EFFECT OF BACTERIAL VOLATILE COMPOUNDS ON PAKCOY (Brassica rapa L.) GROWTH PROMOTION

This research was aimed to test of the ability of MVOC-producing bacteria to increase plant growth of Pakcoy ( Brassica rapa L .). The methodology including testing the ability of MVOC-producing rhizobacteria in Pakcoy plant growth enhancement, MVOC extraction and analysis of compounds in MVOC Extracts using Gas Chromatography. Different bacterial species produce different MVOC . S. maltophilia Sg3 emitted 20 MVOC compounds and MVOC that contribute to increasing plant growth, namely oxalic acid, cyclohexyl undecyl ester, 2-Furancarboxaldehyde, 5- methyl-, 1,2 butanediol, and Piperazine. E. asburiae MjSg48 emitted 12 MVOC compounds and those that contributed to increasing plant growth were oxalic acid, cyclohexyl dodecyl ester and 4-methyl oxazole. E. asburiae TK24 emitted 27 MVOC compounds and those that contributed to increasing plant growth were oxalic acid, isohexyl neopentyl ester, thiazole, Oxalic acid, and cyclohexyl decyl ester. Meanwhile P. rettgeri Al2TT emitted 13 MVOC compounds and those that contributed to increasing plant growth were oxalic acid, diisohexyl ester, and Pyridine, 2,3,4,5-tetrahydro.


INTODUCTION
Rhizobacteria is bacteria that utilize plant root exudate to live as a colony in rhizosphere area.The metabolite products that released by this rhizobacteria is a chemical compound which easily evaporate or frequently categorized as an organic compound with a volatile property that produced by microbes or Microbial volatile organic compound (MVOC) (Kanchiswamy, et al., 2015, Cappellari, et al., 2020).The microbes other than rhizobacteria, such us yeast dan fungi also can produce MVOC.The research on the ability of microbes in producing organic compounds which easily evaporate has been published in many journal (Ramirez, et al., 2010, Schmidt, et al., 2015, Gupta, et al., 2017) .The fresh smells from the pure culture of microbes like yeast and citric or lactic acid bacteria, and the strong smells from various fermented products are a simple proof the existence of compounds from organic acid group as citric acid, acetic acid, lactic acid, propionate, alcohol, ester, mercaptan, pentylfuran and others which are metabolic product of microbes.However, scientific evidence on MVOC activity as a growth inducer and plant resistance was only published in 2003 (Ryu et al. 2003;Ryu et al. 2004).Ryu et al. (2004) reported that Bacillus sp.strain GB03 which emitted 2,3 butanediol and acetoin compounds was able to induce the growth of Arabidopsis thaliana plants when compared to water control and treatment with Escherichia coli strain DH5α.
Since then, the role of the MVOC compounds group for plant growth inducers has received a lot of attention and has attracted the interest of other researchers to explore and study its potential development and applications.
This research is aimed to aimed to test of the ability of MVOC-producing bacteria to increase plant growth of Pakcoy (Brassica rapa L.).Pakcoy is a type of leafy vegetable crops that are very important in Indonesia because it has a high economic value.Pakcoy much in demand as a vegetable because of high nutrient content and it tastes good (Gustiar, et al., 2022).

MATERIALS AND METHODS
The research was conducted in

MVOC Extraction
MVOC was extracted using ethyl acetate, hexane, and methanol.A total of 50 ml of the culture filtrate was centrifuged at 1.610 x g for 30 minutes, the supernatant was separated from the bacterial precipitate, filtered with Millipore filter paper (0.45 mm).
Then it was partitioned with ethyl

Analysis of Compounds in MVOC
Extracts using Gas Chromatography -

Mass Spectroscopy (GC-MS)
The Analysis of compounds that contained in the rhizobacterial filtrate was carried out using Gas Chromatography -

Results
The results of the test of the ability of MVOC-producing bacteria to increase plant growth showed that the MVOC emitted by S.   showed that there were 20 compounds detected shows in Figure 3.    GB03 which emits compounds 3-hydroxy-2butanone, 2,3-butanediol, decanal, dean, tetramethyl pyrazine, and undecane which can increase the growth of A. thaliana (Ryu, 2004, Akhdiya, 2014, Ditengou, et al., 2015).
The results of VOC trapping of B. subtilis G8 which was carried out using SPME fibers and followed by identification using GC-MS  Blom, D., Fabbri, C., Connor, E., Schiestl, F., Klauser, D., Boller, T., et al. (2011).Production of plant growth modulating volatiles is widespread among stated that Acyl homoserine lactone (AHL) is a compound that is emitted by bacterial cells to regulate gene expression when the population has reached a sufficient level of cell density and intermediate chain AHL compounds (C8-C14) such as N-hexanoylhomoserine. lactone, N-3-oxohexanoyl-homoserine lactone, N-octanoylhomoserine lactone, N-decanoyl-homoserine lactone, N-dodecanone-homoserine lactone, and N-tetradecanoylhomoserine lactone can be recognized by plants and are able to change gene expression in plants roots and shoots so that they are able to modulate plant cell growth and medium chain AHL compounds can change root architecture, alter primary root growth, stimulate lateral root formation, and root hairs development in A. thaliana plants.MVOC emitted by Alternaria alternata can trigger an escalation in starch accumulation in potato leaves accompanied by upregulation of sucrose synthase, invertase inhibitors, and starch synthase classes III and IV, glucose-6phosphate transporter, plastidial thioredoxin enzyme, starch breakdown enzymes, and proteins involved in the provision of internal amino acids.This phenomenon is called MVOC-ISAP or MVOC-induced starch accumulation process Octoer until December 2021 in Laboratory of Plant Pests and Diseases, Faculty of Agriculture, Udayana University and in the greenhouse in the Experimental Garden of the Faculty of Agriculture, Udayana University, Bali Indonesia.Figure 1 shows the map of the research location.

Figure 1 .
Figure 1.The Map of the Research Location.
acetate and methanol three times.MVOC compounds can be extracted by partitioning the supernatant using ethyl acetate.The ethyl acetate fraction was then separated from the methanol fraction, then the three ethyl acetate fractions were combined into the ethyl acetate fraction.Furthermore, the ethyl acetate fraction was evaporated using a Vacuum Rotary Evaporator at a temperature of 40 o C. The extract or ethyl acetate fraction was then dissolved with 5 ml of methanol and stored in a refrigerator with minus 20 o C temperature.

Table 1 .
The effect of MVOC on Plant Height, Root Fresh Weight, and Root Dry Weight of Pakcoy Plants.

Table 2 .
The effect of MVOC on Leaf Chlorophyll Content, Plant Fresh Weight, and Plant Dry Weight of Pakcoy Plant.