influence of vulnus and Meloidogyne hapla on the Growth of Rootstocks of Rose~ G. S. SANTO 2 and BERT LEAR '~

Abstract: is involved in a disease of Rosa noisettiana 'Manetti" rose rootstock characterized by darkening of roots, death of feeder toots, and stunting of entire plants. The disease is more severe when plants are grown in silt loam soil than when they are grown in sandy loam soil. The reproduce best in silt loam soil at 20 C. Meloidogyne hapla did not affect the growth of Manetti. Rosa sp. 'Dr. Huey', Manetti, and R. odorata rose rootstocks were found to be good hosts for P. vulnus whereas R. multiflora was less suitable. M. hapla re- produced well on R. odorata, Dr. Huey, and R. multi[lora, but not on Maneni. Key Words: root- lesion , root-knot nematode, reproduction, soil temperature, soil type.

Pratylenchus vulnus Allen and Jensen roses was associated with a reduction in the was first reported from rose roots in Cali- population of M. hapla and Xiphinema fornia in 1953 (14). A 1970 survey of com- arnericanum Cobb as a result of multiple mercial rose greenhouses in northern Cali- applications of 1,2-dibromo-3-chloropro- fornia shows that this nematode is now pane (DBCP) (8). widely distributed (9), and Allen and Jen- Rosa noisettiana Thory 'Manetti' is the sen (l) also report that P. vulnus is widely most popular rootstock used in growing distributed on field-grown roses in southern greenhouse roses for cut flowers. R. odorata California. Sweet and Rosa sp. Dr. Huey (Schafter) Sher (15) demonstrated that P. vulnus rootstocks are used less frequently. is associated with a disease of Rosa sp. 'Dr. Because Manetti is an important root- Huey' rose rootstock. Plants infected with stock and P. vulnus and M. hapla have nematodes are stunted and chlorotic. The been associated with diseases of roses, tests root systems are necrotic and have fewer were conducted to determine their influ- feeder roots than noninfected plants. Sher ence on the growth o[ Manetti. Tests using and Bell (16) found that the disease is most controlled temperature conditions and two severe in plants grown in light sandy soil types of soil were included in the experi- at 29.4C (85F) or 32.2C (90) F). ments with P. vulnus. In addition, four Meloidogyne hapla Chitwood commonly rose rootstocks (Dr. Huey, Manetti, R. occurs and is widely distributed in com- multiflora, R. odorata) were tested to mercial rose greenhouses in northern Cali- compare their suitability as hosts for P. fornia (9). In 1969, severe economic losses vulnus and M. hapla. in the production of 2-year-old field-grown roses (Rosa multiflora japonica) were at- MATERIALS AND METHODS tributed to ,~I. hapla (6). Davis and Jenkins Virus-free Manetti plants were obtained (5) showed that M. hapla causes extensive from the foundation block, Foundation injury to roots of R. multiflora. In Cali- Plant Material Service, University of Cali- fornia, an increase in tire production of cut fornia, Davis. Cuttings 20-cm long were rooted in a mixture of perlite and vermicu- Received for publication 8 May 1975. 1 Portion of a Ph.D. thesis submitted by the senior author lite (2:3). After 6 weeks, rooted cuttings at University of California, Davis 95616. This study was were transplanted to soil in 10-cm diam clay supported in part by Roses. Inc. Appreciation is extended to Dr. George Nyland for supplying virus-free rose root- pots and, 2 weeks later, they were trans- stocks and to Dr. Hassan Mojtahedi for assistance in planted to soil in 7.5-liter plastic pots (un- statistical analysis. -°Former graduate Research Assistant, Department of Nem- less indicated differently). At this time, atology, University of California, Davis; currently Assistant treatments were made and the plants were Plant Pathologist-Nematologist and Assistant Professor of Plant Pathology, Irrigated Am'iculture Research and Ex- arranged in randomized blocks on a green- tension Center, Washington State University, Prosser, house bench or in temperature tanks. Washington 99350. aFormerly Professor, Department of Nematology, University Sandy loam soil (82% sand, 12% silt, of California, Davis; currently Professor, Department of 6% clay) amended with University Cali- Plant Pathology, University of California, Davis, California 95616. fornia (UC) mix (1 part UC mix: 3 parts

18 Pratylenchus and Meloidogyne on Roses: Santo, Lear 19 soil) (2) and/or silt loam soil (50% silt, tile nematodes; (iii) 100+7 P. vulnus; (iv) 28% sand, 22% clay) amended with red- 1,000-+22 P. vulnus; and (v) 10,000+216 wood shavings (1 part shavings: 4 parts P. vulnus. Each treatment was replicated soil) were used in the experiments. The silt eight times. The greenhouse temperature loam soil mixture was obtained from a com- during the experiment ranged from 12.8 mercial rose grower's greenhouse (Enomoto to 24.4 C, with a mean of 20.2 C. and Company, Halfmoon Bay, California). In the second experiment, temperature- All soils were steam-sterilized [3 h at 1.05 controlled water tanks were used to main- kg/cm 2 (15 psi)] at least 3 weeks before tain two temperatures (20 and 25 C). Two they were used. soil types (sandy loam and silt loam) were Tile population of P. vulnus, originally used in 15-cm diam clay pots. For each isolated from prunes, was increased and temperature and soil type, treatments were: maintained on carrot disks (12). M. hapla, (i) complete control (50 ml water); (ii) conL isolated from roses, was increased and main- trol testing the effect of microorganisms as- tained on roots of tomato grown in sand. sociated with the nematodes; and (iii) Nematodes were extracted by placing in- 10,000 +- 192 P. vulnus. Each treatment was fected tissues under a heated intermittent replicated six times. Tile soil temperature mist for 48-72 h. Inoculations were made during the experiment fluctuated between by transferring the desired number of nem- 19 and 23 C in one regime and 24 and 27 C atodes into 50 ml of water and pouring in the other with the higher temperatures this suspension around the roots of the occurring during the day. plants. Meloidogyne hapla: Silt loam soil was In treatments testing effects of associ- used in five treatments: (i) complete con- ated microorganisms, the nematodes from trol (50 ml water); (ii) control testing the the highest inoculum level were removed effect of microorganisms associated with tile by sieving and hand-picking. This treat- nematodes; (iii) 100-+8 M. hapla; (iv) ment was necessary to prove nematode in- 1,000-+15 ill. hapla; and (v) 10,000-+148 volvement (1 I). M. hapla. Each treatment was replicated Experiments were terminated after 4 10 times. The soil temperature during the months (unless otherwise indicated). Fresh experiment ranged from 11.1 to 33.3 C, weights of shoots and roots were deter- with a mean of 23.8 C. After 7 months, the mined, and nematode counts were made plants were harvested in the same manner from soil and roots. Nematodes were ex- as in the experiments with P. vulnus but tracted by placing root systems and a 50- only second-stage larvae were counted. cm:' portion of the mixed soil in separate Host suitability oJ rose rootstocks to P. funnels under heated intermittent mist for vulnus and H. hapla: Four rose rootstocks 7 days (unless otherwise indicated). Using (R. multi[tora, Dr. Huey, Manetti, R. standard cultural procedures, root segments odorata) were tested for their suitability as from each treatment were plated on PDA hosts for P. vulnus and M. hapla. Plants in an attempt to isolate any associated were grown in sandy loam soil in 15-cnl fungi. clay pots to which 1,000 nematodes were Plants were watered with distilled water added. Each treatment was replicated four and fertilized with one-half strength Hoag- times. The soil temperature during the test land's nutrient solution every 2 weeks. ranged from 13.9 to 26.7 C, with a mean of Pratylenchus vulnus: The first experi- 20 C. After 4 months, nematode counts ment was conducted to determine tile path- were made from the soil and roots. The ogenicity of P. vulnus on Manetti. A second centrifugal-flotation method (7) and incu- experiment was run to determine the effects bation under heated intermittent mist were that soil temperature and soil type have on used to extract the nematodes from the soil the population of P. vulnus and the sub- and roots, respectively. Only second-stage sequent effect on growth of Manetti plants. larvae of M. hapla were recovered. In the first experiment, silt loam soil was used in five treatments: (i) complete RESULTS control (50 ml water); (ii) control testing Pratylenchus vulnus: In the pathogenic- the effect of microorganisms associated with ity test with P. vulnus, final fresh weights 20 .Journal of Nentatology, Vohtme 8, No. 1, January 1976

TABLE 1. Reproduction of Pratylenchus vulnus served were similar to those in the previous and its influence on growth of Rosa noisettiana experiment. 'Maneni.' Meloidogyne hapla: In the pathogenic- ity test with M. hapla, no differences in Fresh weight Final number of fresh weight of plants were found among of plantsa nematodes/pota Treatment (g) (in 1,000's) treatments. The numbers of nematodes re- covered per pot were low: 147, 480, and 1,401 nematodes were recovered from pots Complete control 49.4 a 0 CMANb 48.2 ab 0 receiving 100, 1,000, and 10,000 nematodes, 100: 34.2 b 333 respectively; and only a few inconspicuous 1.000 26.3 bd 398 galls were observed on the roots of plants 10.000 17.4 c 212 receiving I0,000 nematodes. Host suitability of rose rootstocks to P. aMean of eight replicates after 4 months growth in vulnus and H. hapla: Dr. Huey, Manetti, silt loam soil. Values in each column not followed R. odorata, and, to a lesser degree, R. by the same letter differ significantly, P=0.01, ac- cording to Duncan's Multiple Range Test. multiflora are good hosts for P. vulnus t,Control testing the effect of microorganisms asso- (Table 3). M. hapla reproduced well on ciated with the nematodes. R. odorata, Dr. Huey, anti R. multiflora, ,'Differs from (;MAN, P=0.02. bttt poorly on Manetti. Dr. Huey, Manetti, and R. odorata root of plants were inversely proportional to the systems infected with P. vulnus were darker number of P. vulnus added (Table 1). and had fewer feeder roots than R. multi- Plants exposed to each level of nematodes flora. Similar symptoms were observed on weighed less than plants with no nema- roots of R. odorata and Dr. Huey infected todes. The control testing the effect of as- with M. hapla but not on roots of R. multi- sociated microorganisms did not differ sig- flora and Manetti. In addition, the roots of nificantly in fresh plant weight from the R. odorata and Dr. Huey had more and complete control. Stunting of tops was also larger galls than those of R. multiflora. No proportional to the number of P. vulnus galls were observed on Manetti. added (Fig. 1). Root systems of infected plants were smaller, darker, and had fewer DISCUSSION feeder roots (Fig. 1). Sloughing off of the cortex was evident on feeder roots. The resttlts show that P. vulnus is in- The final number of P. vulnus recov- volvetl in a disease of Manetti rose root- ered was highest in the pots receiving 1,000 stocks. Sher (15) reported results similar to nematodes (Table 1). The rate of repro- ours with P. vulnus on Dr. Httey rootstock. duction, however, was inversely propor- In the experiment using different num- tional to the number of nematodes added. bers of P. wdnus as inoculum, final weights Cephalosporium, Trichotheciurn, and of plants were inversely related to numbers Monilia species were isolated from the roots of nematodes added. The rate of reproduc- of plants in all treatments. tion was greatest in the lower nematode In the experiment involving the effects inoculum levels. This relationship was ex- of soil type and soil temperature, all plants pected, since there was less competition for infected with P. vulnus weighed less (P= feeding sites. A decrease in the rate of re- 0.01) than those in the complete control production would be expected over a longer or the control testing the effects of micro- period as the competition for feeding sites organisms associated with the nematodes increases. (Table 2). Nematode reproduction was Pratylenchus vulnus reproduced best in greater at 20 C and in the silt loam soil. silt loam soil at 20 C. Nematode damage to The plants grew better in the sandy loam the roots was also more severe in silt loam soil, but they were not affected by tempera- thart in sandy loam soil. Poorer plant ture. growth in silt loam soil without nematodes Regardless of soil temperature, nmre could accentuate damage caused by nema- nematode damage to the roots occurred in todes because plants growing under sub- silt loam soil. The disease symptoms ob- optimal conditions are generally more sus- Pratylenchus and Meloidogyne on Roses: Santo, Lear 21

i-% A B C D E

FIG. 1 (A-E). Influence of PJ'alyle~tchus vul,m~ on the growth of Rosa ~loisettimm 'Manettr in silt loam soil. (A) No nematodes. (B) Contl'ol testing the effed of microorganisms associated with the nema- todes. (C) 100 nematodes. (I)) 1,000 nematodes. (E) 10,00q) nematodes. ceptibfe to nematode damage. These results more rapidly at 25 C than at 20 C and that are significant because silt loam soil and no increase occurred at 30 or 35 C. soil temperatures of 20 C are present in Manetti was not a good host for M. some greenhouse-rose ranges in northern hapla. Berge (3), in France, found Manetti California. growing in moist soil to be a good host for Our results are contrary to those found M. hapla, although under dry conditions by Sher and Bell (16) with P. vulnus on M. hapla failed to maintain itself. Dr. Huey rootstock. They found that rose The resuhs of the host-suitability test plants grew better in heavier soil and that indicate that P. v~tlnus may be an im- nematodes reproduced best in lighter soil portant pathogen on R. odorata rootstock at higher temperatures (29.4 and 32.2 C). as well as on Manetti and Dr. Hney. Dam- Lownsbery et al. (10), working with alfalta age to the root systems of these three root- callus tissue, found that P. vulnus increased stocks was similar. Likewise, M. hapla may 22 Journal o[ Nernatology, Volume 8, No. 1, January 1976

TABLE 2. Influence of soil type and tempera- The fungi (Cephalosporiurn, Tricho- ture on reproduction of Pratylenchus vulnus and thecium, Monilia species) isolated from the on growth of Rosa uoisettiana "Manetti.' P. vulnus experiments are not known path- ogens on roses. Although the probability Fresh that these fungi are involved with P. vulnus weight Final number of of plantsh nematodes/potb in causing a disease of Manetti is low, it "Freatment~l (g) (in 1,000's) should not be overlooked as a factor in nematode-fungus interactions. Several work- 20 C (L): ers have reported that fungi which seem Complete control 97,9 a 0 incapable of invading and damaging roots CMANe 90.1 a 0 do so when roots are predisposed by nema- 10,000 54.4 b 278 todes (4, 13). 20 C (H): Complete control 74.1 a 11 CMAN 84.6 a 0 LITERATURE CITED 10,000 40.3 b 699 1. ALLEN, M. W., and H. J. JENSEN. 1951. 25 C (L): Pratylenchus vulnus, new species (Nematoda: Complete control 87.1 a 0 Pratylenchinae), a parasite of trees and vines CMAN 89.6 a 0 in California. Proc. Helminthol. Soc. Wash. I0,000 44.1 b 57 18:47-50. 25 c (t-i): 2. BAKER, K. F. led.). 1957. The U.C. system for Complete control 78.1 a 0 producing healthy container-grown plants. CMAN 73.6 a 0 University of California Division of Agricul- 10,000 32.7 h 428 tural Sciences. Agric. Exp. Stn.--Ext. Serv. Manual 23, 332 pp. 3. BERGI~, J. 1971. Qualite d'hote de quelques "L=sandy loam soil; H=silt loam soil; Pi of 11),000 portegreffe de rosiers vis-a-vis de leurs prin- nematodes where indicated. cipaux nematodes parasite. Meded. Fac. Laud- bMean of six replicates after 4 months. Values in bouwwet. Ri]ksuniv. Gent. 36:883-888. each column not followed by the same letter differ 4. BERGESON, G. B. 1972. Concepts of nematode- significantly, P=0.01, according to Duncan's Multi- fungus associations in plant disease com- ple Range Test. plexes: a review. Exp. Parasitol. 32:301-314. cControl testing the effect of microorganisms asso- 5. DAVIS, R. A., and ~V. R. JENKINS. 1960. Nem- ciated with the nematodes. atodes associated with roses and the root in- jury caused by Meloidogyne hapla Chitwood, TABLE 3. Reproduction of Pratylenchus vulnus and Meloidogyne hapla on four rose rootstocks. 1959, Xiphinema diversicaudatum (Mico- letzky, 1927) Thorne, 1939, and Helicoty- lenchus nannus Steiner, 1945. Bull. Md. Agric. Final number of Exp. Stn. No. A-106, 16 pp. Treatmenta nematodes/pot ~' 6. HART, W. H., and A. R. MAGGENTI. 1971. (Nematode g: rootstock) (in 1,000's) Control of root-knot nematode, Meloidogyne hapla, on 2-year-old field-grown roses, Rosa multiflora japonica. Plant Dis. Rep. 55:89-92. Pratylenchus vulnus: 7. JENKINS, W. R. 1964. A rapid centrifugal- Rosa sp. 'Dr. Huey' 196 flotation technique for separating nematodes R. noisettiaua 'Mauetti' 162 from soil. Plant Dis. Rep. 48:692. R. odorata 151 8. JOHNSON, D. E., B. LEAR, S. T, M1YAGAWA, R. multiflora 31 and R. H. SCIARONI. 1969. Multiple appli- Meloidogyue hapla: cations of 1,2-dibromo-3-chloropropane for control of nematodes in established rose Rosa sp. 'Dr. Huey' 129 plantings. Plant Dis. Rep. 53:34-37. R. noisettiana 'Manetti' 0.2 R. odorata 170 9. LEAR, B., S. MIYAGAWA, and R. SCIARONI. R. multiflora 78 1970. Rose nematode survey. Flower and Nursery Report for Commercial Growers, November 1970:8. al,000 nematodes were added initially to each pot. 10. LOWNSBERY, B. F., C. S. HUANG, and R. N. bMean of four replicates after 4 months. JOHNSON. 1967. Tissue culture and main- tenance of the root-lesion nematode, Praty- be important on R. odorata and Dr. Huey lenchus vulnus. Nematologica 13:390-394. and, to a lesser extent, on R. multi[lora. 11. MAI, W. F. [Chairman]. 1968. Principles of Plant and pest control. Vol. 4. Control Therefore, these rootstocks should be of plant-parasitic nematodes. Natl. Acad. Sci. avoided in fields infested with either of Natl. Res. Couuc. Puhl. 1696. Washington, these nematodes. D.C. Pratylenchus and Meloidogyne on Roses: Santo, Lear 23

12. MOODY, E. H., B. F. LOWNSBERY, and J. M. lenchidae). Univ. Calif. (Berkeley) Publ. Zool. AHMED. 1973. Culture of the root-lesion 57:441-470. nematode Pratylenchus vulnus tm carrot 15. SHER, S. A. 1957. A disease of roses caused by disks. J. NematoL 5:225-226. a root-lesion nematode, Pratylenchus vulnus. 13. POWELL, N. T. 1971. Interactions between l'hytopathology 47:703-706. nematodes and fungi in disease complexes. 16. SHER, S. A., and A. H. BELL. 1965. The effect Atnm. Rev. Phytopathol. 9:253-274. of soil type and soil temperature on root- 14. SHER, S. A., and M. W. ALLEN. 1953. Revision lesion nematode disease of roses. Plant Dis. of the genus Pratylenchus (Nematoda: Ty- Rep. 49:849-851.

Improved Methods of Hatching Heterodera schachtii Larvae for Screening Chemicals ARNOLD E. STEELE I

Abstract: The rate of hatching of Heterodera schachtii larvae was greatly increased by placing cysts in sieves enclosed by small disposable cups. An apparatus that permitted rapid storage of second-stage larvae at 10 C prolonged the viability of the larvae. Key Words: Hatching ap- paratus, sugarbeet nematode, increased viability.

Syracuse watchglasses have been ex- that enabled rapid inactivation of larvae tensively used to evaluate hatching and by storage at low temperatures (5-10 C) emergence of larvae from cysts of Hetero- soon after hatching was developed, tested, dera schachtii Schmidt (6). The use of and compared with the watchglass method. watch glasses as hatching vessels is adequate This method was used to obtain large num- for relatively small tests of about 50 sanl- bers of hatched L2 which were then used pies. However, this method is not suitable to evaluate the nematicidal efficacies of for bioassay of large numbers of samples oxime carbamates (8). for hatching activity, especially when sam- pie volumes available for bioassay are not MATERIALS AND METHODS greater than 5 ml/sample, and cysts must The watchglass and sieve methods were be transferred to fresh solutions during the evaluated for relative effectiveness in hatch- bioassay. Reports have shown that greatly ing larvae of Heterodera schachtii. Newly increased hatches can be obtained with formed cysts were separated from sugarbeet methods employing sieves (2, 4, 5). roots and soil by floating and decanting Hatched second-stage (L2) larvae of H. suspended debris into screens. Cysts with schachtii rapidly lose viability when stored viable eggs and larvae were selected and at temperatures above 20 C. However, the lnanually separated from soil and root deb- rate of larval hatch may not peak until late ris as previously described (7) and stored in the second week when cysts are incu- 10 days at 8 C. Groups of 20 cysts were bated at 24 C, the optimum hatching tem- incttbated in 15 ml of sugarbeet root dif- perature for H. schachtii populations from fusate (6) or tap water within Syracuse the Salinas Valley of California (Steele, un- watchglasses or in small sieves (Fig. l-C) published). As a result, the majority of (collection cups supplied by Hykro Pet In- hatched L2 that have accumulated may not dustries, Esbjerg, Denmark) enclosed by be viable (1). Consequently, a sieve method plastic portion cups (Thunderbird Con- tainer Corporation, El Paso, Texas 79912) Received for publication 20 June 1975. (Fig. l-A, B). The cups also contained 5 ml l Nematologist, Agricultural Research Service, U. S. Depart- of treatment solution. Treatments were ment of Agriculture, P. O. Box 5098, Salinas, CA 93901. Mention of a proprietary product does not constitute a replicated four times. Cysts were transferred guarantee or warranty of the product by tile U. S. Depart- to fresh test solutions at 5-day intervals, and ment of Agriculture, and it does not imply approval to the exclusion of other products that may also be suitable. the emerged larvae were counted.