Evaluation of the initial growth of Shorea platyclados wildlings through the application of mycorrhizal biofertilizer under different levels of shading

The obstacles to the propagation of Shorea platyclados involve the uncertain flowering and fruiting seasons, the recalcitrant nature of the seeds, and the dependence on shade for growth. Furthermore, a mutualistic symbiosis, called mycorrhizae, usually exists between the roots and fungi, similar to other species of Dipterocarpaceae. This consequently hinders the regeneration process, which in turn may threaten the sustainability of this species in the wild. Ex-situ conservation using wildings is one of the measures that can be taken to overcome this issue. This study aimed to assess the initial growth of S. platyclados seedlings by administering different amountsof mycorrhizal biofertilizers under varying shade conditions at four and eight weeks of age in the nursery. A randomised complete block design was employed with the dose of biofertilizer as the treatment (0, 2.5, 5, 7 and 9 g/polybag) and shade as the group (0, 50, 60 and 70%). Observations were taken in the nursery at four and eight weeks of age. The observation parameters included the survival percentage, height growth, diameter growth, and vigour of the seedlings. There were significant differences in percentage of survival. Still, significant differences in height growth were observed with different doses of biofertilizer and shading at both four and eight weeks of age. Significant differences in diameter growth were observed with different doses of biofertilizer at both four and eight weeks of age, but not with different shades. No significant differences were observed for seedling vigour with different doses of biofertilizer and shade at both four and eight weeks of age. The optimal amount of biofertilizer was 5 g/polybag, resulting in maximum growth. This was achieved in a shade of 60%.


Introduction
The Dipterocarpaceae family exhibits particular attributes regarding its flowering, fertilization, and light necessities during its growth phase.As per reference Schmidt [1], the Dipterocarpaceae family's flowering periodization synchronizes with other species, especially members of the same family in tropical forest areas, occurring every 5 or 10 years.Out of the remaining fruits, 40% were lost to the primary seed predator, Mecysolobus crassus of the Curculionidae family.Fruits that were attacked were selectively aborted during the middle stage of fruit maturation.The final germinating fruits, which accounted for only 3.6% of the total number of flowers, received 47% of the total reproductive effort [2].Another noteworthy feature of Dipterocarpaceae is their mutual symbiosis with fungi in their roots, which is known as mycorrhiza.The presence of mycorrhiza facilitates the absorption of nutrients, thereby maximizing their availability.In 2021, Dipterocarpaceae had mass flowering and fructifications in Southeast Asia, including the Martelu Purba Nature Reserve [3].Thus, there is a high availability of natural seedlings during that time.Given the various characteristics and constraints of S. platyclados, including its conservation status, it is necessary to conduct propagation trials immediately, with a focus on ex-situ conservation efforts.The initial step is to cultivate using natural seedlings/wildings, which are currently abundant.
As a general rule, Dipterocarpaceae require an intensity of 30-50% light to grow [4].Excessive or insufficient light levels will harm the growth of S. platyclados seedlings [5].Thus, regulating shade during growth is crucial to achieve optimal growth rates as it is linked to photosynthesis and evaporation.Additionally, studies have investigated the benefits of applying mycorrhizae on wildlings' growth.Research Anindia et al. [6] on the application of mycorrhiza as biofertilizer indicated significant effects on the diameter and height of Shorea balangeran seedlings.The optimal dose for mycorrhiza was 5 g/polybag.By aiding the absorption of P nutrients bound in the soil, the addition of mycorrhizal biofertilisation results in an increase in the available P content in the roots [7].Building upon previous research, this study aims to assess the initial growth of S. platyclados wildings by testing different doses of mycorrhizal biofertilizers under various shades at 4 and 8 weeks of age in the nursery.

Method
This research began with the wilding collection in the field (Martelu Purba Nature Reserve) and then continued with research activities in the nursery.The phases of activities in this research method are described as follows:

Handling of wildings
Wildings of S. platyclados were collected as wildings at the Martelu Purba Nature Reserve, with an average height of 30-40 cm.To prevent root damage, the roots of the wildings were moistened during collection and planting in the prepared planting media.The planting medium consisted of the topsoil found under S. platyclados stands.Immediate planting on the prepared planting media followed collection of the wildings.To ensure the wildings' survival, transportation was done to the observation location two days after planting.S. platyclados wildings underwent a two-week acclimatization period in the screen house prior to treatment.

Experimental design and data analysis
The study utilized a Randomized Completely Block Design (RCBD) experimental design with shading as the block and mycorrhizal biofertilizer as the research treatment.The shade was achieved by using a paranet with varying densities.The paranet was installed under the roof of the screen house with four levels -0% (no shade), 50%, 60% and 70%, based on previous research [8].The shade frame, made of bamboo, was one meter high from the ground, and the length and width were adjusted to the polybag.The mycorrhizal biofertilizer that was utilized contained Glomus sp. with 53 spores per gram, Enterospora with 52 spores per gram, Gigaspora with 28 spores per gram, Acaulaspora with 36 spores per gram, as well as vermiculite as the carrier material mixed with sterile sand.Different doses of mycorrhizal biofertilizer were applied based on the prior research [6] for each polybag (approximately 3,5 kg soil/polybag).These doses include: Without mycorrhiza; 2.5 grams; 5.0 grams; 7.0 grams; and 9.0 grams.Fertilization was only done once in this observation.This study examined the percentage of survival, height increase, and diameter increase at 4 and 8 weeks in the nursery, as well as seedling vigour.If the ANOVA shows a significant F value, further testing with DMRT (Duncan Multi Range Test) is conducted.

Result and discussion
The research parameters observed included percentage of survival, height growth, diameter growth and seedling vigour at 4 weeks and 8 weeks after treatment applied.Details, as follows:

Percentage of survival
Seedling mortality was low, with only one seedling succumbing to death since its observation at four weeks of age.No further deaths were observed until the 8-week observation.These findings denote that S. platyclados wildlings exhibit a high survival rate and are suitable for cultivation outside their natural habitat.
The application of mycorrhizal biofertilizers to S. platyclados wildlings resulted in a 100% survival rate in each treatment except the 9 g dose of treatment, where the survival rate was 87.5%, owing to the addition of mycorrhiza at a dose of 9 g.This is as high doses of mycorrhiza added to already nutrientrich planting media have limited efficacy.This can be attributed to the limited efficacy of mycorrhiza in high quantities when added to planting media that are already nutrient-rich and fertile.Providing high levels of inoculants (mycorrhiza) reduces the uptake of P [9].The ability of mycorrhiza to infect roots and absorb dissolved P in the soil is thought to be related to competition, thereby leading to a decrease in P uptake when provided in high doses.Providing mycorrhiza at high doses can lead to a decrease in infection degree due to the high number of mycorrhiza in the roots of S platyclados, which causes competition for energy among species [6].
plants survived in the shaded environment except for that with a density of 70%, which had a survival rate of 90%.This demonstrates that wildings of S. platyclados can adapt to shaded conditions while living in full light, provided the shade level does not exceed 70%.According to Panjaitan et al. [10], providing a high-density shade, specifically 75%, can impede the growth of S. selanica.

Increased of height
Early growth in height was evaluated by observing an increase in height at 4 and 8 weeks of age.Anova analysis revealed significant differences between the application of different doses of mycorrhizal biofertilisation and different shading levels.Refer to Table 1 for details, and Figures 1 and 2 for the DMRT test results.The application of 7g of biofertilizer resulted in the greatest height increase at 4 weeks of age, and 9 g of biofertilizer at 8 weeks of age.However, the differences were not significant compared to doses of 2.5g and 5g of biofertilizer.At 4 weeks of age, the best growth was observed in 60% shade, with no significant difference from 0% and 50% shade, as shown in Figure 2. At 8 weeks of age, the best growth occurred in 0% and 60% shade.The mycorrhizal fertilizer used contains various mycorrhizal genera, including Glomus sp, Enterospora, Gigaspora, and Acaulospora, that can promote the growth of natural tiller height.The synergy between Glomus sp and Gigaspora can enhance the uptake of P elements, leading to faster cell division in the meristem tissue and resulting in a height increase in plants [11].The apical meristem, a tissue that supports plant height growth, generates new cells at the root or stem tip, resulting in increased plant height and length.This is because mycorrhiza-forming fungi can generate plant hormones like auxins, cytokinins, and gibberellins and vitamins that accelerate plant tissue growth [12].Shade density is linked to the intensity, quality, and duration of light radiation that plants receive to carry out photosynthesis, and it has a prominent impact on natural sapling height [12].Light intensity is also correlated with temperature and humidity; an increase in light intensity will elevate temperature and reduce relative humidity, thereby influencing the evaporation rate [13].

Increased of diameter
The early growth in diameter was evaluated by observing an increase in diameter at 4 and 8 weeks of age.After carrying out an Analysis of Variance (ANOVA), significant differences were found only at 8 weeks in both the application of various doses of mycorrhizal biofertilisation and various shading.Details are provided in Table 2, followed by DMRT test in Figure 3 and Figure 4. Figure 3 shows that the best diameter increase was observed at the age of 4 weeks with the application of 9 g and 5 g of biofertilizer, but there was no significant difference with 2.5 g and 7 g. Figure 4 shows that there was no significant difference at different shading levels observed at the age of both 4 weeks and 8 weeks.The average diameter growth response is similar across all four mycorrhiza treatment groups.Therefore, economically speaking, adding mycorrhiza with a dose of 5 g can promote the growth of wilding diameter.Adding mycorrhiza with a nutrient absorption rate of 5 grams is adequate to accelerate cambium growth.Mycorrhiza enhances not only P nutrient absorption but also macronutrient uptake including N and K. K plays a role in cell division activity and development of meristematic tissue in plants, resulting in stem enlargement.Mycorrhizae can also increase the necessary uptake of N nutrients in plants.According to Mardiantino et al. [14], better absorption of the N element leads to increased plant diameter.
Maintaining unshaded S. platyclados wildings promotes better diameter growth than under shade, although height growth is better under shade.This suggests that sufficient 5unlight intake is necessary for S. platyclados to achieve proper physiological processes during diameter growth.Shading plants affects metabolic processes within the plant body and ultimately impacts plant growth and production.This is particularly emphasized by the reduced light intensity received [15].The inhibition of plant diameter growth in 70% shade is also caused by the reduced light intensity which reduces hormone activity during the formation of meristematic cells towards stem diameter [16].

Seedling vigour
Seedling vigour is determined by calculating the ratio of seedling height to diameter, which was measured at 4 and 8 weeks in the nursery.According to reference Sudrajat et al. [17], a high seedling vigour ratio value indicates that the seedlings are relatively tall and thin, whereas a low ratio suggests less vigour in the seedlings.There was no significant difference found between the seedling vigour values measured at 4 weeks and 8 weeks, as determined by ANOVA.The seedling vigour values in this study ranged from 9 to 14.This indicates that the seedlings are still relatively thin and not yet fully vigorous.This is likely due to primary growth being more dominant than secondary growth, specifically in terms of diameter.According to Windyarini et al. [18] study, the seedling vigour of S. stenoptera and S. macrophylla was between 7-9.21 at the age of 10 months.BSN specifies the seedling vigour standard for Shorea spp as 11.25 [19].Stimulating the diameter growth is essential to attain this seedling vigour level.The study's findings revealed that the best diameter growth was observed under a lack of shade.These are consistent with [20,22], which argue that Shorea can thrive in shaded environments due to its gap-opportunist character.During the seedling phase, Shorea requires shade, but in the succeeding stage, it necessitates a higher light intensity to sustain growth [21,22].

Conclusion
In the nursery, the survival rate percentage of wildings S. platyclados was relatively high.The most significant height growth was observed at a dose of mycorrhizal biofertilizer of 5 g/polybag and 60% shade.The highest diameter growth was recorded at a concentration of mycorrhizal biofertilizer equal to 2.5 g/polybag and no significant differences on different shades.The vigour of seedlings ranged from 9 to 14 for all doses of mycorrhizal biofertilizer and various shades.In summary, applying 5 g/polybag of mycorrhizal biofertilizer with up to 60% shade is recommended.

Figure 3 .
Figure 3. Increased diameter growth of S. platyclados with various doses of mycorrhiza biofertilizer, 4 and 8 weeks in nursery (mm).

Figure 4 .
Figure 4. Increased diameter growth of S. platyclados in different shades, 4 and 8 weeks in nursery (mm).

Table 1 .
Anova of increased height at 4 weeks and 8 weeks.
Figure 1.Increased height growth of S. platyclados with various doses of mycorrhiza biofertilizer, 4 and 8 weeks in nursery (cm).Figure 2. Increased height growth of S. platyclados in different shades, 4 and 8 weeks in nursery (cm).

Table 2 .
Anova of increased diameter at 4 weeks and 8 weeks.