Variation in Oil Concentration

From first experience, the oil concentration was observed to vary over time (Penfold et al. 1948) and this gave rise to a number of studies that attempted to document and explain the variation (Table 1). Of the 12 studies that measured variation over time, five recorded a variation of more than 100% above the lowest value in the study, six had a variation between 15-57%, and only one recorded no variation. Similar variation is often found with other essential oils or secondary metabolites, as instanced in reviews by Fluck (1963), Wiermann (1981), Harborne and Turner (1984), Lawrence (1986), Gershenzon and Croteau (1991).

Seasonal Variation

The oil concentration is generally highest in summer and lowest in late winter/early spring. Figure 1A shows the average seasonal variation in repeated tests on two plantations (Murtagh 1992; Murtagh and Smith 1996) in the humid subtropical environment of northern New South Wales (NSW). Although insufficient samples were taken during June-August to complete part of the trend line on the plantation that experienced winter frosts, the seasonal range was greater where winters were cooler. The trends shown in Figure 1A are means over a number of years, and while all years have a seasonal trend it can vary both in absolute magnitude and extent of variation (Murtagh and Smith 1996).

Tea tree is also grown in the dry tropics of north Queensland. Here there was almost no seasonal variation in a stand of a low concentration type, but more than 50% variation in a high concentration type (Figure 1B) (Drinnan 1997). Seasonal variation in oil concentration of more than 50% has also been observed in young leaves of Eucalyptus camaldulensis, another myrtaceous species with subepidermal oil glands (Doran et al. 1995).

The upper leaves on a tea tree plant often have a higher oil concentration than lower leaves (Curtis 1996), leading to the suggestion that the seasonal trend in concentration reflects oil losses during autumn-winter, followed by the production of new leaves with

Table 1 A literature survey of the variation in oil concentration in tea tree

Situation

Source of variation

Oil conc.

Reference

A4*

mg/g

%t

Natural

Months

44-57

30

Penfold et al.

a

stand

(1948)

Population

31-64

106

Butcher et al. (1994)

Plantation

Years

39-45

15

Small (1981)

a,c

Stand

Months, high conc.

63-99

57

Drinnan (1997)

a

Months, low

45-59

31

Drinnan (1997)

a

conc.

Months

18-98

444

Murtagh and Smith (1996)

Months

24-57

138

Williams and Home (1988)

d

Weeks

34-81

138

Murtagh (1992)

a

Weeks

25-58

132

Murtagh and

a

Baker (1994)

a,b

Days (Nov.)

23-48

109

Murtagh and Etherington (1990)

Days (Jan.)

64-95

48

Murtagh and Etherington (1990)

a,b

Hours

37-47

27

Murtagh and Baker (1994)

Trees

12-67

458

Williams and Home (1988)

d

Polyhouse

Hours

40

0

List et al (1995)

Glasshouse

Populations

22-36

64

Butcher et al. (1994)

*Factors used to convert units used in published data to mg/g DW; aOil density of 0.9g/ml, bLeaf : twig ratio of 0.68, cLeaf dry weight=36% wet weight, dAssumed published values were %w/w, eRange taken from greatest variation measured within a day. fRange expressed as a percentage of the lowest value.

*Factors used to convert units used in published data to mg/g DW; aOil density of 0.9g/ml, bLeaf : twig ratio of 0.68, cLeaf dry weight=36% wet weight, dAssumed published values were %w/w, eRange taken from greatest variation measured within a day. fRange expressed as a percentage of the lowest value.

a high concentration during the following spring. In other words, the whole-plant oil concentration increases during spring because of the increasing proportion of young leaves rather than an increase in oil concentration in older leaves. List et al. (1995) obtained two pieces of evidence that support this view, that they termed the one-way development path. Their anatomical study suggested that immature glands were lined with metabolically active cells, whereas mature glands were lined with highly vacuolate cells that are unlikely to be involved in oil synthesis. Secondly, they found no variation in oil concentration over 48 hours.

However, not all data supports the one-way development hypothesis. Penfold et al. (1948) found that the oil concentration increased rapidly from the lowest to near the highest value for the year between October and November. This rate of increase was too rapid to be explained by the production of new leaves. A similar result was reported by Murtagh (1988)

Winter frosts

(A) Subtropical

(A) Subtropical

Low conc. selection

(B) Tropical

J FMAMJ JASOND MONTH

Figure 1 Seasonal changes in the oil concentration in (A) the humid subtropical environment of northern NSW, and (B) the dry tropical environment of northern Queensland who, in one year, measured a 62% decline between August and September followed by a complete recovery in October.

Another experiment reported by Murtagh and Baker (1994) sampled plants of different regrowth ages on the same day. The regrowth was aged between 145 and 354 days at one site, and 82 and 292 days at another. The longest regrowth period included autumn—winter when concentrations usually decline but the concentration per unit weight varied more with the specific leaf area rather than the regrowth age. The specific leaf area is a measure of the area per unit leaf weight. Young leaves tend to have a high specific leaf area because they are thin and have less secondary thickening of cell walls. Thus the area, and perhaps the number of oil glands, is greater per unit weight with young leaves leading to a higher oil concentration per unit weight. When this effect was removed by expressing the oil concentration per unit leaf area, there was little difference in oil concentration between regrowth ages at one site, but it was more than double in the older regrowth at the other site. These results illustrate the difficulty of finding a suitable expression of oil concentration when contrasting leaves with very different specific leaf areas, as occurs between young and medium to old leaves.

Daily Variation

Effects due to the leaf age distribution and specific leaf area can be removed from a comparison by sampling over a short period when they would not change. When this was done by sampling at about the same time each day over a sequence, the oil concentration has been shown to vary. In one sequence over 8 days in November, the concentration halved over 2 days and completely recovered by the next (Murtagh and Etherington 1990). There was also a smaller 21% decline, followed by recovery, later in the sequence. These changes were statistically significant and occurred on days that followed the warmest nights (minimum temperatures of 17.7 and 15.9°C) during the sequence (Murtagh 1988). Subsequent sampling during summer at daily intervals on 3 sites also showed significant changes in oil concentration (Murtagh and Etherington 1990), but the changes were not related to temperature (Etherington 1989).

The inverse correlation between night temperature and oil concentration prompted the notion that the concentration declined because warmer nights increased the respiratory load and the oil supplied at least some of the substrate for the process. Monoterpenes are the major constituents of tea tree oil and are thought to be sometimes available for catabolism (Croteau 1988). Warm nights reduced the oil concentration in peppermint (Mentha piperita), especially during the flower initiation stage (Loomis and Croteau 1973). Curtis (1996) also obtained evidence that a warm night can decrease the oil concentration in tea tree. That result is discussed under the Environmental Effects heading.

Diurnal Variation

Studies that examined the diurnal pattern in oil concentration of tea tree have generally found no significant changes. Etherington (1989) in three sequences found no variation. Murtagh and Baker (1994) investigated 14 sequences of which three showed significant variation. List et al. (1995) and Curtis (1996) examined one sequence each and found no variation. The study by List et al. (1995) was done with potted plants in a polyhouse over 48 hours. All other studies were done in the field and a sequence occupied a day.

Another set of diurnal measurements, not included in the above, suggested that diurnal fluctuations were related to the water vapour pressure deficit (VPD) of the atmosphere (Murtagh 1991b). The samples were taken over three consecutive days from two watering treatments; irrigated and rain watered. The soil on the second treatment was dry when sampled. The tea trees were about 1.5m tall, and separate samples were taken from the upper 40cm of the canopy (upper strata), and below 40cm (lower strata). The oil concentration in the upper strata was significantly higher than the lower strata, and it declined significantly within each of the first two days (Figure 2). There was a trend, not always significant, for the concentration in the upper strata to be higher on the rain-watered than the irrigated treatment, but the reverse applied in the lower strata. When data from both strata were pooled, there was virtually no difference between the two watering treatments. There was no change in the percent composition of the major constituents in the oil over the three days.

The weather conditions differed between the three days. Day one was hot and dry and the VPD continued to increase until sampling ceased. Day two was similar in the morning,

Figure 2 Diurnal variation in oil concentration over three consecutive days, in the upper (A) and lower (t) strata of the canopy, on irrigated (solid symbol) and rain-watered (open symbols) treatments. The water vapour pressure deficit (VPD) is also shown

but a sea breeze arrived at noon and lowered the VPD. On this day, the decline in the oil concentration was arrested even before the instruments detected the change in the weather. Day three was overcast with a low VPD throughout. The relation between the oil concentration in the upper strata and VPD was summarised by two intersecting straight lines, that showed a significant decline in the concentration once a threshold VPD was exceeded (Figure 3). Separate relations were fitted to the irrigated and rain-watered treatments.

The threshold VPD were 1.5kPa on the irrigated treatment, and 1.0kPa if rainwatered. Whereas the threshold occurred at a lower VPD on the rain-watered treatment, the decline was greater on the irrigated treatment and equalled 22% at the highest VPD that was measured. The slower rate of decline on the rain-watered treatment suggested that these plants had acclimated to dry conditions.

However subsequent data obtained by Murtagh and Baker (1994) indicated the VPD effect was not the sole factor involved in diurnal fluctuations in oil concentration. In nine of the 14 sequences mentioned above, the VPD exceeded 2kPa at sometime during the day, but only one of the nine sequences showed a significant decline in concentration. It is relevant that the sequence with the decline had the highest oil concentration of the nine sequences, starting the day at 55mg/g. The declines discussed earlier occurred at higher concentrations, suggesting that diurnal fluctuation is more likely at high concentrations. One sequence in the 14 showed a significant increase of 26% in the oil concentration during a day that was warm and particularly humid, with the VPD being less than 0.9kPa for most of the day.

Figure 3 The pattern of change in oil concentration in the upper strata shown in Figure 2 with increasing vapour pressure deficit (VPD) on irrigated (solid symbol) and rain-watered (open symbols) treatments

Post-harvest Oil Concentration

Two studies have shown that the oil concentration remains constant for a considerable period after harvest in treatments that are handled the same whilst fresh. Murtagh and Curtis (1991) exposed samples taken from a common batch of twig material to a range of drying treatments that were designed to provide varying rates of drying, respiration, and opportunities for volatilisation. They found no effect on the oil concentration over 13 days in one experiment, and seven days in another. Twig samples were distilled, and although not presented in their paper, repeated sampling showed that the proportion of leaf in the twig sample was constant throughout each experiment, thus eliminating variation in the leaf to twig ratio as a possible source of error.

Whish and Williams (1996) also tested for post-harvest losses of oil. They distilled leaf samples, and when the leaf was stripped from the fine stems whilst fresh, there was no change in the oil concentration between distillations done immediately or 20 days later. However, when the twigs were air dried before the leaf was removed, the oil concentration was 28% greater. They suggested that oil movement from the stem gave the higher concentration in the latter result, but an alternative explanation is that the striping of green leaves from stems caused some oil loss. As discussed later, detaching branches or rough handling can increase volatilisation losses in other species. Zrira and Benjilali (1991) observed a 62% increase in oil concentration of Eucalyptus camaldulensis after shade drying, but it is not certain if the results were corrected for the varying water contents in the leaves. This issue must be clarified because if oil is lost when leaves are stripped while fresh, there is a strong argument to either distill twigs and adjust the concentration to a leaf basis by separately determining the leaf to twig ratio, or air dry the twigs before stripping leaves.

Oil losses after harvest vary between species, and those with a high water content tend to lose more (Guenther 1948). Fresh tea tree leaves are relatively dry with 26-47% dry matter (Murtagh and Smith 1996), and this combined with the subepidermal position of the oil glands would tend to reduce the loss of tea tree oil after harvest (Fluck 1963; Murtagh and Curtis 1991). However, the relatively thin epidermal cap cell above oil glands could offset the positional protection in both Eucalyptus (Welch 1920) and Melaleuca species (List et al. 1995).

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