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 citrophi- lus. 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 lim- it 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.