Comparison of the Antagonistic Potential of the entomopathogenic Bacterium Serratia nematodiphila GCSR38 with other Effective Microorganisms for the Control of Rice Bacterial Leaf Blight

The efficacy of antagonistic microorganisms and secondary metabolites of entomopathogenic bacteria was evaluated in vitro and in vivo against Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of rice bacterial leaf blight. The effect of the culture filtrates of the microorganisms was tested against Xoo, using the agar well diffusion assay. Trichoderma sp. UB05/3 was the most effective against Xoo. However, the results were not statistically different from those of the entomopathogenic bacterium Serratia nematodiphila GCSR38 with a 22.97 mm and 19.15 mm zone of inhibition, respectively. The effect of the secondary metabolite crude extracts of antagonistic microorganisms (2,000 µg/ml) on Xoo inhibition was tested by the paper disc diffusion method. Only S. nematodiphila GCSR38 was able to control Xoo, with an inhibition zone of 17.60 mm. Minimum inhibitory concentration (MIC) was determined using a 96-well microtiter plate. The MIC of secondary metabolites crude extracts of S. nematodiphila GCSR38 was 1,000 µg/ml and the minimum bactericidal concentration (MBC) was 1,000 µg/ml. The efficacy of the secondary metabolite crude extract of S. nematodiphila GCsR38 against Xoo was tested using the detached leaf technique; the secondary metabolite crude extracts controlled the disease, with 24.45% and 15.56% disease severity when used before and after inoculation with Xoo, respectively. Inoculation with Xoo alone resulted in a disease severity of 44.45%. The secondary metabolite crude extracts of S. nematodiphila GCSR38 can reduce disease severity of bacterial leaf blight in rice by 28.89%, whereas zinc thiazole causes a disease severity of 22.22% and Xoo alone causes a disease severity of 66.67%.


INTRODUCTION
Rice (Oryza sativa L.) is a major economic crop in Thailand, and it is the staple food for more than half of the world's population.In many places worldwide, including the Asia-Pacific region, North America, South America, and Africa, rice is used as the main source of energy.The disadvantage of Thai rice is its high production cost and low yield per hectare, which affect the efficiency of its export in the ASEAN trade market.One major factor affecting the productivity of Thai rice is the bacterium Xanthomonas oryzae pv.][3][4] It occurs in both rainfed and irrigated fields in all regions of Thailand and has a high epidemic potential in many parts of the world, causing severe crop losses of up to 50-60%. 5,6Various disease control strategies have been used to reduce productivity losses and prevent rice disease spread.However, chemical-based strategies were unsuccessful because of differential sensitivity of the rice pathogenic species to the chemicals.In addition, the use of antibiotics and chemicals in the control of BLB is limited because of their toxic residues. 7Currently, BLB in rice is controlled by various chemicals, along with the use of diseaseresistant rice cultivars such as Suphan Buri 60, Suphan Buri 90, Suphan Buri 1, Suphan Buri 2 and Kor Khor 23.However, it is still not possible to control the disease effectively because of genetic variations in the pathogens, unstable resistant rice varieties, sensitivity of pathogen to different antibiotics or other environmental factors, and chemical resistant pathogens, which limit the potential of management strategies.The increasing desire to reduce pesticide use has led to the development of integrated pest management methods that make the best use of natural resources.Therefore, host plant-resistant varieties are the first important strategy for disease control.
Biological controls are on the list of alternative management strategies with high potential.][10][11] To reduce the loss of productivity due to disease, it is necessary to use environmentally friendly methods.Trichoderma spp. is a powerful antagonistic fungus that controls a wide range of plant diseases; it not only acts as a biological regulator, but also stimulates plant defense mechanisms against pathogens and promotes plant growth, resulting in higher crop yields.Biological control mechanisms include mycoparasitism, antibiosis, competition, and stimulation of plant defense mechanisms against pathogens. 12Several studies [13][14][15] have reported the efficacy of Trichoderma in controlling Xoo, the causal agent of BLB in rice, in vitro, under greenhouse conditions, and in the field.For example, a comparison of the antagonism of Trichoderma against Xoo showed that T. harzianum showed the fastest growth with 62.3% mycelial growth after 72 h of incubation.Several species of Trichoderma completely inhibit the growth of Xoo. 13 Volatile compounds produced by T. harzianum resulted in the lowest average Xoo growth radius, followed by T. hamatum.Non-volatile compounds of T. harzianum at a concentration of 6.25% inhibited Xoo growth by 51.1%.Results showed that T. harzianum was the most effective isolate for reducing disease severity, with 48.26% and 59.22% reduction in disease severity, respectively.T. harzianum isolated from the phylloplane of rice can reduce disease severity and lead to higher yields compared to isolates from other sources. 15he most commonly used biological control agents are Bacillus spp.; they control pathogens by mechanisms, including antibiosis, competition, parasitism, and induction of systemic resistance, with antibiosis being the most important mechanism. 16El-Shakh et al. 17 investigated the efficacy of endophytic bacteria in controlling the causal agent of BLB in rice (Xoo).Endophytic bacteria and rhizospheric Bacillus were tested for their antagonistic activity against Xoo in BLB.
In vitro, all tested bacteria were found to have a high potential for antagonism against Xoo.FZB42 to target pathogenic microorganisms in rice, using the antibiotics difficidin and bacilysin.Interestingly, B. amyloliquefaciens FZB42 promoted plant growth and inhibited plant pathogens.The genome of FZB42 has been sequenced, and it contains a large gene cluster that produces several secondary metabolites with antimicrobial activity.The antibacterial activity is mainly due to the non-ribosomal synthesis of polyketides. 19Bacillus D13 also has antagonistic activity against Xoo.The volatile compounds of strain D13 decreased the colony size and motility of Xoo cultured on each plate. 20 e r r a t i a s p p .b e l o n g t o t h e Enterobacteriaceae family.Serratia is a gramnegative bacterium that is rod-shaped, and some species produce red pigments.Some strains of Serratia are harmful to pests, when consumed in large amounts.However, sometimes these species can be virulent even when insects are exposed to small amounts.They can kill insect larvae within 48-72 h, with larvae showing symptoms similar to those of a viral infection. 21n particular, red pigment-producing species have a greater effect on insects than nonpigmented species. 22Serratia sp.EML-SE1 has insecticidal potential that can kill diamondback moths. 23Control of several insect genera, such as Anomala, Costelytra, and Phyllophaga, can be achieved by entomopathogenic strains of Serratia entomophila. 245][26] This confirms that these two species have insecticidal properties. 27Long-term pest management guidelines are required for the importation of exotic microorganisms and their subsequent release to destroy pests.Some insect pathogens are also used in this classical microbial management approach, which is related to antibiotic and siderophore production that can inhibit plant pathogens.The objective of this study was to compare the efficacy of secondary metabolites of the entomopathogenic bacterium S. nematodiphila GCSR38 with that of antagonistic microorganisms in controlling Xoo, the causal agent of BLB in rice, to provide a guide for future management strategies.

Microorganisms
The causative agent of BLB in rice Xoo was obtained from the Plant Protection Research and Development Office of the Ministry of Agriculture and Cooperatives, Thailand.Antagonistic microorganisms, including nine antagonistic bacterial isolates (Bacillus sp.), five antagonistic fungal isolates (Trichoderma sp.), and one entomopathogenic bacterial isolate (Serratia nematodiphila GCSR38) were donated by the Microbiology Laboratory, Department of Biology, Faculty of Science, Mahasarakham University.All isolates were stored in 20% glycerol at -20°C until use.
To confirm the species of GCSR38, the classification was repeated as follows: GCSR38 was grown on nutrient agar (NA) plates for 24 h at 37°C, and colony morphology was observed, such as form, margin, and elevation.They were then Gram stained for microscopic observation.The 16S rRNA gene was classified by sequencing.Genomic DNA of the GCSR38 strain was extracted using the CTAB method. 28Universal bacteria primers 27F 5'-AGAGTTTGATC MTGGCTC AG-3' and 1492R 5'-TACGGYTAC CTTGTTACGACT T-3' were used for 16S rRNA gene amplification.Amplifications were performed in a Gradient DNA Thermal Cycler programmed for the following parameter: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 45 s, and a final incubation at 72°C for 5 min.PCR products were commercially sequenced by Macrogen Inc. (Seoul, South Korea).The 16S rRNA gene sequences of bacterium present in the NCBI database were used for alignment and comparison.Sequence data were obtained using BLAST for phylogenetic tree generation of the GCSR38 strain.Closely related sequences were downloaded and aligned using CLUSTAL X. 29 Maximum parsimony (MP), maximum likelihood (ML), and neighbor-joining (NJ) methods were used for sequence analysis. 30

Efficacy of culture filtrates of effective microorganisms in controlling Xoo
The agar well diffusion method was used to test the activity of the culture filtrate.First, antagonistic fungal culture filtrate was prepared by transferring three mycelia discs (8 mm diameter) of each isolate into 100 ml of potato dextrose broth (PDB), which was then incubated at 28±2°C under static conditions for 7 d.The culture filtrate was collected by separating the fungal mycelia through whatman no. 1 filter paper.The supernatant was centrifuged at 8,000 × g for 15 min to remove suspended particles, and filtered through a 0.22 um sterile membrane filter.Bacterial cultures maintained on NGA slants were aseptically inoculated into 10 ml of nutrient glucose broth (NGB) and then incubated in a shaking incubator at 150 rpm and 28±2°C for 48-72 h.Bacterial cultures were centrifuged at 10,000 × g for 15 min, and the supernatant was collected and filtered through a 0.22 µm sterile membrane filter.Xoo cell suspension (10 8 colony forming unit (CFU)/ml) was swabbed on NA plats, and an 8-mm diameter hole was drilled with a sterile cork borer at four points in a cross pattern.Next, 20 µl of culture filtrate was added to the well.The agar plates were then incubated at 28±2°C, and after 24-48 h a clear zone was observed and measured.There were five replicates for each isolate, and 100 ppm rifampicin 31 was used as a control.

Secondary metabolite extraction
For preparing secondary metabolites, antagonistic bacteria were cultured in NGB medium (500 ml) and incubated under shaking at 150 rpm and 28±2°C for 48-72 h.Antagonistic fungi were cultured in PDB medium (500 ml) under static conditions for 7 d.To remove the bacterial cell pellet or fungal mycelia and spores, the culture flask was centrifuged at 10,000 × g at 4°C for 15 min.The supernatant (350 ml of supernatant was extracted with 350 ml ethyl acetate at a 1:1 ratio) and 5 g of NaCl were added for the first extraction.This extraction step was repeated three times.The supernatant in the upper phase of the separatory funnel was collected and pooled, and the solvents were evaporated in a rotary evaporator at 40°C.

the effect of the antagonistic microorganism secondary metabolite crude extract (AMSMCE) on inhibition of Xoo
To determine the antibacterial effect of AMSMCE against Xoo, the paper disc diffusion method was used.The extract was dissolved in 1% DMSO to a final concentration of 2,000 µg/ ml.A sterile filter paper disc (6 mm) containing 10 µl of extract was placed on the surface of NGA plates already inoculated with Xoo and incubated at 28±2°C for 24-48 h.The inhibitory effect was determined by measuring the inhibition zone on a millimeter scale.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
Determination of MIC was done using the method by Clinical and Laboratory Standards Institute. 32First, serial two-fold dilutions (1.95-1,000 µg/ml) of the AMSMCE were added to 96well microtiter plates and 100 µl of Xoo (10 8 CFU/ ml) was added to each well.Then, 100 µl of Mueller Hinton broth (MHB) was added to well 1 to serve as the negative control, and 100 µl of 100 ppm rifampicin was added to well 12 to act a positive control, and the plate was incubated at 28±2°C for 24 h.The OD600 was then measured using a Asys UVM 340 Microplate Reader (Biochrom) MBC was assessed by placing samples from wells with concentrations above the MIC and MIC values and below the MIC values on an NA Petri plate, and the plate was incubated at 28±2°C for 24 h.The MBC value was assessed when no colony growth was observed on the NA plates.

The efficacy of AMSMCE in controlling rice BLB Detached leaf technique
Healthy leaves of Jasmin Rice 105 were selected for the detached leaf method.Rice leaves were washed with tap water for 5 min, sprayed with 70% ethanol, and air-dried.Wounds were inflicted on the leaves using a sterilized inoculation needle (5 wounds/point).A completely randomized block design experiment was planned, consisting of four treatments as follows: (i) 10 µl of AMSMCE was added 24 h before the addition of 10 µl of Xoo cell suspension (10 8 CFU/ml), (ii) 10 µl of Xoo cell suspension (10 8 CFU/ml) was added 24 h before the addition 10 µl of AMSMCE, (iii) 10 µL of Xoo cell suspension (10 8 CFU/ml) was added, and (iv) 10 µL dH 2 O was added.All plants were incubated in a moist chamber for 7 d.For disease rating, a modified scale was used. 33(Table 1).Disease severity was calculated with the followed formula:   was used along with Xoo spraying. 34The four treatments were as follows: (i) Xoo treatment 24 h before spraying with AMSMCE, (ii) Xoo treatment only, as a negative control, (iii) zinc thiazole treatment as a positive control, and (iv) dH 2 O treatment.The inoculated plants were incubated in a humid chamber for 7 d.Disease severity was scored according to the formula above.

Identification of Serratia nematodiphila GCsR38
The morphology of S. nematodiphila GCSR38 colony on NA plates was as follows: red and circular, with entire margins, umbonate elevation, smooth, gram-negative, short rod, and not endospore-forming.The 16S rRNA sequence showed that GCSR38 had 99.93% similarity with Serratia nematodiphila DZ0503SBS1 (Fig. 1).

The efficacy of the effective microorganism culture filtrate in controlling Xoo
The efficiency of antagonistic bacteria and antagonistic fungi culture filtrate against Xoo was tested using the agar well method.Trichoderma sp.UB05/3 showed the best inhibition of pathogen growth and was not statistically different from 100 ppm rifampicin with inhibition zone diameters equal to 22.97+1.58mm and 32.87+1.89mm, respectively (Table 2).All isolates of the antagonistic microbial culture filters showed Xoo inhibition compared to the inhibition observed in case of dH2O.The inhibition by other seven isolates was not statistically different from that of Trichoderma sp.UB05/3, Trichoderma sp.SK24, UB07 special, UB04/3, WU01, Bacillus sp.The efficacy of BWTM-MSU2, BWWM-MSU16, and S. nematodiphila GCSR38 was tested in the next step.The effect of AMSMCE on the inhibition of Xoo AMSMCE was tested for Xoo inhibition using the paper disc diffusion method.The results showed that only the crude extract of secondary metabolites from S. nematodiphila GCSR38 could inhibit the growth of Xoo with a 17.60+1.06mm inhibition zone, while rifampicin (positive control) showed a 32.70+0.52mm inhibition zone (Table 3).

Determination of MIC and MBC
The MIC of S. nematodiphila GCSR38 secondary metabolite crude extract against Xoo was 1,000 µg/ml.The MBC screening indicated that S. nematodiphila GCSR38 secondary metabolite crude extracts tested against Xoo at 1,000 ug/ml showed no colony-forming ability, indicating that the MBC value for this extract was 1,000 µg/ml (Fig. 2).

The efficacy of GCSR38 secondary metabolite crude extracts against Xoo Detached leaf technique
Seven days after inoculation, the results showed that S. nematodiphila GCSR38 secondary metabolite crude extracts could reduce disease severity with a severity index less than that of the only Xoo treatment.Using the extracts 24 h before and after inoculation with Xoo, we found that the disease severity index was not significantly different and was 24.45% and 15.56%, respectively (Table 4).

Pot experiments
Inoculation of Xoo 24 h before spraying S. nematodiphila GCSR38 secondary metabolite crude extracts reduced the incidence of rice BLB by 28.89%; spraying with zinc thiazol yielded significantly different results compared to those by other treatments, with 22.22% disease severity (Table 5).

DISCUSSION
M o r p h o l o g i c a l a n d m o l e c u l a r characterization of strain GCSR38 indicated that it was S. nematodiphila.This result is similar to that by Zhang et al., 35 who reported the isolation of the bacterium Serratia DZ0503SBS1(T) from the intestine of a nematode (Heterorhabditidoides chongmingensis); it produced red pigment, was gram-negative, mobile, fluorescent, odd-shaped, and had a unilateral flagellum.A comparative analysis of 16S rRNA gene sequences showed that DZ0503SBS1(T) is a novel species named S. nematodiphila sp.nov.The culture filtrates   The results revealed that the dual culture of Trichoderma spp.showed a larger inhibition zone than the monoculture.This indicates that better stimulation of novel secondary metabolites can be achieved by dual culture of beneficial fungi and is better than monoculture. 36Prabawati et al. 37 performed isolation, selection, and identification of phyllosphere bacteria against Xoo.The plug agar method was used to screen the antagonistic activity of 52 isolates of phyllosphere bacteria.The results showed that the seven isolates could inhibit Xoo.The morphology and molecular characteristics of these antagonistic bacteria were used to classify them in the genera Bacillus and Arthrobacter.Nagendran et al. 38 managed BLB using endophytic bacteria.The results of the in vitro study showed a significantly higher inhibition of Xoo, including five isolates of B. subtilis var.amyloliquefaciens.Halim et al. 39 screened and characterized bacterial isolates that had potential antagonistic activity against Xoo.Only 17 endophytic bacterial isolates showed positive antagonistic activity, which was indicated by inhibition zones against Xoo around bacterial colonies on NA plates.While isolating and identifying soil bacteria antagonistic to Xoo, it was found that among 830 isolates, CT-66, CT-78, and CT-88 strains exhibited strong antagonistic effects against the pathogen.These effects are associated with antibiotics.Based on the 16S rRNA gene sequence, morphology, and biochemical properties, strain CT-78 was identified as S. nematodiphila. 40The biochemical synthesis of bacterial secondary metabolites involves multiple enzymatic pathways and structural heterogeneous reactions that give rise to their highly specific mechanisms of action.The genus Serratia lives in a wide variety of environments and has a large amount of plaque, which is considered a source of exotic and biologically important structural secondary metabolites with potential activities include antioxidants, antimicrobial and immunesuppressive properties. 41S. nematodiphila RL2 produces a red pigment called prodigiosin.
Prodigiosin is an antimicrobial agent that is effective in controlling Listeria spp., Pseudomonas spp., Yersinia spp., and Shigella spp. 42he MIC of GCSR38 secondary metabolite extracts against Xoo was 1,000 µg/ml, and the MBC value for this exact was also 1,000 µg/ml.S. nematodiphila GCSR38 secondary metabolite extracts can reduce the disease severity of BLB in vitro and in vivo.This is similar to the findings of Shi et al., 43 who reported the role of Streptomyces roseoverticillatus 63 (Sr-63) against Xoo; disease suppression was accomplished under glasshouse conditions.In addition, carbazomycin B isolated from the fermented broth of strain Sr-63 was found to be active against Xoo, with an MIC of 8 µg/ml.Bioactivity analysis showed that the compounds isolated from Pseudomonas aeruginosa (LN strain) inhibited three Xanthomonas strains under greenhouse conditions.In vivo testing under greenhouse conditions for fractions with antibiotic activity and their bioactivity evaluation is essential.Spraying plants with semi-purified fractions can control Xanthomonas spp. 44 Sutthisa | J Pure Appl Microbiol | 16(1):557-566 | March 2022 | https://doi.org/10.22207/JPAM.16.1.54 SI = [ ∑(Ni x Vi) / (V x N) ] x 100 Ni = number of leaves in each disease scale Vi = disease scale in each leaf V = maximum disease scale N = number of leaves Pot experiments Jasmin rice 105 was used as the test crop.The experiments in the pots were designed as a completely randomized experiment with four replicates, using three 21-d-old seedlings in each pot.The clipping method with sterilized scissors

Table 2 .
Efficacy of effective microorganism culture filtrate to control Xanthomonas oryzae pv.oryzae by agar well diffusion method 1/ Different superscripts within the same column indicate a significant difference (P < 0.05).

Table 3 .
Effect of antagonistic microorganism secondary metabolites crude extracts (AMSMCE) against Xanthomonas oryzae pv.oryzae by using agar well diffusion technique 1/ Different superscripts within the same column indicate a significant difference (P < 0.05).

Table 4 .
Effect of S. nematodiphila GCSR38 secondary metabolites crude extract to control rice bacterial leaf blight disease by using detached leaf technique, 7 days after inoculations

Table 5 .
Effect of S. nematodiphila GCSR38 secondary metabolites crude extract to control rice bacterial leaf blight disease in potted rice plants under greenhouse conditions, 7 days after inoculations Sutthisa | J Pure Appl Microbiol | 16(1):557-566 | March 2022 | https://doi.org/10.22207/JPAM.16.1.54Gangwar and Sinha 14 screened Trichoderma spp.and studied their biocontrol abilities against Xoo isolates obtained from ricegrowing areas of various states.Trichoderma spp.isolated from Pantnagar were able to inhibit the growth of Xoo isolated from Pantnagar and from other locations by 100%.Trichoderma spp.can inhibit the radial growth of Xoo with 89.8 % inhibition for both isolates from Kashipur; however, 100% inhibition was obtained using Trichoderma spp.isolated from Pantnagar and Haldwani.GC-MS was used for further analysis of antibacterial activity against the rice BLB pathogen.