Marigold, Castor Bean, and Chrysanthemum As Controls of Meloidogyne Incognita and Pratylenchus Alleni ~

Marigold, Castor Bean, and Chrysanthemum as Controls of Meloidogyne incognita and Pratylenchus alleni ~

R. W. HACKNEY 2 and O. J. DICKERSON ~

Abstract: Root and soil populations of Meloidogyne incognita were significantly fewer from marigold, castor bean, and chrysanthemum than from tomato roots and soil, but not from fallow soil. Root populations of Pratvlenchus alleni were significantly fewer from marigold, castor bean, and chrysanthemum than from tomato: marigold had the fewest. Root populations of M. incognita and P. alleni from tomato simultaneously cultiwued with marigold, castor bean, and chrysanthemum were significantly fewer than from tomato cultivated alone. Aborted giant cells and dead M. incognita (larvae and females) were observed in roots of marigold and castor bean, but not in chrysanthemum or tomato. Significantly more males than females occurred in castor bean roots. lnfcction sites of P. alleni appeared normal in all hosts. Thin-layer and column chromatography of alcoholic extracts from castor bean revealed no nematicidal thiophenc derivatives, Key Words: resistance, thiophenc dcri,~at ives.

As part of a field experiment, we tested thiophene derivatives similar to those nematicides against Pratylenchus spp. on reported from Tagetes spp. pinto bean (Phaseolus vulgaris L.) in soil where castor bean (Ricinus communis L.) had MATERIALS AND METHODS been grown the previous year. Nematode counts made during the growing season Experimental design: The greenhouse indicated that few PraO'lenchus spp. were in experiment was completely randomized. Data the control plots in that area. Further, were evaluated by a four-way analysis of nematode populations in castor bean soil with variance where the matin effects were: (i) hosts no nematicides were similiar to those in (marigold, castor bean, chrysanthemum, nematicide-treated plots where corn had been tomato, or fallow); (ii) nematodes (P. alleni or grown the previous season and where M. incognita); (iii) controls (tomato, or no populations of PraO'lenchus spp. were high in tomato); and (iv) time (30, 60, or 90 days) nontreated controls. These observations, between transplanting into infested soil and coupled with reports that marigold reduces measuring nematode populations. Each populations of PraO'lenchus spp. and of treatment group consisted of four replicates of Meloidogyne spp. (6, 14, 19, 20, 21), and that each host plant. Roots from one replicate of Chrysanthemum (8) contains a thiophene each treatment were stained with acid fuchsin derivative similiar to those extracted from in lactophenol (I, 7, 10); nematodes were marigold, prompted us to conduct greenhouse extracted from roots of the remaining three experiments to measure effects of marigold, replicates. All bioassay and soil-count data castor bean, and chrysanthemum on were based on four replications. Dwarf French marigold Pratylenchus alleni and Meloidogyne Plant material: 'Lemondrop'), castor bean incognita. Objectives of the study were: (i) to ( Tagetes patula L., measure the effects of marigold, castor bean ( Ricinus communis L., 'Bronz King'), and and chrysanthemum as hosts for M. incognita tomato (Lycopersicon esculenturn L., and P. alleni," (ii) to evaluate the nematicidal 'Rutgers') seeds were germinated in potential of marigold, castor bean, and vermiculite. Two-week-old plants and chrysanthemum grown simultaneously with a chrysanthemum (Chrysanthemum good host for M. incognita and P. alleni; and mor([olium Ramat., 'Escapade') rooted (iii) to determine if castor bean contained cuttings were transplanted into nematode- infested soil in 17.8-cm diameter pots. Nematodes: Greenhouse cultures of Rccci~ed for publication 25 March 1974. Meloidogyne incognita (Kofoid & White) ~Portion of a Ph. D. dissertation submitted by the senior author 1o Kansas State University. Contribution No. 608, Department ol Chitwood on tomato and Prao'lenchus alleni Plant Pathology, Kansas Agricultural Experiment Statfim, Ferris on chrysanthemum were diluted in Manhattan 66506. t'mmcr Assistant Instructor. Department of Plant Pathology, steam-sterilized sandy-loam soil. At Kau,zas State University, Manhattan. Current address: transplanting, the soil contained either an I)cpamncnt of Nematology, University of California, Riverside average of nine P. alleni or twelve M. 925(}2. :Re,catch Ncmatologist, Department of Plant Pathology, Kansas incognita larvae per 25 g soil. Greenhouse State I.;ni~ersit~, Manhattan 66506. temperatures (air and soil) were 24-30 C. 84 Meloidogyne and Pratylenchus Control: Hackney and Dickerson 85

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IABLE 2. Numbers of Pratylenchus alleni extracted from roots, soil around roots, and from bioassay plants grown in soil around 'Lemondrop' marigold, 'Bronz King' castor bean, 'Escapade' chrysanthemum, and 'Rutgers' tomato at three sampling times.

No. per g root dry wt No. per 25 g soil No. per g root dry wt; bioassay~ Treatments Days after transplanting Days after transplanting Days after transplanting 30 60 90 30 60 90 30 60 90 Marigold alone 0 556 69 17 12 6 31,429 12,422 18,125 Marigold + tomato 0 16,152 3,383 11 9 30 19,453 32,979 47,136 Marigold 0 6,917 238 Tomato 0 9,235 3,145 Castor bean alone 6,892 1,122 560 20 8 8 33,125 9,498 9,063 Castor bean + tomato 5,863 10,776 6,054 10 5 8 30,191 60,194 26,11 I Castor bean 4,027 684 617 Tomato 1,836 10,092 5,437 Chrysanthemum alone 1,408 2,704 2,266 32 9 6 I 1,032 27,668 26,563 Chrysanthemum+ tomato 1,056 14,871 4,690 11 9 12 39,958 80,813 33,125 Chrysanthemum 410 3,291 1,788 'tomato 646 11,580 2,902 Fallow 57 11 12 16,436 141,250 12,500 Tomato alone 226 18,105 9,289 23 10 39 20,280 75,052 33,438 'Soil bioassay using 'Rutgers' tomato.

IABLE 3. Numbers of galls and egg masses of Meloidogyne incognila in roots of 'l,emondrop' marigold, 'Bronz King" castor bean, 'Escapade' chrysanthemum, and 'Rutgers' tomato stained with acid fuchsin in lactophenol at three sampling times.

No. galls per 100 cm roots No. egg masses per 100 cm roots Treatments Days after transplanting Days after transplanting 30 60 90 30 60 90 Marigold alone 0 3 0 0 I 0 Marigold + tomato 90 107 200 0 28 54 Marigold 2 1 I 0 0 0 lomato 88 106 199 0 28 54 Castor bean alone 24 28 1 I 0 I I Castor bean + tomato 19 178 161 0 84 52 Castor bean 10 26 29 0 4 3 Tomato 9 152 132 0 80 49 Chrysanthemum alone 44 92 17 0 I 0 Chrysanthemum + tomato 117 120 263 0 1 34 Chrysanthemum 54 50 55 0 0 I Tomato 63 70 208 0 [ 33 Fallow Tomato alone 67 128 282 0 30 31

Nematode counts: P. alleni (all active for microscopic examination were obtained stages) and M. incognita (larvae and males) by randomly selecting 100 cm of root per plant were extracted from roots over a period of 7 from each treatment. Bioassays were days at 27 + ! C using the funnel spray method conducted by removing 500 cc of soil from of Oostenbrink (13). Soil samples were taken around the roots of each treatment and every 30, 60, and 90 days with a 2.5-cm transplanting a 'Rutgers' tomato seedling in it diameter soil probe. Nematodes were at 30, 60, and 90 days after beginning an separated from soil (25 g/sample) with 1.18 experiment. Twenty-one days after beginning sp. gr. sucrose solution and centrifugation (2, the bioassay, tomato roots were processed for 11), and recovered on a 38-#m (400-mesh) P. alleni, or rated with a root-knot index (16) sieve and counted. Specimens of stained roots for M. incognita. Meloidogyne and Pratylenchus Control: Hackney and Dickerson 87 Castor bean root extracts: A crude FABLE 4. Numbers of Pratylenchus alleni in roots of concentrate from castor bean roots was "l,cmondrop' marigold, 'Bronz King' castor bean, "Escapade' chrysanthemum, and 'Rutgers' tomato stained obtained using alcoholic extraction and with acid fuchsin in lactophenol at three sampling times. saponification, as described by Uhlenbroek and Bijioo (19). Crude concentrate was No. P. alleni per 100 cm roots fractionated on a column (57 × 2.2 cm) of, (i) I rcatments Days after transplanting alumina, activated, chromatographic grade, 30 60 90 177-74 ~zm (80- to 200-mesh) using petroleum Marigold alone 2 0 0 ether with increasing quantities of diethyi Marigold + ether as eluent (19); (ii) alumina, activated, to mat o 63 21 16 chromatographic grade, 177-74 /am (80- to Marigold 7 0 9 I omato 56 21 7 200-mesh) with diethyl ether as eluent; and Castor bean (iii) silica gel, 0.73 mm-74 /am (28- to 200- alone 52 0 14 mesh), grade 12, with absolute ethanol as Castor bean + eluent. Fractions of 10 ml were collected, and to mato 133 9 4 Castor bean 51 0 1 the eluate was monitored at 280 rim. An I omato 82 9 3 ultraviolet spectrum was made of all fractions Chrysanthemum (obtained by column chromatography) ahme 32 2 3 showing fluorescence at 280 nm. Chrysanthemum + Thin-layer chromatography, using tomato 75 75 6 Chrysanthemum 35 11 0 Eastman Chromagram Sheets (6062 and 1omato 40 64 6 6061), was used to screen the crude Falh>w concentrate for thiophene derivatives 1 omato alone 82 35 25 according to Curtis and Phillips (3).

RESULTS marigold or chrysanthemum had root-knot indices of 1.0 and 0.5, Host-parasite relationships: Typical M. respectively-significantly lower than that incognita infection sites were observed in (i.9) of tomato grown in soil from around tomato and chrysanthemum, in castor bean castor bean roots. In general, time influenced and marigold, giant cells, females, egg masses, the effectiveness of marigold or and matrix deposition were not typical. In chrysanthemum (Table i). The only castor bean, many giant ceils were small, and statistically significant difference in numbers some had degenerated. A few females reached of M. incognita in stained, infected roots was maturity and eggs were observed, but most an increase in males in castor bean roots, but egg masses were entirely within the gall. No numbers were too few to draw conclusions. ovoviviparity (17) was observed, in marigold, Egg masses were not present 30 days after nematodes developed slowly and in many inoculation, but were present after 60 days. infection sites, giant cells and nematodes were Root populations of P. alleni were degenerating, infection sites originated in the significantly (P ~ 0.05) reduced by marigold, stele. Ninety days after inoculation, some castor bean, and chrysanthemum when females had broken through to the outside of compared with tomato; marigold was the the gall, and small egg masses were produced. most effective (Table 2). Numbers of P. alleni Treatment effects: ...... I) Individual recovered from soil in which the four hosts host.--Tomato was the best host for both M. were grown were not significantly different. ineognita and P. alleni (Tables 1, 2). When Tomato bioassays demonstrated that measured by number of recoverable second- differences in populations of P. alleni were stage larvae from roots (mostly eggs hatched significantly related to time: populations were by the mist system) or from soil, marigold, largest after 60 days and smallest after 90 days castor bean, and chrysanthemum did not on all hosts. No significant differences differ (P ~ 0.05) as host for M. incognita. occurred in numbers of adults, larvae or eggs When measured by tomato bioassay, of P. alleni in stained roots from different chrysanthemum and marigold were hosts. More were present after 60 days than significantly more effective than castor bean after 30 or 90 days. in reducing numbers of M. incognita (Table 2) Host combinations.--Marigold, I). Tomato grown in soil previously planted to castor bean, and chrysanthemum did not 88 Journal of Nematology, Volume 7, No. 1, January 1975 significantly (P~ 0.05) reduce M. incognita or Standard 3-(2-thienyl)-acrylic acid in P. alleni populations in tomato roots with ethanol had maxima at 272 nm, 281 nm, and which they were cultivated simultaneously, 312 nm. Using the same standard on an when compared with tomato grown alone in alumina column and eluting with a mixture of infested soil (Table 1, 2). However, after 30 petroleum ether with increasing days, no P. alleni were recovered from concentrations of diethyl ether produced a marigold roots or from tomato roots grown fraction with maxima at 271 rim, 281 nm, and with marigold. After 60 days, the population 309 nm. from tomato roots was comparable with that Crude concentrate on a column of silica gel from any other treatment, and the number and eluted with absolute ethanol produced a from marigold grown with tomato was higher single fraction with an absorption maximum than that from marigold grown alone (Table at 264 nm. 2). After 90 days, numbers recovered from No fractions obtained from castor bean marigold roots declined. After 90 days, extractions showed the ultraviolet absorption tomato grown with castor bean had 299 M. maxima reported for marigold extracts incognita males per gram root dry wt. containing the nematicidal thiophene compared to only 10/gdry wt. recovered from derivatives (251 nm, 340 nm, and 251 nm, 350 tomato grown with chrysanthemum, and rim) (19). none from tomato grown with marigold or from tomato grown alone (Table I). DISCUSSION AND CONCLUSION in general, more galls, but not more egg masses of M. incognita were produced after 90 Meloidogyne incognita reproduced equally than after 60 days, indicating that the life cycle well on marigold, castor bean, and under our conditions was longer than 30 days chrysanthemum but significantly (P <~ 0.05) (Table 3). Relatively few P. alleni were in less than on tomato. The reduced roots of marigold, castor bean, or reproduction in marigold and castor bean chrysanthemum when compared with obviously resulted from aborted giant cells tomato. Marigold, castor bean, which prevented development of M. chrysanthemum, and fallow reduced the incognita. In chrysanthemum, giant cell number of M. incognita larvae in soil, but development appeared normal, but there were marigold and chrysanthemum did not reduce fewer of them. Fewer egg masses were soil populations when grown with tomato for produced, and thus fewer larvae were 90 days. recovered. In marigold, few developing larvae Chemical extractions: Using thin-layer and no males were recovered from infected chromatography, crude concentrate obtained roots. In castor bean, the number of males was from castor bean roots was compared with 2- relatively greater, and aborted giant cells were methyl-thiophene, 2-thiophene- common. In chrysanthemum a relatively large carboxaldehyde, and 3-(2-thienyl)- number of males was recovered, but the giant acrylic acid. With silica gel and methanol, cells appeared normal. This suggests that the crude concentrate moved with 3-(2-thienyl)- resistance mechanism in the three plants acrylic acid and could not be resolved. No differed. movement was obtained with either alumina Painter (15) thought that resistance and petroleum ether or silica gel and benzene- involved a triad of basic relationships: chloroform (9:1, v/v). antibiosis, tolerance, and preference or Column chromatography with alumina and nonpreference. Aborted giant cells are either a mixture of petroleum ether with common in resistant plants (4, 5) and are a increasing quantities of diethyl ether or form of antibiosis. Sex reversal during periods diethyl ether as eluent produced fractions that of stress, and in unfavorable hosts, also is fluoresced at 280 nm and had ultraviolet known for Meloidogyne spp. (9, 18). Dropkin absorption spectra (solvent ethanol) with (4) pointed out that tolerance should be maxima at 228 nm, 254 nm, 275 nm, 285 nm, distinguished from resistance by considering and 335 nm. Residues from cleansed success of the parasite population as well as petroleum ether used in natural product success of the plant. Whether the antibiosis extractions and chromatography had maxima observed in this study constitutes resistance or at 223 nm, 232 nm, 253 nm, 256 nm,262 nm, tolerance could not be determined because 270 nm, 273 nm, 275 nm, and 288 rim. only parasite productivity was measured. Meloidogyne and Pratylenchus Control: Hackney and Dickerson 89

Based on our data, both M. incognita and P. that T. patula severely suppressed M. hapla, alleni exhibited early nonpreference (as M. incognita, M. arenaria, and M.javanica. indicated by infection during the first 30 days) Yields of main crops after Tagetes were for marigold but did not differentiate among increased by nematicidal action, growth tomato, castor bean, and chrysanthemum. factors, or both, and Tagetes promoted Winoto Suatmadji (2 I) observed that Tagetes growth of apple seedlings in soil with P. patula may resist penetration of P. penetrans penetrans populations up to 167% of fallow- (Cobb); he based his conclusions on the first 4 infested soil, although Tagetes decreased weeks after inoculating with 500 larvae/8 ml growth of apple in uninfested soil. Also, direct of sand and sand-perlite mixture in tube mulch with a "natural dosage of Tagetes culture. After one week, 7 larvae had roots" suppressed P. penetrans much better penetrated the roots, and he recovered 354 than did other mulches or fallow. He from the sand mixture surrounding the roots. recommended, therefore, that simultaneously We recovered no P. alleni from roots of cultivating Tagetes with a main crop would be marigold or tomato grown with marigold 30 effective around and between trees and woody days after transplanting into infested soil; yet ornamentals. after 60 days, these treatment combinations Miller and Ahrens (12) reported that had many P. alleni. We stress recovery rather marigolds suppressed populations of P. than penetration because after 30 days, we penetrans and that their use, in any rotation found about as many P. alleni per unit of where cover crops are grown for an entire stained roots grown with marigold as in season and where nematode control is tomato grown either with another host or desirable, is economically competitive with alone (Table 4). We have no explanation for ethylene dibromide at 56.2 liters/hectare. nonmigration from tomato roots grown with They also reported that marigold interplanted marigold. with strawberry, tomato, and gladiolus did We found no evidence that marigold, castor not increase yields, but acted like a weed in bean, chrysanthemum, or tomato killed competing for water and nutrients. nematodes in soil by nematicidal action. In this study, 'Lemondrop' marigold, Marigold, castor bean, and chrysanthemum 'Bronz King' castor bean, and 'Escapade' did not significantly reduce nematode chrysanthemum reduced populations of M. populations in tomato plants with which they incognita and P. alleni, but not significantly were cultivated simultaneously, compared more than did fallow. In combination with a with tomato plants grown alone in infested good host ('Rutgers' tomato), 'Lemondrop' soil (Tables 1, 2). marigold, 'Bronz King' castor bean, and Oostenbrink et al. (14) found a 90% 'Escapade' chrysanthemum did not reduce suppression of Prao'lenchus by T. patula numbers of M. incognita or P. alleni. 'Bronz compared with fallow soil, and they suggested King' castor bean lacks nematicidal thiophene that Tagetes might alleviate attacks on derivatives present in marigold. perennials and prevent damage to new plantings. Good et al. (6) thought South LII'ERATURE CITED American marigolds were promising as a nematode-reducing cover crop to control M. I. ALVES, L. M., and G. B. BERGESON. 1967. A incognita acrita, M. incognita incognita, and quick destaining procedure for showing contrast others. However, they did not recommend between nematodes and root tissue. Plant Dis. Rep. 51:511. marigolds for reducing populations of M. 2. CAVENESS, F. E., and H. J, JENSEN. 1955. hapla and M. arenaria. Their study showed Modification of the centrifugal-flotation that, of Crotalaria, marigold, beggarweed, technique for the isolation and concentration of hairy indigo, sundangrass, millet, and Coastal nematodes and their eggs from soil and plant tissue. Proc. Helminthol. Soc. Wash. 22:87-89. bermudagrass, Crotalaria and marigold were 3. CURFIS, R. F., and G. T. PHILI,IPS. 1962. Thin- the most effective crops in reducing layer chromatographyof thiophene derivatives. ,1. populations of a wide range of soil nematodes. Chromatogr. 9:366-368. Winoto Suatmadji (21) found that Tagetes 4. DROPKIN, V. H. 1955. The relations between spp. were as effective as, or better than, fallow nematodes and plants, Exp. Parasitol. 4:282-322. 5. DROPKIN, V. H., and P. E. NELSON. 1960. The in suppressing Meloidogyne spp. He noted histopathology of root-knot nematode infections that Meloidogyne larvae were less persistent in soybeans. Phytopathnlogy 50:442-447. that Pratylenchus larvae in fallow soil, and 6. GOOD, J. M., N. A. MINTON, and C. A. 90 Journal of Nematology, Volume 7, No. I, January 1975

JAWORSKI. 1965. Relative susceptibility of 14. OOSTENBRINK, M., K. KUIPER, and J. J. selected cover crops and Coastal bermudagrass to S'JACOB. 1957. Tagetes als Feindpflanzen yon plant nematodes. Phytopathology 55:1026-1030. Pratylenchus-Arten. Nematologica Suppl. 7. GOODLY, J. 13. 1963. Laboratory methods for work 2:424S-433S. with plant and soil nematodes. Ministry of 15. PAINTER, R. H. 1951. Insect resistance in crop Agriculture, Fisheries and Food, London, Her plants. Macmillan, New York. 520 p. Majesty's Stationery Office. Tech. 13ull. No. 2. 72 16. SASSER, J. N. 1954. Identification and host-parasite p. relationships of certain root-knot nematodes 8. GUDDAL, E., and N. A. SORENSEN. 1959. Studies (Meloidogyne spp.). Md. Agric. Exp. Stn. Bull. A- related to naturally occurring acetylene 77.31 p. compounds. Part XXV. The occurrence of methyl 17. SITARAMAIAH, K., R. S. SINGH, and K. P. cis-/3-(5-propynyl-2-thienyl)-acrylate in the roots S1NGH. 1969. Induction of ovoviviparity in of tansy. Acta Chem. Scand. 13:1185-I 188. Meloidogyne javanica by fatty acids. 9. LAUGHLIN, C. W.~ A. S. WILLIAMS, and J. A. Nematologica 15:163-164. FOX. 1969. The influence of temperature on 18. TRIANTAPHYLLOU, A. C. 1960, Sex development and sex differentiation of determination in Meloidogyne incognita Meloidogyne graminis. J. Nematol. 1:212-215. (Chitwood, 1949), and intersexuality in M. 10. MC BEIH, C. W., A. L. TAYLOR, and A. L. javanica (Treub, 1885). Ann. Inst. Phytopathol. S M llH. 1941. Note on staining nematodes in root Benaki, N.S. 3:12-31. tissue. Proc. Helminthol. Soc. Wash. 8:26. 19. UHLENBROEK, J. H., and J. D. BIJLOO. 1958. Investigations on nematicides. 1. Isolation and 11. MILLER, P. M. 1957. A method for the quick structure of a nematicidal principle occurring in separation of nematodes from soil samples. Plant Tagetes roots. Rec. Tray. Chim. Pays-Bas Belg. Dis. Rep. 41:194. 77:1004-1009. 12. MILLER, P. M., and J. F. AHRENS. 1969, 20. UHLEN13ROEK, J. H., and J. D. 131JLOO, 1959. Influence of growing marigolds, weeds, two cover Investigations on nematicides. 2. Structure of a crops and fumigation on subsequent populations second nematicidal principle isolated from of parasitic nematodes and plant growth. Plant Tagetes roots. Rec. Tray. Chim. Pays-Bas Belg. Dis. Rep. 53:642-646. 78:382-390. 13, OOSTENBRINK, M. 1960. Estimating nematode 21. WINOTO SUATMADJI, R. 1969. Studies on the populations by some selected methods. Pages 85- effect of Tagetes species on plant parasitic 102 in J. N, Sasser and W. R. Jenkins, eds. nematodes. Stichting Fonds Landbouw Export Nematology. University of North Carolina Press, Bureau publicatie 47. Veenan und Zonen N.W., Chapel Hill. Wageningen, The Netherlands. 132 p.