Research Journal of Microbiology

Year: 2008 | Volume: 3 | Issue: 4 | Page No.: 225-236
DOI: 10.17311/jm.2008.225.236

Effects of Arbuscular Mycorrhizal Inoculation and Fertilizer on Production of Castanopsis acuminatissima Saplings for Forest Restoration in Northern Thailand

P. Nandakwang, S. Elliott, S. Youpensuk and S. Lumyong

Abstract: Castanopsis acuminatissima is a native tree used to restore forest in Thailand. To accelerate seedling growth experiments were carried out to determine the efficacy of applying to C. acuminatissima. Arbuscular Mycorrhizal (AM) fungi, produced on sorghum, were used as inoculum to investigate the symbiosis on seedlings. The effects of AM inoculation (Acaulospora elegans, Glomus etunicatum, Glomus mosseae) together with phosphate fertilization (KH₂PO₄) on seedlings in a P-deficient soil were studied under greenhouse conditions. Increasing P-application rates greatly enhanced seedling growth (maximum at 250 mg kg^-1 soil). Growth was most rapid with G. etunicatum-colonized plants with P application (40.8 cm), whereas much lower height was found with non-AM plants without P added (14.4 cm). The mycorrhizal effective for C. acuminatissima in previous experiments were confirmed by growing seedlings in a forest soil with slow-release fertilizer (NPK) and combined with AM species under nursery performance conditions. Plant height was significantly enhanced by fertilizer but not by fungi. The greatest height was found in non-AM plants with fertilization (14.5 cm), whereas lower height was found for non-AM plants with no fertilizer added (10.9 cm). AM inoculation greatly enhanced seedling growth in P-deficient soil more than in forest soil due to differences in abilities of AM species to establish a symbiosis. Therefore, in sapling production, the soil properties and level of fertilization should be evaluated keeping secondary effects caused by changed mycorrhizal association.

Keywords: phosphorus fertilizer, mycorrhizal seedling production, Arbuscular mycorrhizal fungi, framework tree species and forest restoration

INTRODUCTION

Forest restoration means the re-establishment of the original forest ecosystem that was present before deforestation occurred. The goals of forest restoration are environmental protection and wildlife conservation (Anonymous, 2006). In 1994, the Forest Restoration Research Unit at Chiang Mai University (FORRU), started to investigate the possibility of restoring forests on degraded sites in northern Thailand by adapting the framework species method (first developed in Queensland, Australia) (Goosem and Tucker, 1995; Anonymous, 2006) to local conditions. FORRU screened indigenous forest tree species to select potential candidate framework species for field trials. In the FORRU^’s research tree nursery, experiments were designed to develop seedling production for high quality planting stock. Castanopsis acuminatissima (Bl.) A. DC. (Fagaceae), was confirmed as a potential framework species that could be used to restore seasonally dry tropical forest in northern Thailand but this species grow relatively slowly and difficult to raise in nursery. To solve such problems, studies have been made on modified potting media and fertilizer application (Anonymous, 2006). Arbuscular Mycorrhizal (AM) fungi have form symbiosis with a wide range of forest tree species (Gai et al., 2006). Such symbioses provide many benefits to host trees and are especially important in development of seedlings grown in nurseries and establishment of saplings planted in deforested sites.

AM symbioses result in increased growth of plants depending on the fungal strains (Pattinson et al., 2004; Youpensuk et al., 2005). AM fungi also protect plants against root pathogens, confer resistance to drought and increase soil aggregation (Dubský et al., 2002; Rilling et al., 2005; Wu et al., 2006). Lack of nutrient availability in tropical soils often limits plant growth. The ability of AM fungi to enhance nutrient absorption, (particularly phosphorus) by hyphal uptake and translocation towards the plant, is an important advantage (Koide and Mosse, 2004). The possibility of using beneficial attributes of AM fungi in planting stock will depend on preliminary assessments of whether inoculation is a suitable management option. AM fungi are obligate symbionts, usually propagated by growing them with living host plants in pot cultures. For starting pot cultures of AM fungi, the combination of appropriate host plant and substrate media for production of mycorrhizal inoculum is crucial (Setiadi, 2000). Pot cultures, which consist of soil, spores and mycorrhizal roots etc., can be used as inoculum for experiments or applied to seedling grown in a nursery or broadcast in the field (Brundrett et al., 1996; Setiadi, 2000; Klironomos and Hart, 2002). Knowledge about the ability of plant species to form symbiosis with AM fungi is very important for restoration success and indicates the need for inoculum in plants cultivated in forest nurseries (Wubet et al., 2003). The purposes of present experiment were to: (1) select appropriate host plants under pot culture conditions for production of indigenous AM fungal inoculum in the nursery, (2) examine the effects of 3 AM species with 6 rates of P application on plant development in P-deficient soil medium under greenhouse conditions and (3) examine the effects of AM fungal inoculation and conventional fertilization on growth of seedlings in forest soil under nursery performance conditions.

MATERIALS AND METHODS

Inoculum Production
The first experiment consisted of 30 treatments with 6 indigenous AM species [Acaulospora elegans Trappe and Gerd., A. mellea Spain and Schenck, A. scrobiculata Trappe, Glomus etunicatum Becker and Gerd, G. mosseae (Nicol. and Gerd.) Gerd. and Trappe and Scutellospora heterogama Walker and Sanders] and 5 host plants [maize (Zea mays L.), marigold (Tegetes erecta L.), soybean (Glycine max (L.) Merr.), sorghum (Sorghum vulgare Pers.) and upland rice (Oryza sativa L. cv. Bue Bang)] with 3 replications. The experiment was undertaken in clay pots (22 cm top diameter, 18 cm bottom diameter and 19 cm depth) with drainage hole containing 3 kg P-deficient soil medium (P-deficient soil and coarse sand ratio 2:1), autoclaved twice at 121°C for 30 min with a 2 day interval. The soil pH (H₂O) was 5.98 and contained 0.041% total N (Kjeldahl method), 1.4 mg kg^-1 available P (Bray II method) and 44.0 mg kg^-1 extractable K (1 M NH₄OAc, pH 7). Seeds were surface-sterilized with 10% sodium hypochlorite for 5 min, rinsed with sterile water and sown in Petri dishes, containing the moist tissue paper for 1 week. Seedlings were transplanted 3 seedlings per pot. AM spores were extracted from the soil samples by wet-sieving and 50% sucrose centrifugation (Brundrett et al., 1996) and collected on a 53 μm sieve. Fifty spores were inoculated into each pot. Seedlings were grown in a greenhouse at the Chiang Mai University (CMU) for 4 months between November 2005 and February 2006. Seedlings were watered once every 2 days with 500 mL of tap water. Twice a month, 80 mL of 4 full strength Hoagland’ solution (Hoagland and Arnon, 1950), without P was added to each pot. At harvest, root samples were separated from soil and cleaned with tap water. The root samples were cleared in 10% KOH at 121°C for 15 min and stained with 0.05% trypan blue in lactoglycerol (Brundrett et al., 1996). Thirty stained root segments from each plant (1 cm long) were taken at random and mounted on microscopic slides to assess mycorrhizal colonization (McGonigle et al., 1990). One hundred gram dried soil of all different treatments were used to determine spore density.

Effects of AM fungi with Phosphorus Fertilizer on Seedling Growth in Greenhouse Experiment
The second experiment consisted of 90 pots with 5 inoculation treatments (no inoculation and AM inoculation: A. elegans, G. etunicatum, G. mosseae and mixed AM species) and 6 levels of P application (KH₂PO₄) (at the rates of 0, 50, 100, 150, 200 and 250 mg P kg^-1 medium) with 3 replications. Spores of AM species were produced on sorghum pot cultures in P-deficient soil medium as shown above and used for inoculation. The experiment was undertaken in clay pots containing 3 kg autoclaved P-deficient soil medium. Seeds of C. acuminatissima were surface-sterilized with 10% sodium hypochlorite for 10 min, rinsed with sterile water and sown in a plastic tray containing the autoclaved forest soil medium (primary evergreen forest soil, coconut husk and peanut husk ratio 2:1:1). The medium pH (H₂O) was 5.60 and contained 0.628% total N, 15.8 mg kg^-1 available P and 132.0 mg kg^- extractable K. Three month-old seedlings (5-6 cm tall) were transplanted one seedling per pot. In each AM treatment, 150 spores were inoculated into each pot. Seedlings were grown in a greenhouse at the CMU for 6 months between December 2005 and March 2006. Seedlings were watered once every 2 days with 500 mL of tap water. Two weeks after transplanting, 6 levels of KH₂PO₄ were added to each treatment. Twice a month, 80 mL of 4 full strength Hoagland’ solution without P was added to each pot. At harvest, height and stem diameter of seedlings were measured. Roots were divided into 2 random sub-samples. Shoot samples and one root sub-sample were oven dried at 60°C for 48 h. Dry samples were analyzed for P content by the dry ashing and molybdovanado-phosphoric acid method. The second root sub-sample was used to determine AM colonization and soil sub-samples were assessed for spore density.

Effects of AM fungi with Slow-Release Fertilizer on Seedling Growth in Nursery Sapling Production
The third experiment consisted of 10 treatments with 5 inoculation treatments (no inoculation and AM inoculation: A. elegans, G. etunicatum, G. mosseae and mixed AM species) and 2 levels of slow-release fertilizer (NPK 14-14-14) (at the rates of 0 and 375 mg kg^- medium) with 28 replications. Slow-release fertilizer has been used successfully at FORRU for many framework species (Anonymous, 2006). The experiment was undertaken in plastic bags (23x6 cm) with drainage holes, containing 800 g autoclaved forest soil medium. Three month-old seedlings of C. acuminatissima were transplanted one seedling per bag. In each AM treatment, 150 spores were inoculated into each pot. Slow-release fertilizer was applied in fertilization treatments at the start of the experimental period. Seedlings were grown at the FORRU^’s nursery for 6 months between April and September 2006. Seedlings were watered once a day with tap water. Every 3 months, slow-release fertilizer was applied to each treatment. At harvest, height and stem diameter of seedlings were measured. Fresh shoot and root samples were treated in the same way as described previously. Dry plant samples were analyzed for P content. Root sub-samples were used to determine colonization percentage and soil sub-samples were assessed for spore density.

Data Analysis
All data were subjected to analysis of variance (ANOVA) for a completely randomized design. Residuals were normally distributed with constant variance. SPSS software version 12.0 was used to conduct the ANOVA. Duncan^’s Multiple Range Test (p<0.05) was used to compare treatment means.

RESULTS

Inoculum Production of Arbuscular Mycorrhizal Fungi
Four months after starting the pot culture, all 5 host plant species inoculated with 6 AM species had developed mycorrhizas. Spore density and colonization percentage varied greatly among the different host species.

Spore density and mycorrhizal colonization were increased by the host species and AM species which interacted (Table 1 and 2). Three fungal species: A. elegans, G. etunicatum and G. mosseae produced significantly the highest spore densities and colonization abilities on S. vulgare and Z. mays. The spore density of these AM species on sorghum was significantly higher than on maize, whereas mycorrhizal colonization on both plants did not differ from each other (93.8-100.0%). The highest spore number were found on sorghum inoculated with G. etunicatum (6148.7 spores/100 g soil), A. elegans (2713.3 spores/100 g soil) and G. mosseae (642.7 spores/100 g soil), respectively. Whilst much lower densities and root colonization of other 3 AM species were found on all host species (Table 1 and 2).

Effects of AM Inoculation with P Application on Growth of C. acuminatissima in P-Deficient Soil Medium
Growth of C. acuminatissima seedlings in the P-deficient soil experiment was highly influenced by both AM inoculation and P-application rates (Table 3). Six months after transplant, plant height, shoot and root dry weights as well as shoot P content were all significantly increased by both factors which interacted, whereas root to shoot ratio and root P content were also increased by both factors but not by their interaction. The only exception was stem diameter, which was only increased by P rates. AM colonization was only increased by fungal inoculation, whereas spore density on the other hand, was increased by both P rates and fungus which interacted (Table 3). Root colonization ranged in the P applied treatments from 36.7-45.4%, which did not differ with P rates (Table 3). In the AM treatments, colonization percentages ranged from 40.1-55.3%. Root colonization in the plants inoculated with the single species of AM fungi was significantly higher than for the mixed species inoculum.

Spore density was significantly increased by P rates and AM fungi which interacted. Spore density in the P applied treatments ranged from 20.9-30.1 spores/100 g soil which reached maximum at 150 mg kg^-1soil (30.1 spores/100 g soil) and continued to decrease with the lowest at 250 mg kg^- soil (20.9 spores/100 g soil). Spore density ranged from 23.1-38.3 spores/100 g soil and the highest density was found in plants inoculated with mixed AM species.

P applications were highly beneficial for growth parameters of plants as measure by height, stem diameter (Table 4), dry weights (Table 5) and P contents (Table 6) and AM inoculations also had significant effects on plant growth. Six months after transplant, non-AM plants grown in P-deficient soil exhibited increasing either height or shoot dry weight to increasing P rates, whereas the other growth parameters showed no such differences. Contrast with all growth parameters of AM plants, tended to increase with increasing P rates (maximum at 250 mg P kg^-1) and mycorrhizal enhancement varied with the different kinds of AM species. In P applied treatments, seedlings generally grew very little and no significant differences between AM plants and non-AM plants were observed for plant height, stem diameter and P contents, whereas dry weights of AM plants significantly trended to be higher than non-AM plants.

AM inoculation significantly increased plant height over non-inoculated controls were found in A. elegans at 200-250 mg P kg^-1, G. etunicatum at 150-250 mg P kg^- and G. mosseae at 200 mg P kg^-1. The maximum height was 1.52 and 1.78 fold higher than the control in A. elegans and G. etunicatum at 250 mg P kg^-1, respectively, whereas, height of G. etunicatum plants was the highest (40.8 cm) (Table 4). Stem diameter was only influenced by P rates. Stem diameter had a 1.45 and 1.32 fold increase over control with no P added in A. elegans and G. mosseae at 250 mg P kg^-1, respectively, which did not differ from one another (0.3 cm) (Table 4).

Only A. elegans plants at the lowest P rate significantly had a higher shoot dry weight than non-AM plants and other AM plants. Whilst at high P rates, AM inoculation significantly increased over non-AM plants were found in most fungus treatments, except for mixed fungal species.

At 250 mg P kg^-1, the maximal shoot biomass exhibited 2.41, 2.56 and 2.34 fold increase over control in A. elegans, G. etunicatum and G. mosseae, respectively, whereas G. etunicatum plants gave the highest shoot biomass (2.7 g plant^-1 ) (Table 5). A. elegans plants with no P added also had a significantly higher root dry weight than non-AM plants and other AM plants. AM inoculation significantly increased over non-AM plants were found in all fungus treatments at the first 50 mg P kg^-1 and continued to increase with further increase in the level of P rates. Root biomass reached their maximum at 250 mg P kg^-1 and exhibited 3.04, 3.05, 2.58 and 2.82 fold increase over non-inoculated controls in A. elegans and G. etunicatum G. mosseae and mixed AM species, respectively, which did not differ from one another (2.8-3.4 g plant^-1 ) (Table 5).

At 50 mg P kg^-1, AM inoculation significantly increased shoot P content over non-AM plants was only found in A. elegans. Whilst at higher P rates, AM inoculation significantly increased shoot P content were found in A. elegans at 200-250 mg P kg^-1, G. etunicatum at 150-250 mg P kg^- and G. mosseae at 250 mg P kg^-1. At the highest rate, the maximal shoot P content exhibited 3.04, 3.05, 2.58 and 2.82 fold increase over non-AM plants in A. elegans and G. etunicatum and G. mosseae, respectively which did not differ from one another (1.4-1.7 mg plant^-1 ) (Table 6). Fungal inoculation significantly increased root P content over non-inoculated controls were found in A. elegans and G. etunicatum at first 50 mg P kg^-1 and still increased at 150 mg P kg^-1. Whilst at 200 mg P kg^-1, only root P content of A. elegans plants were significant increased over controls. At 250 mg P kg^-1, root P content of most AM plants exhibited 5.92, 4.54 and 4.69 fold increase over non-AM plants in A. elegans, G. etunicatum and G. mosseae, respectively which did not differ from one another (1.2-1.5 mg plant^-1) (Table 6).

Effects of AM Inoculation with Slow-Release Fertilizer on Growth of C. acuminatissima in Forest Soil Medium
Growth of C. acuminatissima seedlings in the forest soil measured for 6 months showed consistent effects of slow-release fertilization throughout the experiment (Table 7). Plant height, dry weights and P contents were increased by fertilizer but not by AM fungi. However, fertilization effects on growth parameters were slightly higher it increased growth than non-AM plants without fertilization (controls).

Plant growth was highest in all fertilization treatments for plant height (14.5 cm), shoot and root dry weights (0.6 and 0.8 g plant^-1 , respectively) and shoot and root P contents (0.2 and 0.1 mg plant^-1 , respectively). Whilst significant mycorrhizal effects were only found in stem diameter, which fungal-fertilizer interactions were not found (Table 7). Stem diameter of G. etunicatum and mixed AM species plants with fertilization were highly increased by AM fungi, whereas the highest diameter was found in AM plants with fertilization (0.2 cm). Root colonization and spore density were increased by both fungus species and fertilizer, which interacted (Table 7). Colonization percentages of all AM plants with fertilization were higher than without fertilization. The percentages were high, ranging from 38.4-79.5%, with the highest percentage found in A. elegans plants. Spores in fungal treatments with fertilization, generally recovered in higher number than without fertilization. The spore number ranged from 16.1-74.6 spores/100 g soil and the highest density was found in mixed AM species with fertilization.

DISCUSSION

Pot cultures, using host plants grown in soil diluted with sterile sand, are most commonly used to propagate AM fungi (Brundrett et al., 1996). In our present study, all 6 indigenous AM fungi were recovered from all 5 host plant pot cultures. Variation in spore density and mycorrhizal colonization was increased by host plants and AM fungi. Results presented here for spore density and root colonization are in agreement with those found in the greenhouse and the field showing that AM species, host plant species and soil conditions have been reported to effect on mycorrhizal formation and sporulation in pot cultures (Brundrett et al., 1996; Liu and Wang, 2003). From our observation, most plants inoculated with small to medium sized spores of Glomus and Acaulospora species was generally more successful than inoculation with the larger sized spores of Scutellospora species. The spore abundance must be related to their sporogenous characteristics. It has been reported that Glomus and Acaulospora species usually produce more spores than Gigaspora and Scutellospora species in the same environment conditions, because smaller spores require a short time to produce spores than large spores (Hepper, 1984; Bever et al., 1996). The high success rates of spore density and colonization percentage of at least 3 of 6 AM species were observed on sorghum and maize suggested that these plants are favorable hosts especially for A. elegans, G. etunicatum and G. mosseae compared to other hosts tested. Thus, sorghum and maize pot culture-produced spores as inoculum in P-deficient soil medium are more suitable for large scale production as well as for research purposes and nursery practice.

In the greenhouse experiment, growth of non-AM plants was very stunted in P-deficient soil medium (available P 1.4 ppm). Applying additional P fertilizer to non-AM plants could enhance growth of plants. However, even with the different allotments of P fertilizer, all AM species greatly stimulated plant growth with bigger size than non-AM plants in this unsuitable soil condition. Enhancement effects on plant growth, tended to increase with increasing P rates and more efficiency varied with the different kinds of AM species. Phosphorus responses for the plant growth agreed with that reported for other AM plants grown in a controlled environment (Siqueira et al., 1998a; Youpensuk et al., 2005), thereby confirming mycorrhizal nutritional benefits and the strong interrelationship between P supply and mycorrhizal response under nutrient-stressed conditions. AM inoculation had slightly effects on seedling growth when plants received low P rates at planting, whereas strongly effects were found at higher P rates. The present results suggested that addition 250 mg P kg^-1 to C. acuminatissima seedlings was suitable to produce either non-AM plants or AM plants in P-deficient soil. Although, non-AM plants at maximal P rate were higher than at minimal rate but still significantly lower than AM plants at the same P addition. The greatest height of AM plants was found in G. etunicatum plants with 250 mg P kg^-1, exhibited 1.8 fold over non-AM plants. Contrast with stem diameter of seedlings was not improved by the mycorrhizal symbiosis but diameter was also greatest with the highest P added, exhibited 1.5 fold increase over control with no P added.

The Mycorrhizal Dependence (MD) of C. acuminatissima was high and trended to increase with applying fertilizer into P-deficient soil. In the absence of fertilizer, maximum MD exhibited 43.1% when fertilized with 150 mg P kg^-1, maximum MD exhibited 75.9% (data not shown). P-deficient plants lacking AM symbiosis tend to have a high root to shoot ratios usually associated with nutrient-stressed plants (Pacovsky et al., 1986). Whilst, root to shoot ratios of AM plants in our study were higher than for non-AM plants, especially in the absence of P fertilizer or low P rates, the higher ratios probably resulted in mycorrhizal stimulation of root growth for improving P acquisition under limiting P condition. Plants characterized as inefficient at acquiring soil P, may substantially improve P acquisition by morphological and physical adaptations include changes in P and dry matter partitioning that favor growth of roots over shoots and the induction of a high-affinity P uptake and transport system in roots during the development (Cogliatti and Clarkson, 1983; Marschner et al., 1996).

P contents in AM plants were significantly increased with levels of P application. AM fungi most likely increased nutrient uptake from the soil due to the external hyphae can exploring greater soil volume and delivering nutrients to the host plants (Joner and Jakobsen, 1995; Koide and Mosse, 2004). Root colonization of plant species in many greenhouse experiments is diminished by high soil P availability and concomitant enhanced P concentration in plant tissues (Vaast et al., 1996; Youpensuk et al., 2005). Contrasting with this suppressive effect observed with AM colonization in C. acuminatissima was not differed by increasing P levels application while P status remained unaffected. This experiment showed that AM inoculation of C. acuminatissima seedlings produces large plants with improved P status, thus confirming the high AM-dependency of host plant. This study also indicates the tolerance abilities of selected AM species on P-application rates, resulting from their abilities to promote plant P accumulation.

In the nursery experiment, seedlings grown in forest soil medium with slow-release fertilizer applied were slightly bigger than controls. Most AM plants without fertilizer added grew poorly with growth parameters similar to those of non-AM plants. Plant height, shoot and root dry weights and shoot and root P contents were increased by fertilizer but not by mycorrhiza, whereas only stem diameter was increased by both factors. Higher stem diameter of G. etunicatum and mixed AM species plants with fertilization may result from direct fungal efficiency effects or fungal-fertilizer interactions in such soil condition. AM colonization and P concentrations of C. acuminatissima seedlings were quite high with slow-release fertilizer added. This may be fertilization effect on P accumulation through its influence on AM symbiosis. Heavy application of P fertilizer or sufficient P condition at planting may reduce mycorrhizal formation, sporulation and the MD of host and thus mycorrhizal effectiveness for the seedling growth (Siqueira et al., 1998b). From our observation, growth of AM plants grown in forest soil was less than grown in P-deficient soil. This may the result of plants responding well to mycorrhizas in low or moderate P soils are regarded as mycorrhizal dependent. That is, they depend on mycorrhiza to show their full potential (Haselwandter and Bowen, 1996). The components of forest soil medium (available P 15.8 ppm) were rich in both organic and inorganic nutrients. Thus, AM fungi may be loose their function on stimulating plant growth in this nutrient condition. High dissolved inorganic nutrients in tropical forest soil may make AM fungi unnecessary to meet nutrient (Maffia et al., 1993). The consistent effects of AM fungi on plant growth were diminished or disappeared with nutrient abundance in the soil. Strongly mycorrhizal effects on external P requirement for maximal growth of seedlings were high and consistent in nutrient poor soil but were diminished and varied unpredictably with levels of fertilizer and P requirement of individual plant species. Phosphorus is not only a suppressed factor on AM symbiosis. Further, Youpensuk et al. (2005) reports that application of high rates of P or N can depressed AM colonization and spore formation. C. acuminatissima seedling grown in forest soil medium with nutrient abundance did not respond to AM inoculation although root colonization was high. Without fertilizer added, all AM plants were still equal size with non-AM plants. It may be resulted in reduction of AM symbiosis caused decreasing nutrient uptake ability for growth of mycorrhizal plants.

In addition, mycorrhizal inoculation with selected AM fungi and application of optimal P rate on early plant development are highly advantageous for high quality sapling production in forest tree nurseries and sapling establishment in low nutrient soils in forest restoration areas in Thailand. All AM species tested were greatly effective in promoting growth parameters of AM seedling in nutrient poor medium, but diminished their function in nutrient abundant medium. Differential responses of AM seedlings in these experiments appear to be related to nutrient available (inorganic and organic forms) in medium and form of fertilizer (easy soluble or slow-releasing) by plant and AM fungi. Therefore, the success of AM technology will depend upon dependent mycorrhizal host, optimal soil condition and well-adapted effective fungal strains.

ACKNOWLEDGMENTS

The authors wish to acknowledge financial support from Chiang Mai University Graduate School and Pibulsongkram Rajabhat University for partial support to the first author’s Ph.D. study. This research was supported by the Thailand Research Fund DBG4980004. The authors are grateful Mr. Sittichai Lordkaew and Mrs. Kanjanaporn Lordkaew for plant analysis and the Multiple Cropping Centre, Faculty of Agriculture, Chiang Mai University for experimental facilities.

REFERENCES