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Environmental Sustainability

, Volume 1, Issue 4, pp 341–355 | Cite as

Corn sap bacterial endophytes and their potential in plant growth-promotion

  • Shimaila AliEmail author
  • Joshua Isaacson
  • Yulia Kroner
  • Soledad Saldias
  • Saveetha Kandasamy
  • George Lazarovits
Original Article
  • 476 Downloads

Abstract

Plants interact with many different bacteria at various stages of their life. A mutualistic relationship between a plant and an endophytic bacterium occurs when a plant provides a safe habitat and a secure food supply to the microbe and it, in return, benefits the plants’ capacity to function in its environment. In this study, corn sap was screened for the diversity and functionality of culturable bacterial endophytes, and a total of 437 bacterial endophytes were isolated, identified, and characterized for their possible role as biofertilizers and biocontrol agents. The growth promoting traits that were characterized included siderophore production, phosphate and zinc solubilization, nitrogen fixation, indole acetic acid production, and antifungal activity against several plant pathogens. Most isolates (71.8%) were found to fix nitrogen and solubilize phosphate (66.8%), close to half (42.5%) could solubilize zinc, and 28.3% of the corn bacterial endophytes could sequester iron. Most isolates (77.3%) displayed antifungal activity, and 186 of the 437 isolates tested were found to promote plant growth in a gnotobiotic assay. These results suggest that growth promotion from these bacterial endophytes is the outcome of multiple biochemical and microbiological mechanisms.

Keywords

Corn sap Bacterial endophytes Plant growth promotion Biofertilizer Biocontrol agent 

Introduction

Corn (Zea mays L) is one of the most important cereal crops worldwide and is considered as an essential crop for feeding the world’s growing population (Montañez et al. 2012; Haspel 2015). It is a highly adaptable crop and can be cultivated under a variety of climates from tropical to temperate (Haspel 2015). Numerous microorganisms have been shown to reside on and within corn plants and these actively influence plant growth by imparting resistance or tolerance against phytopathogens, herbicides, and various abiotic environmental stresses (Johnston-Monje and Raizada 2011; Ikeda et al. 2013; Szilagyi-Zecchin et al. 2014; Sandhya et al. 2017; Walitang et al. 2017; Correa-Galeote et al. 2018). To understand the interactions between corn and its associated microorganisms, we need to know their identities and biological activities so that they may be utilized to improve crop yields.

Bacterial endophytes can be defined as a group of microorganisms that can enter and colonize plants but result in no deleterious symptoms (Bacon and White 2000; Reiter and Sessitsch 2006; Ali 2013). Endophytes utilize a number of different mechanisms to promote plant growth, such as nitrogen fixation (Estrada et al. 2002; Doty et al. 2009; Xin et al. 2009; Montañez et al. 2012; Maheshwari et al. 2013), phosphate solubilization (Kuklinsky-Sobral et al. 2004; Forchetti et al. 2007; Dias et al. 2009; Puente et al. 2009; Rajkumar et al. 2009; Palaniappan et al. 2010; Vendan et al. 2010), iron acquisition (Li et al. 2009; Vendan et al. 2010; Lacava and Azevedo 2013; Zhao et al. 2016), zinc solubilization, phytohormone production (Costacurta and Vanderleyden 1995; Apine and Jadhav 2011; Rashid et al. 2012; Duca et al. 2014), and production of antimicrobial compounds. Endophytes by residing within the plant have a greater potential as plant growth promoters as they are more intimately connected to their host compared to those on external plant tissues (Reiter and Sessitsch 2006; Rashid et al. 2012).

In the current study, corn sap was selected as a starting material because very little is known about the sap microbial community. The objectives of the present study were to: (i) identify and assess the phylogenetic diversity of culturable bacterial endophytes from the sap of corn plants grown in 34 different farms across Ontario, Canada and the United States and (ii) screen these endophytes for their plant growth promotion and biocontrol capabilities.

Materials and methods

Corn sampling and sap extraction

A total of thirty-four corn farms from Canada and United States were selected to collect samples. All corn plants were collected at the rapid growth phase i.e. V10 stage (Fig. S1) from each of the participating farms. A 10-cm long segment of corn stem was cut approximately 30 cm above the ground and stored at 4 °C for 24 h. Ten plants (10 stem segments) were sampled from each farm and each stem segment was processed separately. They were surface sterilized as follows: washed for 3 min with tap water, then for 3 min with 70% ethanol, followed by a 3-min immersion in 1% commercial bleach, and finally rinsed three times with sterile water for 1 min each. To ensure that the surface sterilization process was successful, a 100 µl aliquot of the final rinse water was plated onto nutrient agar (NA) and incubated at 30 °C for 3 days. If no bacterial growth was found, the sterile corn stem segment was transferred to a sterile plastic bag, and the sap was collected by crushing the stems using a mechanical device, (Engenho Para Cana B60, Botini®, Industria Brasileira, Brazil), designed to extract sap from sugar cane stalks.

Corn sap plating and enumerating bacterial endophytes

All ten corn sap replicates from each farm were pooled and serially diluted (100–105) in sterile water, and 100 µl of each dilution was plated in duplicate onto NA, Pseudomonas isolation agar (PIA), and phenylethyl alcohol agar (PEA). Plates were incubated at 30 °C for 72 h. Thirty-four pooled sap samples were plated in total. After incubation, the number of colony forming units (CFU) per milliliter of sap were determined. Morphologically different colonies (based on size, shape, and color) were selected. Individual colonies were sub-cultured on respective growth media for further screening and to make glycerol stock cultures which were stored at − 80 °C.

Molecular identification

All isolated bacterial endophytes were identified by partial sequencing of 16S rRNA gene. Bacterial strains were grown in TSB and genomic DNA was isolated using a Soil DNA extraction kit (Norgen Biotek Corp., Thorold, Ontario, Canada) as described by the manufacturer. Using genomic DNA as a template, the bacterial 16S rRNA gene was amplified with the 27F and 1492R bacterial primers (Weisburg et al. 1991). A 50 µl aliquot of the PCR mixture for each strain contained 5 µl of 10 × buffer for Taq DNA Polymerase, 1.5 mM of MgCl2 (Invitrogen, Carlsbad, CA, USA), 0.2 mM of each of the four dNTPs, 0.2 µM of each primer, 30 ng/µl of total genomic DNA as template, 1.25 U of Taq DNA polymerase (Invitrogen), and PCR grade water to make up to the desired volume. A negative control (PCR mixture without DNA template) was included for each PCR run. Amplifications were carried out in a Bio-Rad T100™ thermal cycler using the following conditions: 94 °C for 5 min, 30 amplification cycles consisting of denaturation at 94 °C for 30 s, primer annealing at 53 °C for 30 s, and primer extension at 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. In each case, the PCR product was run on a 1% agarose gel containing Gelred dye and was then isolated and purified using a DNA Clean and Concentrator™-5 kit (Zymo Research Corporation, Irvine, CA, USA). Purified DNA was sent to TCAG DNA Sequencing Facility, University of Toronto. The sequences were analyzed using https://www.ezbiocloud.net/ server for the strain identification with the closely related type strains (Yoon et al. 2017) and subsequently, all sequences were submitted to Genbank.

Biochemical characterization

Siderophore production plate assay

Chrome azurol S (CAS), an indicator dye, forms a blue-colored ferric complex containing iron. The iron is removed from CAS by the siderophores produced by the microorganism (which has higher affinity for iron) and a positive reaction is indicated by a color change of the CAS reagent from blue to orange (Schwyn and Neilands 1987). This assay was done qualitatively on CAS agar plates. Five microliters of a bacterial culture growing in King’s B (KB) medium overnight was spotted onto CAS agar plate and incubated at 30 °C for 4–5 days (Alexander and Zuberer 1989; Rashid et al. 2012).

Phosphate solubilization plate assay

This assay was done qualitatively on Pikovskaya’s (PKV) agar. PKV agar contains insoluble phosphate that can be solubilized by organic acids released from microorganisms growing upon the medium (Kpomblekou-A and Tabatabai 1994). A positive reaction (solubilization of insoluble phosphate) was indicated by a clear zone around the bacterial colony. Five microliters of a bacterial culture grown in tryptic soy broth (TSB) medium overnight was spotted onto PKV agar plates and incubated at 30 °C for 4–5 days (Elias et al. 2016).

Zinc solubilization plate assay

Five microliters of overnight grown cultures (in TSB) were spotted onto a mineral salts agar medium that contains insoluble ZnO (Saravanan et al. 2007) and were incubated at 30 °C for 4–5 days. A positive reaction was indicated by the presence of a clear zone around the bacterial colony (Goteti et al. 2013). Zinc solubilization, like phosphate solubilization, is caused by the release of organic acids by the microorganisms growing upon the medium (Wu et al. 2006; Saravanan et al. 2007).

Nitrogen fixation assay

This assay was done on nitrogen-free malate agar medium (Wright and Weaver 1981). Bacteria were grown in TSB overnight and then five microliters of the bacterial suspension were spotted onto malate agar medium and incubated at 30 °C for 4–5 days. A positive reaction was indicated by a color change from light green to bright blue. Malate agar medium contains bromothymol blue dye that turns bright blue in acidic environments. If the growing bacterium can fix atmospheric nitrogen, the pH of the medium decreases and therefore the color turns blue due to the release of acidic ions by the bacteria.

All the potential nitrogen fixer strains those produced positive results on plate assay were tested for their capacity to fix nitrogen by molecular method. Using genomic DNA as template, the bacterial nifH gene was amplified with nifHf and nifHr (Gaby and Buckley 2012) and Polf & Polr primers sets (Poly et al. 2001). Amplifications were carried out in a Bio-Rad T100™ thermal cycler using the following conditions: 94 °C for 5 min, 30 amplification cycles consisting of denaturation at 94 °C for 30 s, primer annealing at 60 °C for 30 s, and primer extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min.

IAA production assay

A colorimetric based assay (Patten and Glick 2002) was used to measure the production of IAA. Five microliters of overnight cultures in TSB were used to inoculate 1 ml of TSB and 1 ml of TSB supplemented with 200 µg ml−1 of the IAA precursor tryptophan (L-Trp). The inoculated TSB was then incubated at 30 °C for 2–3 days. After the incubation, cultures were centrifuged and 200 µl of supernatant was mixed with an equal volume of Salkowski’s reagent (Gordon and Weber 1951), and incubated for 20 min at room temperature in the dark before the absorbance was measured at 535 nm (Rashid et al. 2012). The concentration of IAA produced by each sample was calculated using a standard curve ranging from 0.78 to 50 µg ml−1 pure IAA (Sigma). Strains that produced significantly higher (P < 0.05) amounts of IAA, when supplemented with l-tryptophan, than no supplementation media were considered positive for Trp dependent IAA production.

Antifungal activity assay

Bacterial antifungal activity was tested against Pythium aphanidermatum, Fusarium oxysporum, Rhizoctonia solani, and Verticillium dahlia using a modified dual culture plating assay. Fungal strains were kindly provided by Dr. Lazarovits of A & L Biologicals. A combination of two media, ¾ potato dextrose agar (PDA) and ¼ NA, was used for this test. For tests with P. aphanidermatum and F. oxysporum, plugs (6 mm) of the fungal inoculant were placed onto the plates on the same day they were inoculated with bacteria while with R. solani and V. dahliae the inoculum plugs were plated 2 and 4 days before bacterial inoculation, respectively. The plug of fungal mycelium was always placed onto the center of the plate and then four bacterial endophytes were streaked on the four sides of the plate and incubated at 25 °C for 1 week. If an inhibition zone was found adjacent to a bacterial colony, a confirmatory test was done in a similar way with that single bacterial strain using the respective fungal culture. Controls were inoculated only with fungal plugs and were used to measure growth in the absence of bacteria.

Plant growth-promotion and colonization assay

A total of 437 newly isolated bacteria were tested for their ability to colonize plant’s interior (true endophyte) and to promote the growth of wheat under gnotobiotic conditions as described by Rashid et al. (2012) with a few modifications. Briefly, test strains were grown in 5 ml of TSB and centrifuged at 5000 rpm for 5 min. The bacterial cells were then re-suspended in sterile water, and the OD600 was adjusted to 0.5 ± 0.02. Eighteen wheat seeds were treated with the diluted bacterial suspension for one hour at room temperature and each paper towel pouch (7 × 5 in2) was inoculated with 6 treated seeds with a total of three pouches per treatment. The three paper towel pouches were then rolled separately, kept in a plastic container in an upright position, and incubated in a growth room. Wheat seeds treated with sterile water were used as a negative control. Observations were recorded 7 days after emergence.

In a separate experimental setting where seeds were treated and grown in similar way, three plants of each treatment were surface sterilized as mentioned above, chopped, serially diluted, and plated on TSA plates. The colonial morphology of the bacteria was compared with the original strain morphology to confirm their ability to colonize plant tissues (Rashid et al. 2012).

Statistical analysis

All statistical analysis was performed using the GraphPad Prism software package 6.04 (GraphPad Software, Inc., San Diego, CA, USA). The data for seedling root growth were analyzed through analysis of variance (ANOVA). To identify which treatments were significantly different (P < 0.05), Tukey’s post hoc tests were performed. A student’s t test was performed to analyze differences in amount of IAA produced with or without L-Trp supplementation (P < 0.05).

Results

Endophytic bacterial enumeration and identification

A total of 437 morphologically different bacteria were isolated from the sap from corn stalks collected from 34 different farms. The 16S rRNA gene sequence for these newly isolated corn sap bacterial endophytes has been submitted to the Genbank and can be found through the accession numbers MG819162 to MG819598. The CFU counts of plated sap for each farm on three different media are presented in Table S1. The 16S rRNA gene sequence alignment data suggest that the endophytic strains belong to 69 different bacterial genera (Table 1), with Pseudomonas spp. being the most common (93 of 437 bacterial endophytes). The prominent genera found in corn sap communities include Enterobacter, Microbacterium, Pantoea, Curtobacterium, and Staphylococcus; however, Acinetobacter spp., Paenarthrobacter spp., Chryseobacterium spp., Erwinia spp., Pedobacter spp., Rahnella spp., Sphingomonas spp., Stenotrophomonas spp., and Variovorax spp. were also well represented. (Tables 1, S2). Although corn sap from Canada and United States shared many taxonomic classes, the microbial composition was found to be somewhat different. Generally, sap of Canadian origin was more diverse than sap from the United States (Tables S2, S3).
Table 1

Frequency of bacterial genera in corn sap endophyte communities and their respective plant growth promotion activities

Bacterial genus

No. of isolates

Zn solubilization

P solubilization

N fixation

Fe sequestration

IAA bioproduction*

Root elongation

Fungal antagonism

F. oxysporum

P. ephenidermatum

R. solani

V. dahliae

Achromobacter spp.

3

0

1

2

0

2

1

0

0

0

0

Acidovorax sp.

1

0

0

1

0

1

0

0

0

0

1

Acinetobacter spp.

10

7

9

9a

1

6

5

2

4

0

6

Aeromonas spp.

3

2

1

2

1

2

1

0

0

0

1

Alcaligenes sp.

1

1

0

1

0

1

1

0

0

0

1

Altererythrobacter sp.

1

0

1

1

0

0

0

0

0

0

0

Arthrobacter sp.

1

1

1

1a

0

0

1

0

0

0

1

Azospirillum spp.

4

0

3

3a

0

2

3

0

0

0

1

Bacillus spp.

9

2

4

6

1

5

6

1

1

0

5

Brevibacterium spp.

4

2

4

0

1

1

3

0

0

0

1

Brevundimonas spp.

3

0

1

1

0

1

2

0

0

0

0

Candidatus sp.

1

0

0

0

0

1

0

1

0

0

0

Carnobacterium sp.

1

0

0

1

0

1

1

0

0

0

1

Caulobacter spp.

4

0

3

3

1

3

3

2

1

0

3

Cedecea sp.

1

1

0

1

0

1

1

0

0

0

0

Cellulomonas sp.

1

0

0

0

0

1

1

0

0

0

0

Cellulosimicrobium spp.

2

2

1

0

0

2

0

0

0

0

0

Cetobacterium sp.

1

0

0

1

1

1

0

0

0

0

0

Chryseobacterium spp.

13

4

9

2

4

10

7

2

1

0

5

Corynebacterium spp.

2

1

1

1

0

2

1

0

0

0

0

Curtobacterium spp.

35

13

29

20a

7

21

15

8

16

2

9

Dankookia sp.

1

0

0

0

0

1

1

0

0

0

0

Delftia sp.

1

0

0

1

1

0

0

0

0

1

0

Enterobacter spp.

21

18

19

19a

4

15

7

5

7

1

5

Erwinia spp.

11

5

9

11

0

5

9

1

1

2

6

Escherichia spp.

2

0

0

2

1

1

0

0

0

0

0

Exiguobacterium spp.

2

0

1

1

1

0

1

0

0

0

0

Fictibacillus sp.

1

0

0

1

0

1

1

0

0

0

1

Flavobacterium spp.

2

1

0

1

0

1

1

0

0

0

1

Janibacter sp.

1

0

0

0

0

1

0

0

0

0

0

Klebsiella spp.

4

4

3

4a

3

4

1

3

1

2

2

Kocuria sp.

1

0

0

0

0

0

0

1

0

0

0

Kosakonia sp.

1

1

0

1

0

0

0

1

0

0

0

Lactococcus spp.

2

0

1

1

0

1

1

0

1

0

0

Luteibacter sp.

1

0

0

1

0

0

0

0

0

0

0

Methylobacterium sp.

1

1

0

1

0

0

1

0

1

0

0

Microbacterium spp.

9

3

5

4

0

2

8

0

0

0

2

Micrococcus sp.

1

0

1

1

0

0

1

0

0

0

0

Morganella sp.

1

0

1

0

0

1

1

0

0

0

1

Mycobacterium sp.

1

0

1

1

0

0

0

0

0

0

0

Mycoplasma sp.

1

1

1

1

1

0

0

0

0

0

1

Nemorella sp.

1

0

1

1

1

0

0

0

0

0

0

Oceanobacillus sp.

1

0

0

0

0

0

0

0

0

0

0

Oerskovia sp.

1

0

0

0

0

0

1

0

0

0

0

Paenarthrobacter spp.

15

12

11

10a

3

7

7

1

0

0

1

Paenibacillus sp.

1

0

0

1

0

0

1

0

0

0

0

Pantoea spp.

10

5

8

10a

2

6

9

2

3

2

6

Pedobacter sp.

15

5

11

6

3

11

4

1

1

0

5

Phenylobacterium sp.

1

0

0

1

0

1

1

0

0

0

0

Plantibacter sp.

1

0

0

0

0

0

1

0

0

0

0

Providencia spp.

2

2

0

0

0

0

2

0

0

0

2

Pseudomonas spp

93

46

73

84a

66

37

31

27

32

18

27

Rahnella spp.

10

6

9

9a

0

6

4

1

8

4

6

Ralstonia spp.

23

13

14

12

3

7

1

4

4

0

10

Ravibacter sp.

1

0

0

1

0

1

1

0

0

0

1

Rhizobium spp.

5

2

3

5

0

3

3

1

0

2

2

Rhodococcus sp.

1

0

1

0

0

0

0

0

0

0

0

Rurimicrobium sp.

1

1

1

0

0

1

1

0

0

0

0

Serratia spp.

4

3

3

4a

1

2

1

1

1

2

2

Shigella spp.

4

2

4

3

1

3

0

0

0

0

1

Shinella spp.

1

0

1

0

0

1

1

0

0

0

0

Siphonobacter spp.

4

2

4

1

2

2

2

0

0

0

3

Sphingobacterium sp.

2

2

1

1

1

1

1

1

0

0

0

Sphingobium spp.

3

2

3

2

1

2

1

1

0

0

2

Sphingomonas spp.

13

3

11a

12

0

7

5

1

1

0

3

Staphylococcus spp.

21

3

10

10

0

13

6

1

2

0

5

Stenotrophomonas spp.

23

4

5a

23

9

20

9

3

4

2

6

Variovorax spp.

13

3

7a

10

3

7

8

0

0

0

1

Xylophilus sp.

1

0

1

1

0

1

0

0

0

0

1

Total

437

186

292

314

124

236

186

72

90

38

138

*Values represent samples that have significantly higher (P < 0.05) l-Tryptophan-dependent IAA production than L-Trp-independent IAA production. A student’s t test (two-tailed test with paired samples) was performed to analyze the differences between IAA production by bacterial endophytes with and without L-Trp supplementation

aValues represent those are positive for the molecular screening of nifH gene

Biochemical characterization

All endophytes were characterized for their capability to release chemical nutrients (Fe, P, and Zn), fix nitrogen, and synthesize the plant hormone IAA. The plate assays (Fe, N, P, and Zn) provided qualitative results based on a score of 0–4, where 0 indicated a negative result and 4 being the most potent positive result. Roughly seventy-two percent (314/437) of the bacterial isolates from corn sap grew on nitrogen free medium (Table 1). These putative nitrogen fixers were mainly from the genera of Pseudomonas, Pantoea, Sphingomonas, Stenotrophomonas, Variovorax, Rahnella, Acinetobacter, Rhizobium, Azospirillum, Brevundimonas, and Curtobacterium (Table 2). However, only 11% (35/314) of these putative nitrogen fixation bacterial endophytes were confirmed to possess nifH gene upon molecular screening. Similarly, 66% (292/437) of the isolates solubilized phosphate (Table 1). The most prominent genera involved in this activity include Acinetobacter, Achromobacter, Paenarthrobacter, Brevindimonas, Chryseobacterium, Curtobacterium, Enterobacter, Microbacterium, Pantoea, Pseudomonas, and Variovorax (Table 2). Forty-two percent (186/437) of the endophytes solubilized zinc (Table 1) and involved Variovorax, Stenotrophomonas, Sphingomonas, Serratia, Rhizobium, Rahnella, Pseudomonas, Pantoea, Klebsiella, Microbacterium, Enterobacter, Chryseobacterium, Acinetobacter, Paenarthrobacter, and Aeromonas (Table 2). Iron sequestration was a less frequent trait as 28% (124/437) isolates displayed this trait (Table 1) and the key genera included Pantoea, Klebsiella, Paenarthrobacter, Chryseobacterium, Pantoea, Pseudomonas, Stenotrophomonas, and Enterobacter (Table 2). Fifty-four percent (236/437) of the isolates synthesized IAA when the growth medium was supplemented with L-Trp. Of the 236 isolates, a little less than half (107) produced significantly higher levels of L-Trp-dependant IAA than the respective control (Tables 1, 2). A group of 19 individual endophytes were found positive for all five traits, and this group was dominated (9/19) by Pseudomonas spp. (Table 3).
Table 2

Occurrence of major corn sap bacterial genera in specific plant growth promotion activity

Genus

Relative proportions (%)

Zinc solubilization

Phosphate solubilization

Nitrogen fixation

Iron acquisition

Antifungal activity

IAA bioproduction

Acinetobacter

70

90

90b

a

80

60

Azospirillum

a

75

75b

a

a

a

Bacillus

a

a

67

a

78

56

Chryseobacterium

a

69

a

a

62

77

Corynebacterium

a

a

a

a

a

100

Curtobacterium

a

83

57b

a

66

60

Enterobacter

86

90

90b

a

62

71

Erwinia

a

82

100

a

64

a

Klebsiella

100

75

100b

75

100

100

Microbacterium

a

56

a

a

a

a

Paenarthrobacter

80

73

67b

a

a

a

Pantoea

a

80

100b

a

90

60

Pedobacter

a

73

a

a

a

73

Pseudomonas

a

78

90b

71

61

a

Rahnella

60

90

90b

a

100

60

Ralstonia

57

61

52

a

57

a

Rhizobium

a

60

100b

a

60

60

Serratia

75

75

100b

a

75

a

Siphonobacter

a

100

a

a

75

a

Sphingobium

67

100

67

a

67

67

Sphingomonas

a

85

92

a

a

54

Staphylococcus

a

a

a

a

a

62

Stenotrophomonas

a

a

100b

a

a

87

Variovorax

a

54

77b

a

a

54

aIndicates < 50 % relative proportion

bValues represent those are positive for the molecular screening of nifH gene

Table 3

Strains that tested positive for multiple plant growth promoting traits

Strain

Identification

IAA bioproductiona µg per ml

Zn solubilization

P solubilization

N fixation

Fe sequestering

AL33

Chryseobacterium taklimakanense

3.64

1

1

1

4

AL34

Pantoea allii

3.83

1

2

3

1

AL57

Pseudomonas stutzeri

3.73

2

4

1

1

AL69

Stenotrophomonas sp.

3.33

2

3

2

3

AL71

Klebsiella michiganensis

11.91

2

1

2

2

AL91

Pseudomonas sp.

3.19

4

3

4

4

AL123

Pseudomonas asplenii

2.12

2

2

4

3

AL166

Enterobacter sp.

6.11

2

3

4

4

AL227

Pseudomonas sp.

3.65

3

3

4a

4

AL238

Pseudomonas sp.

3.41

4

4

4a

3

AL245

Curtobacterium sp.

4.02

1

1

4

2

AL255

Acinetobacter sp.

7.12

1

1

4

1

AL256

Pseudomonas sp.

9.16

1

2

4a

4

AL268

Curtobacterium sp.

3.30

4

4

4

1

AL271

Pseudomonas sp.

2.64

2

1

4

4

AL278

Pseudomonas sp.

2.93

3

1

4

1

AL349

Pseudomonas sp.

2.66

4

3

4

4

AL384

Sphingobium sp.

2.94

1

2

4

1

AL394

Shigella sp.

3.44

2

3

4

3

IAA bio-production test was done quantitatively, and all the other characterizations were scored on a scale from (0) negative, (1) positive/weak, (2) intermediate, (3) strong, and (4) very strong activity

aL-Trp dependent IAA production

Antifungal co-inoculation plate assay

A total of 338 of the 437 isolates produced antifungal compounds on agar plates sufficient to inhibit the growth of at least one fungal species. The most prevalent antifungal activity was found against V. dahliae with 31% occurrence (138/437). Likewise, 90 of 437 (21%) showed antifungal activity against P. aphanidermatum, 72/437 (16%) against F. oxysporum, and 38 of 437 (9%) for R. solani (Fig. 1). Only 23 isolates inhibited the growth of at least three of the phytopathogenic fungi tested, and only two isolates (i.e. AL11 Pseudomonas mandelii and AL12 Klebsiella sp.) inhibited the growth of all four (Table 4).
Fig. 1

Distribution of antifungal activity (a) and the relative proportions of their co-inhibition for one or more fungi (b) among corn sap endophytes. P P. aphanidermatum, F F. oxysporum, R R. solani, V V. dahliae

Table 4

List of corn sap bacterial endophytes that inhibited growth of at least three of the fungi tested

Strain

Identification

F. oxysporum

P. aphanidermatum

R. solani

V. dahliae

AL5

Rhizobium sp.

+

+

+

AL11

Pseudomonas mandelii

+

+

+

+

AL12

Klebsiella sp.

+

+

+

+

AL15

Pantoea sp.

+

+

+

AL25

Stenotrophomonas maltophilia

+

+

+

AL26

Pseudomonas sp.

+

+

+

AL31

Rahnella sp.

+

+

+

AL78

Pseudomonas sp.

+

+

+

AL85

Curtobacterium flaccumfaciens

+

+

+

AL98

Pseudomonas sp.

+

+

+

AL132

Curtobacterium plantarum

+

+

+

AL211

Caulobacter sp.

+

+

+

AL212

Ralstonia sp.

+

+

+

AL217

Erwinia sp.

+

+

+

AL257

Pseudomonas sp.

+

+

+

AL258

Pseudomonas sp.

+

+

+

AL271

Pseudomonas sp.

+

+

+

AL336

Pseudomonas sp.

+

+

+

AL340

Pseudomonas sp.

+

+

+

AL346

Rahnella sp.

+

+

+

AL424

Pseudomonas sp.

+

+

+

AL430

Pseudomonas sp.

+

+

+

AL435

Pseudomonas sp.

+

+

+

Plant growth-promotion assay

All the reported corn sap bacterial endophytes (437) were confirmed as true endophytes in plant tissue colonization assay. Using the paper towel gnotobiotic growth pouch assay, 186 of 437 isolates significantly increased the root length and dry weight vigor index of wheat when compared to untreated controls (Tables 5, S4).
Table 5

Gnotobiotic plant growth promotion assay of wheat

*

**

***

****

*****

Strain

Identification

Strain

Identification

Strain

Identification

Strain

Identification

Strain

Identification

Uninoculated control

AL2

Acinetobacter sp.

AL5

Rhizobium sp.

AL56

Rahnella sp.

AL161

Curtobacterium luteum

AL393

Aeromonas sp.

AL19

Stenotrophomonas sp.

AL7

Enterobacter sp.

AL63

Pseudomonas sp.

AL203

Sphingomonas sp.

AL413

Paenarthrobacter sp.

AL26

Pseudomonas sp.

AL10

Sphingomonas pseudosanguinis

AL66

Alcaligenes faecalis

AL249

Curtobacterium sp.

AL422

Pedobacter sp.

AL33

Chryseobacterium taklimakanense

AL28

Curtobacterium plantarum

AL94

Ralstonia sp.

AL257

Pseudomonas sp.

AL254

Pseudomonas sp.

AL36

Sphingomonas pseudosanguinis

AL35

Stenotrophomonas sp.

AL107

Ralstonia pickettii

AL276

Enterobacter sp.

AL9

Stenotrophomonas sp.

AL40

Stenotrophomonas sp.

AL37

Chryseobacterium haifense

AL148

Siphonobacter sp.

AL289

Pseudomonas sp.

  

AL43

Enterobacter ludwigii

AL39

Rhizobium sp.

AL157

Paenarthrobacter nicotinovorans

AL293

Paenarthrobacter sp.

  

AL54

Chryseobacterium sp.

AL42

Enterobacter sp.

AL200

Variovorax sp.

AL333

Erwinia sp.

  

AL58

Stenotrophomonas sp.

AL44

Ralstonia pickettii

AL213

Rurimicrobium sp.

AL370

Microbacterium sp.

  

AL61

Chryseobacterium sp.

AL46

Variovorax paradoxus

AL228

Curtobacterium sp.

AL373

Escherichia sp.

  

AL72

Enterobacter ludwigii

AL47

Cedecea sp.

AL252

Acinetobacter sp.

AL381

Arthrobacter sp.

  

AL78

Pseudomonas sp.

AL48

Pantoea sp.

AL258

Pseudomonas sp.

AL397

Pseudomonas psychrotolerans

  

AL82

Chryseobacterium sp.

AL51

Sphingomonas parapaucimobilis

AL316

Sphingobacterium sp.

AL401

Variovorax sp.

  

AL85

Curtobacterium flaccumfaciens

AL55

Azospirillum humicireducens

AL339

Pedobacter sp.

AL406

Pseudomonas psychrotolerans

  

AL87

Microbacterium sp.

AL59

Stenotrophomonas sp.

AL356

Variovorax sp.

AL407

Curtobacterium sp.

  

AL90

Caulobacter sp.

AL62

Variovorax paradoxus

AL361

Ralstonia sp.

    

AL97

Curtobacterium sp.

AL64

Curtobacterium plantarum

AL369

Oerskovia sp.

    

AL106

Variovorax boronicumulans

AL77

Erwinia sp.

AL378

Pseudomonas sp.

    

AL108

Brevibacterium sp.

AL83

Pseudomonas sp.

AL382

Aeromonas sp.

    

AL111

Curtobacterium flaccumfaciens

AL93

Enterobacter xiangfangensis

AL386

Rahnella sp.

    

AL114

Pseudomonas psychrotolerans

AL125

Pseudomonas monteilii

AL388

Erwinia sp.

    

AL115

Pseudomonas monteilii

AL129

Caulobacter sp.

AL389

Shigella sp.

    

AL123

Pseudomonas asplenii

AL131

Ralstonia pickettii

AL391

Shigella sp.

    

AL127

Pedobacter sp.

AL135

Paenarthrobacter nicotinovorans

AL394

Shigella sp.

    

AL128

Ralstonia pickettii

AL147

Curtobacterium plantarum

AL395

Variovorax guangxiensis

    

AL133

Pseudomonas mandelii

AL151

Bacillus halotolerans

AL402

Variovorax paradoxus

    

AL134

Erwinia billingiae

AL156

Brevundimonas staleyi

AL403

Ralstonia pickettii

    

AL142

Paenarthrobacter nicotinovorans

AL165

Curtobacterium plantarum

AL409

Paenibacillus sp.

    

AL143

Sphingomonas sanguinis

AL169

Dankookia rubra

AL411

Pseudomonas sp.

    

AL150

Shinella kummerowiae

AL170

Brevibacterium casei

AL412

Bacillus sp.

    

AL159

Ralstonia pickettii

AL171

Ralstonia pickettii

AL415

Pseudomonas sp.

    

AL160

Sphingomonas panni

AL174

Ralstonia pickettii

AL421

Pseudomonas sp.

    

AL162

Microbacterium testaceum

AL176

Methylobacterium radiotolerans

AL428

Pseudomonas sp.

    

AL163

Pseudomonas umsongensis.

AL181

Curtobacterium flaccumfaciens

AL429

Pseudomonas sp.

    

AL173

Staphylococcus saprophyticus

AL182

Staphylococcus succinus

      

AL180

Curtobacterium flaccumfaciens

AL189

Flavobacterium sp.

      

AL185

Pantoea sp.

AL195

Curtobacterium sp.

      

AL188

Erwinia sp.

AL199

Variovorax sp.

      

AL196

Pseudomonas sp.

AL202

Brevundimonas sp.

      

AL197

Bacillus sp.

AL205

Erwinia sp.

      

AL198

Staphylococcus sp.

AL206

Micrococcus sp.

      

AL207

Bacillus sp.

AL212

Ralstonia sp.

      

AL208

Pseudomonas sp.

AL216

Staphylococcus sp.

      

AL223

Rhizobium sp.

AL221

Lactococcus sp.

      

AL230

Staphylococcus sp.

AL222

Enterobacter sp.

      

AL233

Siphonobacter sp.

AL229

Curtobacterium sp.

      

AL241

Pseudomonas sp.

AL232

Fictibacillus sp.

      

AL242

Acinetobacter sp.

AL239

Serratia sp.

      

AL269

Curtobacterium sp.

AL244

Curtobacterium sp.

      

AL285

Erwinia sp

AL245

Curtobacterium sp.

      

AL319

Providencia sp.

AL268

Curtobacterium sp.

      

AL325

Curtobacterium sp.

AL277

Acinetobacter sp.

      

AL327

Exiguobacterium sp.

AL278

Pseudomonas sp.

      

AL347

Chryseobacterium sp.

AL280

Azospirillum sp.

      

AL354

Pedobacter sp.

AL283

Erwinia sp.

      

AL359

Acinetobacter sp.

AL284

Pseudomonas sp.

      

AL362

Curtobacterium sp.

AL291

Staphylococcus sp.

      

AL387

Cetobacterium sp.

AL307

Cellulomonas sp.

      

AL390

Pantoea sp.

AL309

Stenotrophomonas sp.

      

AL420

Bacillus sp.

AL317

Morganella sp.

      

AL432

Pseudomonas sp.

AL321

Ralstonia sp.

      
  

AL332

Corynebacterium sp.

      
  

AL346

Rahnella sp.

      
  

AL375

Achromobacter sp.

      
  

AL376

Ralstonia sp.

      
  

AL392

Erwinia sp.

      
  

AL396

Variovorax paradoxus

      
  

AL405

Stenotrophomonas sp.

      
  

AL408

Curtobacterium sp.

      
  

AL410

Ravibacter sp.

      
  

AL416

Pseudomonas sp.

      
  

AL433

Pedobacter sp.

      

Each treatment was a mean of 18 replications. One-way ANOVA was performed on root lengths and dry weight separately and followed by Tukey’s post hoc test for each test strain and compared with negative controls (P < 0.05). Values that are significantly different than the negative control are represented by “*”

*(P < 0.05)

**(P < 0.005)

***(P < 0.0005)

****(P < 0.0005)

*****(P < 0.00005)

Discussion

Culture dependent screening of a cumulative bacterial population for known plant growth-promoting traits is one of the most common approaches to unveil the potential of beneficial microbes (Finkel et al. 2017; Sandhya et al. 2017). Although several studies have described the isolation of corn endophytes (Ikeda et al. 2013; Pereira and Castro 2014; Mousa et al. 2015; Johnston-Monje et al. 2016; Shehata et al. 2016; Correa-Galeote et al. 2018), to our knowledge this is the first report that characterizes the population, its diversity, and identifies the culturable corn sap bacterial endophytes collected from a large number of diverse farms. We set out to sample corn plants that were grown under different environmental conditions and in different locations to ensure maximal coverage of bacterial endophyte diversity.

Ability of an endophyte to colonize plant interior can be confirmed by labeling and tracking the strain (Miller et al. 2000) inside the host plant and by artificially inoculating the host plant with the potential endophyte (Rashid et al. 2012). In this study, each of the 437 isolates were re-inoculated and re-tested for their ability to colonize the interior tissues of wheat plants to ensure that all the selected strains were true endophytes. Since these bacterial endophytes were isolated from high production sites within each corn farm, we wanted to test if they could promote the growth of other crops as well. For that reason, we selected wheat as an indicator crop and used it to examine how the plants performed under a gnotobiotic colonization assay.

The average total bacterial count of corn sap was 3.6 × 105 CFU per ml of sap expressed from the stem. A very similar trend was described by other groups for corn microbial populations with an average of 103–107 cfu/g fresh weight (f.w.) throughout the growing season (Fisher et al. 1992; McInroy and Kloepper 1995; Rai et al. 2007). More microbes colonize the roots than the shoots (Pereira and Castro 2014). Only microbes that confer selective advantages to the plant are likely to colonize the upper parts of the plant, such as the stem and leaf (Hardoim et al. 2008). These endophytes belonged to 69 bacterial genera and can be classified into four major phylogenetic groups namely Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria (α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria). The diversity of the bacteria identified in the corn sap is similar to what has previously been associated with other corn tissues (Johnston-Monje et al. 2016; Pereira and Castro 2014). The two most dominant groups of bacteria that were found in this study are γ-Proteobacteria (40%) and Firmicutes (30%). Several other studies have also described γ-Proteobacteria and Firmicutes (mainly Bacilli) being the prominent bacterial endophytes in different corn tissues (Johnston-Monje and Raizada 2011; Pereira et al. 2011; Montañez et al. 2012; Ikeda et al. 2013; Pereira and Castro 2014; Szilagyi-Zecchin et al. 2014). Other prominent endophytic groups that were found in this study include Bacteroidetes (largely represented by Chryseobacterium spp., Pedobacter spp., and Siphonobacter spp.), α-Proteobacteria (mostly represented by Rhizobium spp., Brevundimonas spp., Sphingomonas spp., and β-Proteobacteria (mostly represented by Achromobacter spp., Variovorax spp., and Caulobacter spp.).

The composition of Canadian and American corn sap bacterial communities was found to be different. Canadian corn sap had about twice the microbial diversity in its sap compared to the corn from the United States. Corn from the USA was usually sent overnight but sometimes took two days to arrive, so changes in the microbiology may have occurred during transport. The most prominent difference found between the communities was the occurrence of Gram positive bacteria and Bacteroidetes, which were more abundant and diverse in the sap collected from the Canadian farms.

The sap endophytes could be divided into three main groups based on the activities they exhibited: (i) those that displayed multiple plant growth-promotion traits, (ii) those that served as biocontrol agents for at least three or more fungi tested, and (iii) those associated with significantly higher wheat root growth compared to untreated control plants. The first group consisted of 19 endophytes that exhibited positive results for all of five biochemical characterization tests and consisted mainly (47%) of Pseudomonas spp. In addition, 6 of these 19 endophytes also displayed significant wheat root length elongation in growth assays. These six bacterial endophytes were identified as Chryseobacterium aklimakanense (AL 33), Curtobacterium sp. (AL 245), Curtobacterium sp. (AL 268), Pseudomonas asplenii (AL 123), Pseudomonas sp. (AL 278) and Shigella sp. (AL 394). The second group consisted of 23 bacterial endophytes, which were again dominated by Pseudomonas spp. (52%). Within the third group, the bacterial genera that demonstrated higher overall plant growth promotion included Acinetobacter spp., Arthrobacter spp., Chryseobacterium spp., Curtobacterium spp., Enterobacter spp., Erwinia spp., Klebsiella spp., Microbacterium spp., Pantoea spp., Pedobacter spp., Pseudomonas spp., Rahnella spp., Sphingomonas spp., Staphylococcus spp., Stenotrophomonas spp., and Variovorax spp.

The relative proportion of species in each genus displaying a plant growth promoting activity was measured to determine which genera would likely be the strongest at promoting plant growth through each mechanism. In current study, isolates representing bacterial genera Enterobacter, Klebsiella, and Paenarthrobacter performed better in zinc solubilization activity, bacterial strains from genera Acinetobacter, Enterobacter, Rahnella, Siphonobacter, and Sphingobium, have shown stronger phosphate solubilization activity, Klebsiella and Pseudomonas genera were found as better iron sequestration agents among all the genera, Corynebacterium, Klebsiella, and Stenotrophomonas exhibited frequent IAA production, majority of strains from Klebsiella, Pantoea, and Rahnella were found biocontrol agents against phytopathogen fungi tested, and strains from Acinetobacter, Erwinia, Klebsiella, Pantoea, Pseudomonas, Rahnella, Rhizobium, Serratia, Sphingomonas, and Stenotrophomonas were most frequent on nitrogen fixation capacities. However, bacteria genera Klebsiella, Pantoea, Pseudomonas, Rahnella, Rhizobium, Serratia, and Stenotrophomonas were verified by molecular screening methods for the presence of nifH gene. The molecular analysis was only screened for the nifH gene, and it is possible that the endophytes that exhibited positive reactions on nitrogen free media may carry other gene(s) of the nif operon, hence termed as putative nitrogen fixers.

The ability of the newly isolated endophytes was tested for their potential use as biocontrol agents. For that reason, the most destructive, broad host range, and worldwide distributed (particularly in Ontario, Canada) fungal phytopathogens were chosen. For example, vascular wilt caused by V. dahliae and F. oxysporum, root rot and seedling blight caused by R. solani, damping-off disease in nurseries and green house mainly caused by Pythium sp. and R. solani pose huge agriculture problems in Ontario, Canada (Lazarovits, personal communication).

A reasonable portion (31%) of the newly isolated corn sap bacterial endophytes exhibited antifungal activity against V. dahliae, which is a huge threat to potato and tomato crops and is a major disease problem to cotton and olive trees in Spain and elsewhere. The fact that these strains are endophytes could be a benefit for their potential use as biocontrol agents. The bacteria from our collection that sequestered iron on CAS medium also showed antifungal activity. The ability to produce siderophores is considered a major mechanism for allowing bacteria to compete with other microorganisms within the same ecological niche and thereby improve their potential for colonizing plant tissues (Loaces et al. 2011). Siderophore-producing PGPB, including endophytes, thus appear to have a greater chance to act as biocontrol agents, particularly against fungal phytopathogens. Since fungal siderophores generally have lower affinities for iron (III) than bacterial siderophores, the presence of such compounds will reduce the capacity of phytopathogens to proliferate and infect plants (Kloepper et al. 1980; O’Sullivan and O’Gara 1992; Loper and Henkels 1999). Similarly, solubilization of zinc and phosphate by endophytes is an important plant growth-promotion mechanism that is influenced heavily by soil pH and moisture. The beneficial role of endophytes in zinc solubilization and phosphate solubilization has been associated with corn (Goteti et al. 2013), tomatoes (Rashid et al. 2012), cactus (Puente et al. 2009), sunflowers (Forchetti et al. 2007), peanuts, other legumes (Palaniappan et al. 2010), soybeans (Kuklinsky-Sobral et al. 2004), ginseng (Vendan et al. 2010), and strawberry (Dias et al. 2009). Moreover, all members of these genera produced moderate levels of IAA. To be a plant-growth supportive endophyte it is considered essential that bacterium provide levels of IAA that are appropriate to the levels of IAA made by the plant. Excessive concentrations of IAA would be deleterious to plant growth and are often associated with phytopathogens (Davies 1995; Kunkel and Chen 2006).

Many of the bacterial endophytes promoted wheat plant growth in a growth assay and such activity could be linked to their capacities to solubilize phosphate or zinc, fix nitrogen, and produce IAA or siderophores. Some of these isolates will be tested as potential biofertilizers. Endophytes can help promote plant growth and protect against disease and environmental stress by using a combination of traits, at different times under different conditions, which suggests that plant growth promotion is the outcome of multiple mechanisms.

Notes

Acknowledgements

We highly appreciate the assistance of all the participating farmers for allowing us to sample their farms. We would also like to extend our sincere thanks to Kristen Delaney and Jae Min Park for sampling corn farms. We appreciate the help of the A & L Biologicals staff and coop students, especially, Perry Ryersee, Christopher Chiasson, Gabriëlle Zieleman, Stephanie Kerkvliet, Nickolas Werry, and Danika Brook on this project. Finally, the financial assistance provided by Natural Sciences and Engineering Research Council through an Industrial fellowship is highly appreciated. Funding provided by AAFC-Growing Forward was crucial to the successful completion of this project and is greatly appreciated.

Supplementary material

42398_2018_30_MOESM1_ESM.docx (191 kb)
Supplementary material 1 (DOCX 190 kb)

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Copyright information

© Society for Environmental Sustainability 2018

Authors and Affiliations

  • Shimaila Ali
    • 1
    Email author
  • Joshua Isaacson
    • 1
    • 2
  • Yulia Kroner
    • 1
  • Soledad Saldias
    • 1
  • Saveetha Kandasamy
    • 1
  • George Lazarovits
    • 1
    • 2
  1. 1.A&L Biologicals, Agroecological Research Services CentreLondonCanada
  2. 2.Department of BiologyUniversity of Western OntarioLondonCanada

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