Next Article in Journal
Study on Regulation Mechanism of Tomato Root Growth in Greenhouse under Cycle Aerated Subsurface Drip Irrigation
Previous Article in Journal
Effects of Planting Density, Levels, and Forms of Nitrogen Application on the Yield and Nitrogen Utilization of Wheat following Rice in East China
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 12
Issue 11
10.3390/agronomy12112608
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Potential Native Bacilli Reduce Fumonisin Contamination in Maize

by
Mamuna Mahjabin Mita
1,
Muhtarima Jannat
1,
Samrin Bashar
1,
Ismam Ahmed Protic
1,
Plabon Saha
1,
Md. Mostafa Masud
1,
Rummana Islam
1,2,
Nazia Binta Islam
1,
Md. Zahangir Alam
1 and
Md. Rashidul Islam
1,*
1
Plant Bacteriology & Biotechnology Laboratory, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
2
Bangladesh Agricultural Research Institute, Joydebpur, Gazipur 1701, Bangladesh;
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(11), 2608; https://doi.org/10.3390/agronomy12112608
Submission received: 29 August 2022 / Revised: 19 September 2022 / Accepted: 26 September 2022 / Published: 24 October 2022
Browse Figures
Review Reports Versions Notes

Abstract

:
In Bangladesh, Fusarium proliferatum is a prevalent pathogen of maize plants that poses a significant threat to human health and livestock by producing carcinogenic fumonisin. However, the use of the Bacillus species, which can colonize the infected plant parts, has been practiced globally to mitigate fumonisin contamination in maize. In this study, B. subtilis MMM1, a native isolate from the maize rhizosphere, was found to be the most effective antagonist against F. proliferatum, which reduced their mycelial growth and total fumonisin accumulation by 85.31% and 85.12%, respectively, over control through a fivefold reduction in fungal population (1.1 × 104 CFU/g maize grain) in vitro. Furthermore, out of the five bacilli used in this study, B. subtilis MMM1 was able to increase the root and, shoot length, and the vigor index of maize seedlings, by colonizing the fresh roots at 82 × 106 CFU/g root tissue. This suggests that B. subtilis MMM1 might be put forth both as a biocontrol agent and a plant growth promoter in Bangladesh to combat F. proliferatum. Nevertheless, evaluation of its efficacy in reducing fumonisin contamination in maize should be investigated under field conditions.

Graphical Abstract

1. Introduction

Maize (Zea mays L.) (family Poaceae, tribe Andropogoneae) has become one of the most popular cereal crops in Bangladesh due to the fact that 55% of maize here is used for poultry feed, 40% for fish feed, and only 5% for cattle feed [1,2]. However, Fusarium spp. causes severe yield losses and accumulates a wide variety of harmful secondary metabolites in the maize grain known as fumonisins [3]. From a total of 15 major maize growing areas in Bangladesh, Jannat et al. observed that fumonisin levels in samples from 10 districts were above the permissible limit, with a maximum (9.18 mg/kg) level [4].
The primary cause for concern when it comes to maize diseases is that fumonisins found in animal feed samples collected from South Asia, including Bangladesh, indicate that maize-based feed is contaminated [5,6] and this endangers public health and livestock causing severe diseases viz. pulmonary edema and hydrothorax, leukoencephalomalacia, esophageal cancer, and neural tube defects [7,8,9,10]. Several research studies have found evidence of mycotoxins in pregnant women and breast milk [6,11], the subsequent exposure of breastfed children [12], and a negative impact on children’s growth [13].
The current methods of reducing fumonisin levels in maize include the use of synthetic fungicides and photocatalytic detoxification [14,15], the introduction of resistant traits and Cry proteins from Bacillus thuringiensis (Bt) into agronomically valuable maize cultivars by breeding [16,17], and a cultural shift to avoid the pre- and post-harvest attack of fungi [18]. All strategies, however, have consistently proven challenging to utilize. Hence, there is a high demand for biological control methods because of their ability to reduce the primary fungal soil inoculum and the infection rate in the root systems of maize plants [19,20].
Several rhizobacteria have so far demonstrated significant anti-fungal activities toward Fusarium species on maize plants [21,22,23]. Various Bacillus strains reduced fumonisin contamination and mycelial growth of Fusarium species in maize grains, and it was reported as an effective biocontrol agent offering rhizosphere colonization and controlling root diseases [24,25,26]. In Vitro reduction of Fumonisin B1 (a family of toxins known as fumonisins) accumulation with Bacillus species during vertical and horizontal transmission of pathogens has been reported [27,28]. This suggests that using a biocontrol agent, such as Bacillus species, might be a viable alternative to combat F. proliferatum. Nevertheless, the present study was designed to identify the beneficial bacteria from the maize rhizosphere and evaluate the efficacy of rice, maize, and potato-associated Bacillus species, antagonistic to fumonisin-producing F. proliferatum in reducing fumonisin accumulation in maize by in vitro assay. We also aimed to assess the root colonization of these antagonistic Bacillus species in maize, as well as their potential in enhancing the growth of maize seedlings.

2. Materials and Methods

2.1. Bacterial Isolates and Culture Condition

Bacillus species viz. isolate BDISO76MR (B. subtilis) was isolated in this study from maize rhizosphere. BDISO01RR (B. amyloliquefaciens) from rice rhizosphere [29], three isolates from potato rhizosphere BDISO36PR (B. subtilis), BDISO45PR (B. subtilis) and BDISO49PR (B. subtilis) were used in this study [30]. Isolates of bacteria were cultured and maintained in Luria Bertani (LB) media.

2.2. Isolation and Purification of Bacterial Isolates from Maize Rhizosphere

Rhizospheric soil samples were taken from 15 different regions in Bangladesh significant for maize production. Bacteria were isolated from maize rhizosphere soil following concentration up to 10−5 by dilution plate technique. Then, 50 µL of the diluted solution was spread on an LB agar medium and left for two days at 28 °C for incubation. Bacterial colonies grown on the plate were then purified by transferring them into new plates using a sterile toothpick.

2.3. In Vitro Growth Inhibition of F. Proliferatum by Bacterial Isolates

Mycotoxigenic F. proliferatum strains were used as identified by Jannat et al. [4]. The dual culture assay was then followed for screening the potential bacteria. Isolates of bacteria were streaked on potato dextrose agar (PDA) plates using a loop forming a triangular shape. Then, an 8 mm agar block of F. proliferatum was mounted in the middle of the PDA plates. The plates were then incubated at 28 °C for up to 7 days. Plates with F. proliferatum only were used as untreated control, and plates with F. proliferatum and B. subtilis that did not inhibit the growth of F. proliferatum were used as a negative control. Data on the mycelial growth inhibition of mycotoxigenic F. proliferatum were recorded and expressed in percentage over control.

2.4. Identification of Bacillus Species from Maize Rhizosphere

Bacterial isolate BDISO76MR was grown on 5 mL LB broth for 18 h at 28 °C, and Wizard® Genomic DNA purification Kit (Promega, Madison, WI, USA) instructions were followed for the DNA extraction. The primer sets 27F (5′-AGAGTTTGATCMTGG CTCAG-3′) and 1518R (5′-AAGGAGGTGATCCANCCRCA-3′) and PCR conditions were used according to Rahman et al. for 16S rDNA amplification [29]. PCR-amplified 16S rDNA fragments were subjected to partial nucleotide sequencing using 27F primer in the Macrogen Lab, South Korea, via Biotech Concern Bangladesh. The DNA sequences were compared using the BLAST (Basic Local Alignment Search Tool) program to identify their closest relatives.

2.5. Identification of Potential F. Proliferatum by In Vitro Inoculation

2.5.1. Inoculum Preparation of F. Proliferatum

F. proliferatum was grown on PDA plates for seven days and the conidia were harvested with the help of a spatula in sterile water. The suspension of conidia was then filtered through a sterile Cheech cloth. The number of conidia up to 106 spores mL−1 was determined using a Haemacytometer (Marienfeld-Superior).

2.5.2. In Vitro Inoculation of F. Proliferatum with Maize Grain

Maize grain samples were prepared as described previously by Cavaglieri et al. [23]. Briefly, 70 g maize kernel was measured and autoclaved in the 250 mL Erlenmeyer flask for 60 min at 121 °C. The moisture of autoclaved samples was adjusted to 25%. The maize grains were then inoculated with 1 mL of F. proliferatum (1 × 106 spores mL−1) in vitro. Flasks with 70 g of maize grains and only 1 mL of water were used as control (uninoculated maize). In an incubation chamber, flasks were incubated at 25 °C for 35 days. The total fumonisin content was determined by removing three replications of each treatment after 35 days. The fungal isolate (BD Fusarium_4) which produced maximum fumonisin concentration was used for further in vitro inoculation of maize grains and colonization experiments.

2.6. Co-i of Maize Grains with F. Proliferatum and Different Bacillus Species

Maize grain samples prepared as mentioned above were then co-inoculated with 1 mL of the selected potential F. proliferatum (1 × 106 spores mL−1) and 1 mL (1 × 108 cells mL−1) of each Bacillus species. However, the inoculant used on the 70 g sterile maize grains with 1 mL of the strongest F. proliferatum (BD Fusarium_4) in terms of fumonisin production, served as control. Erlenmeyer flasks were then incubated for 35 days at 25 °C in a chamber. Three replicates per treatment were maintained and samples were taken at intervals of 10, 20, and 35 days, for the determination of total fumonisin concentration and the counting of F. proliferatum colony-forming units (CFUs).

2.7. Determination of Total Fumonisin

Total fumonisin concentration in co-inoculated maize grains was detected three times with three replications according to the Celer® FUMO ELISA Test Kit (Eurofins Technologies, Budapest, Hungary) following the manufacturer’s instructions. For this, each representative sample was grounded with a blender and sieved. A clean conical flask was used to weigh out 20 g of ground sample. Then, 100 mL of 70% methanol extraction solution was added to each ground sample. Three minutes of shaking and settling followed the sealing of the flasks, and the extract was filtered to remove the top layer through a Whatman #1 filter paper. Finally, the distilled water was added to the extract at a 1:20 ratio for conducting the Enzyme-linked Immunosorbent Assay (ELISA) by the Celer® FUMO ELISA Test Kit (Eurofins Technologies, Budapest, Hungary) to measure the absorbance at 450 nm.

2.8. Detection of Colony-Forming Units (CFUs) of F. proliferatum

10 g maize grains were blended and added to 90 mL of 0.1% peptone water solution [22]. A series of dilutions from 10−2 to 10−4 was done and three replications of 0.1 mL of concentration were spread on Nash–Snyder agar plates. For seven days, the plates were incubated at 28 °C for the count of total colony-forming units (CFUs).

2.9. Assessment of Plant Growth Promotion

2.9.1. Preparation of Suspension and Seed Treatment

Five bacterial isolates viz. BDISO01RR, BDISO36PR, BDISO45PR, BDISO49PR, and BDISO76MR, were cultured on LB agar medium for 48 h at 28 °C. Then, 2–3 loops of each isolate were taken in five separate Erlenmeyer flasks containing 100 mL sterile water, and cell densities were adjusted to 1 × 108 cells/mL. Flasks with distilled water only were used as control. After that, 30 maize seeds were submerged in each flask with 100 mL of different bacterial suspensions. Flasks with sterilized maize grains and 100 mL of sterile water only were used as an untreated control; none of the aforementioned bacterial isolates were used here. Flasks were then incubated at 25 °C on a rotatory shaker at 70 rpm for 2 h to enable the adherence of bacterial cells to seeds. The excess inoculum was discarded following incubation and the seeds for the pot experiment were immediately sown.

2.9.2. Growth Promotion and Root Colonization Assay

Before sowing the treated seeds, the soil was sterilized with 4% formalin solution @1 cft/500 mL and mixed with the spade, and covered with black plastic sheets for 48 h to ensure that no other bacteria were present in the roots. After 7 days, the plastic sheets were removed and the soil was spade again and left for 2 weeks before potting. The treated seeds were then sown in the sterile soils that had been prepared in plastic pots. For the growth promotion assay, three seeds were sown in each pot and eight replications were maintained for six treatments. The maize seedlings were periodically uprooted at 7, 14, and 21 days after sowing (DAS), respectively, to measure the vigor, root length, and shoot length. The vigor index (VI) was calculated using the following formula:
Vigor Index (VI) = (Mean root length + mean shoot length) × germination (%)
On the other hand, root colonization of each Bacillus species was assessed by surface sterilizing 1 g of freshly harvested roots for each treatment with 1% Clorox for 1 min and ground with 1 mL sterile water to recover the desired bacteria. The suspension was then serially diluted up to 10−8 concentrations. Later, 50 µL of this suspension was sprayed on LB agar media containing rifampicin, so that only rifampicin-resistant desired bacteria could survive. Incubation of the plates at 28 °C for 24 to 48 h was then followed. Data on colony-forming units of each Bacillus species per gram of maize root tissue were taken after 21 days of sowing.

3. Results

3.1. Identification of Potential F. Proliferatum

Eight previously identified fungal isolates were taken in this study for identifying the potential F. proliferatum. The concentration of fumonisin produced by F. proliferatum ranged from 2.03 to 9.16 ppm. Isolate BD Fusarium_4 was identified as a potential candidate for producing maximum (9.16 ppm) fumonisin which is 78.05% over control. (Table S1). Thus, BD Fusarium_4 as the strongest fumonisin producer was used in determining the reduction of fumonisin production by different Bacillus species and root colonization studies in vitro.

3.2. In Vitro Antagonistic Activity of Bacillus Species against F. Proliferatum

At 4 DAI, the minimum (9.25 mm) mycelial growth was observed in the case of BDISO76MR (B. subtilis) which was statistically similar to BDISO01RR (B. amyloliquefaciens) (12 mm) and BDISO45PR (B. subtilis) (12.25 mm) reducing the mycelial growth 70.21%, 61.15%, and 60.52%, respectively over control (Table 1). The maximum (18 mm) mycelial growth was observed by BDISO49PR (B. subtilis), which was statistically similar to BDISO36PR (B. subtilis) contributing the lowest (14 mm) to the mycelial growth reduction by 41.88% and 54.84%, respectively, over control (Table 1, Figure 1).
On the other hand, at 7 DAI, the minimum (11.75 mm) mycelial growth was observed in the case of BDISO76MR (B. subtilis) which was statistically similar to BDISO01RR (B. amyloliquefaciens) (12 mm) and BDISO45PR (B. subtilis) (12.25 mm) reducing the mycelial growth 85.31%, 85%, and 84.69%, respectively, over control (Table 1; Figure 1). However, among the bacterial species, the maximum (18 mm) mycelial growth was observed by BDISO49PR (B. subtilis), which was statistically similar to BDISO36PR (B. subtilis) (14 mm) contributing the lowest to the mycelial growth reduction by 77.5% and 82.5%, respectively, over control (Table 1, Figure 1).

3.3. PCR-Based Identification of Bacillus Species from Maize Rhizosphere

Two hundred bacterial isolates were tested against F. proliferatum for in vitro growth inhibition. Out of these, only one bacterial isolate (BDISO76MR) was found effective in reducing the growth inhibition of F. proliferatum (Figure 2A). This isolate was then confirmed by sequencing PCR products (Figure 2B) of 16S rDNA. Sequenced analysis by BLAST homology search revealed that BDISO76MR showed the highest (100%) homology with B. subtilis MMM1 (accession number OP179630) followed by 98.11% homology with B. subtilis strain PB1 (accession number KU904502.1) (Table S2). This result indicates that BDISO76MR obtained from maize rhizosphere that inhibited the growth of F. proliferatum was B. subtilis MMM1 (Table S2). All these experiments were repeated two times.

3.4. Effect of Bacillus Species on the Reduction o Fumonisin Accumulation in Co-Inoculated Maize Grains

At 10 DAI, the minimum (0.9 ppm) fumonisin concentration was quantified when maize grains were co-inoculated with BDISO76MR (B. subtilis MMM1) and F. proliferatum, which was statistically similar to BDISO36PR (B. subtilis) (1.7 ppm) (Table 2) and reduced the fumonisin concentration by 81.74% and 65.52%, respectively, over positive control (Table 2), while the maximum (3.72 ppm) fumonisin concentration was observed by BDISO49PR (B. subtilis), which were statistically similar with BDISO45PR (B. subtilis) (3.42 ppm) and BDISO01RR (B. amyloliquefaciens) (3.15 ppm) (Table 2) contributing the lowest to the reduction of fumonisin concentration by 24.54%, 30.63%, and 36.11%, respectively, over positive control (Table 2).
At 20 DAI, the minimum (1.29 ppm) fumonisin concentration was measured in BDISO76MR (B. subtilis MMM1) when maize grains were co-inoculated with F. proliferatum followed by BDISO49PR (B. subtilis) (1.36 ppm), which were statistically similar to BDISO36PR (B. subtilis) (1.69 ppm) and BDISO01RR (B. amyloliquefaciens) (3.11 ppm) (Table 2) reducing the fumonisin concentration 76.81%, 75.48%, 69.56%, and 44.12%, respectively, as compared to a positive control (Table 2). However, the maximum (3.42 ppm) fumonisin concentration was observed by BDISO45PR (B. subtilis) (Table 2), which contributed the lowest to the reduction of fumonisin concentration by 38.53% over positive control (Table 2).
At 35 DAI, when maize grains were co-inoculated with F. proliferatum, the minimum (3.02 ppm) fumonisin concentration was determined in BDISO76MR (B. subtilis MMM1), which was statistically similar to BDISO01RR (B. amyloliquefaciens) (3.11 ppm) (Table 2) that reduced the fumonisin concentration by 85.12% and 84.70%, respectively, over positive control (Table 2), while the maximum (5.41 ppm) fumonisin concentration was observed by BDISO49PR (B. subtilis), which were statistically similar with BDISO45PR (B. subtilis) (4.29 ppm) and BDISO36PR (B. subtilis) (3.38 ppm) (Table 2) and contributed the lowest to the reduction of fumonisin concentration by 73.36%, 78.90%, and 83.36%, respectively, over positive control (Table 2).

3.5. Influence of Bacillus Species on the Reduction of F. proliferatum Population in Co-Inoculated Maize Grains

The total counts for colony-forming units (CFUs) of F. proliferatum were made at 10 DAI (Figure S1A), 20 DAI (Figure S1B), and 35 DAI (Figure S1C).
At 10 DAI, the minimum (7.2 × 104 CFU/g maize grain) CFU of F. proliferatum was determined when maize grains were co-inoculated with BDISO36PR (B. subtilis) followed by BDISO49PR (B. subtilis) (8.3 × 104 CFU/g) and 8.8 × 104 CFU/g for both BDISO45PR (B. subtilis) and BDISO76MR (B. subtilis MMM1) over positive control (9.4 × 104 CFU/g). However, BDISO01RR (B. amyloliquefaciens) (12.2 × 104 CFU/g) could not reduce the population of F. proliferatum when maize grains were co-inoculated with the fungus after 10 days of incubation (Table 3).
At 20 DAI, the minimum (4.4 × 104 CFU/g maize grain) CFU of F. proliferatum was measured when maize grains were co-inoculated with both BDISO45PR (B. subtilis) and BDISO76MR (B. subtilis MMM1) followed by BDISO49PR (B. subtilis) (6.6 × 104 CFU/g) as compared to a positive control (10 × 104 CFU/g). On the other hand, after 20 days of incubation, BDISO36PR (B. subtilis) (16.1 × 104 CFU/g) and BDISO01RR (B. amyloliquefaciens) (13.3 × 104 CFU/g) were not able to reduce the fumonisin population over positive control when maize grains were co-inoculated with the fungus (Table 3).
At 35 DAI, the minimum (1.1 × 104 CFU/g maize grain) number of CFU of F. proliferatum was determined when maize grains were co-inoculated with all the bacterial isolates as followed by BDISO01RR (B. amyloliquefaciens), BDISO36PR (B. subtilis), BDISO45PR (B. subtilis) and BDISO49PR (B. subtilis) and BDISO76MR (B. subtilis MMM1) and reduced the population by five times compared with the positive control (5.5 × 104 CFU/g) (Table 3).

3.6. Correlation between Fumonisin Concentration and Population of F. Proliferatum

A strong positive correlation between total fumonisin concentration (ppm) and the population of F. proliferatum (CFU/g maize grain) was observed (Figure 3A). The estimate showed that for a minimum (10,000 CFU/g maize grain) increase in F. proliferatum population, there was a total fumonisin concentration increase of 4 ppm (Figure 3A). On the other hand, for a maximum (50,000 CFU/g maize grain) increase in F. proliferatum population, a 22.22 ppm increase in total fumonisin concentration was observed 35 days after inoculation (Figure 3A).
As the graph shows, a strong negative correlation was observed between the reduction of fumonisin concentration (%) and the population of F. proliferatum (CFU/g maize grain) (Figure 3B). The result revealed that for a minimum (10,000 CFU/g maize grain) increase in the population of F. proliferatum, an 18 ppm decrease of reduced fumonisin concentration was observed 35 days after inoculation (Figure 3B). On the other hand, for the maximum (55,000 CFU/g maize grain) increase in F. proliferatum population, a 99 ppm reduction of reduced fumonisin concentration was observed (Figure 3B).

3.7. Root Colonization of Different Bacillus Species in the Maize Rhizosphere

Rifampicin-resistant bacteria with similar morphology of BDISO76MR (B. subtilis MMM1), BDISO01RR (B. amyloliquefaciens), BDISO36PR (B. subtilis), BDISO45PR (B. subtilis), and BDISO49PR (B. subtilis), were recovered after dilution plate technique on LB media (Figure 4). Colonization was also confirmed by counting the colony-forming units per gram of root tissue (Table 4). The results revealed that the maximum colonization (82 × 106 CFU/g root tissue) of BDISO76MR (B. subtilis MMM1) was recorded followed by BDISO49PR (B. subtilis) (51.6 × 106 CFU/g root tissue), BDISO45PR (B. subtilis) (26.3 × 106 CFU/g root tissue) and BDISO01RR (B. amyloliquefaciens) (15.3 × CFU/g root tissue), while the minimum (3 × 106 CFU/g root tissue) colonization of BDISO36PR (B. subtilis) was recorded in root tissue of maize seedlings (Table 4).

3.8. Influence of Different Bacillus sp. on Average Root and Shoot Length and Vigor Index

At 7 DAS, the maximum (23 cm) root length was observed when maize seeds were treated with BDISO01RR (B. amyloliquefaciens) followed by BDISO45PR (B. subtilis) (18.17 cm), BDISO49PR (B. subtilis) (18.17 cm), BDISO36PR (B. subtilis) (18 cm), and BDISO76MR (B. subtilis MMM1) (15.67 cm) (Table 5). On the other hand, at 7 DAS, maize seeds treated with BDISO36PR (B. subtilis) showed a maximum (13.17 cm) shoot length followed by BDISO45PR (B. subtilis) (9.83 cm), BDISO49PR (B. subtilis) (9.17 cm), BDISO01RR (B. amyloliquefaciens) (8.83 cm), and BDISO76MR (B. subtilis MMM1) (8 cm) (Table 5). At 7 DAS, the maximum (3183.33) vigor index was recorded when maize seeds were treated with BDISO01RR (B. amyloliquefaciens) followed by BDISO36PR (B. subtilis) (3116.67), BDISO45PR (B. subtilis) (2800), BDISO49PR (B. subtilis) (27.33.33), and BDISO76MR (B. subtilis MMM1) (2366.67) (Table 5).
At 14 DAS, maize seeds treated with BDISO76MR (B. subtilis MMM1) demonstrated the maximum (34.17 cm) root length which was statistically similar to BDISO01RR (B. amyloliquefaciens) (34 cm), BDISO36PR (B. subtilis) (31.5 cm), and BDISO49PR (B. subtilis) (31.17 cm) (Table 5). The minimum (19.83 cm) root length was observed for the isolate BDISO45PR (B. subtilis) (Table 5). For shoot length at 14 DAS, the maximum (47.67 cm) shoot length was observed when maize seeds were treated with BDISO01RR (B. amyloliquefaciens), which was statistically similar to BDISO76MR (B. subtilis MMM1) (45 cm), BDISO49PR (B. subtilis) (43 cm), and BDISO36PR (B. subtilis) (40.33 cm), while the minimum (26.83 cm) shoot length for maize seedlings was observed in the seeds treated with BDISO45PR (B. subtilis) (Table 5). In the case of vigor index at 14 DAS, maize seeds treated with BDISO01RR (B. amyloliquefaciens) showed maximum (8166.67) vigor index followed by BDISO76MR (B. subtilis MMM1) (7916.67), BDISO49PR (B. subtilis) (7416.67), BDISO36PR (B. subtilis) (7183.33), and BDISO45PR (B. subtilis) (3888.73) (Table 5).
At 21 DAS, maize seeds treated with BDISO76MR (B. subtilis MMM1) showed the highest (39.67 cm) root length followed by BDISO45PR (B. subtilis) (38.83 cm), BDISO49PR (B. subtilis) (34.67 cm), BDISO1RR (B. amyloliquefaciens) (34.67 cm), and BDISO36PR (B. subtilis) (34 cm) (Table 5) increasing the root length 25.2%, 21.55%, 10.66%, 7.3%, and 8.19%, respectively, over untreated control (Supplementary Figure S2A). However, BDISO49PR (B. subtilis) exhibited the highest (60 cm) shoot length followed by BDISO45PR (B. subtilis) (58.8 cm), BDISO01RR (B. amyloliquefaciens) (55.67 cm), BDISO76MR (B. subtilis MMM1) (53.67 cm), and BDISO36PR (B. subtilis) (53.17 cm), after 21 days of seed sowing (Table 5), which increases the shoot length 40.39%, 36.36%, 30.68%, 24.9%, and 35.74%, respectively, when compared to the untreated control (Figure S2B). In the case of vigor index, at 21 DAS, BDISO49PR (B. subtilis) exhibited the maximum (9466.67) vigor, followed by BDISO76MR (B. subtilis MMM1) (9333.33), BDISO01RR (B. amyloliquefaciens) (9033.33), BDISO36PR (B. subtilis) (8716.67), and BDISO45PR (B. subtilis) (8138.89) (Table 5), increasing the vigor index 55.79%, 50%, 43.16%, 49.47%, and 26.32%. respectively, over untreated control (Figure S2C).

4. Discussion

Native bacterial species of Bacillus associated with potato, rice, and maize roots, were selected for this study in order to examine how different rhizosphere isolates affect the maize F. proliferatum. The most powerful tool for the study of the antagonistic activity of Bacillus species against different Fusarium species is considered to be the dual culture assay [31,32]. In the current study, bacterial isolates from the maize rhizosphere were first screened using this approach for in vitro antagonism towards F. proliferatum revealing only one effective bacterial isolate BDISO76MR (B. subtilis MMM1), which inhibited the growth of F. proliferatum. However, it is worth noting that Figueroa et al. proposed a combination of the liquid antagonistic assay (PDB) with the conventional dual-culture technique, because PDB requires less time to find a potential biocontrol agent than the latter one [33].
The findings of this study clearly indicate that BDISO76MR (B. subtilis MMM1) was the most effective in vitro at inhibiting fungus growth and toxin production. A recent study involving F. udum with Pseudomonas sp. and Bacillus sp. demonstrated a significant reduction of mycelial growth and biomass, and mycotoxin accumulation in vitro [25]. Similar findings were found as reported antagonistic activity of Bacillus species inhibited the radial mycelial growth of various Fusarium species significantly [23,34]. This growth inhibition might be because of the antifungal activity of highly stable lipopeptides viz. iturin from B. subtilis and fengycin produced by B. amyloliquefaciens present in Bacillus genome, which was reported to inhibit fungal mycelial growth by explosive lysis and significant alterations to their secondary metabolism accompanied with hyphal swelling [35,36].
All Bacillus species were tested in this study for their ability to minimize the fumonisin accumulation in maize grain co-inoculated with F. proliferatum. Lizárraga et al. reported that B. cereus from maize rhizosphere is antagonistic to F. verticillioides, resulting in a 93.9% reduction in fumonisin production [37]. This study, on the other hand, determined the lowest fumonisin concentration after 35 days of inoculation. BDISO76MR (B. subtilis MMM1) was found to be highly effective and inhibited the production of fumonisin concentration by 85.12% when they were co-inoculated with F. proliferatum compared to the control. These findings are in accordance with that reported from other fumonisins reduction antagonists, such as B. subtilis, and Serratia marcescens [24,38].
The result of this work also revealed that BDISO76MR (B. subtilis MMM1) significantly inhibited F. proliferatum root colonization in maize at 21 DAS. This observation agrees with previous results about the colonization of different Fusarium sp. On maize root systems [23,39]. It is possible that competition for resources and direct antagonistic interaction involving the secretion of antifungal metabolites by Bacillus are to blame for the decline in fungal colonization [40]. However, the antagonistic relationship between Burkholderia cepacia and F. proliferatum in maize was poorly correlated in vitro [41]. Conversely, this research revealed that the population of F. proliferatum (CFU/g maize grain) was strongly and positively correlated with the total fumonisin concentration (ppm), and inversely correlated with the reduction of fumonisin concentration (%).
As B. subtilis was reported to protect the plant from microbial pathogens through colonizing roots and forming a stable biofilm [42], another focus of this experiment was to assess the root colonization of distinct Bacillus species in the maize rhizosphere. In our study, we found significant colonization of all Bacillus species on the maize root tissues when maize grains were co-inoculated with F. proliferatum. However, BDISO76MR (B. subtilis MMM1) colonized maize roots with high populations of 82 × 106 CFU/g root tissue and demonstrated the most uniform increase in growth parameters of maize seedlings after 21 days of sowing. Earlier reports of this phenomenon can be found in Ndeddy and Babalola [43]. These rhizospheric bacteria increase hormone production like gibberellic acid and indole-3-acetic acid-like, while minimizing the level of ethylene, during colonization, thereby promoting plant growth and suppressing pathogenic fungi [44]. This allows Bacillus sp. to reduce the disease incidence in crops like stalk and ear rot of maize [37], and Verticillium wilt of cotton as well [45]. It appears that rhizospheric bacteria safeguard the plant’s above-ground parts from fungal pathogens in a way that may be caused either by (i) rapid root colonization of bacteria and consequently the movement of pathogens away from the site of infection [46], or (ii) stimulation of systemic induced resistance mechanism in plants, which ultimately leads to the production of compounds with a defensive function [47,48].

5. Conclusions

To conclude, this in vitro study in Bangladesh demonstrated that B. subtilis MMM1, through colonizing the roots, demonstrated a powerful inhibitory effect on the mycelial growth of F. proliferatum and total fumonisin production on maize grain. Therefore, the next phase of this research will focus on how well this strain performs in field conditions for preventing stalk and ear rot of maize caused by various species of Fusarium, as well as fumonisin accumulation in maize.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12112608/s1, Table S1. Detection of potential BD Fusarium_4 by in vitro inoculation of maize. Table S2. Representative B. subtilis isolates identified by homology search of 16S rDNA sequence using BLAST program. Figure S1. Inhibition of the population of F. proliferatum on inoculated maize grain by different Bacillus species at (A) 10 DAI, (B) 20 DAI, and (C) 35 DAI. Figure S2. Percent increase of (A) root length, (B) shoot length, and (C) vigor index of maize seedlings over control at 21 DAS.

Author Contributions

Conceptualization M.R.I.; methodology, M.M.M. (Mamuna Mahjabin Mita), M.J. and M.R.I.; software, M.M.M. (Mamuna Mahjabin Mita), P.S., I.A.P. and M.M.M. (Md. Mostafa Masud); validation, M.R.I., M.J. and M.M.M. (Mamuna Mahjabin Mita); formal analysis, M.M.M. (Mamuna Mahjabin Mita), P.S., I.A.P. and M.M.M. (Md. Mostafa Masud); investigation, M.R.I., M.M.M. (Mamuna Mahjabin Mita) and M.J.; resources, M.R.I., M.M.M. (Mamuna Mahjabin Mita) and M.J.; data curation, M.R.I., M.M.M. (Mamuna Mahjabin Mita) and M.J.; writing—original draft preparation, M.M.M. (Mamuna Mahjabin Mita); writing-review and editing, M.M.M. (Mamuna Mahjabin Mita), M.J., M.R.I., S.B., M.Z.A., R.I. and N.B.I.; visualization, M.M.M. (Mamuna Mahjabin Mita), P.S. and M.R.I.; supervision, M.R.I.; project administration, M.R.I.; funding acquisition, M.R.I. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Bangabandhu Science and Technology Fellowship Trust which was awarded to Muhtarima Jannat and “The article processing charge (APC) was funded by all the authors”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors are thankful to Muhtarima Jannat who purchased the Fumonisin Detection Kit under Bangabandhu Science and Technology Fellowship Trust.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. OGTR (Australian Government Office of the Gene Technology Regulator). The Biology of Zea mays L. spp. mays (Maize or Corn). Australia; 2008. Available online: https://www.ogtr.gov.au/resources/publications/biology-zea-mays-l-ssp-mays-maize-or-corn (accessed on 1 August 2022).
  2. BBS. Statistical Pocket Book of Bangladesh; Bangladesh Bureau of Statistics, Planning Division, Ministry of Planning, Government of the Peoples Republic of Bangladesh: Dhaka, Bangladesh, 2020. [Google Scholar]
  3. Shephard, G.S. Aflatoxin and Food Safety: Recent African Perspectives. J. Toxicol. Toxin Rev. 2003, 22, 267–286. [Google Scholar] [CrossRef]
  4. Jannat, M.; Masud, M.; Nusrat, M.; Bashar, S.; Mita, M.M.; Hossain, M.I.; Alam, Z.; Yeasmin, S.; Islam, R. Aflatoxins and Fumonisins Contamination of Maize in Bangladesh: An Emerging Threat for Safe Food and Food Security. In Maize Genetic Resources: Breeding Strategies and Recent Advances; El-Esawi, M.A., Ed.; IntechOpen: London, UK, 2022; pp. 18–69. [Google Scholar] [CrossRef]
  5. Gruber-Dorninger, C.; Jenkins, T.; Schatzmayr, G. Global mycotoxin occurrence in feed: A ten-year survey. Toxins 2019, 11, 375. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Kyei, N.N.A.; Cramer, B.; Humpf, H.-U.; Degen, G.H.; Ali, N.; Gabrysch, S. Assessment of multiple mycotoxin exposure and its association with food consumption: A human biomonitoring study in a pregnant cohort in rural Bangladesh. Arch. Toxicol. 2022, 96, 2123–2138. [Google Scholar] [CrossRef] [PubMed]
  7. Haschek, W.M.; Gumprecht, L.A.; Smith, G.; Tumbleson, M.E.; Constable, P.D. Fumonisin toxicosis in swine: An overview of porcine pulmonary edema and current perspectives. Environ. Health Perspect. 2001, 109 (Suppl. S2), 251–257. [Google Scholar] [PubMed]
  8. Marasas, W.F.O.; Kellerman, T.S.; Gelderblom, W.C.; Thiel, P.G.; Van der Lugt, J.J.; Coetzer, J.A. Leukoencephalomalacia in a horse induced by fumonisin B₁ isolated from Fusarium moniliforme. Onderstepoort J. Vet. Res. 1988, 55, 197–203. [Google Scholar] [PubMed]
  9. Rheeder, J.P.; Marasas, W.F.; Thiel, P.G.; Sydenham, E.W.; Shephard, G.S.; Van Schalkwyk, D.J. Fusarium moniliforme and fumonisins in corn in relation to human esophageal cancer in Transkei. Phytopathology 1992, 82, 353–357. [Google Scholar] [CrossRef]
  10. Missmer, S.A.; Suarez, L.; Felkner, M.; Wang, E.; Merrill, A.H., Jr.; Rothman, K.J.; Hendricks, K.A. Exposure to fumonisins and the occurrence of neural tube defects along the Texas–Mexico border. Environ. Health Perspect. 2006, 114, 237–241. [Google Scholar] [CrossRef] [PubMed]
  11. Magoha, H.; De Meulenaer, B.; Kimanya, M.; Hipolite, D.; Lachat, C.; Kolsteren, P. Fumonisin B1 contamination in breast milk and its exposure in infants under 6 months of age in Rombo, Northern Tanzania. Food Chem. Toxicol. 2014, 74, 112–116. [Google Scholar] [CrossRef]
  12. Michaelsen, K.F. Feeding and Nutrition of Infants and Young Children: Guidelines for the WHO European Region, with Emphasis on the Former Soviet Countries (No. 87); WHO Regional Office Europe: Copenhagen, Denmark, 2000. [Google Scholar]
  13. Kimanya, M.E.; Shirima, C.P.; Magoha, H.; Shewiyo, D.H.; De Meulenaer, B.; Kolsteren, P.; Gong, Y.Y. Co-exposures of aflatoxins with deoxynivalenol and fumonisins from maize based complementary foods in Rombo, Northern Tanzania. Food Control 2014, 41, 76–81. [Google Scholar] [CrossRef]
  14. UlHaq, I.; Sarwar, M.K.; Faraz, A.; Latif, M.Z. Synthetic chemicals: Major component of plant disease management. In Plant Disease Management Strategies for Sustainable Agriculture through Traditional and Modern Approaches; Springer: Cham, Switzerland, 2020; Volume 13, pp. 53–81. [Google Scholar]
  15. Murugesan, P.; Brunda, D.K.; Moses, J.A.; Anandharamakrishnan, C. Photolytic and photocatalytic detoxification of mycotoxins in foods. Food Control 2001, 123, 107748. [Google Scholar] [CrossRef]
  16. Hammond, B.G.; Campbell, K.W.; Pilcher, C.D.; Degooyer, T.A.; Robinson, A.E.; McMillen, B.L.; Spangler, S.M.; Riordan, S.G.; Rice, L.G.; Richard, J.L. Lower Fumonisin Mycotoxin Levels in the Grain of Bt Corn Grown in the United States in 2000−2002. J. Agric. Food Chem. 2004, 52, 1390–1397. [Google Scholar] [CrossRef]
  17. Lanubile, A.; Ferrarini, A.; Maschietto, V.; Delledonne, M.; Marocco, A.; Bellin, D. Functional genomic analysis of constitutive and inducible defense responses to Fusarium verticillioides infection in maize genotypes with contrasting ear rot resistance. BMC Genom. 2014, 15, 710. [Google Scholar] [CrossRef] [Green Version]
  18. Munkvold, G.P. Cultural and genetic approaches to managing mycotoxins in maize. Annu. Rev. Phytopathol. 2003, 41, 99–116. [Google Scholar] [CrossRef]
  19. Ongena, M.; Jacques, P. Bacillus lipopeptides: Versatile weapons for plant disease biocontrol. Trends Microbiol. 2008, 16, 115–125. [Google Scholar] [CrossRef]
  20. Etcheverry, M.G.; Scandolara, A.; Nesci, A.; Vilas Boas Ribeiro, M.S.; Pereira, P.; Battilani, P. Biological interactions to select biocontrol agents against toxigenic strains of Aspergillus flavus and Fusarium verticillioides from maize. Mycopathologia 2009, 167, 287–295. [Google Scholar] [CrossRef]
  21. Hashem, A.; Tabassum, B.; Abd_Allah, E.F. Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi J. Biol. Sci. 2019, 26, 1291–1297. [Google Scholar] [CrossRef]
  22. Hernández-Morales, A.; Martínez-Peniche, R.A.; Arvizu-Gómez, J.L.; Arvizu-Medrano, S.M.; Rodríguez-Ontiveros, A.; Ramos-López, M.A.; Pacheco-Aguilar, J.R. Production of a Mixture of Fengycins with Surfactant and Antifungal Activities by Bacillus sp. MA04, a Versatile PGPR. Indian J. Microbiol. 2018, 58, 208–213. [Google Scholar] [CrossRef]
  23. Cavaglieri, L.; Orlando, J.R.M.I.; Rodriguez, M.I.; Chulze, S.; Etcheverry, M. Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro and at the maize root level. Res. Microbiol. 2005, 156, 748–754. [Google Scholar] [CrossRef]
  24. Guimarães, R.A.; Zanotto, E.; Perrony, P.E.P.; Zanotto, L.A.S.; da Silva, L.J.; Machado, J.D.C.; Pinto, F.A.M.F.; Medeiros, H.N.; von Pinho, R.G.; de Melo, I.S.; et al. Integrating a chemical fungicide and Bacillus subtilis BIOUFLA2 ensures leaf protection and reduces ear rot (Fusarium verticillioides) and fumonisin content in maize. J. Phytopathol. 2021, 169, 139–148. [Google Scholar] [CrossRef]
  25. Dukare, A.; Paul, S. Biological control of Fusarium wilt and growth promotion in pigeon pea (Cajanus cajan) by antagonistic rhizobacteria, displaying multiple modes of pathogen inhibition. Rhizosphere 2021, 17, 100278. [Google Scholar] [CrossRef]
  26. Ju, R.; Zhao, Y.; Li, J.; Jiang, H.; Liu, P.; Yang, T.; Bao, Z.; Zhou, B.; Zhou, X.; Liu, X. Identification and evaluation of a potential biocontrol agent, Bacillus subtilis, against Fusarium sp. in apple seedlings. Ann. Microbiol. 2014, 64, 377–383. [Google Scholar] [CrossRef]
  27. Bacon, C.W.; Yates, I.E.; Hinton, D.M.; Meredith, F. Biological control of Fusarium moniliforme in maize. Environ. Health Perspect. 2001, 109 (Suppl. S2), 325–332. [Google Scholar]
  28. Al-Masri, S.A.; El-Safty, S.M.; Nada, S.A.; Amra, H.A. Saccharomyces cerevisiae and probiotic bacteria potentially inhibit fumonisin B1 production in vitro and in vivo. J. Am. Sci. 2011, 7, 198–205. [Google Scholar]
  29. Rahman, M.M.; Masud, M.M.; Hossain, M.I.; Islam, N.; Alam, M.Z.; Rashid, M.M.; Khan, M.A.I.; Latif, M.A.; Halder, K.P.; Islam, M.R. Potential Role of Rice Plant Growth Promoting Phylloplane and Rhizospheric Bacteria in Controlling Xanthomonas oryzae pv. oryzae. In Integrative Advances in Rice Research; IntechOpen: London, UK, 2021. [Google Scholar]
  30. Islam, M.H.; Masud, M.M.; Jannat, M.; Hossain, M.I.; Islam, S.; Alam, M.Z.; Serneels, F.J.B.; Islam, M.R. Potentiality of Formulated Bioagents from Lab to Field: A Sustainable Alternative for Minimizing the Use of Chemical Fungicide in Controlling Potato Late Blight. Sustainability 2022, 14, 4383. [Google Scholar] [CrossRef]
  31. Liu, Y.; Teng, K.; Wang, T.; Dong, E.; Zhang, M.; Tao, Y.; Zhong, J. Antimicrobial Bacillus velezensis HC6: Production of three kinds of lipopeptides and biocontrol potential in maize. J. Appl. Microbiol. 2020, 128, 242–254. [Google Scholar] [CrossRef] [Green Version]
  32. He, Y.; Zhu, M.; Huang, J.; Hsiang, T.; Zheng, L. Biocontrol potential of a Bacillus subtilis strain BJ-1 against the rice blast fungus Magnaporthe oryzae. Can. J. Plant Pathol. 2019, 41, 47–59. [Google Scholar] [CrossRef]
  33. Figueroa-López, A.M.; Cordero-Ramírez, J.D.; Martínez-Álvarez, J.C.; López-Meyer, M.; Lizárraga-Sánchez, G.J.; Félix-Gastélum, R.; Castro-Martínez, C.; Maldonado-Mendoza, I.E. Rhizospheric bacteria of maize with potential for biocontrol of Fusarium verticillioides. SpringerPlus 2016, 5, 330. [Google Scholar] [CrossRef] [Green Version]
  34. Xie, S.; Jiang, L.; Wu, Q.; Wan, W.; Gan, Y.; Zhao, L.; Wen, J. Maize Root Exudates Recruit Bacillus amyloliquefaciens OR2-30 to Inhibit Fusarium graminearum Infection. Phytopathology 2022, 112, 1886–1893. [Google Scholar] [CrossRef]
  35. Hanif, A.; Zhang, F.; Li, P.; Li, C.; Xu, Y.; Zubair, M.; Zhang, M.; Jia, D.; Zhao, X.; Liang, J.; et al. Fengycin Produced by Bacillus amyloliquefaciens FZB42 Inhibits Fusarium graminearum Growth and Mycotoxins Biosynthesis. Toxins 2019, 11, 295. [Google Scholar] [CrossRef] [Green Version]
  36. Lei, S.; Zhao, H.; Pang, B.; Qu, R.; Lian, Z.; Jiang, C.; Shao, D.; Huang, Q.; Jin, M.; Shi, J. Capability of iturin from Bacillus subtilis to inhibit Candida albicans in vitro and in vivo. Appl. Microbiol. Biotechnol. 2019, 103, 4377–4392. [Google Scholar] [CrossRef]
  37. Lizárraga-Sánchez, G.J.; Leyva-Madrigal, K.Y.; Sánchez-Peña, P.; Quiroz-Figueroa, F.R.; Maldonado-Mendoza, I.E. Bacillus cereus sensulato strain B25 controls maize stalk and ear rot in Sinaloa, Mexico. Field Crops Res. 2015, 176, 11–21. [Google Scholar] [CrossRef]
  38. Guo, Z.; Zhang, X.; Wu, J.; Yu, J.; Xu, M.; Chen, D.; Zhang, Z.; Li, X.; Chi, Y.; Wan, S. In Vitro inhibitory effect of the bacterium Serratia marcescens on Fusarium proliferatum growth and fumonisins production. Biol. Control 2020, 143, 104188. [Google Scholar] [CrossRef]
  39. Cai, B.P.; Chen, J.Y.; Zhang, Q.X.; Guo, L.D. A survey of arbuscular mycorrhizal colonization in roots of Prunus mume in China. Acta Hortic. Sin. 2008, 35, 599. [Google Scholar]
  40. Köhl, J.; Kolnaar, R.; Ravensberg, W.J. Mode of action of microbial biological control agents against plant diseases: Relevance beyond efficacy. Front. Plant Sci. 2019, 10, 845. [Google Scholar] [CrossRef] [Green Version]
  41. Bevivino, A.; Sarrocco, S.; Dalmastri, C.; Tabacchioni, S.; Cantale, C.; Chiarini, L. Characterization of a free-living maize-rhizosphere population of Burkholderia cepacia: Effect of seed treatment on disease suppression and growth promotion of maize. FEMS Microbiol. Ecol. 1998, 27, 225–237. [Google Scholar] [CrossRef]
  42. Bais, H.P.; Fall, R.; Vivanco, J.M. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol. 2004, 134, 307–319. [Google Scholar] [CrossRef] [Green Version]
  43. Ndeddy Aka, R.J.; Babalola, O.O. Effect of bacterial inoculation of strains of Pseudomonas aeruginosa, Alcaligenes feacalis and Bacillus subtilis on germination, growth and heavy metal (Cd, Cr, and Ni) uptake of Brassica juncea. Int. J. Phytoremediat. 2016, 18, 200–209. [Google Scholar] [CrossRef]
  44. Chen, X.H.; Koumoutsi, A.; Scholz, R.; Eisenreich, A.; Schneider, K.; Heinemeyer, I.; Morgenstern, B.; Voss, B.; Hess, W.R.; Reva, O.; et al. Comparative analysis of the complete genome sequence of the plant growth–promoting bacterium Bacillus amyloliquefaciens FZB42. Nat. Biotechnol. 2007, 25, 1007–1014. [Google Scholar] [CrossRef] [Green Version]
  45. Li, S.; Zhang, N.; Zhang, Z.; Luo, J.; Shen, B.; Zhang, R.; Shen, Q. Antagonist Bacillus subtilis HJ5 controls Verticillium wilt of cotton by root colonization and biofilm formation. Biol. Fertil. Soils 2013, 49, 295–303. [Google Scholar] [CrossRef]
  46. Shafi, J.; Tian, H.; Ji, M. Bacillus species as versatile weapons for plant pathogens: A review. Biotechnol. Biotechnol. Equip. 2017, 31, 446–459. [Google Scholar] [CrossRef] [Green Version]
  47. Ahn, I.-P.; Park, K.; Kim, C.-H. Rhizobacteria-induced resistance perturbs viral disease progress and triggers defense-related gene expression. Mol. Cells 2002, 13, 302–308. [Google Scholar]
  48. Georgelis, N.; Nikolaidis, N.; Cosgrove, D.J. Biochemical analysis of expansin-like proteins from microbes. Carbohydr. Polym. 2014, 100, 17–23. [Google Scholar] [CrossRef] [PubMed]
Agronomy 12 02608 g001 550
Figure 1. In Vitro growth inhibition of Fusarium proliferatum by different Bacillus species on PDA plates at 7 DAI (BDISO01RR: Bacillus amyloliquefaciens, BDISO76MR: Bacillus subtilis, BDISO36PR: Bacillus subtilis, BDISO45PR: Bacillus subtilis and BDISO49PR: Bacillus subtilis).
Figure 1. In Vitro growth inhibition of Fusarium proliferatum by different Bacillus species on PDA plates at 7 DAI (BDISO01RR: Bacillus amyloliquefaciens, BDISO76MR: Bacillus subtilis, BDISO36PR: Bacillus subtilis, BDISO45PR: Bacillus subtilis and BDISO49PR: Bacillus subtilis).
Agronomy 12 02608 g001
Agronomy 12 02608 g002 550
Figure 2. (A) Pure culture of BDISO76MR, an isolate obtained from maize rhizosphere, and (B) PCR amplification of 16S rDNA of BDISO76MR using 27F and 1518R primers (M = DNA marker; W = Water).
Figure 2. (A) Pure culture of BDISO76MR, an isolate obtained from maize rhizosphere, and (B) PCR amplification of 16S rDNA of BDISO76MR using 27F and 1518R primers (M = DNA marker; W = Water).
Agronomy 12 02608 g002
Agronomy 12 02608 g003 550
Figure 3. Linear regression showing the relationship between F. proliferatum population (CFU/g maize grain) with (A) total fumonisin concentration (ppm), and (B) reduction of fumonisin concentration (%) at 35 DAI.
Figure 3. Linear regression showing the relationship between F. proliferatum population (CFU/g maize grain) with (A) total fumonisin concentration (ppm), and (B) reduction of fumonisin concentration (%) at 35 DAI.
Agronomy 12 02608 g003
Agronomy 12 02608 g004 550
Figure 4. Root colonization of different Bacillus species in maize rhizosphere at 21 DAS. (BDISO76MR: B. subtilis, BDISO01RR: B. amyloliquefaciens, BDISO36PR: B. subtilis, BDISO45PR: B. subtilis and BDISO49PR: B. subtilis). Untreated control: seed treatment with only distilled water.
Figure 4. Root colonization of different Bacillus species in maize rhizosphere at 21 DAS. (BDISO76MR: B. subtilis, BDISO01RR: B. amyloliquefaciens, BDISO36PR: B. subtilis, BDISO45PR: B. subtilis and BDISO49PR: B. subtilis). Untreated control: seed treatment with only distilled water.
Agronomy 12 02608 g004
Table
Table 1. In Vitro antagonistic activity of Bacillus species against Fusarium proliferatum.
Table 1. In Vitro antagonistic activity of Bacillus species against Fusarium proliferatum.
TreatmentsRadial Mycelial Growth of F. proliferatum (mm) 1% Reduction of Radial Mycelial Growth Overcontrol
Days after Inoculation (DAI)
4747
Control31 ± 1 a80 ± 0.00 a00
Negative control24.5 ± 2.5 ab42 ± 2.5 b20.6347.5
BDISO76MR9.25 ± 0.75 d11.75 ± 0.75 d70.2185.31
BDISO01RR12 ± 1.00 cd12 ± 1.00 cd61.1585
BDISO45PR12.25 ± 0.75 cd12.25 ± 0.75 cd60.5284.69
BDISO36PR14 ± 0.5 cd14 ± 0.5 cd54.8482.5
BDISO49PR18 ± 0.00 bc18 ± 0.00 c41.8877.5
Level of significance ***--
LSD3.923.71--
CV%0.080.04--
* Indicates significance at a 5% level of significance. 1 Data are the averages of three replications ± SE (standard error). Values with similar letters are statistically similar. (BDISO01RR: B. amyloliquefaciens, BDISO36PR: B. subtilis, BDISO45PR: B. subtilis, BDISO49PR: B. subtilis and BDISO76MR: B. subtilis). Control: F. proliferatum only and Negative control: Bacillus species that could not inhibit the mycelial growth of F. proliferatum.
Table
Table 2. Effect of Bacillus species on the reduction of fumonisin accumulation in co-inoculated maize grains.
Table 2. Effect of Bacillus species on the reduction of fumonisin accumulation in co-inoculated maize grains.
TreatmentsFumonisin Concentration
(ppm) 1		% Reduction of Fumonisin Concentration
Days after Inoculation (DAI)
102035102035
Positive control4.93 ± 0.43 a5.56 + 0.43 a20.32 ± 0.43 a000
BDISO01RR3.15 ± 0.43 ab3.11 ± 0.43 bc3.11 ± 0.43 c36.1144.1284.70
BDISO36PR1.7 ± 0.43 bc1.69 ± 0.43 bc3.38 ± 0.43 bc65.5269.5683.36
BDISO45PR3.42 ± 0.43 ab3.42 ± 0.43 b4.29 ± 0.43 bc30.6338.5378.90
BDISO49PR3.72 ± 0.43 ab1.36 ± 0.43 bc5.41 ± 0.43 b24.5475.4873.36
BDISO76MR0.9 ±0.43 c1.29 ± 0.43 c3.02 ± 0.43 c81.7476.8185.12
Level of significance ****---
LSD1.311.311.31---
CV%0.270.280.12---
* Represents significance at a 5% level of significance. 1 Data are the averages of three replications ± SE. Values with similar letters are statistically similar. (BDISO01RR: B. amyloliquefaciens, BDISO36PR: B. subtilis, BDISO45PR: B. subtilis, BDISO49PR: B. subtilis and BDISO76MR: B. subtilis). Positive control: Sterilized maize grains were inoculated with F. proliferatum only.
Table
Table 3. Influence of Bacillus species on the reduction of F. proliferatum population in co-inoculated maize grains.
Table 3. Influence of Bacillus species on the reduction of F. proliferatum population in co-inoculated maize grains.
TreatmentsThe Population of F. proliferatum
(CFU/g Maize Grain) 1
Days after Inoculation (DAI)
102035
Positive control9.4 × 104 ± 0.64 ab10 × 104 ± 0.77 c5.5 × 104 ± 0.43 b
BDISO01RR12.2 × 104 ± 0.64 a13.3 × 104 ± 0.77 b1.1 × 104 ± 0.43 a
BDISO36PR7.2 × 104 ± 0.64 b16.1 × 104 ± 0.77 a1.1 × 104 ± 0.43 a
BDISO45PR8.8 × 104 ± 0.64 b4.4 × 104 ± 0.77 e1.1 × 104 ± 0.43 a
BDISO49PR8.3 × 104 ± 0.64 b6.6 × 104 ± 0.77 d1.1 × 104 ± 0.43 a
BDISO76MR8.8 × 104 ± 0.64 b4.4 × 104 ± 0.77 e1.1 × 104 ± 0.43 a
Level of significance ****
LSD5.871.371.33
CV%0.120.080.41
* Represents significance at a 5% level of significance. 1 Data are the averages of three replications ± SE. Values with similar letters are statistically similar. (BDISO01RR: B. amyloliquefaciens, BDISO36PR: B. subtilis, BDISO45PR: B. subtilis, BDISO49PR: B. subtilis and BDISO76MR: B. subtilis). Positive control: Sterilized maize grains were inoculated with F. proliferatum only.
Table
Table 4. Root colonization of different Bacillus species in the maize rhizosphere at 21 DAS.
Table 4. Root colonization of different Bacillus species in the maize rhizosphere at 21 DAS.
TreatmentsColony Forming Units
(×106 CFU/g Root Tissue) 1
Untreated control22.6 ± 1.3 d
BDISO76MR82 ± 0.0 a
BDISO01RR15.3 ± 0.6 e
BDISO36PR3 ± 0.0 f
BDISO45PR26.3 ± 0.3 c
BDISO49PR51.6 ± 0.3 b
Level of significance **
CV%2.29
LSD1.96
* Represents significance at a 5% level of significance. 1 Data are the averages of three replications ± SE. Values with similar letters are statistically similar. (BDISO76MR: B. subtilis, BDISO01RR: B. amyloliquefaciens, BDISO36PR: B. subtilis, BDISO45PR: B. subtilis and BDISO49PR: B. subtilis). Untreated control: sterilized maize grains were submerged in sterile water only.
Table
Table 5. Influence of different Bacillus sp. on average root and shoot length and vigor index.
Table 5. Influence of different Bacillus sp. on average root and shoot length and vigor index.
TreatmentDays after Sowing (DAS)Vigor Index
714217DAS14DAS21DAS
Root LengthShoot LengthRoot LengthShoot LengthRoot LengthShoot Length
(cm)(cm)(cm)(cm)(cm)(cm)
Untreated control16 ± 0.679.5 ± 0.8335.5 ± 2.17 a44.67 ± 2.33 a33.33 ± 6.3344.17 ± 7.8325508016.677750
BDISO76MR15.67 ± 1.008 ± 2.3334.17 ± 2.83 a45 ± 1.00 a39.67 ± 3.0053.67 ± 1.332366.677916.679333.33
BDISO01RR23 ±8.008.83 ± 0.8334 ± 3.00 a47.67 ± 3.00 a34.67 ± 1.0055.67 ± 1.333183.338166.679033.33
BDISO36PR18 ±1.6713.17 ± 3.1731.5 ± 1.83 a40.33 ± 2.33 a34 ± 4.0053.17 ± 3.53116.677183.338716.67
BDISO45PR18.17 ±1.679.83 ± 0.1719.83 ± 0.5 b26.83 ± 0.17 b38.83 ± 1.1758.8 ± 2.8328003888.738138.89
BDISO49PR18.17 ± 1.59.17 ± 0.1731.17 ± 0.17 ab43 ± 2.00 a34.67 ± 3.6760 ± 0.332733.337416.679466.67
Level of significance *NSNS**NSNS---
LSD11.865.817.117.0512.7113.05---
CV%0.170.180.080.060.130.003---
* Represents significance at a 5% level of significance. Data are the averages of three replications ± SE. Values with similar letters are not significantly different at a 5% level of significance. (BDISO76MR: B. subtilis, BDISO01RR: B. amyloliquefaciens, BDISO36PR: B. subtilis, BDISO45PR: B. subtilis and BDISO49PR: B. subtilis). Untreated control: seed treatment with only distilled water.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Mita, M.M.; Jannat, M.; Bashar, S.; Protic, I.A.; Saha, P.; Masud, M.M.; Islam, R.; Islam, N.B.; Alam, M.Z.; Islam, M.R. Potential Native Bacilli Reduce Fumonisin Contamination in Maize. Agronomy 2022, 12, 2608. https://doi.org/10.3390/agronomy12112608

AMA Style

Mita MM, Jannat M, Bashar S, Protic IA, Saha P, Masud MM, Islam R, Islam NB, Alam MZ, Islam MR. Potential Native Bacilli Reduce Fumonisin Contamination in Maize. Agronomy. 2022; 12(11):2608. https://doi.org/10.3390/agronomy12112608

Chicago/Turabian Style

Mita, Mamuna Mahjabin, Muhtarima Jannat, Samrin Bashar, Ismam Ahmed Protic, Plabon Saha, Md. Mostafa Masud, Rummana Islam, Nazia Binta Islam, Md. Zahangir Alam, and Md. Rashidul Islam. 2022. "Potential Native Bacilli Reduce Fumonisin Contamination in Maize" Agronomy 12, no. 11: 2608. https://doi.org/10.3390/agronomy12112608

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop