Variation in Radopholus Citrophilus Population Densities in the Citrus Rootstock Carrizo Citrange DAVID T

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Journal of Nematology 18(1):31-34. 1986. © The Society of Nematotogists 1986. Variation in Radopholus citrophilus Population Densities in the Citrus Rootstock Carrizo Citrange DAVID T. KAPLAN 1 Abstract: Seedlings of the hybrid citrus rootstock, Carrizo citrange (Citrus sinensis x Poncirus trifoliata) do not uniformly limit development of the citrus burrowing nematode, Radopholus citrophilus. Variation in nematode population densities in roots of seedlings germinating from the same seed suggests that factors responsible for nematode incompatibility are not functional or are not inherited uniformly among progeny. Seeds which produced a single seedling were more likely to produce plants which suppressed citrus burrowing nematode population increase than were seeds which produced two or three seedlings. Key words: resistance, tolerance, nematode, polyembryony, citrus burrowing nematode.

Spreading decline of citrus (14) caused CBN population densities which persist in by the citrus burrowing nematode (CBN) roots of these rootstocks for prolonged pe- Radopholus citrophilus (5) had been man- riods of time (9; Kaplan, unpubl.; Ford and aged in Florida for more than 25 years O'Bannon, unpubl.). The persistence of through the integration of several control CBN in roots of resistant rootstocks may strategies. These included the "push and have contributed to the development of a treat" program to reduce nematode pop- resistance-breaking biotype (7) isolated ulation densities to nondetectable levels from a sweet orange (Citrus sinensis (L.) Os- prior to replanting (12), physical barriers beck) on Milam (a putative hybrid of rough such as chemically treated fallow buffers lemon, C. limon (L.) Burm. f.) planting with between infested and noninfested groves spreading decline symptoms. Decline to prevent nematode dispersal, and resis- symptoms were observed in isolated plant- tant or tolerant rootstocks. The recent ban ings of sweet orange on Milam lemon and on ethylene dibromide (EDB) and the cur- Carrizo citrange (C. sinensis x Poncirus tri- rent lack of a registered, effective, and eco- foliata (L.) Raf.), rootstocks previously re- nomic substitute for EDB has essentially ported to be resistant or tolerant to R. ci- terminated both the push and treat and trophilus (2,3,10,11). chemical barrier programs. The purpose of this study was to deter- Future programs to manage spreading mine if Carrizo citrange seed sources and decline will continue to use certified CBN- (or) seedlings germinated from the same free citrus nursery stock to prevent spread seed vary in their ability to suppress de- of CBN into noninfested groves, nema- velopment of CBN populations. tode-resistant or tolerant rootstocks to limit nematode reproduction and (or) damage MATERIALS AND METHODS in infested sites, sound cultural practices Comparison of d~fferent Carrizo and Troyer designed to minimize stress in CBN-in- ~citrange seed sources on R. citrophilus repro- fected trees, and environmentally safe duction: Seed was COllected from 25 com- postplant nematicides to reduce CBN pop- mercial seed sources of Carrizo citrange, ulation densities to nondamaging levels. two sources of 'Troyer' citrange, and one Economic and environmental consider- source of rough lemon; Ten 5-month-old ations will probably increase grower reli- plants from each seed source were planted ance on rootstocks to m~Iiimize CBN-re- in 20-cm-d pots containing steam sterilized lated losses. Rootstocks currently available Astatula fine sand (hyperthermic, uncoat- for CBN management generally harbor low ed typic quartzipsamments) and random- ized on a greenhouse bench. Plants were Received for publication 4 March 1985. Research Plant Pathologist, usDA ARS, ~I.S. Horticul- ~elected for uniform vigor and phenotype. tural Research Laboratory, 2120 Camden Road, Orlando, Two months later, the soil in each pot was FL 32803. infested with 100 nematodes (mixed life I thank Dr. J. H. O'Bannon and C. Youtsey foradvice atxd collection of seeds and D. Johnson and J. Kiel for technical cycle stages) froma population previously support. designated as R. citrophilus biotype 1 (7). This paper reports the results of research only. Mention Inoculum was derived from monoxenic of a pesticide in this paper does not constitute a recommen- dation by the U.S. Department of Agriculture nor does it carrot disc cultures (8). Plants were main- imply registration under FIFRA. tained in a greenhouse with soil tempera- 31 32 Journal of Nematology, Volume 18, No. 1, January 1986

TABLE 1. Radopholus citrophilus biotype 1 popu- TABLE 2. Radopholus citrophilus population densi- lation densities in fibrous roots of 25 Carrizo and 2 ties in single, twin, and triplet seedlings arising from Troyer citrange sources and rough lemon (10 plants individual Carrizo citrange seeds. per seed source). Single Nemas/g plants Twin plants Triplet plants root dry Standard Source weight* deviation Percentt 1 1 2 1 2 3

01-21 949 2,048 40 847 7,506 0 3,571 796 699 78-355 792 1,182 50 476 5,665 5,521 2,631 2,558 1,010 469 2,820 141 78-437 642 1,134 50 2,207 1,702 0 01-20 397 836 20 430 3,705 442 2,160 766 75 78-334 370 662 70 270 2,352 0 1,566 202 73 Roughlemon 360 104 80 79 1,653 201 587 98 0 F52-5 (Troyer) 298 446 60 72 1,606 20 560 34 0 9 844 0 283 247 0 F17-20(Troyer) 197 204 50 01-9 194 488 30 0 715 0 282 138 0 01-19 180 569 I0 0 666 0 0 591 0 78-333 175 236 70 01-25 170 537 10 0 441 0 0 405 0 01-26 161 410 20 0 347 141 78-332 154 162 60 78-438 139 169 50 0 192 0 01-12 131 413 10 0 192 0 78-331 81 91 50 0 86 59 01-16 59 127 20 53 34 01-17 54 170 10 16 10 01-7 51 162 10 01-13 40 83 30 Data were analyzed to determine if nema- 01-8 25 80 10 01-22 23 50 20 tode population density was correlated with 01-24 19 59 10 stem diameter or total fibrous root weight. 01-10 0 0 0 Nematode population densities were ex- 01-18 0 0 0 pressed as nematodes per gram root dry * Column means not significantly different according to weight. Duncan's multiple-range test log(x + 1) (P = 0.05). t Percentage of test plants where R. citrophiluswas detected RESULTS in roots. The large standard deviations in Table ture averaging 24 + 5 C. Twelve months 1 show the variation in CBN population after infestation, nematodes were extract- densities associated with plants of Carrizo ed from all fibrous roots from each plant citrange from different seed sources. In for 7 days at 25 +- 1 C (15). Roots were nine seed sources, 50-70% of the plants oven-dried for 48 hours at 76 C and tested supported large CBN population weighed. Data were expressed as nema- densities; in 14 seed sources, 10-40% of todes per gram root dry weight. plants tested supported large CBN popu- CBN population densities in plants germi- lations; and plants selected from only two nated from Carrizo citrange seeds: Seventeen seed sources appeared to uniformly sup- seedlings from seed that produced single press development of large CBN popula- seedlings, 19 sets of twin plants from 19 tions. Consequently, seed sources support- seeds, and 9 sets of triplet plants from 9 ing mean populations as high as 949 seeds, all collected from one seed source nematodes per gram root dry weight could (78-438), were individually planted in As- not be distinguished from sources with no tatula fine sand as before. Soil around each detectable nematodes in their roots by plant was infested with 100 R. citrophilus Duncan's multiple-range test (P = 0.05). derived from monoxenic cultures when Subsequent study of single, twin, and plants were 7 months old. Plants were ran- triplet Carrizo citrange plants indicated domized and maintained in a greenhouse that even though two or three plants may for 15 months. At harvest, stem diameter germinate from the same seed, they often was determined, nematodes extracted from differ in their suitability for CBN popula- fibrous roots, and root weights determined tion increases (Table 2). In this experi- as described in the previous paragraph. ment, CBN was not detected in roots of Variation in Citrus Rootstock: Kaplan 33

52, 23, and 19% of the plants that ger- erant (2), and again as resistant (11). Vari- minated from seeds producing one, two, ation among seedlings of Carrizo citrange or three seedlings, respectively. The like- regarding their influence on CBN popu- lihood of selecting Carrizo citrange plants lation densities may explain why designa- that suppress development of CBN pop- tion of this rootstock as CBN resistant has ulations was negatively correlated with the been difficult. number of seedlings germinating from a Carrizo citrange is a nucellar plant. Nu- single seed (r = -0.91). cellar embryony is unique to Citrus and its Stem diameter and fibrous root weight close relatives (4). Nucellar embryos de- were suppressed in single (r = -0.35, velop asexually from nucellus cells by mi- -0.50), twin (r = -0.50, -0.37), and trip- totic division, and nucellar seedlings are let (r = -0.39, -0.41) plants, respectively, considered genetically identical to their as population densities of CBN increased. seed parents (1,4). Nucellar seedlings may vary from their parent if somatic mutations DISCUSSION (rare) occur or if chromosomes or parts of Some Carrizo citrange seedlings were chromosomes are inherited cytoplasmi- distinctly incompatible with CBN, whereas cally (frequency unknown). Pollination is others supported large numbers of nema- usually essential to nucellar embryo devel- todes. No obvious phenotypic character opment; however, the relationship of fe- (e.g., leaf shape or growth habit) was as- cundation to embryo development is un- sociated with incompatibility to CBN. Some clear (1). Although Carrizo citrange is Carrizo citrange seedlings are primarily considered nucellar (13), the ability to sup- nucellar, but some may be tetraploid or press CBN population increase is not ex- zygotic. Tetraploid and zygotic seedlings pressed uniformly. Plants that suppress can be readily identified by their coarse CBN may be cloned vegetatively through roots, thick leaves, or growth habit. Nei- cuttings to ascertain the planting of a root- ther tetraploid nor zygotic seedlings were stock that will suppress CBN. At present, included in this study. Variation in seedling Carrizo citrange should not be considered vigor was apparent among Carrizo cit- CBN resistant (6). range plants. In the first study, the most Occurrence of spreading decline symp- vigorous and uniform plants from each of toms in Carrizo citrange rootstock plant- 25 Carrizo and 2 Troyer citrange seed ings may reflect the inability of some Car- sources were evaluated for their ability to rizo citrange rootstocks to suppress CBN suppress CBN population densities. On population development. However, occur- evaluation of the data from the first ex- rence of CBN populations that differ in periment, the second experiment was ini- virulence (7) may also play a role when tiated to better document variation toward spreading decline symptoms appear in CBN within Carrizo citrange seed sources. plantings of Carrizo citrange rootstocks. Variability in vigor is common among Car- rizo citrange seedlings, but plants used as LITERATURE CITED 1. Cameron, J. W., and H. B. Frost. 1968. Ge- rootstocks are selected for both uniformity netics, breeding, and nucellar embryony. Pp. 325- and vigor. In the experiment where plants 371 in S. Reuther, L. D. Batchelor, and H.J. Webber, were selected on the basis of the number eds. The citrus industry, vol. 2. Richmond: University of seedlings arising from each seed, vigor of California Press. was disregarded. However, plants with 2. Ford, H.W. 1970. Nematode status of'Carrizo' citrange. Proceedings of the Florida State Horticul- atypical leaves or abnormal growth habit tural Society 83:87-88. were avoided. Some seedlings that would 3. Ford, H. W., and W. A. Feder. 1962. Labora- never be used commercially were used ex- tory evaluation of certain citrus rootstock selections perimentally. Nonetheless, the experiment for resistance to the burrowing nematode. Journal of the Rio Grande Valley Horticultural Society 10:35- clearly demonstrated that seedlings arising 39. from the same seed may differ in their suit- 4. Frost, H. B., and R. K. Soost. 1968. Seed re- ability to suppress CBN populations. production: Development of gametes and embryos. Variation in Carrizo and Troyer cit- Pp. 290-324 in W. Reuther, L. D. Batchelor, and H. J. Webber, eds. The citrus industry, vol. 2. Richmond: ranges has been studied previously (3,13). University of California Press. Carrizo citrange was first designated as a 5. Huettel, R. N., D. W. Dickson, and D. T. Kap- rootstock resistant to CBN (3), then as tol- lan. 1984. Radopholus citrophilus sp. n. (Nematoda), 34 Journal of Nematology, Volume 18, No. 1, January 1986 a sibling species of Radopholus similis. Proceedings of Tylenchulus semipenetrans (Nematoda). Proceedings of the Helminthological Society of Washington 51:32- the International Society of Citriculture 2:544-549. 35. 11. O'Bannon, J. H., and H. W. Ford. 1976. An 6. Kaplan, D. T., and N. T. Keen. 1980. Mecha- evaluation of several Radopholus similis-resistant or nisms conferring plant incompatibility to nematodes. -tolerant citrus rootstocks. Plant Disease Reporter 60: Revue de Nematologie 3:123-134. 620-624. 7. Kaplan, D. T., andJ. H. O'Bannon. 1985. The 12. Poucher, C., R. F. Suit, H. W, Ford, and E. P. occurrence of biotypes in Radopholus citrophilus. Jour- DuCharme. 1967. Burrowing nematode in citrus. nal of Nematology 17:158-162. Florida Department of Plant Industry Bulletin No. 7. 8. Moody, E. H., B. F. Lownsbery, andJ. M. Ahmed. 13. Savage, E. M., and F. E. Gardner. 196,5. The 1973. Culture of the root lesion nematode, Pratylen- origin and history of Troyer and Carrizo citranges. chus vulnus, on carrot discs. Journal of Nematology The Citrus Industry 26:5. 5:225-226. 14. Suit, R. F., and E. P. DuCharme. 1953. The 9. O'Bannon, J. H., and H. W. Ford. 1979. Pos- burrowing nematode and other plant parasitic nema- sible Radopholus similis biotypes reproducing and mi- todes in relation to spreading decline of citrus. Plant grating on resistant citrus rootstocks. Nematropica 9: Disease Reporter 37:379-383. 104 (Abstr.). 15. Young, T. W. 1954. An incubation method 10. O'Bannon, J. H., and H. W. Ford. 1977. Re- for collecting migratory endo-parasitic nematodes. sistance in citrus rootstocks to Radopholus similis and Plant Disease Reporter 38:794-795.

Journal of Nematology 18(1):34-36. 1986. © The Society of Nematologists 1986. Survival and Horizontal Movement of Infective Stage Neoaplectana carpocapsae in the Field GEORGE O. POZ~AR, JR.1 AND ALBERT HOM 2 Abstract: Infective stage Neoaplectana carpocapsae were applied to a furrow of loam soil in the field. At regular intervals over a 7-week period, soil samples were taken at distances of 0, 10, 20, 30, and 40 cm on either side of the furrow. Live, infective nematodes were recovered from the soil at all sampling dates over the 7-week period, and nematodes slowly spread out from the original line of application, averaging a distance of 4.35 cm/day. Key words: entomogenous nematode, dispersal in soil.

Steinernematid nematodes show great has a CEC (cation exchange) of 20.1 meq/ promise as biological control agents of in- 100 g, a pH of 5.2-5.6, and an organic sect pests and are now being commercially matter content of 0.7-1.24%. produced for.this purpose (8). One of the A plot of land 3 m wide by 15 m long important aspects regarding their use is was selected for the experiment. A furrow their survival and distribution after appli- 5 cm deep was made lengthwise through cation in the field. Various workers have the middle of the plot. The experiment was studied the migration of infective stage ju- initiated on 8 October 1984 with dry, bar- veniles of Neoaplectana under laboratory ren soil. The plot was irrigated by a sprin- conditions (1-3,5,6). This study was con- kler on 7 October 1984. ducted to determine the survival and dis- Nematodes used were the 42 strain of tribution of N. carpocapsae infective juve- Neoaplectana carpocapsae Weiser (supplied niles when applied to field soil. We prefer by Biosis, Palo Alto, Calif.). The nema- to maintain the name Neoaplectana carpo- todes had been reared in Galleria mellonella capsae for this nematode. Reasons for this larvae and stored at 8 C for 3 months. In- decision have already been presented (7). fective juveniles suspended in 8 liters of water were poured into the furrow at a rate MATERIALS AND METHODS of 1 million nematodes per linear meter. The experiment was conducted on the Care was taken to agitate the suspension Oxford plots of the University of Califor- during application and to apply the sus- nia at Berkeley. The soil is a Dublin loam pension uniformly in the furrow. (40% sand, 33% silt, 26% clay). The soil Before the furrow was covered, one corn seed was placed every 6 cm in the furrow. Received for publication 28 February 1985. The plants were used as indicators of soil Department of Entomological Sciences, University of Cal- ifornia, Berkeley, CA 94720. moisture content. Only when the plants 2 Biosis, Inc., 1057 E. Meadow Circle, Palo Alto, CA 94303. wilted was the plot irrigated.