Bacillus cereus (OR975563) of maize rhizosphere can promote plant growth and potentially suppress mycotoxin (Fumonisin B1) during storage

doi:10.21203/rs.3.rs-4008304/v1

Seed deterioration and development of toxins are the prime concern of maize seeds for maintaining quality and health during storage in Bangladesh. Endophytic and rhizospheric bacteria of maize were assessed for their growth suppressive potential against mycotoxigenic fungi (Fusarium oxysporum) and reduction of mycotoxin (Fumonisin B1) during storage. In this study, a total of 48 bacterial isolates were obtained and evaluated for their antifungal activity against Fusarium oxysporum. Four rhizospheric (8SISO4, 8SISO5, 14SISO14, and 16SISO16) which were identified as Bacillus velezensis (OR975558), Bacillus amyloliquefaciens (OR975561), Bacillus subtilis (OR975559) and Bacillus cereus (OR975563) and four endophytic (6RISO28, 9RISO31, 11RISO33, and 9RISO32) bacterial isolates which were identified as Bacillus tropicus (PP335187), Bacillus amyloliquefaciens (OR975562), Bacillus zanthoxyli (OR975560) and Bacillus amyloliquefaciens showed in vitro growth suppressive effect against F. oxysporum. Maize seeds were treated 1, 6 and 12 hours by immersing into bacterial suspension. Among the treatments, seeds treated with the isolate 6RISO28 showed the highest mycelial growth suppression (84.0%) against F. oxysporum. After 8 month of storage, the highest seed germination (68.7%), shoot length (5.1 cm), root length (8.1cm), disease incidence (23.3%) with a vigor index of 988.6 were recorded in the T₂₃ (Maize seed dipping for 6 hr in 16SISO16) with the lowest Fumonisin concentration (4.5 ppm) and the highest mycelial growth suppression of Fusarium. Therefore, it can be opined that utilization of antagonistic bacteria can be a promising approach for the reduction of mycotoxin along with the improvement of seed and seedling health of maize during post-harvest storage.

mycotoxin

seed and seedling health

bacteria

endophyte

Maize is one of the major and economically beneficial crops in the world, which is well-known for its adaptability to stress conditions (Rahman and Rahman 2014). Both the acreage and yield of maize is increasing day by day. The demand for maize is enormous in the poultry, fish and animal feed companies in Bangladesh. Despite being the second most significant cereal crop after rice, maize has the highest yield in Bangladesh, at 6.15 t/ha which is below compare to other countries (Alam et al. 2018; BBS 2011a). Cultivation of maize is popular due to its multipurpose use like food, feed, edible oil (Khaiyam et al. 2017) and contains a higher amount of fat, protein, energy, carotene, riboflavin and thiamin than rice (Nuss & Tanumihardjo 2010).

Although maize can be cultivated in different agroclimatic zones due to its genetic diversity (Rahman and Rahman 2014), fungal diseases are major limiting factors of both qualitative and quantitative loss of maize (Lewis 2005) in both pre-harvest and stored conditions (Goko et al. 2021; Nukenine 2010). Besides crop loss, these fungal diseases pose a major threat to public health since they produce mycotoxin (Mitchell et al. 2016). In addition to soil fungus, storage molds can grow to harmful levels in stored grain and may cause up to 5% of post-harvest storage loss (Manandhar and Batsa 2000). Being a tropical country, Bangladesh is congenial for developing molds during storage of seeds and feeds. The three main genera that produce mycotoxins are Penicillium, Aspergillus, and Fusarium. Many mycotoxins such as sterigmatocystin and aflatoxins produced by Aspergillus, deoxynivalenol, trichothecenes A and B, nivalenol, moniliformis, fumonisins, etc. produced by Fusarium and zearalenones & vomitoxin produced by Penicillium (Hussain et al.2013; Ismaiel and Papenbrock 2015; Al-Fakih2014) reduce crop quality, seed rot, seed abortion, reduced germination, seed germination along with human and animal health problem (Leslie et al. 2008). CIMMYT maize program, 2004 reported several economically important maize diseases like smuts, stalk rots, ear rots, and some foliar diseases (Juroszek and Tiedemann 2013). Among them, ear rot caused by Fusarium verticillioides, F. oxysporum, F. graminearum) due to the production of fumonisin mycotoxin (Li et al. 2019) is a significant disease that severely impacts maize production (Yuan et al. 2012).

Numerous methods like host resistance, better cultural practices are followed to promote crop health against fungal attack pre-harvest stage (Cleveland et al. 2003). But, they do not provide satisfactory results (Andriolli et al. 2016). Also, numerous disadvantages to chemical fungicide application like increased cost, difficulties in the application of agrochemicals at the proper stage (Lagogianni & Tsitsigiannis2018), pathogen resistance development (Choi et al. 2017) as well as increasing fumonisin concentration in plants, when seeds are treated with chemicals before planting (Pereira et al. 2007h) restricts their application.

In this regard, the use of antagonistic bacterial agents can be a potential way to control soil-borne and storage fungi (Harman 1992; Hebbar et al. 1992; Bressan 2003). Many plant growth-promoting bacteria are reported to provide resistance against biotic and abiotic stress besides boosting plant growth (Wu et al. 2005). Several bacteria especially Bacillus spp. possess the property to produce various antifungal proteins like protease, chitinase and glucanase which are responsible for their disease suppression ability (Bressan & Fontes-Figueiredo 2010). Simultaneously they also produce various plant growth hormones, proteins and products like siderophore production to promote plant growth (Figueroa-López et al. 2016). The present study was aimed to screen out the rhizopheric microorganisms of maize on the basis of their growth suppression ability in order to treat maize seeds before storage to investigate the mycotoxins production in these seeds and their growth promotion ability.

Collection and preservation of maize root and soil sample

A total of 30 roots from matured maize plants along with adjacent soils (6 inches deep) were collected from 24 villages of three upazilas under two major maize-producing districts of Bangladesh viz. Lalmonirhat and Nilphamari. Samples were kept in clean, sterile zipper bags and preserved at -20ºC for subsequent use at the laboratory of Bio-signaling, Bio-active compounds, and Bioformulations (BBB) Laboratory, Department of Plant Pathology, Bangladesh Agricultural University.

Isolation of rhizospheric and endophytic bacterial isolates

Soil attached to maize roots was separated and serial dilution technique was followed to isolate bacteria. An aliquot of about 100 µl from dilution was spread on nutrient agar (NA) medium for isolation and incubate for 24 hours at 25 ± 2º C. Subsequent streaking on nutrient agar medium was done to obtain pure culture of bacteria (Abiala et al. 2015) while endophytic bacteria were isolated by washing the roots and surface sterilized by 70% ethanol followed by 0.53% Clorox (NaOCl) for 2 minutes and rinsed with distilled water to remove any traces of chemicals according to the method described by Orole and Adejumo (2011). Then 1 g of root tissue was measured and grinded in mortar. Serial dilution from 10^− 4 to 10^− 7 was maintained to isolate bacteria on the NA medium. The incubation temperature was 27ºC for 48 hours (Zinniel et al. 2002). Finally, bacteria were kept at -80º C for long-term preservation in 15% glycerol for future study.

Preparation of pure culture of Fusarium oxsporum

Among the toxigenic fungi of germinating maize seeds was Fusarium which was isolated by agar incubation method. In this method, the germinating maize seeds were observed under stereo-binocular microscope and then Fusarium infected maize seed was placed on acidified PDA medium and incubated at 25 ± 2º C for a week in a incubator (FOC 215I cooled incubator, Velp scientifica, Italy). For multiplication of Fusarium oxsporum,PDA medium supplemented with ampicillin at 250 mg/ml at 22º C for 10 days was maintained (Fovo et al. 2017).

Assessing antagonistic ability of bacterial isolates against Fusarium oxysporum

For assessing the antagonistic ability of bacterial isolates, a modified dual culture method was used (Nielsen et al. 1998). A mycelial disc of 5-mm diameter of test pathogen (F. oxysporum) was placed on one side of a petri plate containing PDA medium and bacterial isolate was streaked at the center of the plate. A plate with only F. oxysporum was used as a control. After incubation at 28°C, the mycelial growth was observed at 4,8,12 and 16 days after incubation (DAI) until the control plate completely covered by the fungus. Inhibition percentages at 16 DAI were calculated by following the formula-

Inhibition percentage \(=\frac{\text{R}1-\text{R}2}{\text{R}1}\) ×100

Here, R₁ = Radial mycelial growth of the pathogen in the control plate

R₂ = Radial mycelial growth of the pathogen in treated plates

Isolates providing the best results in dual culture assay were used for subsequent studies

Molecular identification of bacterial isolates

Molecular identification of selected bacterial isolates was done by amplifying their 16S rRNA sequence. DNA was extracted by Promega DNA extraction kit according to the manufacturer’s instructions. Forward primer 27F (5´- AGAGTTTGATCMTGGCTCAG − 3´) and reverse primer 1492R (5´- TACGGYTACCTTGTTACGG − 3´) were used for PCR (Weisburg et al. 1991). PCR condition and detection of PCR products were done was performed according to the methods stated by Ying et al. (2012). PCR products were sent to National Institute of Biotechnology (NIB), China for PCR purification and sequencing. Finally, bacterial isolates were identified by comparing their sequences with the NCBI database.

Evaluation of selected bacteria for seed treatment

Maize seeds that showed the infection of Fusarium sp. during seed health test were subjected for treatment by different isolates of bacterial antagonists maintaining different treating intervals. Bacterial isolates were initially multiplied on LB medium at 25ºC and 250 rpm for 18 hours (Lizárraga-Sánchez et al. 2015). Before seed treatment, the seeds were surface sterilized by submerging seeds in 2% Clorox (NaOCl) followed by rinsing several times with distilled water. Seeds were dipped in bacterial broth (10⁸) CFU/ml (Almaghrabi et al. 2014). Three different time intervals (1, 6 and 12 hours) were maintained for treating seeds in bacterial suspension. Only LB media without bacteria served as a control. Every month, a sub-sample of each seed lot was removed from storage and determined germination (%), disease incidence (%), shoot and root length (cm) and vigor index. Each treatment was done in triplicate.

Determination of Toxin (Fumonisin) from treated maize seed using ELISA kit

Determination of toxin (Fuminisin B1) of the treated maize seeds was done during storing. For this purpose, the ELISA technique with Fumonisin B1 (FB1) ELISA kit, catalog no: E-T0-E020 was used according to the instructions of the manual. The maize sample was first ground into a fine powder and then mixed with an extraction buffer to extract the Fusarium antigens. The mixture was incubated and centrifuged, and the resulting extract was diluted with a diluent buffer. An ELISA plate was coated with a specific antibody against the Fusarium antigen, and the diluted extract was added to the plate and incubated. The plate was washed to remove any unbound material, and a secondary antibody conjugated to an enzyme was added and incubated. A substrate was then added, which reacted with the enzyme to produce a color. The absorbance of the reaction mixture was measured at a specific wavelength using a microplate reader, and the concentration of Fumonisin in the maize sample was calculated based on a standard curve generated from known concentrations of Fumonisin.

In-vitro screening of antagonistic endophytic and rhizospheric microorganisms against Fusarium sp.

A time dependent in vitro antagonism assay was conducted for the different isolates of endophytic and rhizospheric bacteria where radial mycelia growth of Fusarium sp. was recorded at 4,8,12 and 16 DAI respectively (Table 1& Table 2). In dual culture assay 4 isolates of rhizospheric bacteria viz. 8SIS04, 8SIS05, 14SISO14 & 16SISO16 showed significant suppression of mycelial growth, whereas the highest percentage of inhibition (77.7%) at 16 DAI was recorded in 16SISO16. On the other hand, 4 isolates of endophytic bacteria viz. 6RISO28, 9RISO31, 9RISO32, and 11RISO33 showed superior performance compared to other antagonist bacterial isolates. Among the endophytic bacterial isolates, 6RISO28 showed the highest (84.0%) mycelial growth suppression at 16 DAI.

Table 1

Evaluation of *in vitro* mycelial growth suppression of *Fusarium oxysporum* by different isolates of rhizospheric bacteria in dual culture plate technique
Isolates	Mycelial growth of F. oxysporum (cm) recorded at different days after incubation (DAI)
Isolates	4 DAI	8 DAI	12 DAI	16 DAI	% Inhibition at 16 DAI
Control	19.53 a	33.40 abc	46.56 a	55.77 a	-
1SISO1	19.00 ab	32.56 abc	44.55 a	50.67 ab	9.144
4SISO2	15.73 a-e	36.45 a	45.00 a	49.83 ab	10.65
7SISO3	14.66 a-g	17.00 g-j	18.78 def	19.55 ef	64.94
8SISO4	7.89 h	9.22 j	10.22 cde	13.06 f	76.58
8SISO5	8.67 h	10.00 j	11.55 f	12.62 f	77.37
8SISO6	14.66 a-g	24.33 c-g	26.49 cde	33.33 cde	40.23
9SISO7	15.33 a-f	35.33 ab	45.33 a	52.00 a	6.7
11SISO8	12.44 c-h	15.00 hij	17.67 def	20.56 def	63.13
11SISO9	11.89 d-h	14.11 hij	15.55 def	17.11 f	69.32
12SISO10	10.78 e-h	12.78 ij	14.78 def	15.78 f	71.70
13SISO11	8.44 h	11.55 j	15.22 def	16.39 f	70.61
13SISO12	10.56 fgh	12.78 ij	15.00 def	16.78 f	69.91
14SISO13	12.44 c-h	15.07 hij	16.33 def	19.63 def	64.80
14SISO14	10.00 gh	12.33 ij	13.33 ef	14.89 f	73.30
15SISO15	16.00 a-d	30.78 a-d	40.67 ab	46.78 abc	16.11
16SISO16	12.11 d-h	12.44 ij	12.44 f	12.44 f	77.69
17SISO17	17.33 abc	36.89 a	43.33 a	52.33 a	6.16
18SISO18	9.89 gh	13.45 hij	16.67 def	18.89 ef	66.12
19SISO19	14.55 a-g	21.45 e-i	26.78 cd	35.55 bcd	36.25
22SISO20	14.45 a-g	17.55 f-j	19.74 def	21.67 def	61.14
23SISO21	16.22 a-d	26.44 b-f	34.78 abc	45.33 abc	18.71
24SISO22	16.67 a-d	28.11 a-e	35.22 abc	47.00 abc	15.72
25SISO23	15.78 a-e	22.00 d-h	27.44 bcd	43.78 abc	21.49
26SISO24	14.00 b-g	17.89 f-j	20.44 def	24.89 def	55.37
28SISO25	11.89 d-h	14.74 hij	17.78 def	18.85 ef	66.20
29SISO26	12.89 c-h	15.67 g-j	18.22 def	22.11 def	60.35
30SISO27	10.66 e-h	14.00 hij	15.83 def	19.66 def	64.74
SE (±)	0.93	1.67	2.42	2.89
CV (%)	1.08	2.37	4.1	4.15

Table 2

Evaluation of *in vitro* mycelial growth suppression of *Fusarium oxysporum* by different isolates of endophytic bacteria in dual culture plate technique
Isolate name	Mycelial growth of F. oxysporum (cm) recorded at different days after incubation (DAI)
Isolate name	4 DAI	8 DAI	12 DAI	16 DAI	% Inhibition at 16 DAI
Control	19.67 abc	32.78 ab	47.22 a	55.40 a	0.00
6RISO28	5.44 i	6.00 j	7.56 l	8.89 l	83.95
7RISO29	12.44 d-h	15.67 ghi	17.11 jik	19.56 jk	64.69
8RISO30	17.11 a-e	26.89 bcd	35.39 bcd	48.33 ab	12.76
9RISO31	13.67 a-h	16.33 ghi	17.53 ijk	17.56 kl	68.30
9RISO32	8.44 ghi	11.44 hij	15.61 jkl	16.67 kl	69.90
11RISO33	7.66 a-f	9.87 ij	12.37 kl	14.96 kl	72.99
12RISO34	13.78 a-h	15.30 ghi	22.33 g-j	28.55 g-j	48.46
13RISO35	15.89 a-f	23.56 c-f	29.84 c-g	37.33 d-h	32.61
14RISO36	12.78 d-h	19.45 efg	28.44 c-h	39.44 b-f	28.80
15RISO37	13.44 b-h	15.44 ghi	16.44 jkl	19.89 jk	64.09
17RISO38	12.22 d-h	16.56 f-i	19.50 h-k	22.78 ijk	58.88
18RISO39	12.67 d-h	17.00 fgh	19.11 h-k	23.22 ijk	58.08
19RISO40	9.67 f-i	15.33 ghi	21.22 g-k	27.89 hij	49.65
21RISO41	13.33 c-h	25.55 cdf	32.34 b-f	46.00 a-d	16.96
22RISO42	20.22 a	25.11 cdf	33.78 b-e	38.11 c-g	31.20
23RISO43	15.89 a-f	21.44 d-g	26.33 d-i	35.45 e-h	36.01
24RISO44	13.89 a-h	28.45 a-d	35.89 bc	47.22 abc	14.76
25RISO45	10.89 e-i	16.42 ghi	24.89 e-j	35.11 fgh	36.62
27RISO46	14.33 a-g	19.56 efg	23.55 f-j	32.44 f-i	41.44
28RISO47	20.00 ab	34.67 a	40.44 ab	44.96 b-e	18.84
29RISO48	17.89 a-d	28.55 abc	40.52 ab	45.96 a-d	17.03
SE (±)	1.22	1.29	1.74	1.8
CV (%)	1.08	2.37	4.1	4.15

Identification of antagonistic bacterial isolates using sequenced-based molecular technique and phylogenetic analyses

For confirmation of the identity of the selected rhizopheric and endophytic bacteria 16S rRNA gene based sequence was conducted. In addition phylogenetic analyses were done (Fig. 1). The sequence of 8 bacterial isolates was deposited into NCBI Genbank and got the accession number. The sequence of all eight bacterial isolates were compared with the closest neighbor of NCBI accessions and confirmed as Bacillus. Rhizospheric isolates, 8SISO04, 8SISO05, 14SISO14 and 16SISO16 showed similarity with Bacillus velezensis (OR975558), (98.6%), B. amyloliquefaciens (OR975561) (99.5%), B. subtilis (OR975559) (98.9%) and B. cereus (OR975563) (99.5%) respectively. On the other hand, endophytic isolates, 6RISO28, 11RISO33, 9RISO31, and 9RISO32 showed the highest similarity with Bacillus tropicus(PP335187), Bacillus. zanthoxyli (OR975560) (99.5%), B. amyloliquefaciens (OR975562) (99.8%), and B. amyloliquefaciens (98.1%).

Effect of selected antagonistic endophytic bacteria on different parameters of maize

A set of treatments considering different isolates of endophytic bacteria and seed dipping time were considered for maize seeds before stored them for 8 months. In every month different parameters i.e. germination percentage of seeds, disease incidence, root length, shoot length and vigor index of seedlings were assessed.

Treatment T₂₃ performed the best in yielding the highest germination of maize seeds in every month. The highest germination percentage was 94.7, 90.7, 85.3, 80.0, 79.0, 74.7, 72.7, and 68.7% in 1st – 8th months after storage, respectively. Statistically similar responses were also observed in T₂₀, T₇ and T₁ (Table 3).

Table 3

Effect of seed treatment by different antagonistic bacterial isolates on germination percentage at different time intervals
Treatment	Isolate id	Dipping time of seeds (hr)	Germination (%)
			1st month	2nd month	3rd month	4th month	5th month	6th month	7th month	8th month
T₀	Control		46.67gh	42.67 hi	42.67 gh	33.33 e	32.33 e	28.33 e	24.33 e	21.33 e
T₁	9RISO31	1	93.33a	86.67 ab	81.33 ab	74.67 ab	73.67 ab	71.67 ab	68.67 ab	64.67 ab
T₂	9RISO31	6	76.00c-f	70.67 cde	65.33 cde	61.33 c	59.33 c	57.33 c	53.33 c	48.33 c
T₃	9RISO31	12	25.33i	56.00 fg	50.67 fg	45.33 d	44.33 d	40.33 d	36.33 d	32.33 d
T₄	6RISO28	1	74.67def	70.67 cde	65.33 cde	56.00 c	54.00 c	52.00 c	48.00 c	42.00 c
T₅	6RISO28	6	48.00gh	44.00 hi	38.67 h	36.00 de	34.00 de	32.00 de	28.00 de	22.00 de
T₆	6RISO28	12	42.67h	40.00 i	40.00 h	37.33 de	36.33 de	32.33 de	30.33 de	28.33 de
T₇	9RISO32	1	92.00a	88.00 a	81.333ab	77.33 a	76.33 a	72.33 a	70.33 a	68.33 a
T₈	9RISO32	6	68.00f	62.67 ef	60 ef	58.67 c	56.67 c	50.67 c	48.67 c	43.67 c
T₉	9RISO32	12	72.00ef	66.67 de	61.333 de	56.00 c	55.00 c	50.00 c	48.00 c	41.00 c
T₁₀	11RISO33	1	80.00b-e	72.00 cde	68 cde	64.00 c	62.00 c	58.00 c	54.00 c	51.00 c
T₁₁	11RISO33	6	84.00a-d	76.00 cd	72 bc	64.00 c	62.00 c	59.00 c	54.00 c	48.00 c
T₁₂	11RISO33	12	50.67gh	46.67 ghi	42.667 gh	41.33 de	39.33 de	34.33 de	30.33 de	32.33 de
T₁₃	8SISO4	1	77.33b-f	72.00 cde	64 cde	57.33 c	55.33 c	51.33 c	47.33 c	41.33 c
T₁₄	8SISO4	6	78.67b-f	74.67 cd	69.333 cde	65.33 bc	63.33 bc	57.33 bc	52.33 bc	47.33 bc
T₁₅	8SISO4	12	50.67gh	46.67 ghi	45.333 gh	41.33 de	40.33 de	37.33 de	34.33 de	30.33 de
T₁₆	8SISO5	1	86.67abc	76.00 cd	72.00 bc	64.00 c	63.00 c	58.00 c	52.00 c	47.00 c
T₁₇	8SISO5	6	88.00ab	77.33 bc	70.67 cd	65.33 bc	63.33 bc	59.33 bc	53.33 bc	47.33 bc
T₁₈	8SISO5	12	56.00g	50.67 gh	48.00 gh	45.333 d	43.333 d	38.333 d	32.333 d	28.333 d
T₁₉	14SISO14	1	84.00a-d	74.67 cd	69.33 cde	64.00 c	62.00 c	57.00 c	53.00 c	46.00 c
T₂₀	14SISO14	6	93.33a	89.33 a	84.00 a	78.67 a	77.67 a	73.00 a	70.00 a	67.00 a
T₂₁	14SISO14	12	84.00a-d	74.67 cd	70.67 cd	65.33 bc	63.33 bc	57.33 bc	50.33 bc	48.33 bc
T₂₂	16SISO16	1	80.00b-e	74.67 cd	69.33 cde	64.00 c	62.00 c	58.00 c	52.00 c	47.00 c
T₂₃	16SISO16	6	94.67a	90.67a	85.33 a	80.00 a	79.00 a	74.67 a	72.67 a	68.67 a
T₂₄	16SISO16	12	80.00 b-e	72.00 cde	68.00 cde	64.00 c	62.00 c	57.00 c	51.00 c	47.00 c
SE			2.99	2.72	2.47	2.69	2.54	2.34	2.24	2.54
CV(%)			11.57	10.51	9.56	10.41	10.51	9.54	9.44	10.54

Although there was a steady increment in disease incidence percentage of treated seeds with times, the lowest disease incidence percentage was observed in T₂₃ for every month and continued up to 8th months. The disease incidence range in T₂₃ was 2.67 to 23.33% which was significantly lower than other treatments including control treatment. The disease incidence range was 41.33 to 66.00% in untreated control, which was much higher than all other treatments (Table 4).

Table 4

Effect of seed treatment by different antagonistic bacterial isolates on the incidence of seed-borne disease of maize at different time intervals
Treatment	Isolate id	Dipping time of seeds (hr)	Disease Incidence (%)
			1st month	2nd month	3rd month	4th month	5th month	6th month	7th month	8th month
T₀	Control		41.33 a	50.67 a	52.00 a	56.00a	58.00a	62.00a	66.00a	66.00a
T₁	9RISO31	1	8.00 fgh	10.67 ij	14.67 gh	17.33 g	18.33 g	20.33 g	22.33 g	23.33 g
T₂	9RISO31	6	24.00 b-f	26.67 e-h	33.33 bcde	36.00 def	38.00 def	44.00 def	50.00 def	56.00 def
T₃	9RISO31	12	37.33 ab	40.00 a-e	42.67a-d	45.33 bcd	47.33 bcd	57.33 bcd	62.33 bcd	64.33 bcd
T₄	6RISO28	1	36.00 ab	42.67 abc	44.00 abc	46.67abc	48.67abc	58.67abc	63.67abc	66.67abc
T₅	6RISO28	6	32.00 abc	37.33 a-f	38.67 b-e	42.67 b-e	45.67 b-e	55.67 b-e	61.67 b-e	65.67 b-e
T₆	6RISO28	12	36.00 ab	45.33 ab	44.00 abc	48.00 ab	50.00 ab	55.00 ab	60.00 ab	66.00 ab
T₇	9RISO32	1	6.67 gh	10.67 ij	17.33 fgh	18.67 g	19.67 g	21.67 g	23.67 g	26.67 g
T₈	9RISO32	6	28.00 a-d	32.00 b-h	37.33 b-e	41.33 b-e	43.33 b-e	53.33 b-e	58.33 b-e	60.33 b-e
T₉	9RISO32	12	10.67 e-h	20.00 hij	28.00 ef	33.33 ef	35.33 ef	45.33 ef	50.33 ef	55.33 ef
T₁₀	11RISO33	1	28.00 a-d	32.00 b-h	36.00 b-e	41.33 b-e	44.33 b-e	54.33 b-e	60.33 b-e	66.33 b-e
T₁₁	11RISO33	6	21.33 b-g	32.00 b-h	37.33 b-e	42.67 b-e	44.67 b-e	54.67 b-e	62.67 b-e	62.667 b-e
T₁₂	11RISO33	12	25.33 a-e	32.00 b-h	36.00 b-e	40.00 b-f	46.00 b-f	52.00 b-f	58.00 b-f	61.00 b-f
T₁₃	8SISO4	1	34.67 ab	40.00 a-e	41.33 a-d	44.00 bcd	46.00 bcd	52.00 bcd	56.00 bcd	60.00 bcd
T₁₄	8SISO4	6	25.33 a-e	33.33 b-h	37.33 b-e	42.67 b-e	44.67 b-e	54.67 b-e	58.67 b-e	60.67 b-e
T₁₅	8SISO4	12	36.00 ab	41.33 a-d	45.33 ab	48.00 ab	50.00 ab	56.00 ab	60.00 ab	62.00 ab
T₁₆	8SISO5	1	12.00 d-h	20.00 hij	26.67 efg	30.67 f	32.67 f	40.67 f	44.67 f	50.67 f
T₁₇	8SISO5	6	16.00 c-h	24.00 f-i	30.67 de	37.33 c-f	35.33 c-f	42.33 c-f	47.33 c-f	50.33 c-f
T₁₈	8SISO5	12	30.67 abc	34.67 b-g	37.33 b-e	41.33 b-e	44.33 b-e	52.33 b-e	57.33 b-e	61.33 b-e
T₁₉	14SISO14	1	21.33 b-g	28.00 d-h	32.00 cde	37.33 c-f	39.33 c-f	48.33 c-f	52.33 c-f	51.33 c-f
T₂₀	14SISO14	6	8.00 fgh	10.67 ij	13.33 h	17.33 g	18.33 g	21.33 g	23.33 g	26.33 g
T₂₁	14SISO14	12	13.33 d-h	20.00 hij	30.67 de	37.33 c-f	39.33 c-f	45.33 c-f	51.33 c-f	53.33 c-f
T₂₂	16SISO16	1	25.33 a-e	30.67 c-h	33.33 b-e	38.67 b-f	40.67 b-f	48.67 b-f	53.67 b-f	56.67 b-f
T₂₃	16SISO16	6	2.67 h	8.00 j	10.67 h	17.33 g	18.33 g	19.33 g	21.33 g	23.33 g
T₂₄	16SISO16	12	13.33 d-h	22.67 ghi	32.00 cde	40.00 b-f	42.00 b-f	49.00 b-f	52.00 b-f	54.00 b-f
SE			4.36	3.48	3.31	2.61	2.47	2.57	2.47	2.37
CV(%)			16.87	13.44	12.79	10.09	9.47	9.87	9.67	9.47

Significant growth promotion (shoot length and root length) was observed by seed treatment with antagonistic endophytic bacteria in every month during storing compared to untreated control (Table 5&6). The highest shoot and root length was observed in T₂₃ (6.33 cm and 9.2 cm) followed by T₁ (6.27 cm and 8.33 cm) respectively in the earlier storing time (1st month). In later part of storing (8th month) lower shoot length (5.10 cm and 5.00 cm respectively for T₂₃ and T₁) was found which was still much higher than control treatment. Statistically similar root length (8.1 cm) was observed in T₂₃ and T₁ at 8th month after storage.

Table 5

Effect of see treatment by different antagonistic bacterial isolates on shoot length (cm) of maize seedlings at different time intervals
Treatment	Isolate	Dipping time of seeds (hr)	Shoot length (cm)
			1st month	2nd month	3rd month	4th month	5th month	6th month	7th month	8th month
T₀	Control		2.77 j	2.83 e	3.17 d	3.1 e	3.0 e	2.4 e	2.1 e	2.0 e
T₁	9RISO31	1	6.27 ab	6.03 a	5.43 ab	5.37 ab	5.2 abc	5.2 abc	5.1 ab	5.0 ab
T₂	9RISO31	6	4.2 e-i	3.73 cde	4.17a-d	4.2 a-e	4.0 a-e	3.0 a-e	2.8 a-e	2.6 a-e
T₃	9RISO31	12	4.47 e-h	4.53 bc	4.5 a-d	4.07 cde	3.8 cde	3.4 cde	3.2 cde	3.0 cde
T₄	6RISO28	1	4.77 c-g	4.2 cd	4.5 a-d	4.37 a-d	4.21 a-d	4.01 a-d	4.0 a-d	4.0 a-d
T₅	6RISO28	6	4.4 e-i	4.3 cd	4.2 a-d	3.93 de	3.73 de	3.53 de	3.33 de	3.13 de
T₆	6RISO28	12	3.2 ij	3.5 de	4.1 a-d	4.1 cde	3.8 cde	3.6 cde	3.4 cde	3.3 cde
T₇	9RISO32	1	6.00 abc	5.7 a	5.43 ab	5.3 abc	5.27 ab	5.07 ab	5.01 abc	4.8 abc
T₈	9RISO32	6	5.00 c-f	4.5 bc	3.93 bcd	3.77 de	3.57 de	3.37 de	3.2 de	3.3 de
T₉	9RISO32	12	5.07 b-f	4.2 cd	4.3 a-d	4.17 a-e	4.07 a-e	3.87 a-e	3.67 a-e	3.51 a-e
T₁₀	11RISO33	1	4.97 c-f	4.7 bc	5.2 abc	4.1 cde	3.19 cde	3.09 cde	3.0 cde	2.8 cde
T₁₁	11RISO33	6	4.1 f-i	4.4 bcd	4.5 a-d	4.37 a-d	3.27 a-d	4.07 a-d	3.9 a-d	3.0 a-d
T₁₂	11RISO33	12	4.43 e-i	4.03 cd	4.63 a-d	4.23 a-e	3.03 a-e	4.83 a-e	4.63 a-d	4.0 a-e
T₁₃	8SISO4	1	3.93 f-j	3.93 cd	4.43 a-d	4.03 de	3.83 de	3.63 de	3.43 de	3.23 de
T₁₄	8SISO4	6	4.73 d-g	4.37 bcd	4.07 a-d	4.23 a-e	4.03 a-e	3.83 a-e	3.63 a-e	3.4 a-e
T₁₅	8SISO4	12	3.3 hij	3.77 cde	4.07 a-d	3.87 de	3.47 de	3.47 de	3.27 de	3.17 de
T₁₆	8SISO5	1	4.73 d-g	4.1 cd	3.63 cd	3.97 de	3.67 de	3.57 de	3.37 de	3.3 de
T₁₇	8SISO5	6	5.00 c-f	4.63 bc	5.3 abc	4.13 b-e	4.03 b-e	4.03 b-e	3.87 b-e	3.6 b-e
T₁₈	8SISO5	12	5.03 b-f	4.2 cd	3.97 a-d	3.43 de	3.23 de	3.23 de	3.01 de	2.9 de
T₁₉	14SISO14	1	3.67 g-j	4 cd	4.1 a-d	4.43 a-d	4.13 a-d	4.33 a-d	4.13 a-d	3.9 a-d
T₂₀	14SISO14	6	5.9 a-d	5.33 ab	5.23 abc	5.3 abc	5.1 abc	5.2 abc	5.1 ab	5.0 ab
T₂₁	14SISO14	12	4.63 efg	4.03 cd	3.87 bcd	4.17 a-e	3.77 a-e	4.07 a-e	3.8 a-e	3.5 a-e
T₂₂	16SISO16	1	5.4 a-e	4.23 cd	3.97 a-d	3.93 de	3.43 de	3.73 de	3.53 de	3.3 de
T₂₃	16SISO16	6	6.33 a	6.1 a	5.67a	5.4 a	5.4 a	5.4 a	5.2 a	5.1 a
T₂₄	16SISO16	12	4.73 d-g	4.6 bc	3.87 bcd	4.3 a-e	4.1 a-e	4.1 a-e	3.8 a-e	3.6 a-e
SE			0.32	0.25	0.44	0.32	0.28	0. 24	0. 32	0. 25
CV(%)			1.24	0.97	1.71	1.25	1.15	1.12	1.24	.97

Table 6

Effect of seed treatment by different antagonistic bacterial isolates on root length (cm) of maize seedlings at different storage time
Treatment	Isolate	Dipping time of seeds (hr)	Root length (cm)
			1st month	2nd month	3rd month	4th month	5th month	6th month	7th month	8th month
T₀	Control		3.93 i	5.17 e	5.23 c	5.13 d	5.03 d	4.53 d	4.23 d	4.0 d
T₁	9RISO31	1	8.3 abc	8.53 a	8.67 a	8.77 ab	8.57 ab	8.47 ab	8.27 ab	8.1a
T₂	9RISO31	6	7.07 b-f	5.93 de	6.63 abc	7.17 c	7.07 c	6.17 c	6.0 c	5.6 c
T₃	9RISO31	12	7.53 a-e	6.97 cd	7.07abc	7.07 c	6.07 c	5.37 c	5.17 c	5.0 c
T₄	6RISO28	1	7.17 b-f	6.33 cde	7.17abc	7.23 c	7.03 c	6.43 c	6.23 c	6.0 c
T₅	6RISO28	6	6.97 c-g	6.77 cd	7.43 ab	6.93 c	6.73 c	6.43 c	6.23 c	6.13 c
T₆	6RISO28	12	5.3 ghi	6.23 cde	6.87abc	6.6 c	6.5 c	6.3 c	6.1:c	6.0 c
T₇	9RISO32	1	9.17 a	8.43 ab	8.53 ab	8.83a	8.63a	8.33a	8.13a	8.0 a
T₈	9RISO32	6	7.63 abcd	6.93 cd	6.83 abc	6.3 cd	6.2 cd	6.1 cd	5.6 cd	5.4 cd
T₉	9RISO32	12	7.97 a-d	6.7 cd	7.00 abc	7.4 bc	7.3 bc	7.0 bc	6.5 bc	6.3 bc
T₁₀	11RISO33	1	7.4 b-f	6.97 cd	7.97ab	7.1 c	7.0 c	6.9 c	6.3 c	6.2 c
T₁₁	11RISO33	6	6.53 d-h	6.9 cd	7.3 abc	7.4 bc	7.2 bc	7.0 bc	6.9bc	6.5 bc
T₁₂	11RISO33	12	6.97 c-g	6.93 cd	6.73 abc	7.17 c	7.12 c	7.02 c	6.4 c	6.2 c
T₁₃	8SISO4	1	5.73 fgh	6.3 cde	7.3 abc	7.3 c	7.1 c	6.8 c	6.2 c	6.0 c
T₁₄	8SISO4	6	6.87 c-g	6.9 cd	7.07abc	7.5 abc	7.4 abc	7.3 abc	6.4 abc	5.6 abc
T₁₅	8SISO4	12	5.07 hi	6.5 cd	6.87abc	6.53 c	6.33 c	6.13 c	6.03 c	5.3 c
T₁₆	8SISO5	1	7.87 a-d	6.87 cd	6.4 bc	7.00 c	6.65 c	6.45 c	6.25 c	6.0 c
T₁₇	8SISO5	6	7.90 a-d	7.13 c	7.87ab	6.87 c	6.67 c	6.57 c	6.2 c	5.6 c
T₁₈	8SISO5	12	7.13 b-f	7.07 cd	6.73 abc	6.53 c	6.33 c	6.13 c	6.03 c	5.8 c
T₁₉	14SISO14	1	5.87 e-h	6.63 cd	6.4 bc	7.13 c	7.03 c	6.33 c	6.13 c	6.0 c
T₂₀	14SISO14	6	8.7 ab	8.8 a	8.43 ab	8.8 a	8.6a	8.4a	8.2 a	8.0 a
T₂₁	14SISO14	12	6.83 c-g	7.3 bc	6.73 abc	7.3 c	7.1 c	7.0 c	6.4 c	6.2 c
T₂₂	16SISO16	1	7.00 b-g	7.27 bc	6.73 abc	6.87 c	6.67 c	6.47 c	6.2 c	6.0 c
T₂₃	16SISO16	6	9.2a	8.87 a	8.73 a	8.83 a	8.63 a	8.53 a	8.33 a	8.1 a
T₂₄	16SISO16	12	7.3 b-f	7.23 c	6.87 abc	7.57 abc	7.47 abc	7.37 abc	7.07 abc	7.1 abc
SE			0.45	0.31	0.55	0.36	0.36	0.32	0.31	0.36
CV(%)			1.74	1.20	2.14	1.38	1.28	1.21	1.20	1.28

Like other traits, the vigor index gradually decreased in the treated seeds with endophytic with the progress of storing time (Table 7). However, T₂₃ showed the highest vigor index (1470.9 and 988.6%) at 1st and 8th month of storage followed by T₇, T₂₀ and T₁.

Table 7

Effect of seed treatment by different antagonistic bacterial isolates on the vigor index of maize seeds at different storage time
Treatment	Isolate	Dipping time of seeds (hr)	Vigor index
			1st month	2nd month	3rd month	4th month	5th month	6th month	7th month	8th month
T₀	Control		312.4 k	340.93 j	358.13 k	274.13 f	244.13 f	224.13 f	214.13 f	204.13 f
T₁	9RISO31	1	1333.5 ab	1267.70 a	1165.7 ab	1058.1 a	1008.1 a	988.1 a	968.1 a	948.1 a
T₂	9RISO31	6	854.93 efg	683.2 def	705.73 c-h	697.33 bc	627.33 bc	607.33 bc	567.33 bc	547.33 bc
T₃	9RISO31	12	303.47 k	642.67 efg	585.6 e-k	505.73 cde	405.73 cde	305.7 cde	265.73cde	245.73cde
T₄	6RISO28	1	890.93 d-g	744.27 cde	760.8 cde	650.67 bcd	610.67 bcd	510.7 bcd	480.7 bcd	420.67 bcd
T₅	6RISO28	6	545.87 ij	489.47 hi	449.07 ijk	391.2 ef	331.2 ef	301.2 ef	301.2 ef	281.2 ef
T₆	6RISO28	12	362.93jk	389.33 ij	438.53 jk	400.13 ef	380.13 ef	310.13 ef	300.13 ef	270.13 ef
T₇	9RISO32	1	1420.8 a	1272.4 a	1152.3 ab	1097.9 a	1057.9 a	1007.9 a	987.9 a	967.9 a
T₈	9RISO32	6	858.67 efg	715.87 cde	644.67 d-j	592.93 be	512.93 b-e	492.9 b-e	432.9 b-e	402.93 b-e
T₉	9RISO32	12	942.53 c-f	724.8 cde	690.8 c-i	650.53 bcd	550.53 bcd	510.5 bcd	450.5 bcd	410.53 bcd
T₁₀	11RISO33	1	989.47 cde	838.93 bc	896.27 c	718.67 b	618.67 b	578.67 b	558.67 b	538.67 b
T₁₁	11RISO33	6	892.8 d-g	859.6 bc	852.27 cd	753.33 b	703.33 b	643.33 b	623.33 b	603.33 b
T₁₂	11RISO33	12	577.33 hij	511.73 ghi	484.67 h-k	471.2 def	401.2 def	301.2 def	281.2 def	212.2 def
T₁₃	8SISO4	1	746.13 f-i	739.07 cde	751.47 c-f	650.50 bcd	550.53 bcd	510.5 bcd	480.5 bcd	460.53 bcd
T₁₄	8SISO4	6	912 c-f	841.6 bc	773.33 cde	766.93 b	716.93 b	676.93 b	656.93 b	646.93 b
T₁₅	8SISO4	12	427.6 jk	480.53 hij	496.13 g-k	431.87 ef	411.87 ef	381.87 ef	351.87 ef	331.87 ef
T₁₆	8SISO5	1	1092.4 cd	832.13 bc	721.33 c-h	704.27 bc	684.27 bc	624.27 bc	614.27 bc	601.27 bc
T₁₇	8SISO5	6	1136.1 bc	910.27 b	928 bc	717.33 b	647.33 b	627.33 b	601.33 b	560.33 b
T₁₈	8SISO5	12	678.67 ghi	571.2 fgh	509.33 f-k	453.2 def	413.2 def	353.2 def	333.2 def	313.2 def
T₁₉	14SISO14	1	799.87 e-h	793.87 bcd	731.33 c-g	741.07 b	711.07 b	641.07 b	621.07 b	601.07 b
T₂₀	14SISO14	6	1362.3ab	1261.7a	1149.3 ab	1108.5 a	1058.5 a	1007.5 a	987.5 a	967.5 a
T₂₁	14SISO14	12	963.6 c-f	846.13 bc	748.53 c-f	749.2 b	719.2 b	619.2 b	609.2 b	589.2 b
T₂₂	16SISO16	1	991.33 cde	858.13 bc	741.2 c-f	689.33 bc	609.33 bc	559.33 bc	539.33 bc	519.33 bc
T₂₃	16SISO16	6	1470.9a	1320.5 a	1191.7 a	1129.6 a	1109.6 a	1058.6 a	1008.6 a	988.6 a
T₂₄	16SISO16	12	961.73 c-f	850.53 bc	731.87 c-g	759.2 b	659.2 b	629.2 b	609.2 b	520.2 b
SE			59.05	37.302	63.22	52.22	48.22	42.22	37.22	42.2
CV(%)			228.19	144.15	244.31	201.78	144.78	134.78	144.78	134.78

Evaluation of selected endophytic and rhizospheric bacteria in reducing Fumonisin B1 in stored maize seeds

Maize seeds showing infection by Fusarium were subjected to treat with different endophytic bacteria and stored for 8 months. During storing mycotoxin (Fumonisin B1) was quantified after 8 months after storage to assess the toxin suppression by different endophytic bacteria following ELISA method (Table 8). All four selected endophytic bacteria significantly reduce Fumonisin B1 compared to untreated control. The highest Fumonisin concentration was observed in untreated control (8.8 ppm), whereas the lowest Fumonisin was found in T₂₃ (4.5 ppm).

Table 8

Quantification of mycotoxin (Fumonisin B1) in maize seed using ELISA kit after eight months of storage
Treatment	Isolate ID	Seed dipping time (hr)	Concentration	Value
Control	-	-	8.8 ppm	3.5-4 ppm
T₁	9RISO31	6	4.8 ppm	3.5-4 ppm
T₇	9RISO32	6	4.6 ppm	3.5-4 ppm
T₂₀	14SISO14	12	4.9 ppm	3.5-4 ppm
T₂₃	16SISO16	12	4.5 ppm	3.5-4 ppm

Microorganisms living in close association with crops can be effective biocontrol agents because they provide the first line of defense. Maize is an important cereal crop after rice and can be cultivated in a wide range of climatic zones (Alam et al. 2018; Rahman and Rahman 2014) but it is highly susceptible to various post-harvest diseases and development of mycotoxin can cause havoc in animal and poultry sector (Zain 2011; Katole et al. 2013). Fungal diseases including molds may cause up to 5% grain loss in post-harvest conditions (Alam et al. 2018). Goko et al. (2021) found that maize seeds can be heavily infected by various seed-borne fungi viz: several species from Aspergillus genus, i.e.: Aspergillus flavus, A. parasiticus, A.niger, A. tamarii along with other pathogens like, Penicillium citrinum, Fusarium moniliforme, Rhizopus stolonifer etc. Among them seed-borne Fusarium is a serious problem for causing spoilage and development of mycotoxin like Fumonisisn B1 (Lizárraga-Sánchez et al. 2015). Therefore, reducing deterioration of seeds and avoiding mycotoxins in the storage is a big challenge in a hot and humid country like Bangladesh. This research was therefore aimed to isolate the potential rhizospheric and endophytic bacteria for suppressing various mycotoxigenic fungi especially Fusarium during the post-harvest period.

In total 48 rhizospheric and endophytic bacterial isolates were obtained either from rhizosphere soil or from the maize roots that were evaluated for their potentiality to suppress the mycelial growth of Fusarium oxysporum. Four rhizospheric and four endophytic bacteria showed superior antagonistic properties against Fusarium oxysporum in vitro out of 27 and 21 rhizospheric and endophytic bacteria respectively. For molecular identification of the effective bacterial isolates and phylogenetic study the DNA were amplified using 16S rRNA primers followed by sequencing. This molecular study revealed that the effective bacteria have close similarity with Bacillus. cereus, B. amyloliquefaciens, B. subtilis, B. velezensis and B. zanthoxyli and Bacillus tropicus. Ali et al. (2020) showed in their study that various Bacillus sp. possess the capacity to produce various extracellular and secondary metabolites like lytic enzymes (cellulose, chitinase, lipase, protease) which break the glycosidic linkage of the cell wall of phytopathogens, volatile antibiotics, siderophore, HCN, ACC deaminase which help in boosting their antifungal property. Isolate 16SISO16, 9RISO31, 9RISO32 and 14SISO14 showed superior performances showing IAA and PO₄ solubilization test compared to other bacteria and untreated control in promoting growth and vigor of seedlings as well as reducing infection of seeds at different storage duration. B. amyloliquefaciens, B. cereus and B. subtiliswere reported to produce growth hormones like auxin, indole acetic acid (IAA), siderophore, solubilize phosphate and fix nitrogen (Luo et al. 2022; Ali et al. 2020; Kulkova et al. 2023) which eventually helps plant growth, convert insoluble phosphorus into available forms to plant. Moreover, IAA is an important phytohormone that regulates various important traits like seed germination, and improving plant growth (Egamberdieva, 2008). Simultaneously endophytic bacteria also enhance root surface area and absorption of nutrients (Ali et al. 2020).

A report by Khan et al. (2007) also suggests that phosphate solubilizing bacteria help plants by turning insoluble phosphate into solubilized forms thus reducing nutrient deficiency. Martínez-Viveros et al. (2010) highlighted the importance of siderophore to sequestrate iron (Fe³⁺) and make it unavailable to phytopathogen. Further, siderophore is reported to inhibit the growth of several species of Aspergillus, Fusarium and Pythium and stimulate the growth of root and shoot (Ali and Vidhale 2013; Ahmed and Holmström 2014; Sulochana et al. 2014; Trapet et al. 2016). Besides mechanisms like the production of lytic enzymes, volatile antibiotics, and siderophore, HCN plants also receive the advantage of induced resistance when treated with antagonistic bacteria (Halfeld-Vieira 2006). B. cereus, B. amyloliquefaciens, B. subtilis were also reported to produce antifungal metabolites like fengycin which interacts with phospholipids and sterols in cell membranes of fungi and eventually alters the structure and permeability of membrane (Deleu et al. 2005; Tang et al. 2014) along with bacillomycin, iturin, surfactin production which suppresses fungal growth, eventually deteriorating their toxin production (Kulkova et al. 2023; Tsuge et al. 2005; Zhao et al. 2013). Therefore, these potential endophytic bacteria are excellent candidates for commercial formulation of biopesticide and biofertilizer to reduce storage loss of maize seeds and minimizing Fumonisin toxin contamination in a environmentally safe way.

Acknowledgements: We would like to thank the authority of Department of Plant pathology, and Interdisciplinary Institute for Food Safety, Bangladesh Agricultural University for providing technical support

Author contribution: Md. Atiqur Rahman Khokon conceptualizes the project. Material preparation and data collection were carried out by all authors. The first draft of the manuscript was written by Md. Shariful Islam, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding: The project was funded by the University Grants Commission (UGC) of the Peoples’ Republic of Bangladesh (Grant no. 2022/22/UGC).

Data availability: All data generated or analyzed during this study were included in this manuscript.

Ethics approval and consent to participate: Not applicable.

Consent for publication: Consent was obtained from all individual participants included in this study.

Conflict of interest: The authors declare no competing interests

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No competing interests reported.

Bacillus cereus (OR975563) of maize rhizosphere can promote plant growth and potentially suppress mycotoxin (Fumonisin B1) during storage

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Isolation of rhizospheric and endophytic bacterial isolates

Preparation of pure culture of Fusarium oxsporum

Assessing antagonistic ability of bacterial isolates against Fusarium oxysporum

Molecular identification of bacterial isolates

Evaluation of selected bacteria for seed treatment

Determination of Toxin (Fumonisin) from treated maize seed using ELISA kit

Results

Identification of antagonistic bacterial isolates using sequenced-based molecular technique and phylogenetic analyses

Effect of selected antagonistic endophytic bacteria on different parameters of maize

Evaluation of selected endophytic and rhizospheric bacteria in reducing Fumonisin B1 in stored maize seeds

Discussion

Declarations

References

Additional Declarations

Status:

Version 1