center of the root, probab]y due 11 t o collapse of giant cells. It is evident that the translocation of water and nutrients in roots of this condition would be great]y impaired i f not entire]y stopped. Figure 1. ~ multiflora root system heavily infected with Meloido~ne hapl.a. Many small root galls are evident. Figure 2. !J)ngitudinal section through a Rosa multiflora root - - tip gall infected with Meloidogyne ~la showing a swollen female and several giant cells. Meriste:matic activity in the apical rneristem has ceased (X190). 12 Figure J o Cross section of a root-knot gall on~ ~tiflora showing a Meloido@'.:_ne hapla female and an accunmlated egg mass near the surface of the root (X190). Figure 4. Cross section through a galled root of~ multiflora. Note the large nuclei of proliferated cortical cells (arrow) on one side of the nematode body. Vascular and cortical hyperplasia is evident. At the anterior end of the nematode, a giant cell and two groups of vascular parencl:zyma cells are enclosed by thick walls (X190). 1J Figure 5. Cross section of an uninoculated Rosa multiflora root - approximatezy the same age as those roots illustrated in Figures J and 4 (X95). Figure 6. Cross section through a~ multiflora root gall incited by Meloidogyne hapl.a showing a group of 4 giant cells clustered about the anterior end of the nematode (X500). 14 Figures 7 and 8. longitudinal serial sections through a~ ~iflora root gall infected with Meloi~o~ne hapla illustrating giant cell development in differentia.ted tissues. In Figure 7, a group of vascul&r parenc;y-ma. cells are enclosed by a thick walJ.. Serial sections revealed that the cell walls of these enclosed parenchyma cells disintegrated, their protoplasmic contents coalPsced, and a giant cell developed as illus- trated in F:i.gure 8 (XJ70). 1.5 Figui·e 9. Longitudinz.l section through ~ multiflora root tip showing a Meloidogyne hapla larva, imbedded in the region of elongation, a.nd a developing giant cell. Note J.;ysis of procambial cells bordering the giant cell and the large nuclei. of some of thesE, cells (arrow) (X400 ). Figure 10. longitudinal section through Rosa multiflora root tip infected with Meloidogyne hapl.a showing marzy- nuclei aggregated in the center of a young giant cell (X1200) . - ,.. 16 Figure 11. Cross section through a~ multiflora root tip gall infected with MeloidotQ::ne hapla showi.ng a giant cell nucleus which appears to be partitioned off' into sectors, each containing a nucleolus (X780). Figure 12. Cross section through a~ multiflora root gall infected with Meloidogyne hapla illustratine a donut shaped giant cell nucleus in which nucleoli can be obs9r1ed around the ri.ng (X1750) . 17 Figure 13. Cross section through a ~ nrul.tiflora root gall infectf,d with Meloid?~ne hapla illustrating the disintegration of nuclear membranes of nuclei in a giant cell (X1100). Figure 14. longitudinal section through a ~ multi.flora root gall infected with }'i"eloidogy-ne hapla illus- trating the short, reticulate, :xylem elements (arrow) which have formed from :xylem parencbyma (X5.30) . 18 Figure 15. longitudinal section through a ~ multiflora root gall infected with Meloido~e hapla showing inter- ruption of vessels by giant cells (X575). Figure 16. Cross section of an older Rosa nmltiJ'lora root attacked by Meloido~e hapla. Note cork formation around cavity left by the anterior end of the nematode and the proliferated perid.ermal tissues which enclosed the posterior portfon of the nematode (X100). 19 ' , , I I ,f : Figure 17. Cross section of uninfected P~sa multiflora root of approximate]y the same age as infected root illus- trated in Figure 16 (X100). F'igure 18. Cross section of ~ mul.tiflora root showing 12 or more MeloidofQ'.:ne hapl.a .females and egg masses imbedded in the root. large cavities in the center of the root are probab]y due to collapse of giant cells (x4o). 20 , . I 21 Effects of Xiphinema diversicau laturn.--Micr()scopic examination of sectioned and stained galled roots revealed cytological and histological abnormalities when compared with ungalled control roots (Figs. 19-27 ). GaD_ formation was duE: primarily to a byperplastic response of the cortical tissue. Cortical cells of swollen and curled infected roots showed hyperplasia on the inside of the curve. These cells proliferated to form 15-20 cell layers more than the opposite side of the root (Fig. 19),. In root tips which were swollen but not curled, cortic~.1 h,yperplasia occurred even1y around the root. '!'he epidermis at infection s ites was broken and cortical cells beneath the break stained red (Figs. 21, 22) . Check roots showed norm.al development (Figs. 20, 26) o Cortical cells associated with some infection sites increased in size approxnnate1y 2-.3 times, their walls thickened, and their cytoplasm ap.. peared eranular (Fig. 2.3). These cells were similar in appearance to giant cells produced by root-knot nematodes, except that nuclei eJ Cross section of~ multiflora root attacked by Helicot,ylenchus nannus. Note broken surface and lesion extending into the cortex. Several cortical ceJJ_s beneath the J.esion are deep]y stained (XB?s). 29 Figure JO. Cross section of Rosa nrultiflora root parasitized by Helicotylenchus nan~ illustrating ::if'f ected cortical cells beneath the epidern1is. Note that the epidermis is apparent]y uninjured (X9JO) . JO SUMMARY All samples examined i n a survey of nematodes associated with rose s grmm outdoors contained plant parasitic forms . These forms included the genera Belonolairnus , Criconemoides , Helicotylenchus, Heroicycliophora, Hoplolaimus , Longidor ~ , Meloidoeyne , Paratylenchus, Pratylenchus, Rotylenchus , Trichodor us , ]X.lenchor13lnchus, and Xinhinema. SectioI1E;d and stai ned root-knot galls from rose roots showed the pre s ence of Meloidogyne hapla wit hin the root causing giant cells, hyper- pl,"3.s ia of the cortical and va scular parenchyma, zylem elements formed £'ror.i. vascular parenchy~t , and r etardation of meristematic activity in the root tip. Giant cells i n differentiated tissues were formed from a group of vascular parenchyma cells which wer e first enclosed by thick walls. ',falls of enclosed parenchyma cells disintegrated, the protoplasmic contents coalesced, and a giant cell developed. In undifferent iated t issues , giant cells f ormed from the dissolution of cell walls with a subsequent depo- sition of a thick wall around t he coal esced cytopla sm and nuclei. Giant cell nuclei varied in size and appear ance . Sectioned and stained root galls i ncited by XiEhinema diversicaudatum revealed t hat gall formation was due pri.mar iJ.y t o a hyperplastic response of c ortical cells. Gortical cells a ssociated with some feeding sites in- crease d 2-3 times in size , their cytoplasm appeared granular, and their ·walls t hickened. In some infections , cortical cells ]ying adjacent to, or v ery near, these giant cells contained spherical structures adhering to t he waD.s or clustered i n t he l umen. Meristemat ic activity in infested Jl 32 root tips was retarded and vascular differentiation extended far into the root tip. Rose roots parasiti7.,ed by Helicotylenchus nannus were marked by tiey brown necr otic lesions and discolored areas. S-ections revealed that the surfc1ce of infected roots was broken and several cortical cells beneath the lesion stained deeply. ¼bere no lesions occurred, cortical. cells directly beneath the epidermis were affected without apparent inju:ry to the epidermis. Much of the injury caused by Meloidogyne hapla, Xiphinema diver-___ ....._.,__ - - sicf!.udaturn, and Helicotylenchu~ nannus is apparently due to chemical changes within root tissues, possibly resulting from nematode secretions or a host reaction to nematode feeding. The extensive root injury caused by these nem.i.todes probab]y results in decreased top growth, u.nproductivity, and increased susceptibility to other disease agents . 1. LITERATURE CITED Altstatt, c-. E. 1942. to nematode root-knot. Susceptibility of some conunon rose understocks Plant Disease Reptr. 26:J71o 2. Christie, J . R. 1936. 'lbe development of root-knot nematode gal..1s. Ph;ytopathology 26 :1-22. Crossman, I.ouise, and J . R. Christieo 1937. by miscellaneous plant-infesting nematodes. 144-167. Lists of plants attacked Plant Disease Reptr. 21 : 4 . Golden, A. M. 1956. Taxonorqy of the spiral nematodes (Rotylenchus and Helicotylenchus ), a.nd the developmental stages and host-parasite relationships of R. buxophilus , n. sp., attacking boxwood. Univ. of Md • .Agric. EJcpt. Sta. Bulle .A-85 . 28 P• 5. Goodey, J . B., and Ma:ry T. Franklin. 1956. 'lbe nematode parasites of plants catalogued under their hosts. Connnonwealth Agric. Bur., Farnham Royal, Bucks.,, Englando 140 P• 6. Johansen, D. A. 1940. Plant microtechnique . McGraw-Hill Book Co., Inc., New York, N.Y. 523 p. 7" Kostoff, D., and J . Kendall. 1930. Cytology of nematode galls on Nicotiana roots. Abst. Centralbl. Bakt. II 81:86-91. 8. Krusberg, L. R., and L. w. Nielsen. 1958. Pathogenesis of root-knot nematodes to the Po!~,o Rico variety of sweet potato. Pnytopathology 48:JO-J9. 9. Lini'ord, M. B. 1937. 'Ihe feeding of the root-knot nematode in root tissue and nutrient solution. Phytopathology 27:824-835. 10 Iifle, E. w. 194.3. Nematode resistance in rose understocks. Am. Rose Annual 28 :157- 158. 110 ~..artin, G. c. 1954. Nematodes. Rhodesian Tobacco J. 6(6) :115. 12.. Masse:y, L., M. 1947. Soil fumigants for root-knot control. Am. Rose Annual 32:119-1 24. 1J. 14. Nesbitt , R. B. in California. 19560 New- host plants of plant parasitic nematodes Plant Disease Reptr. 40:276. O:>s tenbrink, M. 1956. O ver de invloed van verschillende gewassen op de vermeerdering van en de schade door Pratylenchus pratensis en iPratylenchus penetrans (Verrnes, Nematoda), mit ve:rmelding van een afwijkend moeheidsverschijnsel bij houtige gewassen. Tijdschr. Plantenziekten 62:189-203. 33 15 16 .. j 8 . 2 0 .. 21 .. Reynolds, H. w. 1950. Root-knot nematode development and root tissue response of the rose. Rl. D. 'Ihesis, Purdue Univ. 56 p. 34 Rohde, R. A., and w. R. Jenkins. 19.57. Host range of a species of Trichodorus and its host-parasite relationships on tomato. Pb0rtopatbology 47:29.5-298. Schindler, A. F. 1956. Nematodes associated with roses in a survey of commercial greenhouses. Plant Disease Reptr. 40:277-278. Schindler, A. F., 1957. Parasitism and pathogenicity of Xiphinema diversicaudatum, an ectoparasitic nematode. Nematologica 2:25-Jl. Scott , ~.arie. 1959. In plant nematodes their bionomics and control, by J . R. Christieo Agric. Expt. sta. Fla. p 15. Seinhorst, Jo w. 1956. The quantitative extraction of nematodes from soil. Nema.tologica 1:249-267. Sher, s. A. 1957. A disease of roses caused by a root-lesion n8rnatode, Pratylenchus vulnus. Rlytopathology 47:70J-706., 22. Van Der Linde, w. J. 1956. The Meloidogyne problem in South Africa. Nema.tologica 1:177-183. Van Gundy, S o D. 1958. The pathogenicity of Hem.icycliophora arenaria on citrus. Hzytopathology 48:399 (Abst. ) VITA 1\fame - Ronald Allan Davis Permanent address.: VF - 12F, College Par k, Ma1yland Degree and date to be conferred: Ma st er of Science, 1959 Date of birth: December 4, 1935 Pl.ace or birth: Penns Grove, New Jersey Seconda1y education: Penns Grove Regional High School, Penns Grove, New Jer sey Collegiate instituti ons attended University of New Hampshire University of Ma.cyla.nd 6ajor:: Plant Pathology (Nem,.:itol ogy ) 11:inor: Botaey Pub1:lca'i::.ions: I:ntes 1953- 1957 1957-1959 Degree B.s. M. S. Date of Dagree June, 1957 June , 1959 1~ Cytological and histologi cal eff ects of Xiphinema diversicaudatmn (Micoletzky, 1927) Thorne 1939 and 11:eloi do~e ~ Chitwood 1949 on rose roots. (Abst. ) Phytopathol ogy ( In press. ) Positions held: 1. Graduate Assistant, Univers ity of Macy-l and. 1957 to pr esent.