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Enhanced biological control of root-knot nematode, Meloidogyne incognita, by combined inoculation of cotton or soybean seeds with a plant growth-promoting rhizobacterium and pectin-rich orange peel

Mohammad K. Hassan
Mohammad K. Hassan
, 
Kathy S. Lawrence
Kathy S. Lawrence
, 
Edward J. Sikora
Edward J. Sikora
, 
Mark R. Liles
Mark R. Liles
 oraz 
Joseph W. Kloepper
Joseph W. Kloepper
   | 22 cze 2021
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Data publikacji: 22 cze 2021
Zakres stron: 1 - 17
DOI: https://doi.org/10.21307/jofnem-2021-058
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© 2021 Mohammad K. Hassan et al., published by Sciendo.
This work is licensed under the Creative Commons Attribution 4.0 International License.

Cotton (Gossypium hirsutum L.) and soybean (Glycine max L.) are economically important crops in the United States and worldwide. In the U.S. alone, cotton yield in 2018 was 18.4 million bales, and soybean yield was 4.54 billion bushels (Anonymous, 2018). Meloidogyne incognita (Kofoid and White) Chitwood, the southern root-knot nematode, is broadly distributed in soils cultivated with cotton (Xiang et al., 2017b) and other crops (Huang et al., 2016), and causes economically significant yield losses annually; for example, in 2018, soybean yield losses due to M. incognita in the southern U.S. were estimated at 11.92 million bushels in total, with 70,000 bushels lost in Alabama (Allen et al., 2018).

Multiple methods are used to reduce M. incognita populations in the field, including chemical nematicides (Basyony and Abo-Zaid, 2018); however, environmental and health concerns have limited the use of chemical nematicides for controlling plant-parasitic nematodes, and there is a need to develop environmentally friendly methods to manage the pathogen, such as the use of biological control agents (Burkett-Cadena et al., 2008).

Plant growth-promoting rhizobacteria (PGPR) are root-colonizing bacteria that enhance plant growth and biological control against multiple plant pathogens (Olanrewaju et al., 2017). Bacillus velezensis is a Gram-positive, rod-shaped PGPR, with some strains reported to reduce M. incognita populations on cotton (Xiang et al., 2017b). Additionally, B. velezensis PGPR strains are able to utilize pectin as a sole carbon and energy source (Hossain et al., 2015); interestingly, citrus peels which are pectin-rich and an abundant and inexpensive agricultural waste product (Rafiq et al., 2018) have been previously demonstrated to increase the efficacy of soybean growth promotion mediated by B. velezensis PGPR strains when co-inoculated with orange peel powder as a seed treatment (Hassan et al., 2019).

In the presence of different carbohydrates (pectin, sucrose, xylan or galactose), Bacillus spp. produce multiple secondary metabolites that inhibit the growth of multiple plant pathogens. For example, Bacillus amyloliquefaciens SQY 162 grown on pectin-amended media increased surfactin production and inhibit bacterial wilt of tobacco caused by Ralstonia solanacearum (Wu et al., 2015). Also supernatants, cell pellet suspensions, and culture broths of B. subtilis strains significantly reduced populations of eggs and J2 of M. incognita under laboratory and greenhouse conditions (Basyony and Abo-Zaid, 2018). In addition, previous studies have reported that the combination of separated cow manure and orange peels (SCM-OP) reduced the number of Meloidogyne javanica populations on tomato roots (Raviv et al., 2005). However, the effect of exogenous orange peel amendments has not been evaluated for enhanced PGPR-mediated biological control of plant pathogenic nematodes such as M. incognita.

The overall goal of this study was to evaluate the combined application of B. velezensis AP203 and orange peel for biological control of M. incognita. More specifically, we investigated (i) the effects of B. velezensis AP203, with and without orange peel amendment, on the mortality of second-stage juveniles (J2) of M. incognita under laboratory conditions; (ii) the effect of orange peel or glucose by B. velezensis AP203 on the expression of secondary metabolite(s) predicted to be responsible for reducing M. incognita populations; (iii) evaluate the efficacy of orange peel and/or B. velezensis AP203 amendments in reducing the number of M. incognita populations on the roots of soybean and cotton under greenhouse conditions.
Materials and methods
Evaluation of nematode killing under laboratory conditions
Preparation of B. velezensis spore and orange-peel suspensions

B. velezensis AP203 was obtained from the Biological Control lab in the Department of Entomology and Plant Pathology, Auburn University, Alabama, USA (retired director Prof. Joseph Kloepper). This strain was originally isolated from a cotton rhizosphere. The cryopreserved culture was streaked onto tryptic soy agar (TSA) and incubated at 28°C for 24 hr. A single colony of the strain was transferred into a spore preparation medium (Zhang et al., 2010), and incubated for seven days at 28°C. Sterilized distilled water (20 mL) was added to each petri plate, and the bacterial mass was transferred to a 50 mL centrifuge tube. The B. velezensis AP203 spore preparation was heat-treated for 20 min at 80°C to kill any vegetative cells, serially diluted, and adjusted to 107 colony forming units (CFU) per mL. The non-organic orange peel powder (Citrus Extracts LLC, Fort Pierce, FL USA) was suspended in sterilized distilled water by a magnetic stirrer at a rate of 1.0 g per 100 mL (1.0% w/v) water and was applied as an aqueous suspension.
Preparation of M. incognita inoculum and mortality determination

M. incognita eggs were isolated and extracted from corn plant roots Mycogen 2H273 (Dow AgroScience, Indianapolis, IN) at the Plant Science Research Center (Auburn University, Auburn, AL) using a modified sucrose centrifugation-flotation method (Jenkins, 1964). The eggs were counted using an inverted TS100 Nikon microscope at 40X magnification. The eggs were hatched for a seven-day period at 30°C in an incubator. In total, 10 µL of J2 M. incognita were counted and transferred into a 96-well plate for the J2 mortality test, with 30 to 40 J2 per well. In total, 90 µL of AP203 alone, citrus extract alone, and AP203 with citrus extract combined were added to each well. The 96-well plate was sealed with parafilm and incubated at room temperature for 48 hr. The number of live J2 were counted at the beginning (0 hr) and at the end (48 hr) of this experiment. The viability of J2 was determined by the sodium hydroxide method (Xiang and Lawrence, 2016) and the mortality percentage was calculated by the equation: [(live J2 at 0 hr‒live J2 at 48 hr)/live J2 at 0 hr] × 100 (Xiang et al., 2017b).
LC-MS experiment
Preparation of B. velezensis AP203

B. velezensis AP203 was prepared as previously described. A single colony of the PGPR strain was transferred into TSA, Tris-Spizizen Salts medium (TSS), or TSS + orange peel powder (OPP; 0.5% w/v) media and grown for 72 hr in a shaking incubator at 28°C. The B. velezensis AP203 cultures were subjected to centrifugation in a Sorvall Legend RT centrifuge (Thermo Scientific, USA) at 10,000 × g for 10 min. The supernatant was collected and passed through a 0.2 µm syringe filter (VWR, Radnor, PA, USA) and was then transferred into a 1 mL microcentrifuge tube for LC-MS analysis.
LC-MS analysis

LC-MS analysis was performed at the Auburn University Mass Spectrometry Center using an ultra-performance LC system (ACQUITY, Waters Corp., USA) coupled with a quadrupole time-of-flight mass spectrometer (Q-Tof Premier, Waters) with electrospray ionization (ESI) in positive and negative mode using Masslynx software (V4.1). A 10 µl sample was injected into a C4 column (Aeris Widepore C4, 3.6 µm, 2.1 × 50 mm, Phenomenex) with a 300 μ L/min flow rate of the mobile phase. In positive mode, the mobile phase was solution A (0.1% formic acid in water) and solution B (95% acetonitrile, 5% H2O, and 0.1% formic acid) was initiated beginning at 0% B, held for 2 min, then linear ramping to 50% B in 18 min, followed by ramping to 100% B in 8 min and held at 100% B for 2.5 min, and then back to 0% B in 0.5 min with 4 min of re-equilibration at 0% B. In negative mode, the mobile phase was solution A (2 mM ammonium formate in water) and solution B (100% acetonitrile) beginning at 2% B, held for 2 min, then linear ramping to 50% B in 18 min, followed by ramp to 95% B in 8 min, held at 95% B for 2.5 min, and back to 2% B in 0.5 min with 4 min of re-equilibration at 2% B. The capillary voltage was set at 3.1 kV in positive mode and 2.8 kV in negative mode, the sample cone voltage was 30 V, and the extraction cone was 4.3 V. The source and desolvation temperature were maintained at 105 and 300 °C, respectively, with the desolvation gas flow at 600 L/hr. The Time of Flight Mass Spectrometry (TOF/MS) scan was 1 sec long from 80 to 1400 m/z with a 0.02 sec inter-scan delay using the centroid data format. The lock mass was used to correct instrument accuracy with a 2.5 µg/mL solution of leucine encephalin (Bachem H-2740). The data were converted to mzXML and analyzed with XCMS Online (Huan et al., 2017).
Greenhouse experiment

The greenhouse test was to evaluate the combined effects of B. velezensis AP203 plus orange peel or glucose for biological control of M. incognita. The treatment groups for the greenhouse study included: (i) TSB-CELLS + M. incognita, (ii) SUPERNATANT-OPP + M. incognita, (iii) SUPERNATANT-Glucose + M. incognita, (iv) TSS-CELLS-OPP + M. incognita, (v) TSS-CELLS-Glucose + M. incognita, (vi) CULTURE-OPP + M. incognita, (vii) CULTURE-Glucose + M. incognita, (viii) TSS + OPP (no bacteria present) + M. incognita, or (ix) TSS + glucose (No bacteria present) + M. incognita, (x) a positive control with M. incognita, and (xi) a negative control without M. incognita.
Preparation of B. velezensis AP203 and orange-peel suspension

B. velezensis AP203 was grown from a cryostock onto Tryptic Soy Agar (TSA) at 28°C for 24 hr. A single colony of this strain was transferred into TSA, TSS + glucose (0.5% w/v), or TSS + OPP (0.5% w/v) media and grown for 48 hr in a shaking incubator at 28°C. The B. velezensis cultures were then subjected to centrifugation in a Sorvall Legend RT centrifuge (Thermo Scientific, USA) at 10,000 × g for 10 min and each was then adjusted to approximately107 CFU/mL based on culture turbidity at an optical density of 600 nm (OD600). The TSA grown strain was suspended in TSS broth based on OD600 and an aliquot (1 mL containing ~107 CFU) of this sample (TSA-CELLS) was applied to each seed. For B. velezensis AP203 grown in TSS media, the carbon source used was either glucose or OPP, and 35 mL of these cultures were subjected to centrifugation at 10,000 × g for 10 min. The supernatant was passed through a 0.2 µm syringe filter (VWR, Radnor, PA, USA) and 1 mL of this sample (SUPERNATANT) was applied to each seed. For the cell pellet suspension, the pellet was suspended in TSS and then subjected to centrifugation again to remove spent media, and then resuspended in 35 mL of TSS. An aliquot (1 mL containing ~107 CFU) of this sample (TSS-CELLS) was applied to each seed. The TSS broth culture (35 mL) without separating cells and supernatant was also prepared (TSS-CULTURE), and 1 mL of the broth culture was applied to each seed.
Preparation of M. incognita inoculum

M. incognita eggs were isolated and extracted from corn roots as described previously and 2,000 eggs/mL populations were inoculated into a 2 cm depth of soil in each cone-tainer (Stuewe & Sons, Tangent, OR, USA) during seed planting and covered with field soil. The M. incognita-inoculated soybean and cotton seeds were kept at room temperature in the greenhouse for 24 hr before transferring to a greenhouse growth chamber at 25 to 35°C.
Soil preparation and seed inoculation

Field soil (Malbis fine sandy loam 59% sand, 31% silt, 10% clay, <1% OM) was collected from the E.V. Smith Research Center of Auburn University (near Shorter, AL) and was mixed with sand at a ratio (2:1). In the greenhouse experiments, the soil/sand mix was placed into each 150 cm3 cone-tainers. Two soybean (DD VSG 75140) and two cotton seeds (DPL1558 NRB2RF) were placed into 2 cm depth of each cone-tainer to ensure seed germination. The different treatment group inocula (i.e. cell pellet suspensions, culture broths, or supernatants) of B. velezensis AP203 were applied on the soybean and cotton seed surface. The seeds were then covered with the soil/sand, kept at room temperature for 24 hr, before transferring to a greenhouse growth chamber (25 to 35°C).
Statistical analyses

All data collected from the in vitro bioassay and greenhouse tests were analyzed with SAS 9.4 software (SAS Institute, Cary, NC, USA) using the PROC GLIMMIX (Generalized Linear Mixed Models) procedure that performs estimation and statistical inference for GLIMMIX at the p ≤ 0.05 level of significance. For the in vitro experiments, the mortality percentages of J2 of M. incognita were analyzed with four treatments and eight replicates. In the greenhouse experiment, plant height, root length, root and shoot fresh weight, and M. incognita eggs/plant data were collected and analyzed. The greenhouse experiments were arranged in a randomized complete block design (RCBD) with eleven treatments and eight replicates. All the in vitro and greenhouse experiments were repeated twice, and the data were pooled.
Results
In vitro antagonistic effects of B. velezensis AP203 with orange peel amendment

A spore preparation of B. velezensis AP203 with orange peel amendment was tested in vitro for its ability to kill M. incognita J2. The mortality percentage of M. incognita J2 ranged from 0 to 100%, with 94% mortality observed for M. incognita eggs inoculated with the combination of B. velezensis strain AP203 with 1.0% (w/v) OPP, which was significantly greater (p < 0.05) than that observed when eggs were incubated with B. velezensis AP203 alone (53%), 1.0% OPP (59%), or the negative control (7%) (Fig. 1). Interestingly, a significant increase in M. incognita J2 mortality relative to the negative control was also observed for B. velezensis AP203 alone (as had been observed previously) as well as inoculation with OPP alone.

Figure 1:

Effects of orange peel power (OPP) amendment on the mortality of J2 of M. incognita in vitro by B. velezensis AP203.
The effects of B. velezensis AP203 cultures grown in different media on M. incognita populations and cotton and soybean growth under greenhouse conditions

Soybean root length of plants treated with culture broth from B. velezensis AP203 with OPP was significantly greater compared to the glucose treatments and M. incognita positive control treatment (Table 1). Soybean root length was also greater in the TSB-growth cells, OPP (supernatant) and OPP (culture) compared to the M. incognita control. OPP (culture) also significantly increased root fresh weight as compared to the M. incognita control (Table 1). Cotton shoot lengths of plants treated with cell pellets from B. velezensis AP203 with OPP was significantly greater compared to the glucose treatments and the M. incognita control (Table 2). Cotton root length was significantly increased by the OPP (supernatant), OPP (cells), and OPP (culture) compared to the M. incognita control (Table 2). Supernatant, cell pellet, or culture broth from B. velezensis AP203 with OPP had a maximum antagonistic activity against M. incognita eggs in soybean (Fig. 2) and cotton (Fig. 3) roots at 45 DAP. The B. velezensis AP203 with OPP (supernatant), OPP (cells), and OPP (culture) all reduced M. incognita populations as compared to the M. incognita grown on both crop plants without any additives. B. velezensis AP203 with glucose amended treatments (supernatant, cell pellet, culture broth from B. velezensis AP203 with glucose) did not significantly reduce M. incognita populations compared to the M. incognita positive control treatment in soybean or cotton at 45 DAP (Figs. 2 and 3).

Effect of B. velezensis AP203 amended with orange peel powder (OPP) or glucose on soybean growth at 45 days after planting in greenhouse trials.

Treatmenta Shoot length (cm)b Root length (cm) Shoot fresh weight (g) Root fresh weight (g)
TSB-grown cells 58.0 a 22.5 ab 6.51 ab 3.91 ab
OPP (supernatant) 67.4 a 21.9 abc 8.12 ab 4.20 ab
Glucose (supernatant) 66.8 a 19.9 cde 7.55 ab 3.98 ab
OPP (Cells) 72.3 a 22.0 abc 9.06 ab 4.92 a
Glucose (Cells) 62.5 a 20.1 bcde 8.43 ab 3.42 ab
OPP (Culture) 72.0 a 22.6 a 10.20 a 4.11 ab
Glucose (Culture) 60.8 a 19.0 e 8.25 ab 4.00 ab
OPP 62.5 a 21.4 abcd 8.12 ab 3.65 ab
Glucose 67.9 a 20.0 cde 5.95 ab 2.53 b
M. incognita nematode
Untreated control

Notes: a B. velezensis AP203 grown in 1.0% (w/v) OPP or glucose-amended TSS medium. M. incognita 2,000 eggs/150 cm3 soil was added to all plants except the untreated control; bmeans with the same letter are not significantly different at p ≤ 0.05 level of significance.

Effect of B. velezensis AP203, amended orange peel powder (OPP) or glucose on cotton growth at 45 days after planting in greenhouse trials.

Shoot length Root length Shoot fresh Root fresh
Treatmenta (cm)b (cm) weight (g) weight (g)
TSB-grown cells 26.0 ab 18.5 abc 2.6 a 1.7 a
OPP (supernatant) 27.3 ab 20.2 ab 2.5 a 1.7 a
Glucose (supernatant) 26.8 ab 18.2 abc 2.5 a 1.6 a
OPP (Cells) 30.7 a 21.2 ab 2.2 a 1.9 a
Glucose (Cells) 27.0 ab 16.1 bc 2.7 a 1.6 a
OPP (Culture) 30.6 ab 20.1 ab 2.4 a 1.4 a
Glucose (Culture) 28.8 ab 18.1 abc 2.5 a 1.6 a
OPP 23.2 ab 17.7 abc 2.1 a 1.7 a
Glucose 22.6 b 16.1 bc 2.3 a 1.1 a
M. incognita nematode 20.0 b 14.3 c 1.7 a 0.9 a
Untreated control 26.2 ab 21.8 a 2.7 a 1.3 a

Notes: a B. velezensis AP203 grew in 1.0% (w/v) OPP or glucose-amended TSS medium. M. incognita 2,000 eggs/150 cm3 soil was added to all plants except the untreated control; bmeans with the same letter are not significantly different at p ≤ 0.05 level of significance.

Figure 2:

Effects of B. velezensis AP203 with an orange peel power (OPP) or glucose amendments on the number of M. incognita eggs on the roots of soybean at 45 days after planting in greenhouse trials.

Figure 3:

Effects of B. velezensis AP203 with an orange peel power (OPP) or glucose amendments on the number of M. incognita eggs on the roots of cotton at 45 days after planting in greenhouse trials.
Secretion of secondary metabolites by B. velezensis AP203 is affected by growth on orange peel powder

Numerous secondary metabolites were detected in the supernatant of B. velezensis AP203 amended with orange peel, which revealed a complex mass spec profile even after removing the metabolites present in orange peel powder (data not shown). Among the mass ions predicted to be expressed by B. velezensis AP203, and not present in orange peel, were four metabolites that had previously been reported to have nematicidal activity: (i) 1,3-Diphenyl-2-propanone, (ii) p-(3,4-Dihydro-6-methoxy-2-naphthyl) phenol, (iii) (E)-1,1,-(1,2-Diethyl-1,2-ethenediyl) bis (4-methoxybenzene), and (iv) 3-(Dimethylamino) propyl benzoate (Table 3) (Huang et al., 2010). The retention times (RT) of these secondary metabolites were 6.69, 6.63, 3.39, and 2.40 min, respectively (Table 3). The product mass to ions charge ratio (m/z) of these secondary metabolites were 211.11, 253.12, 295.17, and 206.12, respectively (Table 3 and Figs. S1-S3). The relative abundances (RA) per colony forming units (CFU) of secondary metabolite 1,3-Diphenyl-2-propanone were 21.11, 25.31, 29.51, and 20.61 (Table 3). The relative abundance (RA) per CFU indicated that (E)-1,1,-(1,2-Diethyl-1,2-ethenediyl) bis (4-methoxybenzene) was produced most abundantly under these culture conditions when grown with OP as a carbon source, followed by p-(3,4-Dihydro-6-methoxy-2-naphthyl) phenol, 1,3-Diphenyl-2-propanone, and 3-(Dimethylamino) propyl benzoate in decreasing order of RA/CFU (Table 3). The complete list of metabolites detected from the supernatant of B. velezensis AP203 grown on orange peel in TSS minimal medium are listed in Supplementary Table 4.

Predicted secondary metabolites found in cell-free supernatants of B. velezensis AP203 after 48 hr growth in 0.5% (w/v) OPP amended Tris Spizizen Salts (TSS) media.

Treatmenta RT (min)b Product ions (m/z) RA/CFUc Predicted metabolites
OPAP203 1 6.69 min 211.11 21.11 1,3-Diphenyl-2-propanone
OPAP203 2 6.63 min 253.12 25.31 p-(3,4-Dihydro-6-methoxy-2-naphthyl) phenol
OPAP203 3 3.39 min 295.17 29.51 (E)-1,1,-(1,2-Diethyl-1,2-ethenediyl) bis (4-methoxybenzene)
OPAP203 4 2.40 min 206.12 20.61 3-(Dimethylamino) propyl benzoate
GluAP203 0 0 0 0
OPP 0 0 0 0
Glucose 0 0 0 0

Notes: aThe in vitro B.velezensis AP203 growth test was repeated twice; bRT indicates retention time; cRA indicates relative abundance/colony forming units.

List of predicted secondary metabolites found in cell-free supernatants of B. velezensis AP203 after 48 hr growth in 0.5% (w/v) orange peel power (OPP) amended Tris Spizizen Salts (TSS) media.

Query ID Query m/z Name of the bioactive compounds Formula Exact mass
1 101.0711 Gyromitrin;Acetaldehyde methylformylhydrazone C4H8N2O 100.0636629
2 103.0559 Indoleacetic acid C10H9NO2 175.0633285
3 103.0559 5-Hydroxyindoleacetaldehyde C10H9NO2 175.0633285
4 104.0549 2-Ethyl-1-hexanol, 9CI; (±)-form, O-Sulfate C8H18O4S 210.0925798
5 104.0568 2-Ethyl-1-hexanol, 9CI; (±)-form, O-Sulfate C8H18O4S 210.0925798
7 104.0585 Histidinyl-Glycine C8H12N4O3 212.0909403
8 104.0585 D-Glycero-D-galacto-heptitol C7H16O7 212.0896029
9 104.0585 Glycyl-Histidine C8H12N4O3 212.0909403
10 104.0707 N-methyl-beta-alanine C4H9NO2 103.0633285
11 104.0707 (2S)-2-Nitrobutane C4H9NO2 103.0633285
13 104.0707 Ethyl carbamic acid methyl ester C4H9NO2 103.0633285
14 104.0707 N-Methyl-L-alanine C4H9NO2 103.0633285
15 104.0707 HBA C4H9NO2 103.0633285
16 104.0707 DL-3-aminobutyrate C4H9NO2 103.0633285
17 104.0707 Mefenamic acid C15H15NO2 241.1102787
18 104.0707 N-[2-(4-Hydroxyphenyl)ethyl]benzamide C15H15NO2 241.1102787
19 104.0707 2-Amino-2-methylpropanoate;2-Aminoisobutyric acid C4H9NO2 103.0633285
20 104.0707 (R,S)-3-Amino-2-methylpropanoate C4H9NO2 103.0633285
21 104.0707 beta-alanine-methyl-ester C4H9NO2 103.0633285
22 104.0707 N,N-Dimethylglycine;Dimethylglycine C4H9NO2 103.0633285
23 104.0707 N-Ethylglycine C4H9NO2 103.0633285
24 104.0707 (R)-2-Aminobutanoic acid;(S)-2-Aminobutanoate C4H9NO2 103.0633285
25 104.0707 1-nitrobutane C4H9NO2 103.0633285
26 104.0707 4-Aminobutanoate;4-Aminobutanoic acid C4H9NO2 103.0633285
27 105.0366 3-methylthiopropanal C4H8OS 104.0295856
28 105.0366 Acutifolane A C16H22O3 262.1569
29 105.0366 tetrahydrothiophene 1-oxide C4H8OS 104.0295856
30 105.0367 3-methylthiopropanal C4H8OS 104.0295856
31 105.0367 tetrahydrothiophene 1-oxide C4H8OS 104.0295856
32 105.0372 3-methylthiopropanal C4H8OS 104.0295856
33 105.0372 tetrahydrothiophene 1-oxide C4H8OS 104.0295856
34 105.0376 3-methylthiopropanal C4H8OS 104.0295856
35 105.0376 tetrahydrothiophene 1-oxide C4H8OS 104.0295856
37 105.044 3-cyanopyridine C6H4N2 104.0374481
38 105.044 2-Cyanopyridine C6H4N2 104.0374481
39 105.044 4-Cyanopyridine C6H4N2 104.0374481
40 105.0441 (+)-18-Hydroxy-7,16-sacculatadiene-11,12-dial C20H30O3 318.2195
41 105.0441 ent-7alpha-hydroxykaur-16-en-19-oic acid C20H30O3 318.2195
42 105.0441 2-Cyanopyridine C6H4N2 104.0374481
43 105.0441 Oxymesterone C20H30O3 318.2194948
44 105.0441 4-Cyanopyridine C6H4N2 104.0374481
45 105.0441 3-cyanopyridine C6H4N2 104.0374481
46 105.0441 8-oxo-5E,9Z,11Z,14Z-eicosatetraenoic acid C20H30O3 318.2195
47 105.0441 9-oxo-5E,7Z,11Z,14Z-eicosatetraenoic acid C20H30O3 318.2195
48 105.0441 11-oxo-5E,8Z,12Z,14Z-Eicosatetraenoic acid C20H30O3 318.2195
49 105.0441 (+)-7beta-Hydroxy-15-beyeren-19-oic acid C20H30O3 318.2195
50 105.0557 Tyrosyl-Tyrosine C18H20N2O5 344.1372218
51 105.0651 Aminoserine C3H8N2O2 104.0585775
52 105.0651 L-2,3-Diaminopropionate C3H8N2O2 104.0585775
53 105.0651 Hydroxyaminoalanine C3H8N2O2 104.0585775
54 105.0662 2,3-Diaminopropanoic acid C3H8N2O2 104.0585775
55 105.0672 4,-O-Methylbavachalcone C22H24O4 352.1675
56 105.0672 Ovalichalcone C22H24O4 352.1675
57 105.0672 Pongagallone A C22H24O4 352.1675
58 105.0672 Candidone C22H24O4 352.1675
59 105.0672 Methylhildgardtol A C22H24O4 352.1675
60 105.0672 Methylhildgardtol B C22H24O4 352.1675
61 105.0672 Xuulanin C22H24O4 352.1675
62 105.0715 Valganciclovir C14H22N6O5 354.1651678
63 105.0733 12-hydroxyjasmonic acid 12-O-beta-D-glucoside C19H30O8 386.1941
64 105.0733 Citroside A C19H30O8 386.1940679
65 105.0733 6,9-Dihydroxy-4,7-megastigmadien-3-one C19H30O8 386.1940679
66 107.0845 p-Xylene;1,4-Dimethylbenzene;p-Methyltoluene C8H10 106.0782503
67 107.0845 Ethylbenzene;Phenylethane;Ethylbenzol;Ethylenzene C8H10 106.0782503
68 107.0845 o-Xylene;o-Dimethylbenzene;o-Methyltoluene C8H10 106.0782503
69 107.0845 m-Xylene;1,3-Dimethylbenzene;1,3-Xylene C8H10 106.0782503
70 107.0848 o-Xylene;o-Dimethylbenzene;o-Methyltoluene C8H10 106.0782503
71 107.0848 m-Xylene;1,3-Dimethylbenzene;1,3-Xylene C8H10 106.0782503
72 107.0848 p-Xylene;1,4-Dimethylbenzene;p-Methyltoluene C8H10 106.0782503
73 107.0848 Ethylbenzene;Phenylethane;Ethylbenzol;Ethylenzene C8H10 106.0782503
74 107.0848 O-6-deoxy-a-L-galactopyranosyl C20H33NO14 511.1901048
75 107.0851 o-Xylene;o-Dimethylbenzene;o-Methyltoluene C8H10 106.0782503
76 107.0851 m-Xylene;1,3-Dimethylbenzene;1,3-Xylene C8H10 106.0782503
77 107.0851 p-Xylene;1,4-Dimethylbenzene;p-Methyltoluene C8H10 106.0782503
78 107.0851 Ethylbenzene;Phenylethane;Ethylbenzol;Ethylenzene C8H10 106.0782503
79 107.0858 p-Xylene;1,4-Dimethylbenzene;p-Methyltoluene C8H10 106.0782503
80 107.0858 Ethylbenzene;Phenylethane;Ethylbenzol;Ethylenzene C8H10 106.0782503
81 107.0858 o-Xylene;o-Dimethylbenzene;o-Methyltoluene C8H10 106.0782503
82 107.0858 m-Xylene;1,3-Dimethylbenzene;1,3-Xylene C8H10 106.0782503
83 107.0858 Daunorubicin C27H29NO10 527.1791462
84 107.0859 m-Xylene;1,3-Dimethylbenzene;1,3-Xylene C8H10 106.0782503
85 107.0859 p-Xylene;1,4-Dimethylbenzene;p-Methyltoluene C8H10 106.0782503
86 107.0859 Ethylbenzene;Phenylethane;Ethylbenzol;Ethylenzene C8H10 106.0782503
87 107.0859 o-Xylene;o-Dimethylbenzene;o-Methyltoluene C8H10 106.0782503
88 109.0287 1,2-Benzoquinone C6H4O2 108.0211294
89 109.0287 Quinone;p-Benzoquinone;Chinone C6H4O2 108.0211294
90 109.0306 Hordatine B glucoside C35H50N8O10 742.3649899
91 109.0306 Hydroxymethylmethylsilanediol C2H8O3Si 108.0242707
92 109.0309 Hydroxymethylmethylsilanediol C2H8O3Si 108.0242707
93 109.0309 Monothioglycerol C3H8O2S 108.0245002
94 109.0315 Hydroxymethylmethylsilanediol C2H8O3Si 108.0242707
95 109.0316 Hydroxymethylmethylsilanediol C2H8O3Si 108.0242707
96 109.0316 Hydroxymethylmethylsilanediol C2H8O3Si 108.0242707
97 109.0321 Hydroxymethylmethylsilanediol C2H8O3Si 108.0242707
98 109.0642 Benzenemethanol;Phenylmethanol;Phenylcarbinol C7H8O 108.0575149
99 109.0642 o-Cresol;2-Hydroxytoluene;o-Methylphenol C7H8O 108.0575149
100 109.0642 3-Cresol;m-Cresol;3-Hydroxytoluene C7H8O 108.0575149
101 109.0642 4-Cresol;p-Cresol;4-Hydroxytoluene C7H8O 108.0575149
102 109.0642 Anisole;Methoxybenzene;Methyl phenyl ether C7H8O 108.0575149
103 111.0434 Resorcinol;Resorcin;1,3-Benzenediol C6H6O2 110.0367794
104 111.0434 Hydroquinone;p-Benzenediol;1,4-Benzenediol C6H6O2 110.0367794
105 111.0434 5-Methyl-2-furaldehyde;5-Methyl-2-furfural C6H6O2 110.0367794
106 111.0434 o-Benzosemiquinone C6H6O2 110.0367794
107 111.0434 Catechol;1,2-Benzenediol;o-Benzenediol C6H6O2 110.0367794
108 111.0434 Benzosemiquinone;p-Benzosemiquinone C6H6O2 110.0367794
109 111.0437 Resorcinol;Resorcin;1,3-Benzenediol C6H6O2 110.0367794
110 111.0437 Hydroquinone;p-Benzenediol;1,4-Benzenediol C6H6O2 110.0367794
111 111.0437 5-Methyl-2-furaldehyde;5-Methyl-2-furfural C6H6O2 110.0367794
112 111.0437 Catechol;1,2-Benzenediol;o-Benzenedio C6H6O2 110.0367794
113 111.0437 Benzosemiquinone;p-Benzosemiquinone C6H6O2 110.0367794
114 111.0438 2-Furanmethanol C5H6O2 98.03677944
115 111.0438 Benzosemiquinone;p-Benzosemiquinone C6H6O2 110.0367794
116 111.0438 Resorcinol;Resorcin;1,3-Benzenediol C6H6O2 110.0367794
117 111.0438 Hydroquinone;p-Benzenediol;1,4-Benzenediol C6H6O2 110.0367794
118 111.0438 5-Methyl-2-furaldehyde;5-Methyl-2-furfural C6H6O2 110.0367794
119 111.0438 o-Benzosemiquinone C6H6O2 110.0367794
120 111.0438 penta-2,4-dienoic acid;beta-vinyl acrylic acid C5H6O2 98.0368
121 111.0438 Catechol;1,2-Benzenediol;o-Benzenediol C6H6O2 110.0367794
122 111.0438 5-Methyl-2(3H)-furanone C5H6O2 98.03677944
123 111.0447 o-Benzosemiquinone C6H6O2 110.0367794
124 111.0447 Catechol;1,2-Benzenediol;o-Benzenediol C6H6O2 110.0367794
125 111.0447 Benzosemiquinone;p-Benzosemiquinone C6H6O2 110.0367794
126 111.0447 Resorcinol;Resorcin;1,3-Benzenediol;1,3-Dihydroxybenzene C6H6O2 110.0367794
127 111.0447 Hydroquinone;p-Benzenediol;1,4-Benzenediol C6H6O2 110.0367794
128 111.0447 5-Methyl-2-furaldehyde;5-Methyl-2-furfural C6H6O2 110.0367794
129 111.0448 o-Benzosemiquinone C6H6O2 110.0367794
130 111.0448 Catechol;1,2-Benzenediol;o-Benzenediol C6H6O2 110.0367794
131 111.0448 Benzosemiquinone;p-Benzosemiquinone C6H6O2 110.0367794
132 111.0448 Resorcinol;Resorcin;1,3-Benzenediol;1,3-Dihydroxybenzene C6H6O2 110.0367794
133 111.0448 Hydroquinone;p-Benzenediol;1,4-Benzenediol C6H6O2 110.0367794
134 111.0451 Catechol;1,2-Benzenediol;o-Benzenediol C6H6O2 110.0367794
135 111.0451 Benzosemiquinone;p-Benzosemiquinone C6H6O2 110.0367794
136 111.0451 Resorcinol;Resorcin;1,3-Benzenediol;1,3-Dihydroxybenzene C6H6O2 110.0367794
137 111.0451 Hydroquinone;p-Benzenediol;1,4-Benzenediol C6H6O2 110.0367794
138 111.0451 5-Methyl-2-furaldehyde;5-Methyl-2-furfural C6H6O2 110.0367794
139 121.0316 Dimethylsulfonioacetate C4H8O2S 120.0245002
140 121.0316 3-(Methylthio)propionic acid;3-Methylthiopropionate C4H8O2S 120.0245002
141 121.0316 sulfolane C4H8O2S 120.0245002
142 121.032 Dimethylsulfonioacetate C4H8O2S 120.0245002
143 121.032 3-(Methylthio)propionic acid;3-Methylthiopropionate C4H8O2S 120.0245002
144 121.032 sulfolane C4H8O2S 120.0245002
145 121.0324 Dimethylsulfonioacetate C4H8O2S 120.0245002
146 121.0324 3-(Methylthio)propionic acid;3-Methylthiopropionate C4H8O2S 120.0245002
147 121.0324 sulfolane C4H8O2S 120.0245002
148 121.0325 Dimethylsulfonioacetate C4H8O2S 120.0245002
149 121.0325 3-(Methylthio)propionic acid;3-Methylthiopropionate C4H8O2S 120.0245002
150 121.0325 sulfolane C4H8O2S 120.0245002
151 121.0325 Dimethylsulfonioacetate C4H8O2S 120.0245002
152 121.0325 3-(Methylthio)propionic acid;3-Methylthiopropionate C4H8O2S 120.0245002
153 121.0325 sulfolane C4H8O2S 120.0245002
154 121.0325 Dimethylsulfonioacetate C4H8O2S 120.0245002
155 121.0325 3-(Methylthio)propionic acid;3-Methylthiopropionate C4H8O2S 120.0245002
156 121.0325 sulfolane C4H8O2S 120.0245002
157 121.037 3-nitro-1-propionate C3H6NO4 120.0296827
158 121.0379 3-nitro-1-propionate C3H6NO4 120.0296827
159 121.05 2,3-dihydroxy-2-methyl-propanoic acid C4H8O4 120.0422587
160 121.05 L-(+)-Erythrose;D-threo-Aldose;D-Erythrulose C4H8O4 120.0422587
161 121.05 3-Deoxytetronic acid C4H8O4 120.0422587
162 121.05 4-Deoxyerythronic acid C4H8O4 120.0422587
163 121.05 L-Erythrulose;L-glycero-Tetrulose C4H8O4 120.0422587
164 121.05 3,4-Dihydroxybutyric acid C4H8O4 120.0422587
165 121.05 D-Threose;D-threo-Tetrose;D-Erythrose C4H8O4 120.0422587
166 121.0516 Purine C5H4N4 120.0435961
167 122.0962 3,4-DIMETHYLANILINE C8H11N 121.0891494
168 122.0962 1-Phenylethylamine;alpha-Phenylethylamine C8H11N 121.0891494
169 122.0962 N-Ethylaniline;N-Ethylbenzenamine C8H11N 121.0891494
170 122.0962 Phenethylamine;2-Phenylethylamine;beta-Phenylethylamine C8H11N 121.0891494
171 122.0962 2,4-Dimethylaniline;2,4-DMA C8H11N 121.0891494
172 122.0962 N,N-Dimethylaniline; N,N-Dimethylbenzenamine C8H11N 121.0891494
173 122.0965 1-Phenylethylamine;alpha-Phenylethylamine C8H11N 121.0891494
174 122.0965 2,6-Dimethylaniline C8H11N 121.0891494
175 122.0965 N-Ethylaniline;N-Ethylbenzenamine C8H11N 121.0891494
176 122.0965 2,5-Dimethylalanine C8H11N 121.0891494
177 122.0965 2-Phenylethylamine;beta-Phenylethylamine C8H11N 121.0891494
178 181.0694 Sorbose;xylo-Hexulose;D-Fructose C6H12O6 180.0633881
179 181.0694 2-Deoxy-D-gluconate C6H12O6 180.0633881
180 181.0694 Ketose C6H12O6 180.0633881
181 181.0996 Methylphophonic acid diisopropyl ester C7H17O3P 180.0915309
182 229.1235 1,1-Bis(4-hydroxyphenyl)propane C15H16O2 228.1150298
183 229.1235 Mansonone C C15H16O2 228.1150298
184 229.1235 Bisphenol A;2,2-Bis(4-Hydroxyphenyl)propane C15H16O2 228.1150298
185 229.1235 dihydropinosylvin monomethylether C15H16O2 228.1150298
186 229.1285 Deoxyguanidinoproclavaminic acid C9H16N4O3 228.1222404
187 229.1298 Deoxyamidinoproclavaminate C9H16N4O3 228.1222404
188 230.058 Lamivudine;3TC;2’,3’-Dideoxy-3’-thiacytidine C8H11N3O3S 229.0521119
189 230.058 Carbonylphophonic acid C9H12NO4P 229.0503944
190 230.058 2,3-Dihydroxy-2’-carboxybiphenyl C13H9O4- 229.0500838
191 357.1718 Rutamarin C21H24O5 356.1623739
191 357.1718 Gingerenone A C21H24O5 356.1623739
192 357.1718 alpha,beta-dihydroxanthohumol C21H24O5 356.1623739
193 357.1718 Kadsurenone;Denudatin B C21H24O5 356.1623739
194 371.0795 Rebamipide C19H15ClN2O4 370.0720347
195 371.0795 Digalacturonate;Digalacturonic acid C12H18O13 370.0747407
196 371.0795 1,2-beta-D-Glucuronosyl-D-glucuronate C12H18O13 370.0747407
197 371.145 Napththalene-2-sulfonamide C20H22N2O3S 370.1351133
198 371.1501 iso-dehydrocycloxanthohumol hydrate C21H22O6 370.1416384
199 371.1501 xanthohumol D C21H22O6 370.1416384
200 371.1501 5’-Prenylhomoeriodictyol;Sigmoidin B 3’-methyl ether C21H22O6 370.1416384
201 371.1501 xanthohumol B C21H22O6 370.1416384
202 371.1501 curcumin C21H22O6 370.1416384
203 371.1501 Alkannin beta,beta-dimethylacrylate C21H22O6 370.1416384
204 371.1501 Sophoraisoflavanone A C21H22O6 370.1416384
205 371.1509 iso-dehydrocycloxanthohumol hydrate C21H22O6 370.1416384
206 371.1509 xanthohumol D C21H22O6 370.1416384
207 371.1509 5’-Prenylhomoeriodictyol;Sigmoidin B 3’-methyl ether C21H22O6 370.1416384
208 371.1509 xanthohumol B C21H22O6 370.1416384
209 371.1509 curcumin C21H22O6 370.1416384
210 371.1509 Alkannin beta,beta-dimethylacrylate C21H22O6 370.1416384
211 371.1509 Sophoraisoflavanone A C21H22O6 370.1416384
212 371.1528 Galactan;Amylose C14H26O11 370.1475117
213 371.1528 Galactan;Amylose C14H26O11 370.1475117
214 371.1541 Galactan;Amylose C14H26O11 370.1475117
215 404.1365 trifluoperazine C21H20F3N3S 403.1330029
216 404.1484 Ampicillin trihydrate C16H25N3O7S 403.1413209
217 405.1191 Spectinomycin dihydrochloride C14H26Cl2N2O7 404.1117066
218 405.1267 Sulfinpyrazone;Sulfoxyphenylpyrazolidine C23H20N2O3S 404.1194632
219 405.1269 Sulfinpyrazone;Sulfoxyphenylpyrazolidine C23H20N2O3S 404.1194632

Figure S1:

LC-MS/MS spectra of the peaks eluted at 6.69 min (m/z of 211.11).

Figure S2:

LC-MS/MS spectra of the peaks eluted at 2.40 min (m/z of 206.12).

Figure S3:

LC-MS/MS spectra of the peaks eluted at 6.63 min (m/z of 253.12) and 3.39 min (m/z of 295.17).
Discussion

This study demonstrated that the PGPR B. velezensis AP203 when used in combination with a pectin-rich orange peel amendment resulted in significantly enhanced M. incognita J2 mortality. The nematicidal activity observed under laboratory and greenhouse conditions suggests that there are nematicidal secondary metabolites produced by B. velezensis AP203 in the presence of a pectin-rich orange peel growth substrate. In the presence of different carbohydrate substrates, the expression of secondary metabolites by PGPR strains has been previously shown to vary considerably (Zhu et al., 2013). The production of surfactin by B. velezensis SQY162 (previously known as B. amyloliquefaciens subsp. plantarum) was significantly increased when this strain was inoculated with an exogenous pectin amendment, resulting in enhanced biological control efficacy against bacterial wilt of tobacco caused by Ralstonia solanacearum (Wu et al., 2015). Previous studies have documented that B. velezensis strains have pectinolytic activity and can utilize pectin as a sole carbon and energy source (Hossain et al., 2015). Hence, exogenous pectin amendments serve as a prebiotic growth substrate that can be utilized by B. velezensis or other pectinolytic microorganisms, thereby enhancing the metabolic activities of the bacteria, which in the case of PGPR B. velezensis strains can include plant growth-promotion and biofilm formation (Beauregard et al., 2013; Hassan et al., 2019). In a similar study, it was reported that chitinolytic bacteria provided with a chitin substrate significantly reduced soybean cyst nematode (Heterodera glycines) populations under greenhouse conditions (Tian et al., 2000). Collectively, these previous studies and the results of this study support the hypothesis that the combination of a prebiotic complex carbohydrate (e.g. pectin-rich citrus peel) with a probiotic bacterium capable of producing beneficial metabolites (e.g. PGPR B. velezensis AP203 and its nematicidal metabolites) may increase the efficacy of disease control in plant hosts, what is also referred to as a ‘synbiotic’ inoculum containing compatible prebiotic and probiotic agents.

The results from the greenhouse tests suggested that cell pellet, culture broth, and supernatant of B. velezensis AP203 with 1.0% (w/v) orange peel amended media significantly increased soybean and cotton plant growth (root length) compared to the M. incognita inoculated positive control. In addition, the numbers of M. incognita eggs of cell pellet, culture broth, and supernatant were reduced in the roots of cotton and soybean compared to the M. incognita inoculated positive control. However, there were no significant differences between cell pellet, culture broth, or supernatant of B. velezensis AP203 amended with orange peel. Previous studies reported that extracts of fresh orange peel significantly reduced M. incognita eggs and J2 in planta and in vitro (Faye, 2017; Raviv et al., 2005; Tsai, 2008). Recent studies also showed that B. velezensis strains were capable of enhancing cotton and soybean yields and reducing M. incognita egg populations in a greenhouse, micro plots, and field experiments (Xiang et al., 2017a, b). This is the first study to evaluate the combination of a PGPR B. velezensis strain and an orange peel amendment in antagonizing M. incognita populations. Agricultural waste can be an environmental problem, and waste management is an enormous challenge worldwide. The use of orange peel to promote the efficacy of PGPR-mediated plant pathogenic nematode control may reduce the need for chemical nematicides and improve plant and soil health. The present findings indicate the potential for B. velezensis AP203 and orange peel amendments to reduce M. incognita population density and increase yield under field conditions, thereby providing an alternative option to chemical nematicides. In addition to improving plant health and suppressing plant-parasitic nematodes, orange peel amendment to soils may also enhance soil nutrient levels. A recent study showed that agricultural waste orange peel significantly enhanced soil nutrient levels and regenerated tropical forest vegetation in Costa Rica (Treuer et al., 2018). Taken together, the results of this and other studies suggest that this synbiotic treatment may be a cost-effective strategy of benefit to sustainable agricultural practices.

The four metabolites that were specifically produced by B. velezensis AP203 when grown on an orange peel substrate may have direct inhibitory effects on M. incognita eggs and J2 viability. A previous study reported that the volatile organic compounds phenol, propyl benzene, propanone, and 1-ethenyl-4-methoxy benzene produced by B. megaterium YFM3.25 showed nematicidal effects and significantly reduced M. incognita eggs in pot experiments (Huang et al., 2010). Orange peel contains pectin, limonene, and phenolic compounds that have antioxidant properties and exerts beneficial effects on plant health (Bocco et al., 1998; Rafiq et al., 2018). The increased M. incognita mortality observed in this study may be due to a combination of metabolites producing by B. velezensis AP203 and derived from orange peel. These data support the conclusion that the metabolome of B. velezensis AP203 is significantly altered when this strain is grown on OPP, resulting in the expression of previously identified nematicidal compounds which were not observed when this strain was grown on glucose as a sole carbon source. No doubt, the chemical milieu of citrus peel includes a greater complexity of carbon sources and organic compounds, relative to growth on glucose alone, may elicit changes in B. velezensis gene expression. Further experiments should explore the changes in secondary metabolite expression for B. velezensis AP203 and other bioactive PGPR strains within the rhizosphere, as affected by the presence of pectin-rich amendments or other compatible prebiotic complex carbohydrates. This study is the first report that a B. velezensis strain in combination with an orange peel prebiotic amendment can inhibit the viability of M. incognita eggs and J2, mediated at least in part through the production of bioactive secondary metabolites. Thus, treatments that combine B. velezensis strains with nematicidal potential along with an orange peel amendment are predicted to more effectively reduce plant-pathogenic nematode population density in planta.

In conclusion, B. velezensis AP203 when combined with an orange peel amendment significantly reduced M. incognita populations in vitro and in planta. Hence, the combined use of B. velezensis strains and orange peel represents a promising and sustainable biological control technique for plant-parasitic nematodes.
eISSN:
2640-396X
Język:
Angielski
Częstotliwość wydawania:
Volume Open
Dziedziny czasopisma:
Life Sciences, other
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