Effect of an organic plant fertiliser on the establishment of rooted cuttings of two species of Cyclopia (honeybush)

Abstract

The effect of different application rates of an organic plant fertiliser (Nitrosol^®) on the establishment and early growth of rooted cuttings of Cyclopia (Fabaceae) species was evaluated. Rooted terminal and subterminal cuttings of C. intermedia and C. genistoides were transplanted into pots (576 cm³) and treated with Nitrosol^® fertiliser at application rates of 0 (control), 1.67 and 3.33 ml Nitrosol^® l^-1 water. To enhance the formation of nodules the cuttings were inoculated with symbiotic Rhizobium bacteria. The different species differ in their response to the fertiliser treatment, with C. genistoides showing a larger response than C. intermedia, especially at the higher application rate of 3.33 ml Nitrosol^® l^-1 water. This study therefore suggests that Nitrosol^® fertiliser applications can be recommended to enhance the establishment of rooted cuttings of Cyclopia species, but optimum application rates may differ between species. Although a significant specie × cutting position interaction was shown with regard to plant height and number of shoots per plant, cutting position in general did not have an effect on the establisbment of the species used in this study.

Keywords:

• Cutting position
• Cyclopia genistoides
• Cyclopia intermedia
• Nitrosol^®
• root growth
• shoot growth

Honeybush (Cyclopia species) is a shrub belonging to the Podalyrieae tribe, which is a subdivision of the Fabaceae family, that grows in the Fynbos biome (Schutte 1997). It grows naturally in the coastal and mountainous areas of the Western and Eastern Cape provinces, from Darling to Port Elizabeth, being bounded in the north by the Cedarberg, Koue Bokkeveld, Klein Swartberg, Groot Swartberg and Kouga mountain ranges (du Toit and Joubert 1998). Honeybush tea is produced from the leaves and flowers of most Cyclopia species and is one of the traditional herbal teas with a long history of regional use (du Toit and Joubert 1998, Joubert et al. 2008).

Unlike rooibos (Aspalathus linearis) tea, honeybush tea remained a cottage industry until the mid-1990s (Joubert et al. 2011). A review of South African herbal teas, including honeybush, focused mainly on their phenol composition and biological properties (Joubert et al. 2008), essential factors in establishing honeybush as a health drink. According to Joubert et al. (2011), in the 1990s it was decided that, for cultivation and production of Cyclopia spp., the use of organic production principles would be in the interest of the industry as the cost of registering chemicals to control pests, diseases or weeds would be prohibitive for such a small industry. Organic production principles are widely practiced, but registered farmers are very few, mainly because of the high cost of certification. According to Joubert et al. (2011), little is known about honeybush cultivation except that most species naturally occur on acid, quartzitic, sandy to loamy soils containing low levels of mineral nutrients such as nitrogen and phosphorus and that populations occasionally survive wildfires. Honeybush is a symbiotic legume and forms a symbiosis with Rhizobium bacteria for the fixation of nitrogen (Spriggs et al. 2003). According to McEwen (1970) and Dean and Clark (1980), most legumes require small amounts of nutrient elements. Spriggs et al. (2003) further indicated that some legumes do not require any additional fertiliser to enable formation of nodules and N₂ fixation.

The purpose of this study was to evaluate the effect of Nitrosol^®, a commercially available organic fertiliser, on the establishment and growth parameters of rooted cuttings of two Cyclopia species (C. intermedia and C. genistoides). These two species are amongst the Cyclopia species that are currently cultivated for production purposes (Joubert et al. 2011). The effect of species and the origin of the cutting position on growth parameters and establishment were also studied.

This study was conducted in a glasshouse at Welgevallen experimental farm at the University of Stellenbosch, Western Cape, South Africa (33°56′ S, 18°52′ E; altitude 119 m). The plants used in this study were obtained from the rooted cuttings of C. genistoides and C. intermedia with two cutting positions (terminal and subterminal). The original cuttings were obtained from cultivated mother stock plants grown at the Agricultrural Research Council (ARC) Infruitec/ Nietvoorbij research farm (33°56′53.84″ S, 18°51′40.09″ E; altitude 156 m). Rooted cuttings with similar root development (length and number) were carefully transferred to pots (576 cm³) filled with washed coarse sand and allowed to acclimatise under controlled glasshouse environmental conditions for four weeks. This acclimatisation process was done to ensure that all the plants had regained some stability before the commencement of the experiment. During this acclimatisation period, no fertiliser was applied to the plants; however, they were irrigated with water using mist blowers. Misting was scheduled to irrigate for 30 s every hour from 7:00 to 18:00. Misting as the method of irrigation was maintained throughout the course of the experiment. The temperature of the glasshouse was set at 25/13 °C (day/ night). Rhizobium bacterial strain number PPRICI3 from the South African Rhizobium Collection selected by the Plant Protection Research Institute (PPRI) of the ARC was applied once as a suspension for infection of the roots at the onset of the experiment. To prepare the suspension, 50 g of the inoculant was diluted in 10 l water as recommended by the supplier. About 50 ml of the inoculant suspension was added directly to each pot in all treatments, including the control, 24 h before the first fertiliser treatment application to ensure thorough infection of the roots by the bacteria. Any excess solution was allowed to leach out of the pot.

Nitrosol^®, a commercially available natural organic plant fertiliser that contains 80 g kg^-1 N, 20 g kg^-1 P, 58 g kg^-1 K, 7 g kg^-1 Mg, 6 g kg^-1 Ca, 4 g kg^-1 S (macroelements) and 6 mg kg^-1 Fe, 1 mg kg^-1 Zn, 1 mg kg^-11 Cu, 40 mg kg^-1 Mn, 23 mg kg^-1 B and 15 mg kg^-1 Mo (microelements) was used in this study. To prepare the fertiliser solution, 3.33 ml and 1.67 ml of commercial concentrated Nitrosol^® were diluted in 1 l water. For treatment 1, 40 ml of the 3.33 ml Nitrosol^® l^-1 water was applied to the pots. For the second treatment, 40 ml of 1.67 ml Nitrosol^® l^-1 water was applied, whereas no Nitrosol^® was applied to the third treatment (control). The treatments were applied at the beginning of the experiment and every second week until the end of the experiment after 10 weeks (5 × 40 ml = 200 ml pot^-1).

After 10 weeks, fresh and dry shoot mass, fresh and dry root mass, plant height, number of nodules per plant (only active nodules were counted) and number of shoots per plant were determined. The plant material was dried in an oven at 80 °C for 48 h to determine dry shoot and root mass. Nodules were excised and all the nodules that showed a pink-reddish colour were regarded as active, whereas those with a blue-brownish colour were regarded as inactive and not counted (Sharma and Ambasht 1986, Kumar Rao and Dart 1987, Dubach and Ruselle 1993).

Analysis of variance was performed using the GLM (General Linear Model) procedure of SAS statistical software version 9.1 (SAS Institute 2000). The Shapiro-Wilk test was performed to test for normality (Shapiro and Wilk 1965). Student's t-least significant difference (LSD) was calculated at the 5% level to facilitate the comparison between treatment means.

All growth parameters measured in this study were significantly affected by fertiliser (Nitrosol^®) treatment and species, but not by cutting position (Table 1). Interactions between the species and fertiliser treatments were significant for all parameters measured with root fresh mass the only exception. Significant interactions between the species and cutting position were found with regard to plant height and number of shoots per plant, whereas interactive effects between species, fertiliser treatment and cutting position were only significant with regard to number of shoots per plant. However, data for this last mentioned interaction (species × fertiliser treatment × cutting position) are not shown, because the interaction was significant for one parameter (number of shoots per plant) only and trends shown in this interaction were similar to that shown for species × fertiliser treatments.

Table 1 : Significance level (Pr > F) and coefficient of variance (CV) of the main effects and interactions for the growth parameters of two Cyclopia species

	Pr > F
Source	Fresh shoot mass	Dry shoot plant mass	Fresh root mass	Dry root mass	Plant height	Number of nodules	Number of shoots
Fertiliser (N)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Species (S)	<0.0001	<0.0001	0.0015	<0.0001	<0.0001	<0.0001	<0.0001
Cutting position (P)	ns	ns	ns	ns	ns	ns	ns
S×P	ns	ns	ns	ns	0.0154	ns	0.0483
N×S	<0.0001	0.0002	ns	0.0053	<0.0001	0.0052	0.0018
N×P	ns	ns	ns	ns	ns	ns	ns
N×S×P	ns	ns	ns	ns	ns	ns	0.0333
CV (%)	20.82	32.25	36.96	35.38	12.15	61.65	29.89

The results (Table 2) clearly showed that with the exception of plant height and number of shoots, establishment and early growth of the rooted cuttings of the different species did not differ significantly where no fertiliser (Nitrosol^®) was applied (control treatments). In the case of C. genistoides all parameters, except for fresh root mass, were significantly increased in response to the application of fertiliser (Nitrosol^®), whereas no significant response was recorded in C. intermedia cuttings with regard to fresh root mass, dry root mass and number of nodules per plant. Species also differed in response to an increase in fertiliser rate from 1.67 to 3.33 ml Nitrosol^® l^-1 water with C. genistoides showing significant increases for all parameters except fresh root mass and number of nodules per plant. Significant increases in C. intermedia in response to a similar increase in fertiliser (Nitrosol^®) application rate were only recorded for fresh shoot mass, plant height and number of shoots per plant. It can thus be concluded that C. genistoides are more responsive to especially the higher fertiliser (Nitrosol^®) application rates than C. intermedia. The differences could be attributed to the differences between the two species in relation to their growth behaviour or the utilisation of the available nutrients. Berry (2006) stated that, although plants of the same species respond similarly to nutrient stress, species may show significant differences in growth rate, root distribution, phase of development and efficiency of nutrient uptake and utilisation.

Table 2 : Response of Cyclopia spp. to Nitrosol^® fertiliser with regard to growth and establishment of cuttings. Means followed by the same letter within a column are not significantly different (LSD = 0.05)

Species	Nitrosol^® (ml l^-1water)	Fresh shoot mass (g plant^-1)	Dry shoot mass (g plant^-1)	Fresh root mass (g plant^-1)	Dry root mass (g plant^-1)	Plant height (mm)	Number of nodules per plant	Number of shoots per plant
C. genistoides	3.33	1.83^a	0.47^a	2.35	0.40^a	149.30^a	6.95^a	4.23^a
C. genistoides	1.67	1.28^b	0.34^b	1.79	0.30^b	121.15^b	5.38^a	3.35^b
C. genistoides	0	0.48^e	0.12^d	0.98	0.19^c	73.65^d	1.53^b	0.80^d
C. intermedia	3.33	1.00^c	0.23^c	1.51	0.14^c	89.38^c	1.08^b	2.19^c
C. intermedia	1.67	0.71^d	0.18^cd	1.19	0.16^c	62.68^de	1.43^b	1.04^d
C. intermedia	0	0.54^de	0.11^d	0.83	0.13^c	57.53^e	0.10^b	0.00^e

A similar trend was also shown with regard to the significant species × cutting position recorded for plant height, because C. genistoides showed significant differences, whereas C. intermedia showed no significant differences because of cutting position (Table 3). However, in contrast C. intermedia showed a significant response to cutting position with regard to number of shoots per plant, whereas no response was recorded for C. genistoides. The results showed that C. genistoides outyielded C. intermedia with regard to both parameters, most probably because C. genistoides is known as a vigorous grower, whereas C. intermedia is commonly known as a slow-growing species (Joubert et al. 2011).

Table 3 : Response of Cyclopia spp. to different cutting positions with regard to growth and establishment of cuttings. Means followed by the same letter within a column are not significantly different (LSD = 0.05)

Species	Cutting position	Fresh shoot mass (g plant^-1)	Dry shoot mass (g plant^-1)	Fresh root mass (g plant^-1)	Dry root mass (g plant^-1)	Plant height (mm)	Number of nodules per plant	Number of shoots per plant
C. genistoides	Terminal	1.26	0.31	2.35	0.40^a	149.30^a	6.95^a	4.23^a
C. genistoides	Subterminal	1.13	0.30	1.79	0.30^b	121.15^b	5.38^a	3.35^b
C. intermedia	Terminal	0.69	0.14	1.19	0.16^c	62.68^de	1.43^b	1.04^d
C. intermedia	Subterminal	0.82	0.21	0.83	0.13^c	57.53^e	0.10^b	0.00^e

Although plants from both species responded positively to fertiliser (Nitrosol^®) applications, plants from C. genistoides cuttings showed larger responses than those of C. intermedia with regard to most plant parameters tested and especially at the higher application rate. In the case of C. genistoides, application rates of 3.33 ml Nitrosol^® l^-1 water gave the best results in all parameters except fresh root mass and number of nodules per plant. In the case of C. intermedia no significant differences between fertiliser application rates of 1.67 and 3.33 ml Nitrosol^® l^-1 water were shown with regard to fresh root mass as well as dry shoot and root mass. This study therefore suggests that Nitrosol^® fertiliser applications can be recommended to enhance the establishment of rooted cuttings of Cyclopia species, but optimum application rates may differ between species. Cutting position did not have a large effect on the establishment and early growth of rooted cuttings of species used in this study.

Acknowledgements

The authors acknowledge the Agricultural Research Council (ARC), Agribusiness in Natural African Plant Products (ASNAPP) and the Department of Agriculture, Forestry and Fisheries (DAFF) for funding this study.