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Meloidogyne enterolobii-induced Changes in Guava Root Exudates Are Associated With Root Rotting Caused by Neocosmospora falciformis

Journal of Nematology
Volume 55 (2023): Issue 1 (February 2023)
By:
Open Access
|Dec 2023

Figures & Tables

Figure 1:

Effect of extracts and fractions of guava root exudates on the pathogenicity of Neocosmospora falciformis to guava seedlings. Root exudates (coded NP) were collected from plants cultivated in a growth chamber and parasitized by Meloidogyne enterolobii. A) Uninoculated seedling in glass tube filled with autoclaved sand (bioassay 1). B) Seedling in an Eppendorf tube, inoculated with a fungus-colonized agar-water plug positioned in the collar region (bioassay 2). C) Damping-off in a seedling inoculated with the fungus and watered with an aqueous solution prepared from the extract NP-03. D) Damping-off and fungal growth in a seedling watered with an aqueous solution prepared from the fraction NP-03-F2. E) Fungus-inoculated seedlings watered with an aqueous solution prepared from NP-03-F2 (left) or sterile, distilled water (right). F) From left to right: fungus-inoculated seedlings watered with sterile, distilled water or fractions NP-03-F2, NP-03-F2-3, NP-03-F2-4, NP-03-F2-5 or NP-03-F2-6 (bioassay 3).
Effect of extracts and fractions of guava root exudates on the pathogenicity of Neocosmospora falciformis to guava seedlings. Root exudates (coded NP) were collected from plants cultivated in a growth chamber and parasitized by Meloidogyne enterolobii. A) Uninoculated seedling in glass tube filled with autoclaved sand (bioassay 1). B) Seedling in an Eppendorf tube, inoculated with a fungus-colonized agar-water plug positioned in the collar region (bioassay 2). C) Damping-off in a seedling inoculated with the fungus and watered with an aqueous solution prepared from the extract NP-03. D) Damping-off and fungal growth in a seedling watered with an aqueous solution prepared from the fraction NP-03-F2. E) Fungus-inoculated seedlings watered with an aqueous solution prepared from NP-03-F2 (left) or sterile, distilled water (right). F) From left to right: fungus-inoculated seedlings watered with sterile, distilled water or fractions NP-03-F2, NP-03-F2-3, NP-03-F2-4, NP-03-F2-5 or NP-03-F2-6 (bioassay 3).

Figure 2:

Concentration (mg/mL) of amino acids (A), carbohydrates (B), sucrose (C), phenols (D), and alkaloids (E) in extracts of guava root exudates. The guava plants were cultivated in a growth chamber, and they were parasitized by Meloidogyne enterolobii (coded NP) or nematode-free (NF). The solvents used to obtain the extracts were methanol (coded -03) or water (-04). Values are means of two quantifications with three replicates each. Different letters on top of the columns indicate difference according to Tukey test at 5%. In the columns, numbers are standard deviation × 10−5.
Concentration (mg/mL) of amino acids (A), carbohydrates (B), sucrose (C), phenols (D), and alkaloids (E) in extracts of guava root exudates. The guava plants were cultivated in a growth chamber, and they were parasitized by Meloidogyne enterolobii (coded NP) or nematode-free (NF). The solvents used to obtain the extracts were methanol (coded -03) or water (-04). Values are means of two quantifications with three replicates each. Different letters on top of the columns indicate difference according to Tukey test at 5%. In the columns, numbers are standard deviation × 10−5.

Figure 3:

Representation of the most stable conformation of 1,5-dinitrobiuret according to DFT calculations at the theoretical level B3LYP/6-31G(2df,p), implicitly considering the solvent DMSO. A) two-dimensional representation; B) 3D representation with the open chain; C) 3D representation with aligned carbonyls.
Representation of the most stable conformation of 1,5-dinitrobiuret according to DFT calculations at the theoretical level B3LYP/6-31G(2df,p), implicitly considering the solvent DMSO. A) two-dimensional representation; B) 3D representation with the open chain; C) 3D representation with aligned carbonyls.

Figure 4:

Calculated affinities of 1,5-dinitrobiuret and its tautomers (DNB*); biuret and its tautomers (BIU*); and different protonation states of N-formyl-D-aspartic acid (ASP*) for biuret hydrolases.
Calculated affinities of 1,5-dinitrobiuret and its tautomers (DNB*); biuret and its tautomers (BIU*); and different protonation states of N-formyl-D-aspartic acid (ASP*) for biuret hydrolases.

Supplementary Figure 1:

Chemical structure of 1,5-dinitrobiuret and all its tautomers, which were submitted to computational calculations with dimethylsulfoxide (DMSO) as the solvent.
Chemical structure of 1,5-dinitrobiuret and all its tautomers, which were submitted to computational calculations with dimethylsulfoxide (DMSO) as the solvent.

Supplementary Figure 2:

Chemical structure of 1,5-dinitrobiuret and its tautomers, which were submitted to computational calculations with water as the solvent.
Chemical structure of 1,5-dinitrobiuret and its tautomers, which were submitted to computational calculations with water as the solvent.

Supplementary Figure 3:

Chemical structure of biuret and its tautomers, which were submitted to computational calculations with water as the solvent.
Chemical structure of biuret and its tautomers, which were submitted to computational calculations with water as the solvent.

Supplementary Figure 4:

Protonation states of N-formyl-D-aspartic acid that were considered during computational calculations.
Protonation states of N-formyl-D-aspartic acid that were considered during computational calculations.

Supplementary Figure 5:

Examples of alignment of the most stable conformations of 1,5-dinitrobiuret (containing hydrogen atoms) to the structure of biuret (without hydrogen atoms), experimentally complexed to biuret hydrolase 6azq.
Examples of alignment of the most stable conformations of 1,5-dinitrobiuret (containing hydrogen atoms) to the structure of biuret (without hydrogen atoms), experimentally complexed to biuret hydrolase 6azq.

Supplementary Figure 6:

Examples of alignment of the most stable conformations of biuret (containing hydrogen atoms) to the structure of biuret (without hydrogen atoms), experimentally complexed to biuret hydrolase 6AZQ.
Examples of alignment of the most stable conformations of biuret (containing hydrogen atoms) to the structure of biuret (without hydrogen atoms), experimentally complexed to biuret hydrolase 6AZQ.

Supplementary Figure 7:

Examples of alignment of the most stable conformations of different protonation states of N-formyl-D-aspartic acid (containing hydrogen atoms) to the structure of N-formyl-D-aspartic acid (without hydrogen atoms), experimentally complexed to biuret hydrolase 6AZS.
Examples of alignment of the most stable conformations of different protonation states of N-formyl-D-aspartic acid (containing hydrogen atoms) to the structure of N-formyl-D-aspartic acid (without hydrogen atoms), experimentally complexed to biuret hydrolase 6AZS.

Supplementary Figure 8:

Chromatogram obtained by analyzing the fractions NP-03-F2-3+4-ET-02 (top) and NP-03-F2-3+4-ET-05 (bottom) by high performance liquid chromatography, using a Luna 5-μm column (phenyl-hexyl, 250 by 4.60 mm), with water as the eluent and the UV detector set at 190 nm.
Chromatogram obtained by analyzing the fractions NP-03-F2-3+4-ET-02 (top) and NP-03-F2-3+4-ET-05 (bottom) by high performance liquid chromatography, using a Luna 5-μm column (phenyl-hexyl, 250 by 4.60 mm), with water as the eluent and the UV detector set at 190 nm.

Supplementary Figure 9:

13C NMR spectra of 1,5-dinitrobiuret. Top: experimental signal obtained for the fraction NP-03-F2-3+4-ET-05; Bottom: signal obtained from simulation with the software MestReNova 6.0.2.
13C NMR spectra of 1,5-dinitrobiuret. Top: experimental signal obtained for the fraction NP-03-F2-3+4-ET-05; Bottom: signal obtained from simulation with the software MestReNova 6.0.2.

Dry mass and relative proportion of extracts obtained from guava (Psidium guajava) root exudates after extraction with organic solvents and water_ The plants, cultivated in a growth chamber, were either parasitized by Meloidogyne enterolobii (coded NP) or nematode-free (NF)

Extract codeSolvent usedDry mass (g)Relative mass (%)a
NP-01Hexane0.010.04
NF-01 0.0180.13
NP-02Ethyl acetate0.1450.69
NF-02 0.0860.6
NP-03Methanol12.7260.42
NF-03 7.44351.8
NP-04Water8.17738.84
NF-04 6.82447.5

Bioassay 3_ Effect of fractions of guava root exudates on the pathogenicity of Neocosmospora falciformis to guava seedlings_ Root exudates were collected from plants parasitized by Meloidogyne enterolobii and submitted to solvent extractions and silica-column fractionation_ Column chromatography of NP-03-F2 resulted in fractions -3 through -6, which were used to prepare the seedlings’ watering solutions_

TreatmentsRootlets’ rotten fresh massRootlets’ unrotten, fresh massFresh shoot mass
Inoculated seedlings watered with sterile, distilled water (control)0*b36.3a141a
Inoculated seedlings watered with NP-03-F2 (control)12a13.7b95.1b
Inoculated seedlings watered with NP-03-F2-38.1a11.4b93.4b
Inoculated seedlings watered with NP-03-F2-412.2a10.8b94.6b
Inoculated seedlings watered with NP-03-F2-50b36.4a157.7a
Inoculated seedlings watered with NP-03-F2-60b36.6a150.4a
CV%22.313.312.6

Dry mass and relative proportion of fractions obtained from chromatographic fractionation of NP-03-F2 with different eluents_ NP-03-F2 is a fraction of the methanol-soluble extract of root exudates of guava (Psidium guajava) plants cultivated in a growth chamber and parasitized by Meloidogyne enterolobii_

Fraction codeEluentColumn fractionsDry mass (g)Relative mass (%)a
NP-03-F2-1Ethyl acetate/methanol (7:3)1–40.00040.03
NP-03-F2-2Ethyl acetate/methanol (7:3)5 and 60.0513.5
NP-03-F2-3Ethyl acetate/methanol (7:3)7–90.44830.73
NP-03-F2-4Ethyl acetate/methanol (7:3)10–140.50734.77
NP-03-F2-5Ethyl acetate/methanol (7:3)15–190.1157.89
NP-03-F2-6Ethyl acetate/methanol (3:7)20–230.32322.15
NP-03-F2-7Ethyl acetate/methanol (3:7)24–290.0060.41
NP-03-F2-8Watersingle0.0080.55

Bioassay 1_ Effect of extracts of guava root exudates on the pathogenicity of Neocosmospora falciformis to guava seedlings_ Root exudates were collected from plants cultivated in a growth chamber that were either parasitized by Meloidogyne enterolobii (coded NP) or nematode-free (NF)_ The solvents used were hexane (extracts -01), ethyl acetate (-02), methanol (-03) and water (-04)_ The extracts were used to prepare the seedlings’ watering solutions_

TreatmentsRootlets’ unrotten, fresh massFresh shoot mass
Uninoculated seedlings watered with sterile, distilled water (control)64.4*a185.2a
Inoculated seedlings watered with sterile, distilled water (control)64.7a198.9a
Inoculated seedlings watered with NP-0163.5a198.6a
Inoculated seedlings watered with NP-0267.1a163.2a,b
Inoculated seedlings watered with NP-0311.5b84.9c
Inoculated seedlings watered with NP-0415.9b79.6c
Inoculated seedlings watered with NF-0174.1a149.7b
Inoculated seedlings watered with NF-0266.1a167.7a,b
Inoculated seedlings watered with NF-0365.7a184.2a
Inoculated seedlings watered with NF-0467.2a189.7a
CV%12.17.7

Bioassay 2_ Effect of fractions of guava root exudates on the pathogenicity of Neocosmospora falciformis to guava seedlings_ Root exudates were collected from plants parasitized by Meloidogyne enterolobii_ The methanol-soluble extract NP-03 was submitted to silica column fractionation with the eluents ethyl acetate/methanol (fraction F1), methanol (F2), water (F3), water/acetic acid (F4) and aqueous hydrochloric acid (F5)_ These fractions were used to prepare the seedlings’ watering solutions_

TreatmentsRootlets’ unrotten, fresh massFresh shoot mass
Uninoculated seedlings watered with sterile, distilled water (control)64.4*a185.2a
Inoculated seedlings watered with sterile, distilled water (control)64.7a198.8a
Inoculated seedlings watered with NP-03-F170.1a159.7a
Inoculated seedlings watered with NP-03-F236.2b87.7b
Inoculated seedlings watered with NP-03-F365.7a184.2a
Inoculated seedlings watered with NP-03-F467.2a189.7a
Inoculated seedlings watered with NP-03-F563.5a198.6a
CV%15.19.7

Dry mass and relative proportion of fractions obtained from extraction of NP-03-F2-3+4 with different solvents_ NP-03-F2-3+4 is a chromatographic fraction of root exudates of guava (Psidium guajava) plants cultivated in a growth chamber and parasitized by Meloidogyne enterolobii_

Fraction codeSolvent usedDry mass (mg)Relative mass (%)a
NP-03-F2-3+4-DIDiethyl ether4.91.32
NP-03-F2-3+4-ETEthanol291.678.42
NP-03-F2-3+4-MEMethanol7219.37
NP-03-F2-3+4-INInsoluble3.30.89

Dry mass and relative proportion of fractions obtained from chromatographic separation of NP-03-F2-3+4-ET_ NP-03-F2-3+4-ET is an ethanol-soluble fraction of root exudates of guava (Psidium guajava) plants cultivated in a growth chamber, and parasitized by Meloidogyne enterolobii_

Fraction codeColumn fractionsDry mass (mg)Relative mass (%)a
NP-03-F2-3+4-ET-011–1040.713.6
NP-03-F2-3+4-ET-0211–1436.212.1
NP-03-F2-3+4-ET-0315–2045.115
NP-03-F2-3+4-ET-0421–2522.67.5
NP-03-F2-3+4-ET-0526–3289.429.8
NP-03-F2-3+4-ET-0633–5016.65.5
NP-03-F2-3+4-ET-0751 (methanol)45.815.3
DOI: https://doi.org/10.2478/jofnem-2023-0055 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X
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Language: English
Submitted on: Nov 6, 2022
Published on: Dec 31, 2023
Published by: Society of Nematologists, Inc.
In partnership with: Paradigm Publishing Services
Publication frequency: 1 issue per year
Keywords:
1,5-dinitrobiuret,
disease complex,
guava,
guava decline,
nematode-fungus interaction,
root exudate,
root-knot nematode
Related subjects:
Life sciences,
Life sciences, other

© 2023 Ricardo M. Souza, Denilson F. Oliveira, Vicente M. Gomes, Abraão J. S. Viana, Geraldo H. Silva, Alan R. T. Machado, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution 4.0 License.

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