Archives of Microbiology

https://doi.org/10.1007/s00203-020-02113-5

ORIGINAL PAPER

Siderophore production by Bacillus subtilis MF497446

and Pseudomonas koreensis MG209738 and their efficacy in controlling
Cephalosporium maydis in maize plant
Nasr Ghazy1 · Sahar El‑Nahrawy2

Received: 15 July 2020 / Revised: 7 October 2020 / Accepted: 1 November 2020

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Late wilt disease, caused by Cephalosporium maydis in maize plant, is one of the main economical diseases in Egypt.
Therefore, to cope with this problem, we investigated the potentiality of plant growth promoting rhizobacteria in control-
ling this disease. Six strains (Bacillus subtilis, B. circulance, B. coagulanse, B. licheniformis, Pseudomonas fluroscence and
P. koreensis) were screened for siderophore production, and using dual plate culture method and greenhouse experiment,
antagonistic activity against C. maydis was studied. Using two superior strains, single and dual inoculation treatments in
maize were applied in field experiment during the 2018 and 2019 seasons. Results indicated that B. subtilis and P. koreen-
sis strains had shown the most qualitative and quantitative assays for siderophore production and antagonistic activities.
In greenhouse, the most effective treatments on the pre- and post-emergence damping off as well as growth promotion of
maize were T3 treatment (inoculated with B. subtilis), and T8 treatment (inoculated with P. koreensis). In field experiment,
T5 treatment (inoculated with a mixture of B. subtilis and P. koreensis) showed significant increases in catalase (CAT), per-
oxidase (POX) and polyphenol oxidase (PPO) activities, as well as total chlorophyll and carotenoids than control treatments
during the two growing seasons. In the same way, the highest effect in reducing infection and increasing the thickness of
the sclerenchymatous sheath layer surrounding the vascular bundles in maize stem was observed and these results were a
reflection of the increase in yield and yield parameters.

Keywords Late wilt disease · Maize · Siderophore production · PGPR · Antioxidant enzymes · Stem histological
differences

Introduction

Maize (Zea mays L.) is one of the most important cereal

crops for nutrition in the global population and is a source
of protein and micronutrients (Nuss and Tanumihardjo,
Communicated by Erko Stackebrandt.
2010; Zhao et al. 2020). In Egypt, harvested has reached
about 935,778 ha for that production quantity 7.3 million
Electronic Supplementary Material The online version of this tons of grains (FAOSTST 2018). Worldwide, diseases on
article (https​://doi.org/10.1007/s0020​3-020-02113​-5) contains maize plants lead to detrimental effects on the economy and
supplementary material, which is available to authorized users.
the food supplies and pose serious health hazards to both
* Sahar El‑Nahrawy humans and animals. One of these fungal diseases in maize
sahar.elnahrawy@yahoo.com is late wilt, caused by Cephalosporium maydis, which is
1
one of the main economical diseases in Egypt (Samra et al.
Maize and Sugar Crops Dis. Department, Plant Pathology 1963), and the infection incidence leads to decrease in grain
Research Institute, Agricultural Research Center, Giza,
Egypt yield to 40% (Labib et al. 1975). This pathogen enters tissue
2 from root and colonizes the xylem (Sabet et al. 1970), and
Department of Agricultural Microbiology, Soils, Water
and Environment Research Institute, Agricultural Research during tasseling formation stage, late wilt disease appears in
Center, Giza, Egypt

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leaves and then develops into stalks (El-Shafey and Claflin biomass of maize plants under greenhouse and field condi-
1999). tions. Sheng et al. (2020). showed that Brevibacillus brevis
To protect against soilborne diseases, it is better to GZDF3 (PGPR strain) isolated from the rhizosphere soil
use chemical pesticides in controlling these diseases but, of Pinellia ternate could produce siderophores with strong
these are expensive as well as not environmental friendly. antagonistic activity to Candida albicans. Also, P. aerugi-
So, many researchers have used plant growth-promoting nosa and P. fluorescens strains could be used to develop
rhizobacteria (PGPR) as biological control against late active biocontrol agents of tomato bacterial wilt caused by
wilt disease associated with the maize rhizosphere which Ralstonia solanacearum (Mohammed et al. 2020).Similarly,
could improve plant health, reduce chemical pesticides, and Larkin (2020) suggested that bacterial inoculation with B.
develop efficient biological control strategies (El-Mehalowy subtilis GB03 can be used as biocontrol against two strains
et al. 2004 and Ashour et al. 2013; Omara et al. 2017, 2018). of Rhizoctonia (Rhs1A1 and Bs69) under organic production
Therefore, recently, biological control by PGPR has been practices over three field seasons.
adopted on a commercial scale by using a number of experi- The purpose of this research was to evaluate some strains
mental approaches. These microorganisms can be found in of PGPR to siderophore production, and their ability to con-
the rhizosphere, rhizoplane or in association with roots, trol late wilt disease, growth promotion and yield of maize
which improve plant growth promotion in the absence of plant under laboratory, greenhouse and field conditions.
pathogens (direct mechanism) or reduce the deleterious
effects of pathogens on crop yield (indirect mechanism) by
antibiosis, HCN production, competition, induced systemic Materials and methods
resistance and siderophore production (Shivakumar 2007;
Mohammed et al. 2020; Larkin 2020). Microorganisms and growth conditions
Siderophore production by microorganisms has received
great attention due to their application in different branches Six bacterial strains, Bacillus subtilis MF497446, B. circu-
of the agriculture sector such as soil science, plant pathol- lance NCAIM B.02324, B. coagulanse NCAIM B.01123, B.
ogy and environmental sciences. Siderophores are organic licheniformis Sh8, Pseudomonas fluroscence SARS 5 and P.
compounds with low molecular weight (< 10 kDa) and high koreensis MG209738, were used in the present study. These
specific affinity to chelate iron (Oswald 2010). On the other were obtained from Bacteriology Laboratory, Sakha Agri-
hand, there are four groups of siderophores produced by cultural Research Station, Kafr El-Sheikh, Egypt. All bacte-
bacteria (catecholates, hydroxamates, salicylate and car- rial strains were maintained on Nutrient Broth (NB) medium
boxylates) (Rajkumar et al. 2010), which play a vital role containing (g l−1): peptone 5.0, beef extract 3.0, NaCl 5.0,
in the accumulation of iron from different organic materi- pH 6.8–7.2 (Anonymous 1957).
als. Common genera of siderophore-producing bacteria On the other hand, Cephalosporium maydis fungi were
are Rhizobium, Arthrobacter, Azospirillum, Pseudomonas, obtained from Maize and Sugar Crops Dis. Dept., Plant
Azotobacter, Bacillus Acinetobacter, Alcaligenes, Beijer- Pathol. Res. Inst., Agric. Res. Center, Giza, Egypt. C. may-
inckia, Burkholderia and Enterobacter (Bashan et al. 2014; dis was grown on potato dextrose medium supplemented
Ghavami et al. 2016; Grobelak and Hiller 2017; El-Nahrawy with 0.2% yeast extract (Yassin 2000).
et al. 2019; Sinha and Parli 2020).
Previously, some studies have reported the role of sidero- Evaluation of siderophore production
phores in the suppression of Pythium species and enhance-
ment of the growth of wheat by P. fluorescent (Becker and The production process of siderophore depends on the
Cook 1988). Also, pyoverdin production by P. aeruginosa 7 preparation of the cell growth medium that is sufficiently
NSK 2 was able to increase the yield of barley, wheat, maize, Fe deficient. So, all glassware used was washed in acid (e.g.,
cucumber and spinach (Hofte et al. 1991). Suryakala et al. 6 M HCl) overnight then rinsed thoroughly in distilled water
(2004) suggested that trihyobroxamate type pyoverdines of and sterilized by autoclaving before use. The siderophore
siderophores forming hexadentate ligands with F ­ e+3 ions. production capability of tested strains was evaluated by
These siderophores might be exploited as potent biocontrol both qualitative and quantitative methods. Also, detection
compounds against plant pathogens. of siderophore production by Arnowʼs, Csáky and ­FeCl3
Lately, most studies support the siderophore theory of tests was determined.
biological control by PGPR. López-Reyes et al. (2017)
showed that A. brasilense, isolated from teosinte (Zea mays Qualitative assay
L. ssp. mexicana), produced bacterial metabolites such as
siderophores and indoles which are used in the suppression Chrome azurol sulfonate (CAS) agar plate method (Schwyn
of Alternaria, Bipolaris and Fusarium as well as increase the and Neilands 1987) was performed for qualitative assay

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siderophore production. Briefly, all tested strains were (1% w/v) prepared in acetic acid 30% (v/v) with 0.5 ml
grown in Nutrient Broth medium for 48 h on a rotary shaker iodine (1.3% w/v) was mixed and this mixture was incubated
(120 rpm) at 30 °C and then 0.05 ml of the resultant bacte- for 5 min at room temperature. Further, excess iodine was
rial suspension containing 9 × 108 cells ml−1 was spotted removed by adding 1 ml of trisodium arsenite (­ Na3AsO2)
over CAS-agar plates in triplicate and incubated for 5 days (2% w/v) prepared in water. Finally, 1 ml solution of
at 30 °C. On CAS blue agar, an orange halo formed around α-naphthylamine (0.3% w/v) prepared in acetic acid 30%
colonies which indicates the ability of the bacterial strains (v/v) was added to the solution resulting in change of color
to produce siderophore. The size of the halo diameter/colony from orange to red.
diameter ratios were screened and the results are shown as
the mean ± standard deviation (SD). FeCl3 test

Quantitative assay According to Arnow (1937), 0.5 ml of 2% F ­ eCl3 (freshly

prepared) was mixed with 1 ml of the culture supernatant
In culture supernatant extracts, CAS solution was used to and observed for the presence and absence of red color.
quantify siderophore activity (Schwyn and Neilands 1987).
So, all tested strains were grown in a Nutrient Broth medium Bioassay against fungal pathogen
for 48 h on a rotary shaker (120 rpm) at 30 °C and then cen-
trifuged for 15 min at 3000 rpm. 0.5 ml (9 × 108 cells ml−1) All the six strains were tested in vitro for their biocontrol
of the supernatant was mixed with 0.5 ml CAS solution activity against plant pathogenic fungi Cephalosporium
and 10 μl shuttle solution (sulfosalicylic acid). After 2 h maydis. C. maydis fungi was grown on a PDA plate till
of incubation at room temperature, the absorbance of the it covered the whole surface of the agar. A disc of fungal
mixture was read using UV/Vis SPECTROPHOTOMETER growth from this plate was taken by sterile cork borer and
(Bibby Scientific Ltd, Dunmow, Essex. UK, Model 6705) placed at the center of the fresh PDA plate. In triplicate, each
at 630 nm. Simultaneously, the reference samples (blank) bacterial strain was then streaked parallelly on either side of
were prepared with Nutrient Broth medium, CAS solution, the fungal disc and kept for incubation at 28 °C for 7 days.
and shuttle solution. The percentage of siderophore was esti- The reduction in the growth of C. maydis was calculated,
mated using the following formula: using the following formula: RG (%) = C − T/C × 100, where
C is the growth of the test pathogen (cm) in the absence of
(Ar − As)∕Ar × 100, the antagonistic strain, and T is the growth of the test patho-
gen (cm) in the presence of the antagonistic strain.
where Ar is the absorbance at 630 nm of the reference, and
As is the absorbance at 630 nm of the samples. Experiment
Pot experiment
was performed in triplicate, and the results are shown as the
mean ± standard deviation (SD).
Soil from the experimental farm (Sakha Agric. Res. Station,
Kafrelsheikh Gov.) was collected for the pot experiment.
Arnowʼs assays The soil was air-dried, passed through a ten mesh sieve,
analyzed for physical and chemical properties Table 1, and
Arnowʼs assays was used to confirm catecholate siderophore then transferred to polyethylene bags filled with 4 kg of soil
production (Arnow 1937; Lacava et al. 2008). The culture after being sterilized. In 0.5 L Erlenmeyer flasks, C. may-
supernatants obtained from quantitative assay were sub- dis fungi were inoculated into sterilized 250 ml potato dex-
jected to determine. 3 ml of the culture supernatant was then trose broth medium supplemented with 0.2% yeast extract,
mixed with 0.3 ml of 5 N HCl solution, 1.5 ml of Arnow’s then incubated for 14 days at 28 °C. After that, flasks were
reagent (10 g N­ aNO2, 10 g N ­ a2MoO4.2H2O dissolved in thoroughly shaken, and 20 ml of the suspension was poured
50 ml distilled water) and 0.3 ml of 10 N NaOH. Then the into 1 L Erlenmeyer flask, containing 2/3 wet autoclaved
mixture was incubated for 10 min to complete the reaction in grain sorghum and kept at 28 °C for 4 weeks. Soil infesta-
which the presence or absence of pink colour was observed. tion was carried out 1 week before planting by mixing 120 g
of inoculum to the soil in every pot, followed by irrigation
Csáky test El-Shabrawy and Shehata (2018).
The grains of susceptible maize (Z. mays cv. Baladi)
Csáky test was used for assay of hydroxamate siderophore were surface sterilized with 70% ethanol and 5% sodium
production Cśaky (1948). One milliliter of sample culture hypochlorite solution for 5–7 min, then washed five times
broth with 1 ml of ­H2SO4 (6 M) was mixed and autoclaved with sterile distilled water. Each pot was seeded with four
for 30 min at 121 °C. Upon cooling, 1 ml of sulfanilic acid seeds and plants were thinned to two plants per pot. The

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Table 1  Some physicochemical properties of soil used in pot and treatments B. subtilis and P. koreensis were applied. The
field experiments treatments were as follows: T ­ 1: uninoculated (control), T
­ 2:
Soil properties Pot experiment Field experiment grains treated with fungicide (METAZED), ­T3: inoculated
with B. subtilis, ­T4: inoculated with P. koreensis and ­T5:
2018 2019
inoculated with a mixture of B. subtilis and P. koreensis
Soil texture Clayey Clayey Clayey (1:1), with arrangement in three replicates. Fifteen millilit-
pH (1: 2.5 water suspension) 8.22 7.90 7.72 ers of each culture (1 × 108 CFU ml−1) was mixed with 30 g
EC (dS m−1) 3.22 3.16 3.28 of sterilized carrier, and then treated carefully with grains
Organic matter % 1.12 1.24 1.36 of maize using a sticking material before sowing. Each plot
Available P mg Kg−1 8.24 9.33 10.03 (3 × 4 m) consisted of five ridges, 4 m in length and 60 cm
Available NH4 mg Kg−1 9.70 12.60 15.66 apart, and the grains were sowed at the rate of 3 grains/hole
Available K mg Kg−1 304 350 372 with 20 cm space and the space between replications 1 m.
Fe mg Kg−1 2.54 2.62 2.67 Thereafter, they were thinned to one plant/hole. According
to the agricultural recommendations for maize by the Egyp-
Physical and chemical analyses of soil used were determined by
Department of Soil Chemistry, Soils, Water and Environment tian Ministry of Agriculture and Land Reclamation, irriga-
Research Institute, Agricultural Research Center tion and mineral fertilizers were applied.

grains were inoculated with 1.0 mL of the grown cultures of Measurements

tested bacterial strains, B. subtilis, B. circulance, B. coagu-
lanse, B. licheniformis, P. fluroscence and P. koreensis, con- Antioxidant enzymes and photosynthetic pigments
taining 3 × 109 cells mL−1. The cultures had been grown on
Nutrient Broth medium on a rotary shaker for 48 h at 30 °C. At 60 days after sowing (DAS), three antioxidant enzyme
Six pots were used for each strain, and sterile grains treated activity catalase (Aebi (1984), peroxidase (Hammerschmidt
with non-inoculated culture media as well as other grains et al. 1982), and polyphenol oxides (Malik and Singh 1980)
treated with fungicide (METAZED 38% SC, 3 cm/1 kg were determined under this investigation. Also, total chloro-
grains, Kafr El-Zayat pesticides and chemicals Co.) were phyll and carotenoids were determined according to Mousa
used as control. At 21 days after planting, urea (46% N) as et al. (2007).
a nitrogen fertilizer was added at 300 mg N/kg soil and the
plants were irrigated regularly. The maize plants were har- Disease incidence and stem histological differences
vested at 35-day age by mulching the plants from the pots.
A replicate of three plants was used to measure pre- and Disease incidence and reduction % of late wilt were recorded
post-emergence percentage (Nawar 2007). Also, vegetative at 85 DAS. Also, the histology of the stem of maize plants
growth such as fresh and dry weight (g plant−1), length of was measured according to the methods used by Abd El-
shoots and roots (cm), as well as areal root, seminal root and Ghani (1987). Briefly, from the second lower internodes,
stem diameter (cm) were determined. stem segments were taken and fixed for 36 h in a mixture of
acetic acid, formalin, ethyl alcohol 70% and water at the rate
Field trials of 5, 10, 50 and 35 ml, respectively, then transferred to etha-
nol 70% until use. The drying process was performed with
Under field conditions, the impact of single and dual inocu- an increase in the concentrations of ethanol and N-butanol
lation on the incidence of maize late wilt, vegetative growth series, then embedded into paraffin wax (58 °C). By a rotary
and yield of susceptible maize plant (Baladi cv.) were microtome, cross sections were cut (15 μm thickness) and
studied in a disease nursery at Sakha Agriculture Research fixed on microscopic slides with Haupt`s adhesive (1 g gela-
Station, Plant Pathology Research Institute, Agriculture tin + 2 g phenol + 15 ml glycerol + 100 ml water), as men-
Research Center, Kafrelsheikh Governorate, Egypt, during tioned by Sass (1961). Slides were left to completely dry in
2018 and 2019 growing seasons. This nursery was infested the oven at 40ºC for 1 day. Sections were stained with 1%
artificially with C. maydis (4 clonal lineages) that causes late safranin and 1% light green dyes, cleared in xylene, mounted
wilt of maize and commonly used in Egyptian maize breed- in Canada balsam and examined microscopically.
ing programs (Zeller et al. 2002). The physical and chemi-
cal properties of soil used in this experiment are shown in Yield and yield component
Table 1. Plots were arranged in the field using a randomized
complete block design. From the above-mentioned treat- During the harvest period, quantitative and qualitative maize
ments in pot experiment, the most two tested bacterial strain yield such as ear length (cm), ear diameter (cm), number of

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rows per ear, 100-grains weight (g) and yield (ton ha−1) were were considered positive for siderophore production.
evaluated during the 2018 and 2019 seasons. Among the strains, intensity of orange halo and diameter
showed wide variation which ranged from 1.62 cm (B.
Statistical analysis subtilis) to 0.36 cm (B. licheniformis). So, B. subtilis and
P. koreensis strains were identified as producer of more
Statistical analysis of data was conducted, using SPSS 14.0 avid iron chelator. On the other hand, two strains of B.
for windows and the mean values were compared by Dun- subtilis and B. coagulanse showed siderophore positive in
can’s multiple range test at P ≤ 0.05 (Duncan 1955). Arnow’s assay. On the contrary, three strains of B. licheni-
formis, P. fluroscence and P. koreensis showed siderophore
positive in Csáky test. ­FeCl3 test was siderophore positive
Results for B. subtilis and B. coagulanse strains as compared to
other tested strains.
Qualitative and quantitative assays for siderophore Using the dual culture technique, six bacterial strains
production were tested for antagonistic activity against C. maydis
(Fig. 2 and Supplementary Figure 1). After 7-day cultiva-
In the present investigation, six strains of B. subtilis, B. tion on PDA medium, all strain treatments inhibited the
circulance, B. coagulanse, B. licheniformis, P. fluroscence growth of C. maydis in dual culture compared to the con-
and P. koreensis were screened by five different siderophore trol. The antifungal activity of B. subtilis and P. koreensis
assays, viz., CAS-agar assay, CAS-liquid assay, Arnow’s strains reduced the growth of C. maydis by 4.36 and 4.26 cm
assay, Csáky test and ­FeCl3 test (Table 2; Fig. 1). (Fig. 2a), and the reduction % attained was 51.55 and 52.66
All tested strains exhibited an orange halo after 5 days (Fig. 2b), respectively, compared to other tested strains. In
of incubation at 30 °C on CAS agar plate and, therefore, addition, antifungal activity of B. licheniformis strain was

Table 2  Quantitative and Stains CAS-agar ­(cm−1) CAS-liquid (%) Arnowʼs Csáky test FeCl3 test
qualitative assays of siderophore assays
produced by different bacterial
strains B. subtilis 1.62 ± 0.06 18.14 ± 0.64 + − +
B. circulance 0.92 ± 0.04 9.02 ± 0.14 − − −
B. coagulanse 0.72 ± 0.03 8.10 ± 0.30 + − +
B. licheniformis 0.36 ± 0.03 7.22 ± 0.14 − + −
P. fluroscence 1.09 ± 0.07 11.50 ± 0.19 − + −
P. koreensis 1.50 ± 0.02 16.03 ± 0.18 − + −

Values are average of three replications; values after ± represent standard deviation. Antagonistic activity
tests
+ siderophore positive, − siderophore negative

Fig. 1  Comparison of siderophore production in different bacte- detected in CAS liquid, C: control; 1: B. subtilis, 2: B. circulance, 3:
rial strains, a siderophore production in the six strains was detected B. coagulanse, 4: B. licheniformis, 5: P. fluroscence and 6: P. koreen-
on CAS agar plates, b siderophore production in the six strains was sis

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A 10 B
9 80
8 70
7 60
Growth (cm)

Reducon (%)
6 50
5
40
4
30
3
20
2
1 10
0 0
C B1 B2 B3 B4 B5 B6 C B1 B2 B3 B4 B5 B6
Strains Strains

Fig. 2  Mycelial growth inhibition of C. maydis by tested bacterial culance, B3: B. coagulanse, B4: B. licheniformis, B5: P. fluroscence
strains in dual culture test on PDA after 7 days of incubation at 28 °C: and B6: P. koreensis. Values are average of three replications and the
a linear growth; b reduction %; c control; B1: B. subtilis, B2: B. cir- results are shown as mean ± SD

less than that of other strains and recorded 2.26 cm growth Data of Fig. 3 showed that all inoculation treatments
and 74.44% reduction. decreased damping off % and increased healthy plants com-
pared with the control treatments (T1 and T2). The lowest
pre-emergence damping-off plants (15 days) were recorded
Pot experiment for the treatments T3 and T8 (11.66%) compared to other
treatments. Also, the post-emergence percentages of damp-
Antagonistic effect and growth promotion studies ing-off plants (30 days) showed notable decreases due to
the different studied bacterial strains; therefore, the most
The effects of antagonistic bacterial strains (B. subtilis, B. effective treatments were T3, T7 and T8 which completely
circulance, B. coagulanse, B. licheniformis, P. fluroscence prevent post-emergence. Concerning the growth promo-
and P. koreensis) on the pre- and post-emergence damping tion parameters (Table 3), T3 treatment (grains inoculated
off of C. maydis and growth promotion studies of maize with B. subtilis) attained an increase rate of 37.42% in fresh
plants in greenhouse are shown in Fig. 3 and Table 3. weight, 84.79% in dry weight, 76.29% in root length, 21.47%

Pre Post
70 a
60 a
50
b b
40 b
%

30 c c
20 b d d
d c c
10
0
T1 T2 T3 T4 T5 T6 T7 T8
Treatments

Fig. 3  Effect of antagonistic bacterial strains on the pre- and post- culance; T5: grains inoculated with B. coagulanse; T6: grains inocu-
emergence damping off of C. maydis under greenhouse conditions. lated with B. licheniformis; T7: grains inoculated with P. fluroscence;
T1: uninoculated (control); T2: grains treated with fungicide; T3: T8: grains inoculated with P. koreensis. Values are average of three
grains inoculated with B. subtilis; T4: grains inoculated with B. cir- replications and the results are shown as mean ± SD

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Table 3  Effect of antagonistic bacterial strains on growth promotion studies of maize plant in soil infested with C. maydis under greenhouse
conditions
Treatments Fresh weight (g Dry weight (g Root length (cm) Shoot length (cm) Areal root Seminal root Stem diameter (cm)
­plant−1) ­plant−1)

T1 62.33 ± 2.08e 19.66 ± 2.08f 19.66 ± 2.08f 63.66 ± 1.52e 4.33 ± 0.57c 13.00 ± 1.00e 4.60 ± 0.10de
T2 65.66 ± 2.08d 22.66 ± 1.52e 24.00 ± 1.00e 66.33 ± 1.52d 4.33 ± 0.57c 14.33 ± 0.57cde 4.73 ± 0.11cd
T3 85.66 ± 2.08a 36.33 ± 2.08a 34.66 ± 2.08a 77.33 ± 1.52a 6.66 ± 0.57a 19.00 ± 1.00a 5.53 ± 0.15a
T4 75.66 ± 1.52b 29.00 ± 1.00c 29.00 ± 1.00bc 74.33 ± 0.57b 5.66 ± 0.57ab 15.00 ± 1.00bcd 5.13 ± 0.05b
T5 70.66 ± 2.08c 25.00 ± 1.00de 26.66 ± 1.52cd 67.33 ± 1.52d 4.33 ± 0.57c 13.66 ± 0.57de 4.80 ± 0.10c
T6 65.33 ± 2.08e 23.00 ± 1.00e 25.00 ± 1.00de 62.00 ± 1.00e 4.33 ± 0.57c 13.00 ± 1.00e 4.50 ± 0.10e
T7 75.66 ± 0.57b 27.33 ± 1.15cd 30.00 ± 1.00b 70.00 ± 1.00c 5.00 ± 1.00c 15.33 ± 0.57bc 5.06 ± 0.11b
T8 83.66 ± 1.52a 32.33 ± 2.08b 33.33 ± 0.57a 74.00 ± 1.00b 6.66 ± 0.57a 16.33 ± 0.57b 5.40 ± 0.10a

Values are average of three replications and the results are shown as mean ± SD; the letters show significance at P ≤ 0.05
T1 uninoculated (control), T2 grains treated with fungicide, T3 grains inoculated with B. subtilis, T4 grains inoculated with B. circulance, T5
grains inoculated with B. coagulanse, T6 grains inoculated with B. licheniformis, T7 grains inoculated with P. fluroscence, T8 grains inoculated
with P. koreensis

in shoot length, 53.81% in areal root, 46.15% in seminal root all treatments, T5 (inoculated with a mixture of B. subti-
and 20.21% in stem diameter compared to control treatments lis and P. koreensis) induced the activity of POX enzyme
(T1). Of the six studied antagonists, B. subtilis and P. kore- (mM H2O2 g−1 FW min−1) recording higher values than
ensis were observed to perform better in antagonistic effect control, which changed significantly from 107.33 (control)
and growth promotion parameters. to 172.66 (T5) in the season 2018. Likewise, in the season
2019, POX activity recorded 115.66 (control) to 180.00 (T5)
(Fig. 4). In the same way, the highest values of PPO enzyme
Field experiment activity (µM tetra-guaiacol g−1 FW min−1) recorded 0.28
and 0.32 for T5 treatment, followed by 0.22 and 0.26 for
Antioxidant enzymes, total chlorophyll T3 treatment and 0.18 and 0.22 for T4 treatment during the
and carotenoids 2018 and 2019 seasons, respectively, compared to control
treatment (Fig. 4).
The presented results in Fig. 4, show that the antioxidant Our findings for total chlorophyll and carotenoids was sta-
enzymes’ activity (CAT, POX and PPO), as well as total tistically significant (P ≤ 0.05). Data showed that an increase
chlorophyll and carotenoids significantly improved in plants in total chlorophyll and carotenoids was observed with T5
exposed to inoculation treatments under soil infested con- treatment (inoculated with a mixture of B. subtilis and P.
ditions with C. maydis at 60 days during the two growing koreensis) resulted in 2.28 and 0.79 mg g−1 FW, followed
seasons. by 2.07 and 0.68 mg g−1 FW for T4 treatment (inoculated
T h e h i g h e s t v a l u e s o f C AT a c t i v i t y with P. koreensis) and 1.88 and 0.63 mg g−1 FW for T3 treat-
(mM H2O2 g−1 FW min−1) in treated plants were determined ment (inoculated with B. subtilis) compared to control in the
to be increased from 9.00 (control) to 12.66 (grains treated season 2018, respectively. A similar trend was observed in
with fungicide), 18.66 (inoculated with B. subtilis), 22.33 the season 2019 (Fig. 4).
(inoculated with P. koreensis) and 27.00 (inoculated with a
mixture of B. subtilis and P. koreensis) in the season 2018, Efficiency of treatments on maize late wilt disease
whereas in the season 2019 the same treatments increased and stem histological differences
CAT activity from 11.33 (control) to 14.00 (grains treated
with fungicide), 19.66 (inoculated with B. subtilis), 23.66 Under artificial soil infestation, data presented in Figs. 5 and
(inoculated with P. koreensis) and 28.66 (inoculated with 6 showed that grains treated with bacterial inoculation either
a mixture of B. subtilis and P. koreensis) significantly as alone or in combination significantly reduced the infection
shown in Fig. 4. percentage with late wilt disease and increase in reduction
Additionally, soil infested with C. maydis caused a percentage compared to the control treatment. For disease
reduction in POX activity as found in untreated maize incidence, treatment with a mixture of B. subtilis and P.
plants, while treating plants with bioinoculation alleviated koreensis (T5) gave the highest effect in reducing infection
the detrimental effect of soil infested with C. maydis on 13.33 and 14.66%, followed by treatment with B. subtilis
antioxidant capacity represented in POX activity. Among (T3) 23.00 and 20.55% compared to control treatment (T1)

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Fig. 4  Effect of inoculation CAT 2018 2019

with B. subtilis and P. koreen- 35
sis alone or in combination on 30 a
a
activities of catalase (CAT), b b

mM H2O2 g-1 FW min-1

25
peroxidase (POX), polyphenol c c
20
oxidase (PPO), total chlorophyll
15 d d
and carotenoids of maize plants e
e
at 60 days in the 2018 and 10
2019 seasons. T1: uninoculated 5
(control), T2: grains treated 0
with fungicide, T3: inoculated T1 T2 T3 T4 T5
with B. subtilis, T4: inoculated Treatments
with P. koreensis and T5:
inoculated with a mixture of B. POX 2018 2019
subtilis and P. koreensis. Values
are average of three replica- 200

mM tetra-guaiacol g-1 FW min-1

a a
180
tions and the results are shown b b
as mean ± standard errors and
160 c c
140 d d
the letters show significance at 120 e
e
P ≤ 0.05 100
80
60
40
20
0
T1 T2 T3 T4 T5
Treatments

PPO 2018 2019

0.4 a a
b
µM tetra - guaiacol g-1 FW min-1

c
0.3
b c
0.2 d
d
0.1 e
e

0
T1 T2 T3 T4 T5
Treatments

Total Chlorophyll 2018 2019

2.5 a a
b b
2 c c
d d
e e
mg g-1 FW

1.5

0.5

0
T1 T2 T3 T4 T5
Treatments

Carotenoids 2018 2019

1
a a
0.8 b b
c c
d d
mg g-1 FW

0.6
e e
0.4

0.2

0
T1 T2 T3 T4 T5
Treatments

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Disease incidence % Reduction %

80
a a
70
b b
60 b
b a
50 a
b b
40
30 c c c c
c c
20 d d
10
0
T1 T2 T3 T4 T5 T1 T2 T3 T4 T5
2018 2019

Fig. 5  Effect of inoculation with B. subtilis and P. koreensis alone or subtilis, T4: inoculated with P. koreensis and T5: inoculated with a
in combination on the incidence of late wilt of maize grown under mixture of B. subtilis and P. koreensis. Values are average of three
field conditions during 2018 and 2019 seasons, T1: uninoculated replications and the letters show significance at P ≤ 0.05
(control), T2: grains treated with fungicide, T3: inoculated with B.

45.66 and 49.66% during the 2018 and 2019 seasons, respec- respectively (Table 4). In regard to 100 grain weight, a dif-
tively. Similar trend was observed in reduction % in the sea- ferent increase rate was noticed between single and dual
son 2018 and 2019 with the studied treatments following the inoculation treatments compared to control, where 21.38
descending order of T5 > T3 > T4 > T2 > T1 (Fig. 5). and 21.08% at T5 treatment (inoculated with a mixture of
On the other hand, the cross section of maize plant stem B. subtilis and P. koreensis), 4.11 and 3.86% at T4 treatment
at 85 days after sowing, in Fig. 6, showed increase in the ((inoculated with P. koreensis), and 7.67 and 7.46% at T3
thickness of the sclerenchymatous sheath layer surround- treatment ((inoculated with B. subtilis) were recorded during
ing the vascular bundles in bacterial inoculation treatments the 2018 and 2019 seasons, respectively (Table 4).
(T3, T4 and T5) compared to uninoculated treatments (T1 On the other hand, the positive effect caused by dual inoc-
and T2). Also, it is worth to note the presence of C. may- ulation treatment on the yield of maize plants which was
dis aggregates in the xylem vessels of control treatments 10.04 and 10.12 ton ha−1 recorded an increase rate of 60.24
(Fig. 6a, b), whereas these did not appear in the xylem ves- and 51.27% during the 2018 and 2019 seasons, respectively,
sels of inoculation treatments (Fig. 6c–e). compared to the control treatment (Table 4).

Yield and yield components

Discussion
A perusal of results (Table 4) revealed that inoculation treat-
ments either alone or in combination showed significant It is important to understand the mechanisms involved in
influence for yield and yield-related parameters (ear length, the interactions between microorganisms, pathogens, and
ear diameter, number of rows/ears and 100 grain weight) on host plants for the better use of natural resources in crop
soil infested with C. maydis, whereas uninoculated treat- productivity management (Thomashow and Weller 1991).
ments had no significant effect. One of these mechanism are siderophores production, which
The combination treatment (T5) caused the maximum are defined as low molecular weight organic compounds
values of ear length (cm), ear diameter (cm) and number (< 10 kDa) that are excreted by microorganisms living in
of row/ear recorded as 23.00, 4.58 and 13.46, respectively, low iron conditions (Oswald 2010), and is one of the mecha-
compared to the control (T1), recorded as 20.43, 4.16 and nisms of antagonism and biocontrol agents (Sinha and Parli
10.73 in the season 2018. Likewise, in season 2019, T5 treat- 2020) that has been proved in plant growth-promoting bac-
ment recorded 23.20, 4.61 and 13.66 compared to T1 treat- teria (Yang et al. 2011). In the present study, a total of six
ment (control) that recorded 20.66, 4.25 and 10.80 for ear PGPR strains were tested in laboratory to produce sidero-
length (cm), ear diameter (cm) and number of rows/ears, phores using CAS-agar assay, CAS-liquid assay, Arnowʼs

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Fig. 6  Transverse section (X 400) in the second stem internode of lis (T3), d inoculated with P. koreensis (T4), and e inoculated with a
maize plants at 85 days from sowing during 2019 season; a control mixture of B. subtilis and P. koreensis (T5)
(T1), b grains treated with fungicide (T2), c inoculated with B. subti-

assay, Csáky test and F­ eCl3 test. Two strains, B. subtilis and salicylate and carboxylate. These siderophores play a vital
P. koreensis, were found to be the most qualitative and quan- role in the accumulation of Fe from various organic materi-
titative assays for siderophore production (Table 2; Fig. 1). als (Balagurunathan and Radhakrishnan 2007).
Rajkumar et al. (2010) showed that bacterial cells produce The antagonistic activity tested in our study effectively
many types of siderophores, i.e., hydroxamate, catecholate, suppressed the mycelial growth of C. maydis in in vitro

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Table 4  Effect of inoculation Treatment Ear length (cm) Ear diameter (cm) No. of row/ear 100 grain weight (g) Yield
with B. subtilis and P. koreensis (ton ­ha−1)
alone or in combination on
some parameters of yield and 2018
yield component of maize
T1 20.43 ± 0.3 e 4.16 ± 0.15 c 10.73 ± 0.2 e 30.11 ± 0.09 e 6.64 ± 0.07 e
plants grown under field
conditions during 2018 and T2 21.26 ± 0.3 d 4.40 ± 0.1 bc 11.26 ± 0.25 d 30.51 ± 0.14 d 7.29 ± 0.1 d
2019 seasons T3 21.86 ± 0.05 c 4.33 ± 0.15 ab 12.16 ± 0.15 c 32.42 ± 0.1 c 8.41 ± 0.1 c
T4 22.36 ± 0.15 b 4.50 ± 0.1 ab 12.80 ± 0.1 b 31.35 ± 0.18 b 9.52 ± 0.09 b
T5 23.00 ± 0.2 a 4.58 ± 0.7 a 13.46 ± 0.37 a 36.55 ± 0.22 a 10.04 ± 0.02 a
LSD 0.05 0.442 0.215 0.467 0.237 0.106
2019
T1 20.66 ± 0.2 d 4.25 ± 0.13 c 10.80 ± 0.1 e 30.26 ± 0.08 e 6.69 ± 0.03 e
T2 21.63 ± 0.05 c 4.41 ± 0.09 bc 11.36 ± 0.25 d 30.74 ± 0.02 d 7.31 ± 0.15 d
T3 22.56 ± 0.57 b 4.37 ± 0.13 bc 12.30 ± 0.1 c 32.52 ± 0.03 c 8.49 ± 0.07 c
T4 22.60 ± 0.1 b 4.54 ± 0.08 ab 12.86 ± 0.05 b 31.43 ± 0.03 b 9.54 ± 0.08 b
T5 23.20 ± 0.26 a 4.61 ± 0.03 a 13.66 ± 0.2 a 36.64 ± 0.02 a 10.12 ± 0.02 a
LSD 0.05 0.529 0.180 0.328 0.080 0.103

T1: uninoculated (control), T2: grains treated with fungicide, T3: inoculated with B. subtilis, T4: inocu-
lated with P. koreensis and T5: inoculated with a mixture of B. subtilis and P. koreensis. Values are aver-
age of three replications and the results are shown as mean ± standard errors. The presented data are the
mean ± standard errors, and the letters show significance at P ≤ 0.05

assays (Fig. 2). Data indicated that B. subtilis and P. kore- P. fluorescens had a significant reduction in late wilt disease
ensis proved to be the most effective antagonist than other under greenhouse conditions.
tested strains which reduced the growth of C. maydis by Influence of the tested bacterial strains on the disease
4.36 and 4.26 cm (Fig. 2a), and the reduction % attained expressions of maize plants grown in soil infested with C.
was 51.55 and 52.66 (Fig. 2b), respectively. The suppres- maydis under greenhouse conditions are studied by pre- and
sion of mycelial growth of C. maydis was mainly due to the post-emergence damping off in this study Fig. 3. Results
diffusible antifungal substances such as hydrolytic enzymes, showed that the most effective treatments were T3 (grains
antibiotics or some other secondary metabolites (Beneduzi inoculated with B. subtilis), T7 (grains inoculated with P.
et al. 2012). The antagonistic activities of Bacillus and fluroscence) and T8 (grains inoculated with P. koreensis)
Pseudomonas were recognized by many researchers. Fer- which completely prevent pre- and post-emergence. These
reira et al. (1991) showed that different Bacillus spp. can results may be due to the effective antibiotics (PCA, PLT,
produce 66 types of antibiotic compounds against bacteria DAPG and PLN) and lipase, pectinase, amylase and pro-
and fungi such as bacillomycin, fengycin, mycosubtilin, and tease enzymes as well as siderophores (pyoverdine and pyo-
zwittermicin, which are effective in suppressing the growth chelin) that are produced by P. fluorescens, which showed
of target pathogens in vitro (Kim et al. 2008). Also, many antagonistic activity toward C. maydis (Ebrahim 2010).
biocontrol agents of Pseudomonas spp. can produce extra- Also, Bacillus spp. have modes of action such as antibiosis,
cellular secondary metabolities such as cepacins, testin, parasitism, colonization, competition and induced systemic
2,4-diacetylphloroglucinol, pyoluteorin, phenazine, pyrrol- resistance (Jacobsen et al. 2004). Similar findings against
nitrin, pyoverdine and altericidines that inhibit the growth C. maydis by actinomycetes and yeast fungi were stated by
of some soilborne fungal pathogens (Mavrodi et al. 2001; El-Mehalawy et al. (2004). Hamza et al. (2013) reported that
Subagio and Foster 2003), and are attributed to the produc- the formulations of B. subtilis, B. pumilus, P. fluorescens and
tion of siderophores, volatile compounds, cell wall-degrad- Epicoccum nigrum caused noticeable reduction in maize late
ing molecules, extracellular chitinase, and protease enzymes wilt. Also, El-Shabrawy and Shehata (2018) found that sig-
activity (Mavrodi et al. 2001). Likewise, under in vitro con- nificant reduction in the disease incidence was recorded by
ditions, Sandani et al. (2019) showed that Burkholderia and maize grains treated with P. fluorescens, either individually
Pseudomonas bacteria are effective antagonists of C. trun- or in combination with B. subtilis. Concerning the growth
catum causing anthracnose. Also, Ali et al. (2018) studied 40 promotion parameters, results showed that grains inoculated
PGPR strains which are able to produce siderophores as well with B. subtilis (T3) and grains inoculated with P. koreensis
as have a significant growth suppression of pathogenic F. (T8) attained significant increase in fresh weight, dry weight,
oxysporum and R. solani. El-Shabrawy and Shehata (2018) root length, shoot length, areal root, seminal root and stem
demonstrated that maize grain treated with B. subtilis and diameter compared to control treatments in soil infested with

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 Archives of Microbiology

C. maydis under greenhouse conditions (Table 3). These (inoculated with a mixture of B. subtilis and P. koreensis)
results reflect the role of the tested bioagent inoculants in recorded 7- and 4.75-fold in the 2018 and 2019 seasons,
their effects on growth parameters and control plants such respectively (Fig. 4). It is known that the process of oxi-
as stimulating cell division, elongation, formation of dried dizing phenols to quinones substances, which are toxic to
tissues in plants and production of plant growth regulators pathogens due to PPO enzyme. Also, PPO enzyme can
(López-Reyes 2017; Shi et al. 2018; Zhang et al. 2019; Khan function in the following ways: (1) quinines substances
et al. 2020). Similarly, Laslo et al. (2012) reported that the produced by PPO may alkylate essential amino acids
rapid colonization of maize plant roots due to the produc- which decrease plant nutritional quality, (2) sometimes,
tion of siderophores by Pseudomonas strains stimulates and through redox cycling, quinones may produce oxida-
plant growth and is often due to the reduction in the fungal tive stress in the gut lumen and (3) from phenolic oxida-
population in the roots (Penyalver et al. 2001). tion, quinone substances produced may be absorbed and
Under artificially infested field conditions, grains treated have toxic effects on herbivores (Helmi and Mohamed
with rhizobacterial strains significantly decreased disease 2016). Chandrasekaran and Chun (2016) indicated that B.
incidence and improved yield and yield component of maize subtilis CBR05 treatment showed a significant increase
during the 2018 and 2019 seasons. In the current study, in PPO activity in the leaves of tomato infested with soft
antioxidant enzymes’ activity (CAT, POX and PPO) sig- rot disease (Erwinia carotovora subsp. carotovora). Also,
nificantly improved in plants exposed to inoculation treat- PPO enzyme activities have been well established by Kaur
ments with B. subtilis and P. koreensis either alone or in et al. (2017). They found that infested wheat plants with
combination, which in turn reflected in the reduced disease aphid showed a significant increase in PPO activity (1.4-
incidence. We observed that plants inoculated with dual fold) at 28 days after emergence (DAE) as compared to
inoculation showed higher (3 and 2.5 fold) CAT activities the respective uninfested plants. Also, the content of pho-
than control plants in the 2018 and 2019 seasons, respec- tosynthetic pigments showed a significant increase under
tively, at 60 days after sowing (Fig. 4). These increases in the combined inoculation with B. subtilis and P. koreensis
CAT activity were associated with higher growth rates of the (Fig. 4). In this regard, the severity of late wilt disease
most infective fungus. Therefore, CAT plays a vital role in is associated with chlorophyll catabolism; therefore, the
the regulation of intraradical fungal growth. Also, surveying pathogen causes chlorophyll deterioration and organelle
with ­H2O2 by CAT is an effective mechanism for mitigating damage and cell death (Kariola et al., 2005; Bagy et al.
plant defense responses (Wu et al. 1997). These findings are 2019). The inoculation treatments with B. subtilis and
supported by those reported for other crops grown under P. koreensis individually or in combinations may induce
infested soil conditions such as maize (Kumar et al. 2009), disease resistance through photosynthetic pigment resto-
eggplant (Altinok et al. 2013), tomato (Chandrasekaran and ration to reduce disease development. Our results are in
Chun 2016), cotton (Selim et al. 2017), soybean (Zilli et al. agreement with the findings of Borkar and Yumlembam
2018) and potato (Bagy et al. 2019). (2016) and El-Shabrawy and Shehata (2018), concern-
A similar trend was also observed in the activities of ing the efficiency of inoculation treatments on maize late
POX. The higher activity of peroxidase was recorded by wilt disease and stem histological differences (Figs. 5, 6).
the application B. subtilis and P. koreensis (T5) which Treatment with a mixture of B. subtilis and P. koreen-
showed an increase rate recorded 55–60% during the two sis (T5) gave the highest effect in reducing infection and
growing seasons (Fig. 4). Vidhyasekaran (1997) showed increase in thickness of the sclerenchymatous sheath layer
that peroxidase enzyme may be involved in the regulation surrounding the vascular bundles. These results may be
of plant cell elongation, polysaccharide cross-linking, phe- due to the production of siderophores, non-volatile diffus-
nol oxidation and IAA oxidation as well as associated with ible metabolites which are related to significant inhibition
disease resistance in plants and increases in host plants fol- and biocontrol of the pathogen (El-Mehalowy et al. 2004;
lowing pathogen infection (Scott-craiz et al. 1995). Several Selim et al. 2017; Bagy et al. 2019). Also, mycelial growth
previous researches confirmed that addition of PGPR to was clearly found in the bundles of stems in 85 days maize
plants induces the activity of POX enzyme and, therefore, plants infected with C. maydis (control treatments). On the
enhances the plant’s disease resistance (Ramamoorthy contrary, all inoculation treatments showed more layers
et al. 2001; Paul and Sarma 2005; Renuka et al. 2009; of sclerenchymatous cells surrounding the xylem vessels.
Kaur et al. 2017; Zilli et al. 2018; Bagy et al. 2019). In These findings are supported by (Kumar et al. 2009; Boon
the case of PPO activity, we observed that maize plants et al. 2012; Hajiboland et al. 2012; Ghazy et al. 2017).
inoculated with single inoculation showed 2.75- and For yield and yield parameters, inoculation treatments
3.14-fold higher activity for T3 (grains inoculated with either alone or in combination showed significant influ-
B. subtilis) and 5.5- and 4.75-fold for T4 (grains inocu- ence compared to control (Table 4). These increase in
lated with P. koreensis), whereas dual inoculation for T5 yield due to improve the availability of essential nutrients,

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production of phytohormones, improved Fe availability Bagy HMK, Hassan EA, Nafady NA, Dawood MF (2019) Efficacy of
through chelating soil iron by production of siderophores arbuscular mycorrhizal fungi and endophytic strain Epicoccum
nigrum ASU11 as biocontrol agents against blackleg disease
and enhanced plant resistance system. These results cor- of potato caused by bacterial strain Pectobacterium carotovora
roborate with those of , , , and ). subsp. atrosepticum PHY7. Biol Control 134:103–113
Balagurunathan R, Radhakrishnan M (2007) Microbial Sidero-
phores-gateway for iron removal. Envis Centre Newsletter. https​
://www.envis​madra​suniv​.org/nl200​07art​icles​%20sid​ephor​e.html
Conclusion Bashan Y, De-Bashan LE, Prabhu SR, Hernandez JP (2014)
Advances in plant growth-promoting bacterial inoculant
technology: formulations and practical perspectives (1998–
Our findings indicate that B. subtilis MF497446 and P. 2013) (a marschner review). Plant Soil 378:1–33. https​://doi.
koreensis MG209738 were found to be the most effective org/10.1007/s1110​4-013-1956-x
PGPRs as shown by qualitative and quantitative assays for Becker JO, Cook RJ (1988) Role of siderophores in suppression of
Pythium species and production of increased growth response of
siderophore production and individually or in combination wheat by fluorescent pseudomonads. Phytopathol 78:778–782
inoculation condition, leading to induce the disease resist- Beneduzi A, Ambrosini A, Passaglia LM (2012) Plant growth-pro-
ance of late wilt caused by C. maydis in maize plant and moting rhizobacteria (PGPR): their potential as antagonists and
improvement of growth promotion and yield. Further inves- biocontrol agents. Genet Mol Biol 35:1044–1051
Boon EJMC, Struik PC, Engels FM, Cone JW (2012) Stem charac-
tigations are required to study the interactions at the level of teristics of two forage maize (Zea mays L.) cultivars varying in
gene related to siderophore production in PGPR, pathogenic whole plant digestibility. IV. Changes during the growing sea-
fungi or host plant. son in anatomy and chemical composition in relation to fermen-
tation characteristics of a lower internode. NJAS Wageningen J
Acknowledgements We thank all staff members and colleagues in The Life Sci 59(1–2):13–23
Bacteriology Research Laboratory and Maize and Sugar Crops Dis. Borkar SG, Yumlembam RA (2016) Bacterial diseases of crop
Dept., Sakha Agricultural Research Station, Kafrelsheikh, Egypt, for plants. CRC Press, Boca Raton
their valuable cooperation which made completion of this work possi- Chandrasekaran M, Chun SC (2016) Expression of PR-protein genes
ble. Also, this work is supported in cooperation between SWERI, ARC, and induction of defense-related enzymes by Bacillus subtilis
and Egyptian maize breeding programs, PPRI, ARC, Egypt. CBR05 in tomato (Solanum lycopersicum) plants challenged
with Erwinia carotovora subsp. carotovora. Biosci Biotechnol
Biochem 80(11):2277–2283
Authors contributions SE conceived of and designed the study. SE and Cśaky TZ (1948) On the estimation of bound hydroxylamine in bio-
NG analyzed and interpreted the data. All authors gave final approval logical materials. Acta Chem Scand 2:450–454
of the version to be published. Duncan BD (1955) Multiple ranges and multiple F. test. Biometrics
11:1–42
Compliance with ethical standard Ebrahim EMT (2010) Relation of fluorescent bacteria in controlling
some root diseases of sugar beet. Ph.D. Thesis, Faculty of Sci-
Conflict of interest The authors declare that they have no conflict of ence, Bani-Seuf University, Egypt, 195p
interest. El-Mehalowy AA, Hassanein NM, Khater HM, Daram El-Din EA,
Youssef YA (2004) Influence of maize root colonization by
Rhizosphere actinomycetes and yeast fungi on plant growth
and on the biological control of late wilt disease. Int J Agric
Biol 6:599–605
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