CAMELLIA SINENSIS PARASITISM OF SIX NEAAATODE SPECIES ON. At least 2 attempts were made to establish. vesting. The introduction of mechanical tea

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1 410 FLORIDA STATE HORTICULTURAL SOCIETY, 1964 Summary and Conclusions A nitrogen source-fertilizer rate experiment was initiated in 1957 to study the fertilization of lychees on Lakeland fine sand. Results of the first five years showed no significant difference in tree growth due to nitrogen source or fertilizer rates. In general, growth rate was lowest in plots where sludge was the only source of nitrogen. This usually was accompanied by a chlorotic ap pearance of the trees during cold, dry weather. Greatest growth was obtained from trees which received the medium rate of fertilizer with nitro gen derived from a combination of ammonium nitrate and sludge. It seems that the low rate of fertilization was ample for these young trees. Three years data on bloom and yield showed no consistent trend due to nitrogen source except in 1960 when trees under the sludge-only treat ments bloomed and yielded signicantly less than those under other nitrogen sources. In 1961 highly significant increases in bloom occurred with increasing rate of fertilization in the sul phate of ammonia, ammonium nitrate and sludgeonly treatments. The nitrogen content of leaves from plots where sludge was the only source of nitrogen was less, with highly significant differences, than from the other nitrogen sources at a given rate of fer tilization. The potassium content of leaves from sludge-only plots was significantly higher than those from other nitrogen sources. The calcium and magnesium contents of leaves were significantly lower where nitrogen was de rived from sulphate of ammonia than from other sources. LITERATURE CITED 1. Cobin, Milton, and R. Bruce Ledin The lychee in Florida. Fla. Agr. Exp. Sta. Bui DuCharme, E. P., and R. F. Suit Immunity of lychees to the burrowing nematode. Fla. State Hort. Soc. Proc. 68 : Goldweber, Seymour Observations on lychees grown in pot culture. Fla. State Hort. Soc. Proc. 72: Joiner, Jasper N., and R. p. Dickey Effects of nitrogen, potassium and magnesium levels on growth and chemical composition of the lychee (Litchi chinensis Sonn) grown in sand culture. Proc. Amer. Soc. Hort. Sci. 77: Lynch, S. J., and Roy O. Nelson Care of young lychee trees. Florida Grower. Jan. 1953, p Reitz, H. J., et al. (In press) Recommended fertilizers and nutritional sprays for citrus. Fla. Agr. Exp. Sta. Bui. 536B. 7. Young, T. W., and J. C. Noonan Influence of nitrogen source on cold tolerance of lychees. Proc. Amer. Soc. Hort. Sci. 73 : PARASITISM OF SIX NEAAATODE SPECIES ON CAMELLIA SINENSIS H. V. Morton1 Research Assistant Department of Entomology University of Florida Gainesville Introduction At least 2 attempts were made to establish commercial acreages of Chinese varieties of Ca mellia sinensis (L.) in the United States during the last century. These gardens were situated in South Carolina. According to Mitchell (12), the failure was attributed to the high costs of har vesting. The introduction of mechanical tea harvesting in other countries has again stimu- Florida Agricultural Experiment Stations Journal Series No lthis work was conducted as partial fulfillment of the Master of Science in Agriculture degree while the author was a Research Assistant in the Department of Fruit Crops of the University of Florida. lated interest in commercial production of the higher yielding Assam-Chinese hybrids of tea in Florida. Since tea harvest involves regular "plucking" of the top 2 leaves and a bud, any limitation of the continual development of the vegetative growth adversely effects yields. One widespread group of micro-organisms limiting root growth, and thus vegetative growth, is the nematodes. As effective control of plant parasitic nematodes is extremely difficult under a permanent cropping system, it was decided to establish the host status of 6-month-old Assam-Chinese hybrid seedlings in Florida to the following 6 nematode species: Radopholus similus (Cobb, 1893) Thorne, 1949; Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949; Meloidogyne javanica (Treub, 1885) Chitwood, 1949; Pratylenchus coffeae (Zim merman, 1898) Filipjev and Stekhoven, 1941; Belonolaimus longicaudatus Rau, 1958; Hoplolaimus coronatus Cobb, These species were selected because of their prevalence in Florida and, in the case of the first

2 MORTON: NEMATODES ON CAMELLIAS 411 4, their parasitism on tea as noted in the host list compiled by T. Goodey (6) and supplemented by J. B. Goodey et al. (5). Zimmerman (18) first reported a nematode species as parasitising tea, when he found R. similis on stunted, wilted seedlings in nursery beds in Java. Ever since 1939 when Gadd (4) reported Pratylenchus loosi as parasitising tea in Ceylon, work there has centered on this species. Injury is manifested in plantings as patches of unthrifty bushes. Scattered throughout these areas were bushes that did not appear to be af fected. The infected bushes put on little new growth, the leaves were smaller, and the internodes reduced in length (witches-broom). A pro nounced tendency toward flowering was often evi dent. An investigation of the roots showed a gen eral lack of feeder roots and bark lesions on the storage roots. Hutchinson and Vythilingham (7) found that P, loosi recovered from soil in excess of 125 per 100 grams were usually associated with severe injury to tea, while 70 or more would generally cause severe injury. At the lower limit, no obvious injury was found with 5 P, loosi per 100 grams. Soil temperature is an important environmental factor concerning distribution, the optimal soil temperature being 65 to 70 F. In Assam and Africa (13, 11) Meloidogyne spp. are the primary nematodes parasitic on tea, in particular M. incognita and M. javanica. Loos (10) reported that tea is very susceptible to root-knot while in the young seedling stage, but that a form of resistance develops with age until an apparent immunity is acquired. This research has been confined by Mukerjea (13) and Martin (11) although there is some discrepancy as to the age at which this resistance develops. The importance of nematode races or biotypes has still not been elucidated and their oc currence has led to many conflicting reports in the literature. Steiner (16) concluded that all experiments on nematode resistance, immunity or susceptibility of plants should be made only with populations of which the history is well-known for at least a number of generations. Materials and Methods Open-pollinated Assam-Chinese hybrid tea seed imported from the Kiganga Estates in Tan ganyika were germinated in sphagnum moss kept at 70 F. The seedlings were then grown in 6 x 18 inch polyethylene bags containing a 75:25 mixture of peat and perlite under 70 percent shade. Three-month-old plants of similar size were selected for each treatment and transplanted into 8-inch cans containing Arredondo topsoil which had been fumigated with methyl-bromide. The plants were then transferred into a growth chamber at 78 F and a light photoperiod of 8 hours per day. Soil temperatures ranged between 72 and 78 F. For inoculations involving Meloidogyne spp. 10 grams of infected roots from a known source were cut into one-inch lengths and thoroughly mixed with the soil around each of the 5 tea plants making up the respective treatments. The endo-parasites, R, similis and P. coffeae, were collected using a food blendor as recommended by Taylor and Loegering (17); the ectoparasites, H. coronatus and B. longicaudatus, from soil samples using the Baermann funnel technique described by Christie and Perry (2). Samples of 100 of each of the nematode species were trans ferred with a pick into Syracuse dishes of dis tilled water for each replicate. The water sus pensions of the parasites were then poured into holes near the tea roots. The holes were then sealed. Three months after inoculation the plants were extracted from the cans. The soil and nema tode adhering to the roots were washed into a quart container. The roots were carefully ex amined under a binocular microscope for any ex ternal symptoms of parasitism. Further processing of the roots for the pur pose of counting nematode populations varied ac cording to the type of nematode. Root-knot in fected roots were placed in a solution of boiling lactophenol-acid fuchsin for 3 minutes and then cleared with lactophenol. Counts were made on the number and type of galls. A pint of soil was collected from around the roots which had been inoculated with ectoparasitic nematodes and processed, using the Baer mann funnel, along with the water which had been used in washing the roots. Soil from around the endoparasitic infected plants was treated in the same way. In addition, root samples, which weighed 5 grams each, of endoparasitic infected plants were comminuted in a food blendor. Counts of the nematodes were made by screening the filtrates and washing the nematodes off the screen into centrifuge tubes with 10 millimeters of water. After centrifuging, one milliliter was drawn off and placed in a Peters counting slide. The damage caused by parasitism was evalu ated by recording the heights of both control and inoculated plants at the time of inoculation, and

3 412 FLORIDA STATE HORTICULTURAL SOCIETY, 1964 Table lo Comparison of average plant height measurements expressed as a percentage of that of the control. Treatment In1tial percentage Three months after inoculation Percent gain or loss Control R«simi1 is P* coffeae B, lonqicaudatus Ho coronatus M# javanica M* incognita , * ^ ^.9 3 months after inoculation when the plants were removed for examination. At the time of removal, a record was also made of root and top weights. In the pathogenicity work on the galls pro duced by M, incognita, the tertiary butyl alcohol dehydrating process recommended by Johansen (8) was used prior to embedding. Longitudinal and transverse sections were cut at 10 to 15 ^ The slides were stained with a slightly modified safranin-fast green technique and mounted in balsam. Results and Discussion Examination of the roots disclosed that M. in cognita, M. javanica and R. similis parasitised the seedlings. Tables 1 and 2 show that the aver age growth, top weight and root weight were closely interrelated within the treatments. The Meloidogyne spp. had a definite deleterious effect on the growth of the plants, while inoculations of the other species had little or no effect on plant growth, with the exception of those inocu lated with H. coronatus. The smaller height attained by H. coronatusinoculated plants cannot be explained on the basis of parasitism. No root damage was noted. In fact, no nematodes were recovered 3 months after inoculation. Galls produced by the Meloidogyne spp. were plainly visible, many of them being as much as 3 times the normal diameter of the root. This is contrary to Martin's findings in Rhodesia (11), where the root-knot nematodes produce galls which are often difficult to observe under field conditions. The M. incognita-infected roots formed a bunched-matted system around the tap root, with many of the root tips being necrotic. This necrosis often terminated at a gall. It was not uncommon to find egg masses protruding Table 2.--Mean root weight and top weight (grams) of tea plants 3 months after inoculation with 6 species of nematodes. Treatment Mean root weiqht Mean top weiqht Control Bo lonqicaudatus P. coffeae R. similis Ho coronatus Mo Javanica M. incognita k ,

4 MORTON: NEMATODES ON CAMELLIAS 413 from galls, especially the compound galls. M. javanica-vcdected root systems were more open and normal, compound galls were more prevalent but less necrosis and fewer visible egg masses were observed. Several eruptions, which Martin (11) termed "warts," were found on many of the tap roots at the junction of the feeder roots and tap root near the soil surface. Few females and egg masses were found in these "warts." Rau (14) pointed out that great care should be taken in selecting a nursery site for tea be cause of the world-wide distribution of the rootknot nematodes, and thorough tests should be conducted for these nematodes prior to planting. The Department of Fruit Crops at the University of Florida has established nurseries both under shade in the field, and in flats containing a mix ture of peat and perlite in a 50 percent shade house. The superiority of the latter medium re sulted in plants 2 to 3 times the size of those in the field seed beds. Although the raising of nur sery plants in a peat-perlite mixture is a more costly method of propagation, an additional ad vantage is elimination of the dangers of rootknot nematode infections. The R. similis infected root systems were not reduced as a result of parasitism. This is con trary to Zimmerman's (18) findings. This para site appeared to enter in or near root tips since the lesions were predominantly found in this area. However, lesions up to an inch in length could also be seen some distance from the tips. Obser vations indicated that in these areas of infection the outer vascular tissue had turned brown and could easily be separated, in conjunction with the cortex, from the inner vascular system, which appeared healthy and intact. Of interest was the fact that this outer vascular necrotic mani festation extended ahead of that of the cortex. One possible reason for this was that this tissue was preferred by R. similis while feeding. The R. similis counts ranged from a fourfold to a fourteenfold increase, showing tea to be a good host for this population of burrowing nematodes. In his taxonomic studies, Loof (9) proposed a new species, Pratylenchus loosi, from the popu lations he received from Ceylon which had been classified as P. coffeae. This could account for the lack of parasitism by P. coffeae and should be further investigated as a means of classifying these 2 species of Pratylenchus. Healthy Root. The tea plant, a dicotyledon, produces a taproot with lateral roots. These are covered with a corky layer that has its origin in the pericycle, Solereder (15), which is impermable to water. Absorption is via innumerable root hairs and fine roots which are of a semi-perman ent nature and continually replaced during the growing season. The short (± 25 ^) root cap is made up of isodiametric-like parenchyma, usually 5 cells thick. The promeristem and procambium are characterized by cells with prominent nuclei. The procambium is lined with tannin-filled cells which apparently give rise to the endodermis. In crosssection the young primary feeder root has an epi dermis, 9 layers of parenchymatous cells form ing the cortex, a tannin-filled endodermis and a central cylinder containing a tetrach arrangement of xylem and phloem. As the root becomes older, the cortex becomes reduced, and suberization of the tannin-filled endodermal cells takes place. Initiation of cork cells can be seen in a multiseriate pericycle toward the interior. The ex arch arrangement of the xylem persists but the center is filled with parenchymatous cells. After the cortex sloughs off, a root with secondary growth is encompassed by 3 to 4 cork cells. Beneath these lies a multiseriate pericycle through which are scattered tannin-filled cells. Inside of the pericycle are bundles of primary phloem fibers embedded in large, thin-walled cells of secondary phloem. The vascular cambium is very obscure, but a change to safranin-stained sclerified thicker cell walls demarks the secondary xylem. Interior to this is the metaxylem layer composed of small thickened cells surrounding the center of the root which is made up of larger metaxylem cells. Distinct uniseriate rays are seen traversing the secondary xylem and phloem in which starch reserves abound. Root-knot Infected Root. Without exception the pyriform females were located within the vascular cylinder with the preferred orientation being longitudinal. The central cylinder was dis rupted and disorganized. Except in those cases where the egg masses protruded to the epidermis, the endodermis remained intact. Hypertrophy was limited to the giant cells and endodermal cells, which were markedly en larged. The cortical cells were slightly elongated. Hyperplasia on the other hand was very preva lent in the pericycle, both in close proximity to and some distance removed from the parasites and the giant cells. Examination of the crosssection of compound galls frequently disclosed that the vascular cylinder was almost entirely filled with hyperplastic cells, the main factor bringing about gall production. This multiplica tion resulted in an interruption of the xylem

5 414 FLORIDA STATE HORTICULTURAL SOCIETY, 1964 vessels and displacement of many of the tracheids. Numerous lateral roots were seen to arise from this pericyclic proliferation. The young giant cells had a dense, granular, maroon-colored cytoplasm after staining in which were located numerous, distinct nuclei. By con trast, older giant cells were substantially larger with vacuolated, olive-colored cytoplasm and thin cell walls. According to Bird (1), vacuolation develops when the nematode stimulus is removed, the cytoplasm being most dense when the nema tode produced its egg sac and eggs. Using Dropkin's (3) classification, the older giant cells would rank as type 3. The phenomenon observed by Loos (10) of the inability of the second stage (infective) lar vae to moult to the third stage on mature tea bushes was not observed on these 6-month-old seedlings. Summary A parasitism study was conducted on Assam- Chinese hybrid seedlings of tea, which originated from the Kiganga Estates in Tanganyika, with the nematodes R, similis, M. incognita, M. Javanica, P. coffeae, H. coronatus and B. longicaudatus. R. similis, M. incognita and M. javanica were found to parasitise tea with the Meloidogyne spp. having a severe stunting effect on the growth of the tea seedlings. Because of the wide distri bution of this genus, it is recommended that the seedlings be grown either in sterilized nursery beds or preferably in 10-inch deep flats con taining sterilized peat and per lite. Healthy tea feeder roots have a small root cap, a primary root with a tetrach arrangement of xylem and phloem and a typical dicotyledonous woody secondary root covered by cork produced in the multiseriate pericycle and internally disected by uniseriate rays. High tannin and starch re serves are present. The roots inoculated with M. incognita pro duced galls up to 3 times the diameter of the normal root. There was pronounced hyperplasia of the pericycle and severe disruption of the vascular system. Hypertrophy was limited to the endodermal region. The latter in conjunc tion with the cortex remained intact except where the egg masses protruded to the epidermis. The contents of the giant cells were dense and granu lar when young; as they aged, they became more sparse and vacuolated. Resistance to M. incognita and M. javanica has been reported to develop at various ages in young tea; hence studies on parasitism in Florida utilizing plant materials older than those experi mented with here are in progress. LITERATURE CITED 1. Bird, A. F The inducement of giant cells by M. javanica. Nematologica 8: Christie, J. R. and V. G. Perry Removing nematodes from soil. Proc. Helminthol. Soc. Wash., D.C. 18: Dropkin, V. H The histopathology of rootknot nematode infections in soybeans. Phytopathology. 50: Gadd, C. H A destructive root disease of tea caused by the nematode Anguillulina pratensis. Tea Quart. 12: Goodey, J. B., Mary T. Franklin and D. J. Hooper Supplement to the nematode parasites of plants catalogued under their hosts. Commonwealth Agr. Bur., Farnham Royal, England. 66p. 6. Goodey, T. Revised by J. B. Goodey and Mary T. Franklin The nematode parasites of plants cata logued under their hosts. Ed. 2. Commonwealth Agr. Bur., Farnham Royal, England. 14Op. 7. Hutchinson, M. T. and M. K. Vythilingam The distribution of Pratylenchus loosi Loof among tea estates in Ceylon, with particular reference to altitude. Tea Quart. 34: Johansen, D. A Plant Microtechnique. New York: McGraw-Hill. 523p. 9. Loof, P. A. A Taxonomic studies on the genus Pratylenchus (Nematoda). Tijdschr. PI. Ziekt. 66: Loos, C. A Meloidogyne brevicauda, n. sp. a cause of root-knot of mature tea in Ceylon. Proc. Hel minth. Soc. Wash. 20: Martin, G. C Population levels of nematodes m roots, and soil around the root zones of tea, sugarcane, tobacco and wheat grown in the Federation of Rhodesia and Nyasaland. Rhodesia Agric. Jour. 59: Mitchell, G. F The cultivation and manufac ture of tea in the United States. The U.S.D.A. Bureau Plant Industry. Bull. 234: Mukerjea, T. D Personal communication. 14. Rau, S. A A practical method of testing tea nursery soils for the root-knot celworm. Plant. Chron. 39: Solereder, H Systematic anatomy of the dicotyledons. Transl. by L. A. Boodle and F. E. Fritsch. 2 vols. Oxford: Clarendon Press. 16. Steiner, G The problem of host specialization of certain plant-infesting nemas and its application in the study of nemic pests. Phytopathology 15: Taylor, A. L. and W. Q. Loegerin Nematodes associated with root lesions of Abaca. Turrialba 3: Zimmerman, A Tylenchus acutocaudatus Zn. Thee Teysmannia 10: