All microorganisms produce volatile organic compounds (VOCs) as part of their normal metabolism (Tilocca et al., 2020; Żuchowska and Filipiak, 2023), and it has been discovered that microbes interact often through the VOCs they release (Tirranen and Gitelson, 2006; Garbeva et al., 2014). The importance of VOCs emitted from the plant growth-promoting rhizobacteria (PGPR) and their contribution to the plant growth promotion and induction of systemic resistance (ISR) to pathogens has already been established (Ryu et al., 2003, 2004). Bacillus, Pseudomonas, Serratia and Stenotrophomonas are some of the bacterial genera that are known to produce VOCs affecting on the plant growth. Acetoin and 2,3-butanediol from Bacillus spp. are identified to enhance the plant growth (Ryu et al., 2003) while the biological functions of several other compounds, including short chain alcohols, aldehydes, acids, esters, ketones, alkanes, alkenes and hydrocarbons are yet to be elucidated (Kai et al., 2009). Such VOCs can act as info chemicals because they occur over a range of concentrations in the biosphere and function over distant spaces (Wheatley, 2002). The VOCs also function as signals, affecting rhizosphere microorganisms and indirectly influencing plant growth (Mhlongo et al., 2022). In addition to improving the plant growth, their ability to elicit induced systemic resistance (ISR) in plants indicates the potential contribution of VOCs toward the effect of PGPR on plants.

The Bacillus vallismortis strain EXTN-1 (EXTN-1) was initially isolated from red pepper and has been identified as a potential PGPR for growth promotion and ISR against a variety of pathogens in rice, tobacco, cucumber, tomato and potato (Park et al., 2001, 2006a; 2006b; Park et al., 2007). The ISR was verified by the up-regulation of defense-related gene expression and structural modifications in host plants, as well as the absence of direct antagonism in the antibiosis assay (Park et al., 2001; Ahn et al., 2002). This PGPR strain is capable of eliciting ISR against fungal, bacterial, and viral diseases through salicylic acid (SA), jasmonic acid (JA) and ethylene (ET)-mediated pathways (Park and Kloepper, 2000; Park et al., 2006b). The role of VOCs is yet to be investigated, despite the fact that various aspects of the mechanisms of interaction between EXTN-1 and the host plants have been investigated. The application of PGPR is significantly impeded by the inconsistency that arises under a variety of conditions. Consequently, it is imperative to examine all of the primary mechanisms and determine their appropriate application for agriculture purposes. VOCs are produced by microorganisms, animals, and plants and are distinguished by their high vapor pressure, hydrophobicity, and low molecular weight at ambient temperature. These compounds are capable of performing a variety of vital functions within and across diverse groups of living organisms due to their capacity to traverse porous soils and air without water as a medium (Schulz-Bohm et al., 2018; del Rosario-Cappellari et al., 2019; Suárez-Estrella et al., 2023).

In order to enhance seed germination, seedlings vigor, and to mitigate abiotic stress, a variety of seed priming techniques may be implemented (Nedunchezhiyan et al., 2020; Devika et al., 2021). In addition to these advantages, the biopriming method offers the additional benefit of managing biotic stress. The term “biopriming” typically denotes the utilization of beneficial microorganisms, in particular plant growth-promoting bacteria (PGPB), which are capable of surviving under a variety of challenging environmental conditions (Fiodor et al., 2023). A previous study demonstrated that the use of microbial inoculants containing plant growth-promoting bacteria (PGPB) can increase crop production by 12–20% (Pérez-Jaramillo et al., 2016). Additionally, the biopriming of seeds with a variety of beneficial bacteria, particularly PGPB, can enhance the plant’s resilience and effectiveness in adverse conditions (Chakraborti et al., 2021). The concept of priming plants with PGPR to promote growth and induce eventual defense responses on pathogen attack has been an interesting topic of research (Kandasamy et al., 2009).

The majority of VOCs investigations are conducted under controlled conditions, with a recent shift in focus to field applications. However, several parameters, including soil nutrient status, must be analyzed to produce consistent results in the field environment. Therefore, the current investigation was conducted to evaluate the significance of bacterial culture media and the impact of VOCs emitted by EXTN-1 in the I-plate assay. We also endeavored to investigate the impact of priming seeds with VOC on growth promotion under greenhouse conditions, as the mode of application of VOC for growth enhancement is a critical factor in commercial success. The primary compounds responsible for the effect on plant growth were identified through chemical analysis of VOCs from EXTN-1 in various media. Additionally, some lesser-known VOCs were identified as potential plant growth promoters.

Previous research has explored the significance of VOCs in the context of ISR and plant growth (Ryu et al., 2004; Tahir et al., 2017). Differential VOC emission from rhizosphere bacteria has the potential to influence the architecture of the root system and promote plant growth (Almeida et al., 2023). Our research suggests that VOCs are essential for the beneficial impact of EXTN-1 on plants; however, the impact on plant growth is contingent upon the bacterial culture media. It has been reported that the number and spectrum of volatiles produced by bacteria are dependent upon the availability of nutrients and the growth conditions (Weise et al., 2012; Garbeva et al., 2014; Rani et al., 2023). In sugar-enriched media, the VOCs from EXTN-1 promoted plant growth, whereas in nitrogenous media, VOCs inhibited plant growth by causing leaf chlorosis. This is a consequence of the alkalinization of the plant medium by ammonia. Previous research has demonstrated that ammonia is a significant bioactive compound in bacterial volatiles, which is responsible for significant growth inhibition of plants (Weise et al., 2013). It has been reported that a rapid influx of NH₃ into plant cells occurred when the external pH was high. This phenomenon may result in a variety of ammonia-induced changes in the cells, such as reduced growth and chlorosis (Plieth et al., 2000; Liu and von Wirén, 2017). Soil pH changes can have a significant impact on iron uptake by hosts since niche pH affects this process (Zhang et al., 2009). Bacterial VOCs may play a pivotal role in dictating the dynamics of rhizosphere microbial community; given that soil pH is critical for microbiome composition (Lakshmanan et al., 2014). A recent study found that the growth of plants was indirectly influenced by the alteration of the rhizosphere microbiome by PGPR and VOCs such as indole (Yaghoubi Khanghahi et al., 2024). However, the negative effect was not observed when the ammonia was confined with phosphoric acid. Conversely, in the absence of ammonia, VOCs from EXTN-1 had a beneficial effect on exposed plants, irrespective of the culture media. This is an important aspect of the investigation, as the fundamental experiment utilizes the I-plate assay to evaluate the effect of VOCs from PGPR on plant growth. The outcome may be either positive or negative, as the growth media plays a significant role in the VOCs emitted by bacteria. This may result in the elimination of certain potential bacterial sources of VOC based on an experiment that utilizes only one media. In certain instances, the impact of beneficial VOCs may be obscured by other volatiles, such as ammonia in this case. The potency of bacterial volatiles necessitates a comprehensive investigation. In this study we have selected some of the most frequently used media for PGPR strains in the laboratory, particularly in the I-plate assay to identify VOCs such as TSA, NA and LB. Additionally, we have included media such as PDA, which is used for antifungal analysis. Because VOCs differ between culture media, they may alter laboratory assays used to characterize the PGPR strain. Furthermore, soil nutrient level may alter the production of VOCs, determining the ultimate outcome of PGPR strain application under field conditions.

When seedlings were transferred to greenhouse after 10 days of exposure, their growth consistently increased compared to the control group. The results of this study show that initial exposure is an important and sufficient to carry on the positive effect. When VOCs were used to prime seeds for 24 and 48 h, the result was the same with faster germination of seeds as compared to the control. When it came to fresh and dry weight, seedlings that were exposed for 48 h did better than seeds that were exposed for 24 h. When EXTN-1 was grown in NA and TSA, the negative effect observed in I-plates in seedlings exposed to VOCs was not observed in seed-primed plants under greenhouse conditions. There was a statistically significant increase in growth, in comparison to the control group. The leaves were in good health and did not exhibit any symptoms of chlorosis. Our research indicates that the growth of plants is facilitated by the short-term priming of them with VOCs, despite the fact that additional research is required to confirm this effect. This outcome is crucial for the practical implementation of VOCs in field conditions. Seed-priming with live cells of PGPR is a well-established phenomenon. It has also been reported that the treatment of roots with suspensions of VOC from PGPR results in induced resistance against biotic stress (Farag et al., 2013). However, this is the initial report of seed priming with VOCs, to the best of our knowledge. Further analysis of seed exposure at various time points, as well as the concentration of VOCs required for priming, will identify the threshold level.

The chemical profile of VOCs from EXTN-1 demonstrated the emission of several bioactive compounds, including previously reported growth-modulating VOCs such as acetoin, 1-butanol, and 2,3-butanedione (Ryu et al., 2003, 2004; Scala et al., 2013). Acetoin was the most prominent peak in all media (excluding NA), and it can be inferred that it is the growth-promoting VOC from EXTN-1 in these media. The growth improvement in plants was significant in KB plates, despite the fact that EXTN-1 produced a similar quantity of ammonia in KB and NA media. Conversely, the growth in NA plates was reduced. One potential explanation is the duration of time required to alkalize the medium. The pH of the plant medium in KB increases to 6 over an 8-day period when exposed to EXTN-1. However, in NA, the pH reached 7 on the 3rd day (detailed data not shown). The pH increases abruptly on the 2nd day for TSA and LB. Normal growth is not impeded in KB plates, as the plants are provided with a suitable medium for growth for a period of 8 days, Acetoin contributes to the growth improvement.

The peak for acetoin was not detected in the VOCs of EXTN-1 grown in NA. However, upon exposure to plants, the VOCs increased plant growth in the I-plate assay once the ammonia was contained (Figure 1). The primary peak in NA and KB media has been identified as 1-butanol. Acetoin and 2,3-butanediol were not detected in NA media, so we tested some of the other compounds identified in the spectrum to isolate the VOC related to growth promotion. The peaks were highest in NA and TSA media, with the exception of benzaldehyde, dimethyl sulfide and heneicosane that were exclusively detected when EXTN-1 was grown in NA medium. Benzaldehyde has been identified as a microbial VOC that inhibits plant growth (Kanchiswamy et al., 2015). Arabidopsis was differentially affected by the emission of benzaldehyde by lemon rhizobacteria (Guitierrez-Luna et al., 2010). Therefore, further in-depth analysis of VOC concentration is required. Dimethyl disulfide has been identified as a catabolic product of the sulfur-containing amino acids methionine and cysteine. It has been reported to promote growth in Nicotiana attenuata by enhancing sulfur nutrition (Meldau et al., 2013), the same effect is not observed in our experiment. With the exception of the 50 ng concentration, which only slightly increased fresh weight of tobacco in comparison to the control, the other concentrations did not have positive impact on the plant growth.

Other research has suggested that heneicosane may be a contributing factor to the antimicrobial properties of the sea urchin Temnopleurus alexandri and the medicinal plant Pulicaria crispa (Elshiekh and Mona, 2015). Currently, there is no report available regarding the possible enhancement of plant growth by heneicosane as a VOC from PGPR. Our in vitro findings imply that heneicosane is a promising VOC that promotes tobacco growth. The issue would be to identify the concentration needed to provide a similar growth-promoting effect across diverse plant species and under field conditions. Several long-chain decanes were detected in the VOCs of EXTN-1, including hexadecane, tertadecane, and tridecane. Hexadecane and tridecane from Paenibacillus polymyxa were reported earlier to be responsible for ISR in Arabidopsis against Pectobacterium carotovorum and Pseudomonas syringae (Lee et al., 2012; Park et al., 2013). The positive function of long-chain decanes is suggested by the fact that tetradecane increased the fresh weight of plants at low concentrations in this study. The results of this investigation suggested that the growth of plants was influenced by the concentration of VOC that was administered. The growth promoting effect is dose-dependent, and the quantity of VOC produced by PGPR is a determining factor in the resultant effect of plants, as indicated by a similar earlier report (Bailly and Weisskopf, 2012; Bitas et al., 2013).

In conclusion, our research demonstrates the significance of VOCs emitted by EXTN-1 in the promotion of tobacco growth through the short-term priming of seeds or seedlings, which can be exploited for field application. It has been observed that the majority of the VOCs released by EXTN-1, including acetoin, carbon dioxide, long-chain decanes, benzaldehyde, and dimethyl disulfide, have the ability to promote plant growth in various conditions. Another important factor in deciding the effect of on plant growth is the concentration of VOC. With the identification of heneicosane as one of the promising VOCs that contribute plant growth, further analysis is needed to optimize the concentration and conditions required. Consequently, the results obtained under controlled conditions may not precisely reflect the actual picture of the impact of VOCs from PGPR in the field. However, factors such as the influence on competing microbial communities and the impact on the environment must be thoroughly investigated prior to practical application in the field. Further research is required in several areas, including the optimal concentration of VOCs, effects on plant physiology, and consistency in different crops. Nevertheless, the present results can be taken as basic analysis for further detailed experiments that are essential for understanding the diverse factors involved in the mechanisms during the interaction of PGPR and/or VOCs with host plants.