Quantification of FEND and ITUDI Anti-fungal Lipopeptide Gene Expression in Bacillus megaterium using RT-qPCR

Phytopathogenic diseases are a major concern in modern agriculture, and for decades, pesticides have been used to prevent potential damage. Bacillus megaterium is proposed as a biological controlling agent, and gene expression of the lipopeptide genes FEND and ITUDI was assessed using RT-qPCR. Inhibition effects of B. megaterium on Alternaria sp. and Botrytis sp. were examined over a period of nine days, which confirmed the potential use of this bacterium to counteract these two pathogens. In addition, expression of FEND and ITUDI genes was assessed over nine days in the aforementioned dual cultures and inhibition tests. FEND expression in B. megaterium increased 20.16-fold in response to Alternaria sp., and ITUDI expression increased 3.20-fold in response to Botrytis sp. on day five of incubation. These results were corroborated by gene expression data obtained from B. megaterium during fermentation, where FEND and ITUDI gene expression increased 95.14and 18.70-fold, respectively. In conclusion, B. megaterium can increase lipopeptide synthesis when exposed to these particular phytopathogens and can significantly increase the respective expression during fermentation.

understand the underlying mechanisms and to design targeted studies on the efficiency of biological controllers [8][9][10] .
B. megaterium has been shown to contain antifungal genes, which can inhibit growth of Alternaria sp. and Botrytis sp. 11 ; however, it is important to examine the respective expression patterns and to assess how they change over time and under certain conditions so as to determine the factors that induce expression and thereby increase the pathogen-controlling potential. Therefore, using RT-qPCR, we aimed to assess expression of FEND and ITUDI antifungal lipopeptide genes in B. megaterium in response to Alternaria sp. and Botrytis sp. and during fermentation.

Inoculum preparation
Microbes that had been identified using molecular genetic means as B. megaterium, Alternaria sp. and Botrytis sp. were used in this study. The cultures, cryopreserved at -80°C, were obtained from the Laboratories of Life Sciences of the Universidad Politécnica Salesiana, Quito, Ecuador 12 . First, a culture medium was prepared using 22 g of Nutritional Agar and 1 L distilled water, in which the bacteria were incubated at 36°C. For fungal cultures, potato dextrose agar (PDA) culture medium was prepared at the same concentration, which was then used for incubation at 26°C.

Dual culture assays to test antagonistic activity
In order to test inhibitory effects of B. megaterium on Alternaria sp. and Botrytis sp., a dual culture method was used, in which each pathogenic fungus was exposed to B. megaterium in PDA culture medium where the fungus was placed at the center of the box, and the battery was inoculated through a 3-mm striation between bacteria and fungus. Incubation was continued at 36°C for nine days 13,14 . Genetic material and data for calculating the proportion of inhibition were obtained after this step. Data were collected on days 1, 5, and 9. Bacteria cultured without fungi were used as a control. The proportion of radial growth inhibition was calculated using the following equation 15 : Where R1 is the diameter of radial fungus growth and R2 is the diameter of radial growth of fungus exposed to B. megaterium.

Liquid Fermentation Preparation of the inoculum and fermentation
Nutritional Agar medium was used to reactivate cryopreserved B. megaterium for incubation at 35°C for 24 h. Next, the bacterial concentration was recorded as a starting point. For this purpose, a suspension was prepared from normal saline that was set to a standard of 0.5 using spectrophotometry and the McFarland scale, thus representing a concentration of 10 5 CFU mL -1 . After preparing the initial inocula, the fermentation process was initiated, starting with the preparation of 100 mL bacterial suspension (10 5 CFU mL -1 ) per Erlenmeyer flask using Triptocasa Soja Broth as the liquid culture medium. B. megaterium was incubated at 25°C under agitation at 100 rpm for 120 h. RNA extraction Extraction and quantification of Bacillus megaterium RNA from dual cultures RNA was extracted from dual cultures of Botrytis sp. and Alternaria sp. exposed to B. megaterium on days 1, 5, and 9 using the PureLink RNA Mini-Kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, RNA was quantified in µg/µL using a Qubit RNA BR Assay Kit (Thermo Fisher Scientific). Extraction and quantification of RNA from Bacillus megaterium in fermentation RNA was extracted from bacterial cultures fermented in broth for 24, 48, 72, and 120 h, as specified above.

Reverse transcription and quantification
A Superscript® III First-Strand Synthesis SuperMix for RT-qPCR (Thermo Fisher Scientific) kit was used to obtain cDNA from the RNA templates, according to the manufacturer's instructions. Briefly, 5 µg/µL RNA were used, and the reaction was incubated in a thermal cycler at 25°C for 10 min, 50°C for 30 min, and 85°C for 5 min before cooling to 4°C. Then, 1 µL E. coli RNase H was   added, and the reaction mixture was incubated at 37°C for 20 min. cDNA was quantified using a Qubit ssDNA Assay Kit (Thermo Fisher Scientific).
A LightCycler 2.0 (Roche, Basel, Switzerland) and 2 µg/µL cDNA were used to perform RT-qPCR with primers and Fast Sybr Green Master Mix (Thermo Fisher Scientific) at a final volume of 20 µL, according to the manufacturer's instructions. Thermocycling was performed as follows: 95°C for 20 s, followed by 40 cycles of 95°C for 3 s, 60°C for 30 s, and 72°C for 30 s, after which a melting curve was generated at 95°C for 0 s, 65°C for 1 min, and 95°C for 0 s. The reaction was stopped by cooling to 4°C. To calculate expression of FEND and ITUDI, the initial concentration of gene copies (N0) was required, including the respective efficiencies (E) of each reaction, which was computed using LinRegPCR version 11.0, 16 which uses the 2 −∆∆ct method 17,18 . Data were standardized with the 16S housekeeping gene as a reference, according to the following equation 19 : N 0 (normalizado)= N 0 gen problema N 0 gen normalizador Gene expression was calculated from standardized data by comparing all treatments with the control, which in this case was cultivation of B. megaterium without fungi, using the following equation 19 : Relative expression level= N 0 gene day n N 0 gene day 1

Statistical analyses
FEND and ITUDI gene expression in B. megaterium were considered the response variables, and presence of the phytopathogenic fungi Botrytis sp. and Alternaria sp. and incubation time (1, 5, or 9 days) were used as independent variables. A completely randomized design was used for a 3 X 3 factorial analysis. The 27 experimental units were categorized according to incubation time and growth using three replicates of exposure of bacteria to each pathogen, plus the respective controls. Gene expression data obtained from dual cultures were compared with data collected at 24, 48, 72, and 120 h of bacterial fermentation under optimal growth conditions. For this, an analysis of variance (ANOVA) was performed, followed by Tukey's post hoc test at 5%, using the statistical software, Infostat (2018).

RESULTS AND DISCUSSIoN Radial growth inhibition
ANOVA showed a highly significant difference for both growth and incubation time (p < 0.0001), and the interaction between these variables was significant (p = 0.0395). Therefore, radial growth inhibition differed between the two fungi and depended on incubation time. Tukey's test (Fig. 1) showed three ranges of significance, A, B, and C, with range C representing antagonism of Alternaria sp. on day 5 of incubation with an average inhibition of 76.45% and day 9, with inhibition averaging 98.48%, cultures that presented the highest inhibition, which indicated

FEND gene expression in B. megaterium
When B. megaterium was not challenged with pathogenic fungi, no increase in relative expression of FEND by more than 2 was observed, which was not considered significant. Lengyel (2018) 24 reports that molecules involved in gene expression are an inevitable source of chance because they are found under certain conditions in the cells, which causes proteins to be produced randomly and at minimal quantities, which is termed as the "noise" of expression. However, when B. megaterium was exposed to Alternaria sp., a significant increase in gene expression was evident on day 5 of incubation. ANOVA showed a highly significant difference in growth between pathogens and incubation times, and the interaction between the two variable was also significant (p < 0.0001, each); thus, relative expression of FEND depended on the fungus and time of incubation.
In Fig. 2, according to Tukey's test, four ranges of significance are presented: A, B, C, and D; within range A, the test presented the lowest expression, and range D includes the effect of Alternaria sp. on B. megaterium on day 5, which showed the greatest increase in FEND expression since day 1. FEND expression was down-regulated over time in the control. A similar phenomenon was observed when B. megaterium was exposed to Botrytis sp.
The results of the current study showed that B. megaterium may be a promising biocontroller of Alternaria sp. as it showed upregulation of FEND expression when challenged with this phytopathogenic fungus. Knight et al. (2018) reported that B. subtilis, a species very similar to B. megaterium, is an effective controller of the phytopathogens, Alternaria sp. and Fusarium sp., with upregulated FEND expression on day 3 of incubation. A different study using in vitro assays found that B. subtilis inhibited the growth of Alternaria sp. and Fusarium sp., and on day 4 of incubation confirmed the presence of genes such as ituA, bamC, and sfp which encode iturine A, bacilomycin D, and surfactin, respectively 26 .

ITUDI gene expression in B. megaterium
No significant changes in ITUDI expression over time were observed; however, B. megaterium exposed to Botrytis sp. and Alternaria sp. showed ITUDI upregulation on day 5. Exposure to Botrytis sp. led to a 3-fold increased expression, compared to that on day 1 (Table 3).
Only moderate changes in ITUDI expression were observed; however, the strength of inhibitory effects depends on several metabolites, including other lipopeptides (fengicins, surfactins, bacilliomycins, etc.), suggesting that each metabolite is required. Thus, both fengicins and surfactins depend on the expression of iturins, as they play an important role for effective inhibitory activity 27,28 .
Even so, gene expression differed between incubation conditions. For this purpose, an ANOVA was carried out considering each variable of interest separately, as well as the interactions between them. No significant difference was found regarding growth, whereas the effect of incubation time showed 1.91-fold increased expression until day 5, compared to that on day 1. In Fig. 4, data are shown as a logarithm to the base 2, and in Fig. 5, amplification curves until  day 5 are shown. These results indicated that ITUDI expression by B. megaterium exposed to Botrytis sp. increased by 3.20-fold until day 5, compared to that on day 1. Such a response was expected as B. megaterium can reach the exponential growth phase after 48 h; thus on day 9, the bacteria would have already entered the dying phase 29 . In addition, production of lipopeptides by several species of the genus Bacillus to which inhibitory effects of Botrytis sp. are attributed suggest the presence of iturines, among other lipopeptides. Stable production occurs from 120 h to 168 h. Thus, on day 5, it increases by up to three fold 30 . Moreover, fengicines 31 can also be produced during the stationary phase which occurs from 48-72 hours of incubation; however, inhibitory effects of B. megaterium are attributed to the synthesis of lipopeptides, as observed previously in microbiological test including exposure to Alternaria sp. and Botryris sp. 32 .

FEND and ITUDI gene expression in fermenting B. megaterium
FEND and ITUDI expression during fermentation of B. megaterium was evaluated using RT-qPCR on days 1, 3, 5, and 9 (Table 4), and we observed that expression of both genes significantly increased until day 5 of fermentation; however, it was necessary to perform an ANOVA. Tukey's post hoc test showed a significant difference in gene expression between days of incubation. Gene expression of both lipopeptides increased 56.93-fold until day 5. Regarding differences in expression between genes, the results showed stronger upregulation of fengicines with a 95.14-fold increase, and an 18.71-fold increase in iturines. This indicates that lipopeptide synthesis increases during fermentation. Fig. 6 shows data of logarithmic phase 2 and Fig. 7 shows the amplification curve during fermentation.
The fermentation environment is more appropriate for bacterial growth 33 . In addition, with respect to time, the production of iturines is associated with the early stationary phase, and fengicines accumulate during the later stationary phase, which should occur after 72 hours, as reflected in the observed changes in gene expression 34 . During fermentation, the results were more promising, regardless of whether liquid or solid culture media are used 35 . Large commercial producers use this route of production because of its strong performance and high yield of metabolites such as antibiotics, bioregulators, and toxins for use in different fields including agriculture. B. subtilis, which is closely related to B. megaterium, showed considerable production of iturines and fengicines from days 2-5 of fermentation in our study, and similar results have been obtained previously 36 . Therefore, B. megaterium apparently increases the expression of both lipopeptides independently when exposed to pathogens and during fermentation.

CoNCLUSIoNS
We conclude that B. megaterium inhibits Alternaria sp. growth by 98.48% during 9 days of incubation. FEND gene expression increased 20.16-fold until day 5 of incubation when B. megaterium was exposed to Alternaria sp., which was the maximum of upregulation, suggesting that B. megaterium is a promising controlling agent to counteract specifically Alternaria sp. Regarding iturines, gene expression in B. megaterium increased three times more when exposed to Botrytis sp., and incubation time is a key factor for the synthesis of this lipopeptide, as evidenced by our results. Fermentation was associated with a significant increase in expression of both genes as under optimal growth conditions, this bacterium could produce metabolites at higher yields. Both genes were upregulated rapidly, but upregulation of the FEND gene was stronger. B. megaterium can be considered an promising candidate for agricultural use as it produced excellent results in in vitro microbiological and molecular tests; specifically, it may be used as a biological controlling agent to counteract Alternaria sp. and Botrytis sp.

ACKNowLEDGMENTS
Thank you To Engineer Johana Meneses, for her support in the Research.

CoNFLICT oF INTEREST
The authors declare that there is no conflict of interest.

AUThoRS' CoNTRIbUTIoN
VC authorship and direction of the project, management of resources for research