Effective Spore Density of Glomus mosseae , Arbuscular Mycorrhiza ( AM ) , for Inoculation of Rooted Cuttings of Black Pepper ( Piper nigrum Linn . )

Introduction of mycorrhizal associations is a complementary alternative to improve fertilizer absorption efficiency of black pepper (Piper nigrum Linn.). Therefore, as an initial step, this experiment was carried out to select a suitable spore density of the Arbuscular Mycorrhize (AM) fungus Glomus mosseae for inoculation of rooted cuttings of black pepper in the nursery. Four mycorrhizal inoculum levels of G. mosseae, namely 25 g (T1), 75 g (T2), 150 g (T3) and 300 g (T4) were tested with a control (T5) after quantification of the initial spore density values. The respective quantities of inoculum were incorporated separately to standard size polythene bags (21 cm diameter x 13 cm height; gauge 150) filled with a sterilized standard potting mixture. A two nodal cutting of black pepper (local selection GK 49) was planted in each polythene bag and weeding and watering were done as required. Observation of darkly stained AM hyphae at the 2 month after inoculation in all the inoculated treatments confirmed the success of inoculation. The heaviest infection of mycorrhizal fungi was observed in roots of T3 treatment showing root colonization, along with mycelium (100 %), vesicles (94 %), and spores (769/50 g) at the 6 month after inoculation. Significantly higher (p<0.05) shoot biomass (5.07 g) and root length (2740 cm) were observed at the 6 month in the plants in T2 than in the control (3.28 g and 1952 cm of roots). Incorporation of the AM at the rate of 75 g for one standard size polythene bag before planting rooted cuttings of black pepper was suitable to obtain good quality planting materials for field planting at the 6 month of growth in the nursery.


INTRODUCTION
Mycorrhizae are highly evolved symbiotic associations formed between soil fungi and plant roots.Arbuscular Mycorrhizae (AM) is the most common subgroup coming under endomycorrhizae.The AM fungi inoculated to crop plants colonize the plant root system and increase the growth and yield of crop plants including pepper (Rao, 1993;Thanuja, 2002, Durgapal et al., 2002;).The AM associated improvement of plant growth is attributed to various mechanisms such as increased uptake of nutrients and water, production of plant growth promoting substances, tolerance to drought and salinity and resistance to plant pathogens (Dalpe and Monreal, 2004).Glomus mosseae, an AM fungus, forms large asexual chlamydospores at the hyphal tips, usually one per tip, which are highly infective to genera of herbaceous and woody plants in a wide range of conditions.It has a high reproduction 1 Research Station, Department of Export Agriculture, Matale, Sri Lanka 2 Department of Plant Science, Faculty of Science, University of Colombo, Colombo 7, Sri Lanka ability mediated through the production of spores.At maturity, the spore contents are separated from the attached hypha by a septum or by occlusion with deposits of wall material.Spores are borne singly in soil and also formed in the root cortex or in sporocarps (James and Schenck, 1984).The G. mosseae can form mycorrhizal associations with many plant species with significant effects (Xioutang, 1994).Moreover, there is a considerable potential for the development of inoculum of G. mosseae for tree crops.
Black pepper (Piper nigrum Linn.) is one of the most important export agricultural crops in Sri Lanka and is considered as the king of the spices (Anon., 2003).One of the main problems faced by the pepper farmers is the high cost of production due to increasing trends of using inorganic fertilizer.The problem becomes more complex, as black pepper is a high nutrient demanding crop.Among the various fertilizers applied, phosphorous fertilizers have a very low absorption efficiency in plants, due to its fixation and low mobility in soil (Liu et al., 2004).Therefore, it is important to develop cost effective methods to fulfill the nutrient demand of black pepper.As organic farming is also becoming popular among pepper growing farmers, inoculation and promotion of mycorrhizal associations would be a complementary alternative to increase the efficiency of fertilizer absorption under black pepper cropping systems with special reference to phosphorous fertilization.Therefore, as an initial step towards this goal, the present experiment was carried out with the objective of selecting a suitable spore density of AM fungus Glomus mosseae for inoculation of rooted cuttings of black pepper at the nursery stage.

MATERIALS AND METHODS
This experiment was carried out in the nursery of the Research Station of the Department of Export Agriculture (DEA), Matale, Sri Lanka from March to October, 2008.Four mycorrhizal inoculum levels of G. mosseae (25 g, 75 g, 150 g and 300 g) were tested with a control after quantification of initial spore density value of each inoculum using the wet sieving and decanting technique with sucrose centrifugation (Brundrett et al., 1996).Inoculum levels and corresponding quantifications are shown in Table 1.The experiment was laid out in a randomized complete block design with 5 replicates.Each replicate consisted of 5 polythene bags having one cutting in each bag (black polythene bag, 21 cm diameter × 13 cm height; gauge 150).The sterilized (autoclaved at 121 °C for 15 minutes) standard potting mixture consisting of equal parts of top soil, cow dung, coir dust and sand was used (Anon, 2003).The initial nutrient composition and the pH of the standard potting media were measured.The quantity of corresponding inoculum (mycorrhizal spores and structures with sorghum (Sorghum bicolor L.) roots and moist soil were obtained by harvesting AM (Glomus mosseae) regeneration bed that was assigned to each treatment (Table 1).The innoculum was well mixed with the standard potting mixture required to fill rest of the polythene bags.Each inoculum treatment was incorporated into 25 polythene bags.A two-nodal cutting of black pepper (Piper nigrum Linn.) local selection GK 49 was planted in each polythene bag and these pots were kept in an airtight humid chamber for rooting.In order to find out the equivalent dry weight of a moist soil sample, the moisture factor was adopted using a sample for determination of moisture content on a dry basis.The humid chamber was opened at the end of the 3 rd week after planting.The cuttings were gradually hardened by opening the polythene cover for few hours every day for about two weeks, and the cover was totally removed at the 5 th week after planting.General agronomic management practices were adopted throughout the growing period as per recommendations of the DEA (Anon, 2003).
Casualties of cuttings were counted at the time of complete opening of the humid chamber.Thus, number of leaves, height of the new shoots and the diameter of new shoots were recorded at 2 nd , 4 th and 6 th month after inoculation.Leaf area, root length and root and shoot dry weights were measured using destructive methods at the 2 nd , 4 th and 6 th month after inoculation by uprooting five rooted cuttings from each treatment.The roots of the uprooted plants were thoroughly washed with running water.Thereafter, the roots were carefully separated using a surgical scissor and the total root length of each sample was measured using the modified line intersection method described by Tennant (1975).The shoot and root dry weights of the plants were also taken after drying the plant samples at 70 °C for 48 hours.
In order to ascertain the success of AM inoculation, pepper root samples were stained using the procedure described by Jarstfer and Silvia (2001).The root samples from each treatment were cut into 1 cm segments and were treated with 10% KOH overnight (about 12 hours).Then, they were rinsed in 30% H 2 O 2 at 50 °C.As soon as the roots were bleached white, they were rinsed with several changes of tap water.Then the roots were dipped in freshly prepared 0.1% Trypan blue for two hours and de-stained by two changes of tap water.Finally, the root segments were mounted on glass slides and covered with glass slips and observed under a light microscope (10 × 40).
The percentage mycorrhizal colonization of roots of successfully inoculated rooted cuttings using root samples was measured using an arbitrary scale as proposed in the modified technique described by Philips and Hayman (1970).The per cent of AM colonization was assessed by counting either the presence or absence of hyphae and vesicles and arbuscules in each segment of root and was expressed as percentage of colonization according to the method described by Kapoor and Paroda (2005).
Rhizosphere soils under each inoculum level were also assessed for AM spore population by the wet sieving and decanting method (Brundrett et al., 1996).The spores were counted using a stereomicroscope.The data of different observations were statistically analysed using GLM procedures of the SAS package (SAS, 1999).

RESULTS AND DISCUSSION
Initial chemical properties of the potting media were 0.19 % total nitrogen, 2.2 mg kg -1 available phosphorous, 1109 mg kg -1 exchangeable potassium, and 576 mg kg -1 magnesium with a pH of 5.55 (1:2.5 water), although, chemical properties of the potting media were not tested after the experiment, as it is beyond the objectives of this experiment.The number of live cuttings at the opening of the humid chamber at 21 days after planting revealed that the overall success was very high with 75 % survival, irrespective of treatment effects.
Darkly stained AM hyphae were observed in the samples of rooted cuttings at the 2 nd month after inoculation at all inoculum levels except in T5 (control).Neither vesicles nor arbuscules were seen in the roots of control plants.This confirms the successful inoculation of G. mosseae in this experiment.Furthermore, extra-radical, intra-cellular and inter-cellular hyphae were observed during the 6 month period of inoculation (Plates 1a and 1b).

Plate 1b. Stained black pepper root segment at 4 th month after inoculation showing vesicle formation (indicated by an arrow) within root tissues
Different mycorrhizal structures such as globose and sub-globose vesicles with hyphal connections were observed in the root samples of treated cuttings.These structures were recognized with reference to the illustrations of Brundrett et al., (1996) and Brown and King (1982).Preddy et al., (2003) also reported observing similar mycorrhizal structures in root sample of different genotypes of turmeric.It indicates that the time taken to produce vesicles and arbuscules is longer in black pepper roots than the time taken for the same process in S. bicolor, which was only two months (Kumari et al., 2008).This is comparable with studies of Brundrett et al., (1996).The heaviest infection of mycorrhizal fungi was observed in roots of cuttings in treatment T3 having the maximum root colonization, along with mycelium, vesicles, and spores at the 6 th month after inoculation (Table 2).Replacing one third of the nutrient-rich potting media with inoculum (top soil base material) in T4 may be one of the reasons for relatively low infection of AM in T4 when compared to that in T3.The introduction of high number of infective propagules at T4 could result in relatively low infection density in the pepper rhizosphere due to competitive effect.

Shoot biomass
The shoot biomass of plants increased with time irrespective of inoculation treatments (Fig. 1).The treatment effect was not significant (p>0.05) for shoot dry weight until the 4 th month after inoculation.Shoot dry weight became significant (p<0.05) at the 6 th month after inoculation with the maximum shoot biomass (5.07 g) in the treatment T2 (75 g inoculum) (Fig. 2).This suggests the possibility of a temporary growth retardation in the inoculated cuttings at the initial stages of AM infection, which gradually disappeared at the 6 th month.
According to Linderman and Hendrix (1982), the AM fungi initially act as parasites, absorbing organic nutrients from its host plant until it develops effective nutrient and water absorbing external mycelium.
Initial competition due to AM infection in pepper rooted cuttings at higher inoculation levels has also been reported by Wimalaratne (2005).Xioutang (1994) found that inoculation of AM fungi increased the shoot dry weight and root dry weight of Mangifera indica plants.

Root length
The root length of cuttings increased with time irrespective of inoculation treatments (Fig. 2).
In contrast to shoot biomass, the treatment effect was significant (p<0.05) for root length from the 4 th month of inoculation.During 4 th to 6 th month of inoculation, a significant root elongation was observed and the order of magnitude with reference to treatments also changed showing almost similar root length values for each of the inoculated treatments.All the inoculated rooted cuttings had greater root length values than the non-inoculated plants at the 4 th and 6 th month and the minimum root length was observed in the non-inoculated control (T5).Nevertheless, the differences in root length among T2, T3 and T4 were not significant.The narrow differences in root as well as shoot growth response to different inoculation levels may be attributed to the high nutrient status of the standard potting mixture.In general, the mycorrhizal induced growth improvements in higher plants are prominent under low nutrient conditions.The root length improvement observed in this study indicates the effectiveness of AM inoculation on initial growth of the rooted cuttings of black pepper at the nursery stage.Thanuja (2002) also reported the beneficial effects of inoculation with AM fungi, which resulted in enhanced rooting and root growth in black pepper cuttings.The improvement in rooting of black pepper cuttings would be beneficial in acquisition of drought resistance and to reduce transplanting shock at the post-nursery field establishment stage of black pepper cultivation.Improvement in nutrient absorption capacity as well as enhancement of plant vigor can also be expected with improved root growth in rooted cuttings of black pepper.

Other growth parameters
The mean values of other growth parameters at the 2 nd , 4 th and 6 th month after inoculation are shown in Table 3. Significant changes were not observed (p>0.05) in root biomass, leaf area and number of leaves among the treatments.Similarly, some of the other selected growth parameters also did not show any trend in response to treatments, showing mean values of 0.39 cm, 0.54 cm and 39.2 cm for new shoot diameter, old shoot diameter and height of new shoot, respectively, at the 6 th month after inoculation.This indicates that, these growth parameters may not be useful as sensitive indicators for a nursery stage study of rooted cuttings of black pepper.
The field planting of rooted cuttings of black pepper is done by farmers using 4-6 months old nursery plants, and this depends on factors such as onset of rainfall in the growing season, availability of well developed rooted cuttings in the nurseries and availability of labour and other resources.Overall observations of this experiment indicated that, adoption of mycorrhizal inoculations seems to be a beneficial practice for pepper nursery management.

CONCLUSIONS
Inoculation of pepper rooted cuttings with inoculum containing the mycorrhizal (G.mosseae) spores and fungal structures with sorghum (Sorghum bicolor L.) roots and soil, led to a successful inoculation of black pepper rooted cuttings (Piper nigrum Linn).Incorporation of 75 g of inoculum (795 mean number of spores) at the preparation of nursery bags was found to be adequate for a potting mixture of approximately 900 g, which is required to fill one standard polythene bag of size of 21 cm diameter x 13 cm height, to enhance root length and shoot dry weight of the plants.Further experiments to reduce the dependency of the high value components such as cow dung and coir dust in the standard potting mixtures along with mycorrhizal inoculum as a supplementary potting media component is proposed.

Fig. 1 .
Fig. 1.Effect of AM inoculation on mean shoot biomass of black pepper (each vertical bar indicates LSD at 0.05 probability) Fig. 2. Effect of AM inoculation on mean root length of black pepper with time after inoculation, (each vertical bar indicate LSD at 0.05 probability)