Evaluation of the application of rice husk biochar and various types of microbes on physiological and characteristics of shallot bulbs (Allium ascalonicum L.)

Shallot is a popular and widely developed horticultural crop in Indonesia. This study aims to evaluate the application of rice husk biochar and various types of microbes on the physiological and bulbs characteristics of shallot (Allium ascalonicum L.). The research design used a split plot design. The main plot was rice husk biochar with three levels, namely control 0 t ha−1, 2 t ha−1, and 4 t ha−1. As subplots, there were three types of microbes namely without microbes/control, Trichoderma asperellum, Beauveria bassiana, and Metarhizium anisopliae. The results optained that there was an interaction between the of rice husk biochar 4 t ha−1 with the Trichoderma asperellum which gave the best results for the chlorophyll index (19.70). The application of various types of microbes namely in the of Trichoderma asperellum gave the best results on bulb height (2.75 cm) and the number of bulbs per plant (1.61).


Introduction
Shallot is a spice vegetable that is in great demand and cultivated in Indonesia.This spice vegetable has many advantages and high selling value.Shallots are usually much needed as a spice in cooking and traditional medicine.The demand and need for shallots are increasing from year to year due to population growth and diversification of needs, as well as the people's large purchasing power.
Harvested area of 159,195 ha in 2019 and grew 17.4% to 186,900 ha in 2020.The increase in shallot harvested area in production was followed by a significant increase, but productivity decreased.As a result, national shallot production in 2019 was 1,580,247 tons with a productivity of 9.93 t ha -1 .In 2020, production will increase to 1,815,445 tonnes with a productivity of 9.71 t ha -1 [1].This shows that shallot production increases with the harvested area, but productivity does not increase significantly.
One of the problems in shallot cultivation is the excessive use of chemical inputs.Now, produce shallots with high yields generally rely heavily on inorganic fertilizers.This impacts the environment, especially soil conditions, and microorganisms in it.According to Hawayanti and Palmasari [2], inorganic fertilizers cause many environmental damage problems and decreased agricultural land productivity [3].This condition causes shallot plants not to grow optimally and impacts the physiological state of plants and shallot bulbs.Increasing organic matter content is a way to increase soil fertility.Adding organic matter in the form of rice husk biochar is an alternative to reducing the use of inorganic fertilizers.Rice husk biochar is one of the uses of agricultural waste that can increase soil fertility.According to Lehmann et al., Widyantika and Prijono [4,5], the residue from processing grains such as rice husks can be used to make biochar.Bearing in mind that rice husk waste is not maximized, for now, husk as rice milling waste accounts for 20-23% of the grain.Therefore, biochar is one of the main parts of agricultural waste management, and biochar can increase soil fertility and shallot growth [6].With the use of rice husk biochar as an organic material and soil conditioner so that it can stimulate physiological conditions and shallot bulbs.
In addition to the addition of organic matter, various types of microbes are widely used to help plant growth.For example, besides acting as a biological control agent antagonistic to fusarium wilt [7], Trichoderma asperellum can also stimulate physiological mechanisms plant [8].Besides that, apart from being used as pest control, Beauveria bassiana and Metarhizium anisopliae can stimulate physiological mechanisms that enhance plant nutrition and growth [9,10].
Therefore, the combination of rice husk biochar and various types of microbes is intended to can increase organic matter source and land repairer so that shallot can grow optimally.The presence of rice husk biochar and various types of microbes is expected to influence the physiological conditions and characteristics of shallot bulbs.

Location and research design
The research was helded at Experimental Farm, Faculty of Agriculture, Hasanuddin University, Makassar.The research was helded from July to November 2022.The tool used in this research was the CCM-200 plus tool and microscope.The materials used in this study were the Maserati variety of shallot seeds, rice husk biochar, and various types of microbes (Trichoderma asperellum, Beauveria bassiana, and Metarhizium anisopliae).The research design uses a Split Plot Design.The main plot was rice husk biochar with three levels: without biochar/control (b 0 ) = 0 t ha -1 , b 1 = 2 t ha -1 , and b 2 = 4 t ha -1 .As subplots, there were three types of microbes: without microbes/control = m 0 , m 1 = Trichoderma asperellum, m 2 = Beauveria bassiana, and m 3 = Metarhizium anisopliae.There were 12 treatment combinations with three replications to produce 36 experimental plot units.

Research implementation 2.2.1. Biochar preparation.
Making rice husk biochar begins with putting rice husk waste into the combustion furnace.The combustion process is carried out in a pyrolysis furnace (incomplete combustion where oxygen in the air does not participate in the combustion reaction).Rice husk burning is carried out in a tightly closed furnace to reduce oxygen availability during combustion at 300°C for 3-4 hours.After the material is entirely black, the fire under the stove is extinguished and allowed to stand until the temperature of the biochar decreases.Furthermore, the biochar is removed from the combustion furnace and ready to be applied according to the treatment dose.

Microbes preparation.
The technique described by Siahaan et al. [11] was used to cultivate Trichoderma asperellum, Beauveria bassiana, and Metarhizium anisopliae microbial strains.Microbial isolates were grown on a petri dish containing potato dextrose agar (PDA).Following 14 days of inoculation (DAI), the microbial isolates were multiplied on rice media to attain the maximum yield of spores.Rice is a growing medium used to reproduce microbial isolates.As a candidate for growing media, rice was previously washed using water until clean.Afterwards, the rice is immersed in water for a duration of two hours.After soaking, put it in a plastic bag and tie it.The rice substrate was disinfected in an autoclave at 121°C for 30 minutes and subsequently cooled.Subsequently, each plastic pouch containing the rice substrate was seeded with a fungal solution on 100 g of the medium.

Observation parameters
The variables examined in this investigation included chlorophyll index, stomatal opening area (μm 2 ), bulb height (cm), and the number of bulbs per plant.The examination of observational data was conducted through the utilization of analysis of variance.In the event that the outcomes display a noteworthy impact, an additional test known as Least Significant Difference (LSD) is executed with a significance level of α 0.05.The entire procedure of analyzing data was executed utilizing the Microsoft Excel 2019 application.

Results
The results optained a significant interaction between the treatment of rice husk biochar with various types of microbes on the chlorophyll index.Rice husk biochar treatment had no significant effect on stomata opening area, bulb height, and the number of bulbs.Whereas the treatment of various types of microbes, namely the treatment of Trichoderma asperellum had a significant effect on bulb height and the number of bulbs per plant but had no significant effect on the area of stomatal openings.

Clorophyll index.
Observing the average chlorophyll index of shallot plants and analysis of variance optained a significant interaction between the treatment of rice husk biochar with various types of microbes on the chlorophyll index of shallot plants.The average chlorophyll index of shallot plants is presented in Table 1.
The LSD test at 0.05% level in Table 1 shows that the average chlorophyll index of shallot plants was highest in the rice husk biochar 4 t ha -1 with Trichoderma asperellum, namely 19.70.In comparison, the lowest average chlorophyll index was in the treatment without microbes and the application of rice husk biochar, namely 8.73.  2 ).The analysis of variance optained no interaction between the rice husk biochar with various types of microbes in the area of stomatal openings.Individually, the treatment of rice husk biochar and various types of microbes had no significant effect on the stomatal opening area of shallot plants.The average stomatal opening area of shallot plants treated with rice husk biochar and various microbes is presented in Figure 1.Based on data on the average stomatal opening area of shallot plants in Figure 1, it shows that the treatment of rice husk biochar 4 t ha -1 with Trichoderma asperellum had the largest average stomatal opening area of 756.74 μm 2 .While the treatment without biochar of rice husk and the treatment without microbes had the smallest average stomatal opening area of shallot plants, namely 330.75 μm 2 .

Bulb height (cm).
The results of the analysis of variance optained that there was no interaction between the treatment of rice husk biochar with various types of microbes on bulb height.Individually, the treatment of rice husk biochar had no significant effect, while the treatment of various types of microbes significantly affected the height of shallot bulbs.The results of observing the average height of shallot bulbs treated with rice husk biochar and various microbes are presented in Table 2.
Based on the LSD test at the 0.05% level in Table 2, it shows that the highest average shallot bulb height was in the Trichoderma asperellum treatment (2.75 cm), which was not significantly different from Beauveria bassiana treatment, but significantly different from Metarhizium anisopliae treatment and the without microbes.On the other hand, the lowest average bulb height was in the treatment without microbes (2.59 cm).Note: Means with the same letter are not statistically different for a significance level of p ≤ 0.05 as per the LSD multiple comparison test.

Number of bulbs per plant.
The analysis of variance optained no interaction between the treatment of rice husk biochar and various types of microbes on the number of bulbs per plant.Individually, the treatment of rice husk biochar had no significant effect.In contrast, the treatment of various types of microbes had a significant effect on the number of bulbs per shallot plant.The results of observing the average number of bulbs per shallot plant treated with rice husk biochar and various microbes are presented in Table 3.
Based on the LSD test at the 0.05% level in Table 3, it shows that the highest average number of bulbs per shallot plant was Trichoderma asperellum treatment (1.61), which was not significantly different from the Beauveria bassiana and Metarhizium anisopliae treatments, but significantly different from the without microbes.On the other hand, the lowest average number of bulbs per plant was the treatment without microbes (1.42).

Table 3. Number of bulbs per plant.
Note: Means with the same letter are not statistically different for a significance level of p ≤ 0.05 as per the LSD multiple comparison test.

Discussion
The results showed an interaction between the treatment of rice husk biochar 4 t ha -1 with Trichoderma asperellum, which was the best on the chlorophyll index of shallots.This proves that the combination of rice husk biochar with Trichoderma asperellum can increase the physiological response, especially on the chlorophyll index of shallot plants.The increase in chlorophyll index is closely related to the role of biochar which can maintain the stability of the N element in the soil so that it is not leached and remains available for plants.This is by what was stated by Nguyen et al. [12], that biochar has a high-water holding capacity so that the N nutrient is maintained and not leached, making it more available to plants.Utilizing of rice husk biochar could improve the physicochemical properties of the soil and increase plant growth [13].This is to what was stated by Abukari [14] that adding rice husk biochar 4 t ha -1 into the soil can enhance its water retention ability owing to the abundance of micro and macro pores, resulting in a sturdy and efficient capillary force.Moisture readiness and nutrient retention capacity are essential for optimal plant growth.Furthermore, biochar has the ability to enhance soil dampness and pH levels, which in turn can encourage the processes of N mineralization and nitrification, leading to a boost in plant absorption.The same thing was conveyed by Karam et al. [15], rice husk biochar can function as a soil conditioner because it can absorb and retain nutrients so that it can increase soil fertility.One of the roles of the element N is as an essential macronutrient for plants which functions as a constituent of green leaf matter (chlorophyll) [16].In addition, the findings of Tchameni et al. [17] reported that adding Trichoderma asperellum could increase the rate of chlorophyll, P uptake, and acid phosphatase activity.Therefore, the application of rice husk biochar and Trichoderma asperellum increase the chlorophyll index in shallot leaves.
The application Trichoderma asperellum significantly affected bulb height and the number of bulbs per plant but had no significant effect on the stomata opening area.One of the roles of Trichoderma asperellum is its ability to dissolve phosphorus that are still bound in the soil [18].Microbes that have the ability to solubilize phosphorus can release phosphatase enzymes that aid in breaking down organic P into inorganic P [19,20].Additionally, phosphorus is known to stimulate root growth, which results in the formation of a robust root system that is beneficial for plants [21].With the ability of the Trichoderma asperellum related to the availability and process of absorption of these nutrients so that plants can absorb the elements optimally.This indicated that the Trichoderma asperellum was able to increase bulb height and the number of bulbs per shallot plant.

Conclusion
Based on the results of the study, it can be concluded that there was an interaction between the of rice husk biochar 4 t ha -1 with the Trichoderma asperellum which gave the best results for the chlorophyll index (19.70).The application of various types of microbes namely in the of Trichoderma asperellum gave the best results on bulb height (2.75 cm) and the number of bulbs per plant (1.61).

Acknowlegment
The investigator expresses gratitude to Lembaga Penelitian dan Pengabdian Masyarakat (LPPM), Hasanuddin University, for the economic backing provided in carrying out this investigation.

Figure 1 .
Figure 1.Average stomatal opening area of shallot plants.