Philippine Journal of Science 132 (2): 89-94, December 2003 ISSN 0031 - 7683

Response of philippinensis Benth. & Hook (Philippine ) Cuttings to Applied Mykovam and Biocore

Yolina Tarranco-Castañeto* and Minda P. Follosco-Edmiston1

Nueva Vizcaya State University, College of Forestry Bayombong, 3700 Nueva Vizcaya 1Biology Department, College of Science De La Salle University-Manila 2401 Taft Ave., Manila

Tectona philippinensis locally known as Philippine teak is an indigenous species belonging to the family . This species had been declared as endangered due to rapid population decline. Because of this, there is an urgent need to propagate and the species to save it from future extinction. Successful rooting using stem cuttings was already achieved and therefore further study was conducted to determine the response of Philippine teak to added Mykovam and Biocore in the nursery. Results of the statistical analysis revealed significant effect of the treatments on height, stem diameter, % organic matter and % nitrogen of the rooted cuttings. Tallest height of rooted cuttings was observed using Biocore at 20 g/seedling (36.97 cm) giving 82.30% increase over that of control seedlings (20.28 cm). Likewise, seedlings applied with combination of Biocore + Mykovam at 5 g/seedling had 25.30 cm height. Seedling diameter was also affected by the application of treatments. Biocore applied at 20 gram/seedling gave significantly biggest seedling diameter among other treatments. Moreover, analysis of tissues revealed significant differences in % organic matter and % nitrogen content among different treatments. Percent organic matter and % N was found 12.16% and 11.32% higher using Biocore 20 g/seedling over that of the control seedlings. From the results, seedlings applied with Biocore 20 g improved seedling height, diameter, % organic matter and % nitrogen over other treatments and that of the control seedlings. Application therefore of Biocore at 20 g/rooted cutting of the Philippine teak is recommended for better growth and survival of seedlings in the nursery.

Keywords: biofertilization, rooted cuttings, saplings, stem cuttings, wildings

The serious attempt of the Philippine government of quality seeds, the use of other plant parts like to restore degraded forest lands calls for a cohesive stem as planting propagules is a feasible alternative. and comprehensive approach to abate the dwindling Vegetative propagation by stem cuttings is becoming status of forest resources. However, one of the major an important tool for forest tree improvement activities problems in restoration is the inadequate supply of and for the establishment of clonal plantation. It is also quality seeds for massive planting. In the absence an effective way of saving endangered species like Philippine teak for genetic conservation and upland *Corresponding author: [email protected] rehabilitation. Successful rooting using stem cuttings

89 Tarranco-Castañeto & Follosco-Edmiston has been done and the species is found easy to root provide a balance of elements needed by a variety of (Follosco-Edmiston 2002). crops. The organic formulation has been amended with Philippine teak is an endemic forest tree species nitrogen-fixing bacteria isolated from grass (talahib) and belongs to family Verbenaceae. It is a small to roots. It provides a buffering capacity because it has medium tree reaching 15 m high and 50 cm dbh. a pH of more than 7. It also acts as slow-release Its yellowish or creamy-white textured wood is used fertilizer. It contains partially decomposed organic in the construction of bridges, wharves, railway materials which also conditions the soil by improving carriage, ship decks, in wood carving and in general its physical and chemical microbial properties through carpentry. In addition, decoction of fresh dried time (Garcia 1989, Garcia 1990). It is one of the leaves is prescribed for menstrual disorders and promising biofertilizers that can be used for . It hemorrhages. The oil from its nuts has good odor and proves to be very effective in enhancing better growth is used as hair grower or applied to the skin to soothe of crops and forest trees and minor forest products irritation (De Guzman et al. 1986). Philippine teak is as well. Biocore used in the experiment was obtained very important because it is one of the three known from Dr. Mercedes U. Garcia. Application of Biocore teak species in the world (Rojo 1981). However, in dagwey (Saurauia subglabra Merr.) an indigenous due to slash and burn cultivation, firewood gathering species enhanced four times increase in height and land conversion to other uses, there was a rapid and three times increase in diameter of seedlings decline of the population of species (Madulid & Agoo planted in the field (Baculanta & Castañeto 2000). 1990). The global conservation status of the species In Dendrocalamus strictus, culm height was six times has been assessed and designated endangered both better for seedlings applied with Biocore as compared in the 1997 IUCN Red List of Threatened Species to the control (Espiritu et al., 2001). In another and the World List of Threatened Trees (Oldfield et experiment using Swietenia macrophylla rooted al. 1998, Walters & Gilleth 1998) as cited by Follosco- cuttings grown in the nursery revealed significant Edmiston (2002). differences in height, stem diameter, length of root, With this current status, conservation efforts root and shoot biomass were higher for seedlings should be immediately undertaken to save the species applied with Biocore (Castañeto, 2001). With the from future extinction. Successful macropropagation positive response of the different species to added using stem cuttings was found feasible and were biofertilizers, species like T. philippinensis needs to successfully done already. However, nursery culture be studied immediately. technique like biofertilization needs to be tried to It is therefore the ultimate objective of the study enhance growth and to produce vigorous seedlings. It to determine the effect of Mykovam and Biocore and will also lessen the dependence to inorganic fertilizer. its combination on the growth of. T. philippinensis. Among the biofertilizers available in the market, Inoculation of Mykovam and Biocore as a nursery Mykovam and Biocore and its combinations were technique aims to produce vigorous seedlings for tested on growth of T. philippinensis. field planting operations and at the same time as an ex-situ conservation. Specifically, the study aims Mykovam is a soil-based biofertilizer contaning to evaluate the response of T. philippinensis rooted effective species of mycorrhizal (VAM) fungi. It is cuttings to added Mykovam and/or Biocore and its a soil inoculant containing spores and mycorrhizal combination. It also determines which of the two propagules commonly used to inoculate nursery- biofertilizers is best and the application dosage best grown seedlings (BIOTECH 1995, Lapitan & Garcia for T. philippinensis in the nursery. 1994). It assists in the absorption of nutrients and water from the soil and can be used for agricultural crops, fruits trees and forest trees. Moreover, the existence of mycorrhiza affects the effectiveness Materials and Methods of roots. It improves water and nutrient uptake, increases resistance to drought and root disease and Collection and maintenance of wildlings survival of newly planted and established trees (Darr, 1996). It is composed of a cocktail of 3 arbuscular Philippine teak wildlings, between 1 to 2 year-old mycorrhizal fungi of Glomus etunicatum, Glomus with a height between 24.50 cm to 35.56 cm were macrocarpus and Gigaspora margarita (Zarate et al., earth-balled from the steep ridges of a secondary forest 1999). It is produced and marketed at the Institute and thickets in Lobo, Batangas. These were planted of Molecular Biology and Biotechnology (BIOTECH), individually, in a black polyethylene bags filled with University of the Philippines, Los Baños, Laguna. garden soil, watered daily, fertilized once and placed Biocore, on the other hand, comprise a scientific under shaded area until they were carefully transferred formulation produced from carefully decomposed to bigger seedling bags to allow proper rooting. The agricultural wastes, biochemically processed to wildlings were used three months after collection.

90 Response of T. philippinensis cuttings to Applied Mykovam and Biocore

Preparation of cuttings the treatments similar, its application dosage was also doubled. The different inoculants were weighed based Young stem cuttings measuring 7.62 cm – 12.7 on the treatments identified and placed in separate cm were collected from apical shoots and lateral papers for use in the experiments. branches of phenotypically superior T. philippinensis 4’ x 6’ black polyethylene bags were filled with 1/3 wildlings maintained in the nursery. Cuttings were prepared potting media. Afterwhich the different amount collected early in the morning when temperature is low of Biocore and Mykovam were placed in the respective to minimize transpiration. The collected cuttings were polyethylene bags with prepared labels as follows: cut into two-node cuttings for rooting experiment and T1 – Control (untreated) placed in a container with water to avoid dessication T2 – Biocore (10 g) and to wash off dust. Leaves from the lower half of the T3 – Mykovam (10 g) cuttings were removed to prevent them from rotting T4 – Biocore and Mykovam (5 g each) while remaining leaves were trimmed in half. The T5 – Biocore (20 g) cuttings were soaked in 5% Benlate solution (Benomyl There were 100 rooted cuttings/treatment used. at 500 g/kg active ingredients), a fungicide, for 30-45 A total of 500 rooted cuttings with approximately the minutes. Soaked cuttings were then bundled up to 30 same length of sprouts and roots were utilized in the pieces, with the basal part at the same end and soaked experiment. The rooted cuttings were planted in the in 1,000 ppm indolebuteric acid (IBA), rooting hormone individual polyethylene bag and filled with potting for 30-45 minutes, without rinsing in tap water (Follosco- media, watered and place inside the plastic chamber Edmiston, 2002). and maintained in a shaded area. Maintenance was done by regular watering of the Preparation of rooting/potting media planted cuttings and gradually opening the plastic The rooting/potting media is a mixture of coconut chamber within a month. The following month, the coir dust, sand and garden soil at 2:1:1 ratio. The rooted cuttings were taken out of the chamber, placed mixture was sterilized in an autoclave (Market Forge: in the nursery, cared and maintained for hardening. Sterilmatic) for 20 minutes at 1210C (15 psi) in three separate times. Hiko trays were also submerged in Parameters Measured a basin with prepared 10 ml of commercial bleaching Evaluation of treatment effects was done after four solution containing 2.5% by weight sodium hypochlorite months and the following parameters were obtained: (Chlorox/Purex)/100 ml overnight. The sterilized media height (cm), stem diameter (mm), % organic matter, was placed in Hiko trays, wetted, and treated cuttings and % total Nitrogen of tissues. were planted in Hiko trays. Cuttings in Hiko trays were Height as used in this study is the length of the new kept in polyethylene chambers which were placed shoots of the cutting which originated from the node. It in greenhouse. The planted cuttings were watered was measured using a foot rule from the point where regularly or as the need arises using a water hose shoot originated from the shoot tip. with a mist sprayer. After three to four weeks, cuttings Stem diameter was measured using vernier caliper, produced adventitious roots and were ready for transfer by measuring the new shoot and not the original to individual 4” x 6” black polyethylene bags. planting stocks used in the experiment. A destructive sampling process was done to analyze Preparation of inoculants and the plant tissues specifically the % organic matter and treatments application % total Nitrogen. The following formulas were used: Mykovam was bought at BIOTECH UPLB College, Laguna while Biocore was obtained from Dr. Mercedes % organic matter = % dry weight - % ash weight U. Garcia, UPLB College, Laguna. The two inoculants (De Jesus & Mencias-Ragragio 2000) were both in loose form and ready for use. For Mykovam, % Total N = % organic matter X 0.50 the prescribed dosage is 5-g/rooted cutting. It has a shelf life of only six months. The effect of the applied The experiment was laid out in a simple Completely Mykovam is through infection of the microorganism on Randomized Design (CRD) replicated four times. The the host plant and to hasten its effect on the growth data gathered on the different parameters mentioned of T. philippinensis, the dosage was doubled. Unlike above were subjected to Analysis of the Variance inorganic fertilizers, over dose is not a problem with (ANOVA) to determine significance of results while the Mykovam. comparison of treatment means, for parameters yielding Biocore, on the other hand, was also a ready significant differences was done using the Duncan’s to use biofertilizer, so there is no more preparation Multiple Range Test (DMRT) (IRRISTAT, Version needed for this type of inoculant. The prescribed 3.1, Biometrics Unit, International Rice Research dosage of application is 10 g/rooted cutting, To make Institute).

91 Tarranco-Castañeto & Follosco-Edmiston

Results and Discussions Stem Diameter Assessment of growth in T. philippinensis rooted Stem diameter was significantly increased by the cuttings in the nursery was influenced by the application addition of Biocore (20 g). Biocore (20 g) inoculated of Mykovam and Biocore. ANOVA results showed seedlings had the largest diameter (19.33 mm) while significant effects on height, stem diameter, % organic control seedlings had the smallest (15.17 mm) among matter and % nitrogen as shown on Table 1. treatments giving a percent increase of 27.42% over

Table 1. The average seedling height, stem diameter, % organic matter, and % nitrogen of Philippine teak seedlings at different treatments.

Treatments Height (cm) Diameter (mm) % Organic Matter %Total N

T1 Control 20.28a 15.17a 21.05ab 1.06ab T2 Biocore (10g) 18.80a 16.33ab 20.57ab 1.03ab T3 Mykovam (10g) 19.42a 15.67ab 19.83a 0.99a T4 Bio + Myko (5 g each) 25.30b 17.83bc 22.01bc 1.10bc T5 Biocore (20g) 36.90c 19.33c 23.61c 1.18c Fc 74.81** 5.18* 5.75* 5.62* CV 6.30% 7.70% 4.90* 4.90%

Means followed by the same letter are not significantly different * Significant ** Highly signifiant

Seedling Height the control seedlings (Table 1). Application of Biocore (10 g) and Mykovam (10 g) did not yield significant ANOVA revealed highly significant effects of differences as compared with the control seedlings. Mykovam and Biocore inoculation on seedling height Similar results were obtained using tissue cultured (Table 1). Seedlings applied with Biocore at 20 g strictus bamboo. Culms diameter was improved three had the highest height (36.97 cm) over the control times when applied with Biocore over control seedlings seedlings (20.28 cm). This treatment gave an increase (Espiritu et al., 2001). Addition of Biocore (10 g) and in height equivalent to 82.30%. Application of combined Mykovam (10 g) on T. philippinensis seedlings did Biocore + Mykovam (5 g each) on the other hand, not yield significant differences between control, and gave a significant increase of 24.75% over the control diameter of untreated seedlings was comparable to seedlings. The height of untreated seedlings was those treated with Biocore (10 g) and Mykovam (10 g). comparable with those applied with Biocore (10 g) and However, doubling the dosage of Biocore (20 g) yielded Mykovam (10 g) as shown in Table 1. The results show the biggest stem diameter of seedlings. that application of biofertilizers influenced the height growth of T. philippinensis. The superiority of height of Percent Organic Matter the seedlings applied with Biocore (20 g) jibed with the findings of Baculanta and Castañeto (2000) on their There was a significant effect on the organic matter study of Saurauia subglabra Merr. (dagwey), an endemic content of seedlings due to applied treatments. The species and on Vitex parviflora (molave) where the highest percent organic matter was observed on seedlings grown in the nursery and applied with Biocore seedlings applied with Biocore (20 g) amounting to were the tallest (Castañeto 2001). These results can 23.61% while the lowest was recorded from seedlings be explained by the fact that Biocore provided the treated with Mykovam (10 g) with 19.83% as presented seedlings an appreciable amount of 1.18% nitrogen in Table 1. Control, Biocore (10 g), Mykovam (10 g) thus helping the plants to grow vigorously and taller, treated seedlings were not significantly different from since nitrogen is one of the most important nutrients for each other, while combined Biocore + Mykovam (5 g growth and development. According to Brady (1985), each) and Biocore (20 g) are significantly different from nitrogen is essential for carbohydrate utilization within each other yielding 22.01% and 23.61%, respectively. plants and stimulates root growth and development as well as nutrient uptake. Of the macronutrients usually Percent Total Nitrogen applied, nitrogen seems to have the quickest and the Analysis of variance on % N revealed significant most pronounced effect. It encourages above ground differences due to treatments (Table 1). The vegetation and imparts dark green color of the leaves. seedlings with highest nitrogen were those seedlings

92 Response of T. philippinensis cuttings to Applied Mykovam and Biocore treated with Biocore (20 g) with 1.18% N and the Baculanta DA & YT Castañeto. 2000. Growth of lowest are those seedlings treated with Mykovam dagwey (Saurauia subglabra Merr.) an endemic (10 g) with .99%. Untreated seedlings, Biocore species applied with biofertilizers. In: proceedings of (10 g) and Mykovam (10 g) treated seedlings were the 8th BIO-REFOR. Kathmandu, Nepal (eds. Bista not significantly different from each other in terms MS, RB Joshi, SM Amatya, AV Parajuli, MK Adhikari, of nitrogen content of tissues. However, Biocore HK Saiju, R Thaku, K Susuki, K Ishii). + Mykovam (5 g each) and Biocore (20 g) were Brady NC. 1985. The Nature and Properties of Soils. significantly different from the three treatments with New York: McMillan Publishing Company. 1.10% and 1.18%, respectively (Table 1). From the above results, Biocore at 20 g was a better Castañeto YT. 2001. Growth response of molave (Vitex biofertilizer for T. philippinensis cuttings than Mykovam parviflora Juss.) seedlings to Biocore and Mykovam. in the nursery phase. This can be explained by the fact Philippine Journal of Science. 130 (2): 113-118. that Biocore is made from decomposed agricultural Castañeto YT. 2002. Growth response of mahogany wastes and the readily available nutrients in Biocore can (Swietenia macrophylla King.) cuttings to applied foster better growth earlier as compared with the effect Mykovam and Biocore. In: Proceedings of the 10th of Mykovam which is dependent of the germination and BIO-REFOR Tokyo, Japan (eds. I. Katsuaki, M. infection of fungal spores and later on assist the plant Masumori and K. Suzuki). in absorbing nutrients from the soil. Chaturvedi OP, AN Jha & DK Das. 1996. Vegetative propagation of Acacia auriculiformis by stem cuttings. In Forest, Farm and Community Tree Conclusion and Recommendations Research Report. Winrock International Institute The study was conducted to determine the for Agricultural Development. 1: 41-44. growth response of T. philippinensis rooted cuttings Darr S. 1996. Tree Fertilization Reasons and Methods. to Mykovam and Biocore inoculation in the nursery Arbor Age Magazine. condition. Results revealed significant effects in height, stem diameter, % organic matter and % De Guzman ED, Umali RM & Sotalbo ED. 1986. nitrogen due to inoculation. Between Biocore and Guide to the Philippine Flora and Fauna. Natural Mykovam, Biocore at 20 g/seedling worked best for Resources Management Center, Ministry of Natural T. philippinensis. The seedlings had better height, root Resources and University of the Philippines, Diliman, collar diameter, and higher % organic matter and % Quezon City. 3:360. N over other treatments. With the positive response De Jesus JB & EF Mencias-Ragragio. 2000. Laboratory of the species, the application of Biocore at 20 g is Manual in General Botany. Department of Biology, recommended for better growth of T. philippinensis College of Science, De La Salle University. Manila. cuttings in the nursery. With improved growth, sturdy and vigorous nursery grown seedlings will make good Dela Cruz RE, NS Aggangan & JT Zarate. 1991. planting stocks in the field for restoration activities, Biotechnology application for growth and development that will ensure better survival of outplanted seedlings. of reforestation species. In: Improved reforestation However, it is highly suggested that other fertilizers technologies in the Philippines. PCARRD/DOST Los and combination of dosages be studied further to Baños, Laguna 65-78 pp. know the optimum requirements of T. philippinensis for Espiritu CP, ET Castañeto & YT Castañeto. 2001. Better maximum growth and development. Field trials should growth of strictus bamboo (Dendrocalamus strictus also be conducted immediately to determine response Nees.) through bio-organic fertilization. Meristem Vol and adaptability of the species in other areas outside 1 (1). NVSIT Bayombong, Nueva Vizcaya. its natural habitat in Lobo, Batangas and Iling Island in Mindoro to facilitate conservation of the endangered Follosco-Edmiston M. 2001. Clonal propagation through T. philippinensis. rooting of stem cuttings in Philippine Teak (Tectona philippinensis) Benth. and Hook. f Verbenaceae). In: Proceedings of the 10th BIO-REFOR Tokyo, Japan (eds I Katsuaki, M. Masumori & K Suzuki). References Follosco-Edmiston M. 2002. Rooting of stem cuttings in BIOTECH. 1995. Mykovam: Inoculant for Agriculture Tectona philipinensis Benth. & Hook. (Verbenaceae). fruit trees and forest tree crops. National Institute Philippine Journal of Science. 131(1): 17-21. of Molecular Biology and Biotechnology, Ext. Com. Unit Univesity of the Philippines at Los Baños. Garcia MU. 1989. Role of biological inoculants and organic College, Laguna. matter in forestry. Training Materials in Agroforestry Course.

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Garcia MU. 1990. Application of biological fertilizer technology in reforestation. In Improved Reforestation Technologies in the Philippines. PCARRD/DOST. Los Baños, Laguna. 49-63 pp. Lapitan PG & MU Garcia. 1994. Application of Biotechnology in Forestry: Prospects and Progress. A paper presented during the continuing Professional Education for foresters Program. A short Course on Recent Developments in Forestry and Environments. IFC College of Forestry. UPLB College, Laguna. Madulid DA and EMG Agoo. 1990. Conservation Status of Tectona philippinensis Benth. & Hook. A Threatened Philippine Plant. Acta Manillana. 38:41 – 55. Rojo JP. 1981. Philippine teak an endangered tree. Canopy International. 7:16. Zarate JT, RE Dela Cruz & DS Iringan. 1999. Response of six Acacia species to arbuscular mycorrhizal mycorrhizal fungal inoculation. In BIO-REFOR Proceedings of Manila Workshop. (eds RE Dela Cruz, M Follosco, K Ishii, & K Susuki) Manila, Philippines. 90-97 pp. Umali-Garcia M. 1989. Clonal propagation in short rotation Forestry:Progress and potentials. In Proc. Joint Meeting on Short Rotation Forestry. College of Forestry and Natural Resources UPLB College, Laguna. March 3-7, 1999.

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