154 Journal o/Nematology, Volume 9, No. 2, April 1977 10. PAIN, B. F., and N. G. M. HAGUE. 1971. The cysts of Heterodera schachtii. J. Am. Soc. effect of organophosphate and carbamoy|- Sugar Beet Technol. 11:528-532. oxime nematicides against the potato cyst 14. STEELE, A. E. 1976. Improved methods of eelworm, Heterodera rostochiensis Woll. hatching Heterodera schachtii larvae for Meded. Fac. Landbouwwet. Rijksuuiv. Gent. screening chemicals. J. Nematol. 8:23-25. 36:161.173. 15. STEELE, A. E. 1976. Effects of oxime carbamate 11. REDDY, D. D. R., and A. R. SESHADRI. 1971. nematicides on development of Heterodera Studies on some systemic nematicides. I. schachtii on sugarbeet. J. Nematol. 8:137-141. Evaluation for systemic and contact action 16. STEELE, A. E., and L. R. HODGES. 1975. In- against the root-knot nematode, Meloidogyne vitro and in-vivo effects of aldicarb on incognita. Indian J. Nematol. 1:199-208. survival and development of Heterodera 12. SPURR, H. W. 1966. Nematode cholinesterases schachtii. J. Nematol. 7:305-312. and their inhibition by nematocidal carba- 17. STEUDEL, W. 1972. Versuche zum Einfluss yon mates. Phytopathology 56:902-903 (Abstr.). Aldicarb auf den Schliipvorgang bet Zysten 13. STEELE, A. E. 1961. The effect of nabam yon Heterodera schachtii nach liingerer solution on the emergence of larvae from Einwerkung. Nematologica 18:270-274. Effects of Pratylenchus vulnus on the Growth of Sour Orange R. N. INSERRA and N. VOVLAS ~ Abstract: Pratylenchus vulnus suppressed the growth of sour orange seedlings in greenhouse experiments. Growth retardation (in height, in trunk diameter, and in dry top and root weights) was observed in inoculated plants growing in two soil types. Population density, 13 months after inoculation, averaged more than 1,000 specimens/gin of fresh root. Anatomical studies showed that P. vulnus prefers to attack the cortex and causes cavities among the cortical cells. Key Words: lesion nematode, population density, soil type. Pratylenchus spp. are reported as being 13) and with declining sour orange seed- pathogenic to citrus in greenhouse experi- lings in nurseries in Italy (5). ments and as having a wide host range No information is available on the among citrus species (3, 7, 8). PratyIenchus pathogenicity of this species to Citrus. This coffeae (Zimmermann) Filipj. 8c Schuur.- paper reports the pathogenic effects of P. Stekh. causes citrus decline in Florida (10), vulnus to sour orange (Citrus aurantium Japan (15), and India (12). Stunting, die- L.) under greenhouse conditions. back, and poor growth with feeder-root lesions are the symptoms observed in P. METHODS AND MATERIALS col~eae-infected citrus trees. Pratylenchus Two soils, a river sand and Massafra vulnus Allen 8¢ Jensen has been associated sandy clay loam, were used in this study occasionally with citrus in California (1, 2, conducted in Italy. Each soil had previously been steam sterilized and stored for 40 days. Received for publication 9 September 1976. Physical and chemical properties of the soil a Nematologists, Laboratorio di Nematologia agraria of the C.N.R., 70126 Bari, Italy. are given in Table 1. TABLE 1. Physical and chemical characteristics of soils utilized. Clay Silt Sand Gravel Organic (<~0.002 mm) (0.002-0.05 ram) (0.05-2 mm) (~2 ram) matter Soil type pH (%) (%) (%) (%) (%) Massafra sandy clay loam 7.60 22.62 10.76 62.20 4.42 1.70 River sand 8.90 0.72 4.11 65.89 29.28 0.02 Pratylenchus vulnus on Citrus: Inserra, Vovlas 155 TABLE 2. Influence of Pratylenchus vulnus on the growth of sour orange seedlings 13 months after inoculation. Soil type and P. vulnus Total Trunk Dry root Dry top initial inoculum height diam. weight weight level (cm) (ram) (gin) (gin) Sandy clay loam y 100 93.9a 8.1a ll.6a 16.7ab 250 81.4a 7.2b 9.7a 11.1a 500 78.0a 7.8ab ll.5a 13.7a Control 126.8b 9.2c 15.1b 21.3b River sand z 100 6.8 8.8 Control 8.9" 20.9"* YMeans followed by the same letters are not significantly different (P ~_~ 0.05) according to Duncan's Multiple Range Test. "Asterisks (% **) indicate significantly greater than inoculated plants at P = 5 and 1% level, respectively. One-year-old sour orange seedlings were incubation method (16). Pots were random- selected for uniformity and transplanted ized on a glasshouse bench and plants were into 18-cm clay pots, each containing one of grown for 13 months at 24-26 C. the soils. Plants growing in Massafra sandy Root samples were collected for nema- clay loam were inoculated 1 month later by tode population counts and plant height pipetting water suspensions of I00, 250, or measnrements were taken at 6, 11, and 13 500 P. vulnus into four holes spaced evenly months after inoculation (always from the in the soil around the base of each plant. same pots). Individual root samples were Each inoculum level was replicated 10 cut into segments, washed in tap water, and times, one seedling/replicate. Five replicates incubated moist in half-liter jars in the of plants growing in the river sand received dark at 22-24 C (16). Nematodes that 100 nematodes/plant 1 month after being emerged were counted at 2 and 4 days. At transplanted with the same inoculation harvest, top and root oven dry weights and procedure. Noninoculated plants served as stem diameter measurements were taken. controls. Nematode inoculum was obtained Results were anaIyzed by either Duncan's from infected seedling roots collected in a multiple range test or Student's 't' test. citrus nursery by using the Young's jar So that roots could be examined micro- scopically, root segments were washed free TABLE 3. Mean numbers of Pratylenchus of soil, fixed in FAA (formalin-acetic acid- vulnus/gm of sour orange fresh root. alcohol) for 48 h, dehydrated in tertiary butyl alcohol, embedded in paraffin, sec- Soil type and tioned at 10-15 p,m, stained in safranin-fast P. vulnus No. nematodes green, mounted in Permount, and examined initial inoculum (months after inoculation) with a compound microscope (6). For level 6 11 13 examination with a scanning electron microscope, longitudinal and cross sections Sandy clay loam y of roots were cut at 20/~m, washed twice in 100 230 1,745 1,167 benzene to remove paraffin, washed in 250 305 901 1,185 acetone, metalized with gold, and examined 500 614 !,733 1,313 (4). River sand = 100 3,719 1,145 1,959 RESULTS AND DISCUSSION YMean of 10 replicates. Growth of seedling roots infected with =Mean of 5 replicates. P. vulnus in the sandy clay loam soil was 156 ]ournal o[ Nematology, Volume 9, No. 2, April 1977 FIG. 1-3. 1) Effects of Pratylenchus vulnus (PV) on roots of sour orange seedlings growing in fine tex- tured soil (sandy clay loam). Three plants at left ("INF") = infected; three plants at right ("NON INF") = controls. 2) Longitudinal section of sour orange root showing coagulated cortical cells around a specimen of P. vuInus (N), (scanning electron microscope). 3) Longitudinal section of sour orange root showing P. vulnus in a cavity in the cortical tissue. severely suppressed (Fig. 1). Plant height, density was observed 6 months after inocula- trunk diam., and dry root and top weights tion in the roots of seedlings grown in river of inoculated plants were (P --~ 0.05) less sand than in sandy clay loam. This result than noninoculated controls (Table 2). In might indicate that coarser soils favour early river sand, statistically significant growth re- and rapid root invasion by P. vulnus. No tardation of inoculated seedlings occurred, substantial differences between nematode in comparison with that of noninoculated populations were detected in the two soil seedlings (Table 2). The trunk diam. and types at 11 and 13 months after inoculation dry root weights of inoculated plants were (Table 3). These similar populations in the respectively 23 and 57 % less titan controls. two soil types were tile result of the damage Tile numbers of P. vulnus extracted being similar in both soils. from plants grown in sandy clay loam at 6, Cross sections of infected feeder root 11, and 13 months were not significantly showed that P. vulnus usually invaded different among the three inoculum levels cortical tissue, an action which resulted in (Table 3). A higher nematode population large cavities. Cells adjacent to cavities had Pratylenchus vulnus on Citrus: Inserra, VovIas 157 coagulated protoplasm which took the 5. INSERRA, R., and N. VOVLAS. 1974. Damage safranin stain (Fig. 2, 3). Occasional dam- by Pratylenchus vulnus to sour orange in age to the epidermal tissue was observed. Apulia. Nematol. Medit. 2:183-185. 6. JOHANSEN, D. A. 1940. Plant microtechnique. No evidence of damage to stelar tissue was McGraw-Hill Book Co., Inc. N.Y., 523 pp. noted. 7. O'BANNON, J. H., and R. P. ESSER. 1975. Pratylenchus vulnus caused stunting of Evaluation of citrus hybrids and relatives as sour orange seedlings similar to that caused hosts of Pratylenchus coffeae, with comments on other hosts. Nematol. Medit. 3:113-120. by other Pratylenchus spp., especially P. 8. O'BANNON, J. H., R. P. ESSER, and R. N. co~eae. The behaviour of P. vulnus in INSERRA. 1975. Bibliography of nematodes relation to sour orange is similar to that of citrus. ARS-S-68, U.S. Dep. Agric. 41 p. reported for P. coffeae (11). Both nema- 9. O'BANNON, J. H., J. D. RADEWALD, A. T. todes are localized in cortical tissue, seldom TOMERLIN, and R. N. INSERRA. 1976. Comparative influence of Radopholus similis invade vascular tissue, and suppress growth.