tion of tenlined June beetle larvae by using 10 lb A1 per acre of 10 G carbofuran, and we observed suppression with 5.0 lb A1 per Melaleuca alternifolia: new acre of 10 G carbofuran and 10 and 5 lb A1 per acre of 14 G diazinon (table 1).In trial 1, when insecticides were applied on Novem- crop for California? ber 7,1985, the first evaluation on January 8, 1986, showed no significant difference be- Roy M. Sachs Q Choong I. Lee 0 Sue A. Carhnrright Q Michael S. Reid tween treatments. However, when we re- evaluated the trial on April 25,1986, we Colin Smith observed significant mortality. Thus, the larvae must be feeding actively, which they do when soil temperatures warm in the spring, in order for the insecticides to be effective. In trial 2, when insecticides were applied on April 25,1986, significant mor- tality was observed 7weeks after treatment. In the 1988 trial, significant reduction of Researchers say tea trees can we to %foot centers under drip irrigation (one tenlined June beetle larvae was achieved grown successfully and with emitter per seedling) in April 1986 at UC with 2.8 lb A1 per acre of diazinon 14 G Davis. One hundred pounds of elemental when applied commercially in large plots. profitable oil yields in California's nitrogen was applied as ammonium sulfate These studies indicate that suppression Central Valley. Based on an analy- in the spring of the second year of cultiva- but not complete control of tenlined June sis of seedlings from one seed tion. We observed considerablevariation in beetle larvae can be achieved with granular source, the yield from a seedling the vigor of seedlingswithin a few months applications of diazinon, which is regis- plantation would be about half that of planting, and many seedlings became tered for use in almond in California. Car- bofuran, if it becomes registered for this use of a clonalplantationderived from in the future, will be an alternateinsecticide. the best seedlings. Conclusion Foliage of Melaleuca alternifolia [Cheell, a From our studies, the tenlined June beetle woody perennial commonly known as the appears to have limited dispersal because tea tree and native to northern New South mated females do not move far, and the Wales, Australia, contains approximately 1 beetle has only one generation every 2 to 2%(by fresh weight) essentialoil. This oil, years. Thus, infestations will spread very because of its demonstrated antibacterial slowly. Controlling the adult beetles with and antifungal activity, has commercial foliar insecticides does seems unlikely be- value in the treatment of skin lesions and is cause of the prolonged adult male emer- used in the manufacture of shampoos and gence and female behavior after mating. other health-related products. The market The most suitablestage for control would be for tea tree oil in the United Statesis increas- larval, with application of soil insecticides ing, and demand currently exceeds supply. in September. This timing would corre- Prices for the oil imported from Australia spond to egg hatch, and newly emerged exceed $10 per pound. Research on the ag- first instars would be most susceptible to ronomic and horticulturalcharacteristics of control. A second application would come the crop was considered an essential pre- in May, before third instar pupation. liminary step to determining its economic These two application timings would potential in California. occur when the larvae are actively feeding Plant materials. We sowed seeds of M. in warm soil. Controlling the larvae with alternifolia densely in rows spaced 1 cm soil insecticideswould permit suppression apart in a flat of a fine-grained sand. The of the population without costly and de- surface was kept moist by misting at 2.5- structive tree removal and fuegation. minute intervals during daylight hours. Flats were maintained at 75°F. The gennina- Robert A. Van Steenwyk is Extension Ento- tion rates of three seed lots were between 10 mologist, Department of Entomological Sci- and 20% over several weeks. Emergence in ences, University of California, Berkeley; the flats occurred at 2 weeks. About 6 weeks Donald Rough is Farm Advisor (retired) and after emergence, the most vigorous seed- Paul S. Verdegaal is Farm Advisor, Cooperative lings were transplanted to peat pots in a Extension, Sun JoaquinCounty. peat-sand mixture, which were then main- Theauthors gratefully acknowledge the assis- tained in a greenhouse at 75°F during the tance of B. A. Burr, L. W. Barclay, and C. F. day and 65°F during the night for another 5 8 Fouche, Cooperative Extension, University of to 6 weeks. Plants were then moved to a lath 2 California,Berkeley,and R. Freeman and D. M. house for a week of hardening before field y" Havens, Cooperative Extension, Stockton, for planting. ; their help in this study. They also gratefully Plantation establishment and Per- Tea tree oil floats on water in this graduated test acknowledge Van Dyken Farms' provision of a f0rmanCe. Using about 250 of the most tube. The antibacterial and antifungal proper- study site. This research was supported in part vigorous seedlings, we established a 0.2- ties of this oil make the tea tree a valuable crop by the Almond Board of California. acre plantation, with plants spaced on 2.5- for medical and hygene applications. CALIFORNIA AGRICULTURE, JULY-AUGUST 1990 27 1 5 -% 9 1 f .5 After initial rooting, establishment in the greenhouse, and hardening in the - lathhouse, a tea tree clone is ready to be planted in the field. Twice-a-year u" harvesting of the 12-3 clone planted at 5,000 trees per acre could yield 4 368 pounds of oil. In the laboratory, a foliage tissue sample is prepared for steam distillation. chlorotic. The plantation canopy closed same as for seed germination) for 2 to 3 fraction collected from the second distilla- sometime during April 1987. The most vig- weeks, after which time most cuttings had tion was discarded, and the remainder was orous seedlings were 9 to 10 feet tall with begun root initiation. Rooting percentage collected in a tared, 15 ml graduated test stem diameters of up to 1%or 2 inches' di- exceeded 70%for all seedlings tested from tube. The volume of the oil layer suspended ameter 18 inches from the ground. April to July. Rooted cuttings were trans- on top of the aqueous layer was recorded, Plant sampling and yield determina- planted to peat pots in a peat-sand potting along with that of the remaining water tion. At 12to 14months after planting, indi- mix, maintained in a greenhouse for one to layer. The tube and contents were weighed. vidual seedlingswere cut approximately 18 two weeks, and then moved into a lath- The oil weight was determined by subtract- inches from the ground, and branches with house for hardening before field planting. ing the new tare weight (which included the leaves were stripped from the main stem. Clones were field planted on 3-foot centers small aqueous fraction) from the total tube The stem was weighed and discarded. The (about 5,000 plants per acre)in28 to 35 plant weight. We calculated the specific gravity of branches were weighed, and 0.2-pound blocks, and each block had double borders. the oil from the weight and volume data. samples were taken for steam distillation. Yields of foliage with small branches were The following equation gives the percent- Propagating potential high-yield determined for three clones as described age oil in the sampled plant tissue: seedlings. We made 10 cm stem cuttings above. from lateral branches of the eight seedlings Steam distillation. The steam distilla- % oil = (oil weight/fresh weight plant with the highest oil percentage (table 1). tion apparatus consisted of a sterilizer with sample) x 100 After dipping the bases for 10 seconds in a a separate compartment constructed to 4,000 mg per liter indole butyric acid solu- contain the plant tissue sample and a steam Leaves and small branches analyzed were tion, we stuck them into a 5050 perlite-ver- outlet that led directly to a condenser and about 35%dry weight, so the oil percentage miculite mix in flats. The flats were placed collection funnel. Distillate was collected on a dry-weight basis could be computed by under mist benches with bottom heat (the for up to 2 hours in a 500 ml separatory dividing by 0.35. funnel. Gross oil yield. Biomass accumulation, W 1,8 Cineole 0 Terpinene 4-01 Cohobation is the practice of recycling oil content, and oil yield varied widely Em the aqueous distillate back into the steam among seedlings. Data on leaf and stem chamber. Since our still was not fitted to biomass and percentage oil for the top eight permit automatic recycling of the aqueous seedlings of the more than 200 analyzed are fraction, we had to distill two plant samples ranked by oil content in table 1. The specific when we wanted to practice cohobation. gravity of the oil isolated was between 0.89 Most of the lower aqueous fraction was and 0.91 for all seedlings. R518 R715 R104 Rll 5 discarded unless cohobationwas practiced. Initially, we performed all distillations Clones The aqueous fraction collected from the without cohobation, with washed glass- Fig. 1. Ratios of terpinene-4-01to ceneole for first distillation, usually in excess of 500 ml, ware. Some values for oil content were50% oils from four clones analyzed by gas chroma- was used as the charge for distilling the lower than when we employed cohobation tography. All samples were taken in fall, 1988. second sample. The bulk of the aqueous and pre-rinsed the glasswarewith oil. Five

28 CALIFORNIA AGRICULTURE, VOLUME 44, NUMBER 4 samples were distilled for each selected This spacing permits approximately 5,000 Soil, water quality, and climate. Small clone to determinethe oil yield as a function plants per acre, and should prove sufficient plantations of Melaleuca alternifolia seed- of each of three distillation techniques: to give canopy cover in 12 to 18 months. At lings and clones should be established at (1)no cohobation, (2) total cohobation with higher densities, expect faster cover, several locations in California for studies on oil-coated glassware, and (3)total cohoba- smaller plants (which may prove more sat- yield and quality as functions of site and tion with oil-coated condenser glassware. isfactory for optimum foliage yield), and climate. The clones were damaged during Small stems were included in distilla- increased early yields. An economic analy- the hard freezes at Davis in February 1989, tions designed to simulate a commercial sis that includes the costs of plant materials and this accounts for somewhat lower harvest; the plants sampled were shrubs, and field planting, and the expected in- yields in 1989 than in 1987. Mild winter not trees. We ran replicate distillations for crease in yields for the first year or two as a temperatures appear to be important. Sea- some clones, but the distillations in table 2 function of planting density, must be used sonal variations in oil quality and yield were not replicated sufficientlyfor statisti- to determine the optimum density. should be examined. The plants at Davis cal analysis. Variance was on the order of Harvest times and frequencies. With produced more oil in late summer than in 5%(changeof O.l%inoilcontent)forrepeat growth rates equivalent to those obtained early spring, a possible indication of the distillations. Cohobated distillations following a March-April field establish- importance of high summer temperatures yielded significantly more oil, and further ment, first harvests could begin 3 to 5 or low atmospheric humidity. Water qual- increases became apparent when all of the months after planting, when the plants are ity, particularly pH, is significant. glassware used was pre-rinsed with oil about 3 feet tall. The first cut should be made Genotypic variation. Additional seed- from prior distillations. Note that cohoba- 1 foot above ground level to encourage ling blocks will help us examine further tion is of critical importancefor the qualita- branching from the crown. All harvesting seedling variations in vigor, oil quality, and tive analysis of tea tree oil because of the protocols should promote branching and yield, as well as adaptation to high pH. Our extreme water-solubility of terpinene-4-01, small stem diameters, since oil yield is neg- initial screening of 250 seedlings for vigor the most vital ingredient in tea tree oil. ligible in large-diameterstems and is high- and oil yield suggest that further research Although there is danger in extrapolat- est in the foliage. Harvests could be re- could lead to (1) better-yielding clones, ing commercialyields from small test plots, peated as soon as the new shoots added perhaps with higher-grade oil, and (2) a the projected annual yields for clonal plan- about 3 feet of growth. Frequent harvesting more accurate picture of the genetic deter- tations derived from the top eight seedlings, is likely to keep many herbaceous shoots on minants of yield and oil content.More rapid with about 5,000 plants per acre (assuming the plants; hence, an alfalfa or silage mower distillation and gas chromatographictech- growth rates equivalent to that of the test may prove suitable for initial harvest trials. niques for seedling analyses would facili- seedling), suggest that some clones of this tate such a study. species would be outstanding candidates Economics. At $10 per pound of oil for commercial exploitation. These clones wholesale, projected annual farm gate val- have relatively high foliage yield and oil ues for the oil derived from clones yielding percentage, resulting in a high per-acre oil about 5 tons of foliage per acre per year yield. However, qualitative analyses sug- exceed $1,800 per acre. Cultural costs are gest that only two clones produce oil that estimated at $600 per acre for the first year, would meet current world standards for tea and approximately $200 to $300 per acre trees. The Australian specifications for tea (depending on irrigation and harvest costs) tree oil are 1-8cineole not greater than 15% throughout the life of the plantation. Distil- and terpinene-4-ol not less than 30%. lation costs are estimated at $40 per ton of Gas chromatographic analysis of oils. biomass processed, or about $200 per acre. A temperature programmable gas chroma- Plant materials would cost about $2,500 for tograph fitted with a megabore capillary anacre of clonal plantationand about $1,000 column and a flame ionization detector and for an acre of seedling plantation; both are integrator was used to analyzeall distillates. one-time costs. Plantation longevity is un- The analyses indicate that two of the eight known, but may exceed 25 years. Some high oil clones, 5-18 and 7-15, produce an oil Australian plantations have been harvested with approximately40%terpinene-4-01 and for longer than that. Positive cash flow less than 10%cineole (fig. 1). would be expected in the second or third Soil pH. Many field-grown seedlings year of cultivation. were highly chlorotic and grew poorly. All of the field-planted materials were of a Roy M. Sachs and Michael S. Reid are Profes- lighter green than greenhouse-grownseed- sors, Choong I. Lee is Staff Research Associate, lings and clones. The two field plantations Sue A. Cartwright is Post-Graduate Research were grown in soils of pH 7.3 to 7.8. The Horticulturist, and Colin Smith was formerly peat-sand soil mix for greenhouse plants National Science Foundation Scholar, Depart- had a pH between 6.5 and 7 over three ment of Environmental Horticulture, Univer- months of observation. Foliar applications sity of California,Davis. of ferrous sulfate to field-grown plants re- Melaleuca alternifolia clonal material is sulted in somewhat darker green foliage, available from the Department of Environ- although we observed no substantial differ- mental Horticulture, UC Davis. ence in plant growth. Growth of seedlings Melaleuca Estates of America, Los Angeles, in solution culturewas severelyinhibited at California,provided support for part of this re- pH 8, but vigorous at pH 6 to 7. search. A contract between Melaleuca Estates of America and Plant Clones of Davis, as part of a Future research Phase4 USDASmall Business Innovation Planting density. Relatively high-density ResearchProgram,financedpart of thegas chro- plantations of 3 x 3 feet are recommended. matographic analyses.

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