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

Ricardo M. Souza
Ricardo M. Souza
, 
Denilson F. Oliveira
Denilson F. Oliveira
, 
Vicente M. Gomes
Vicente M. Gomes
, 
Abraão J. S. Viana
Abraão J. S. Viana
, 
Geraldo H. Silva
Geraldo H. Silva
 und 
Alan R. T. Machado
Alan R. T. Machado
   | 31. Dez. 2023
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Article Category: Research Paper
Online veröffentlicht: 31. Dez. 2023
Seitenbereich: -
Eingereicht: 06. Nov. 2022
DOI: https://doi.org/10.2478/jofnem-2023-0055
Schlüsselwörter
© 2023 Ricardo M. Souza et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

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 code Solvent used Dry mass (g) Relative mass (%)a
NP-01 Hexane 0.01 0.04
NF-01 0.018 0.13
NP-02 Ethyl acetate 0.145 0.69
NF-02 0.086 0.6
NP-03 Methanol 12.72 60.42
NF-03 7.443 51.8
NP-04 Water 8.177 38.84
NF-04 6.824 47.5

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.

Treatments Rootlets’ rotten fresh mass Rootlets’ unrotten, fresh mass Fresh shoot mass
Inoculated seedlings watered with sterile, distilled water (control) 0*b 36.3a 141a
Inoculated seedlings watered with NP-03-F2 (control) 12a 13.7b 95.1b
Inoculated seedlings watered with NP-03-F2-3 8.1a 11.4b 93.4b
Inoculated seedlings watered with NP-03-F2-4 12.2a 10.8b 94.6b
Inoculated seedlings watered with NP-03-F2-5 0b 36.4a 157.7a
Inoculated seedlings watered with NP-03-F2-6 0b 36.6a 150.4a
CV% 22.3 13.3 12.6

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 code Eluent Column fractions Dry mass (g) Relative mass (%)a
NP-03-F2-1 Ethyl acetate/methanol (7:3) 1–4 0.0004 0.03
NP-03-F2-2 Ethyl acetate/methanol (7:3) 5 and 6 0.051 3.5
NP-03-F2-3 Ethyl acetate/methanol (7:3) 7–9 0.448 30.73
NP-03-F2-4 Ethyl acetate/methanol (7:3) 10–14 0.507 34.77
NP-03-F2-5 Ethyl acetate/methanol (7:3) 15–19 0.115 7.89
NP-03-F2-6 Ethyl acetate/methanol (3:7) 20–23 0.323 22.15
NP-03-F2-7 Ethyl acetate/methanol (3:7) 24–29 0.006 0.41
NP-03-F2-8 Water single 0.008 0.55

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.

Treatments Rootlets’ unrotten, fresh mass Fresh shoot mass
Uninoculated seedlings watered with sterile, distilled water (control) 64.4*a 185.2a
Inoculated seedlings watered with sterile, distilled water (control) 64.7a 198.9a
Inoculated seedlings watered with NP-01 63.5a 198.6a
Inoculated seedlings watered with NP-02 67.1a 163.2a,b
Inoculated seedlings watered with NP-03 11.5b 84.9c
Inoculated seedlings watered with NP-04 15.9b 79.6c
Inoculated seedlings watered with NF-01 74.1a 149.7b
Inoculated seedlings watered with NF-02 66.1a 167.7a,b
Inoculated seedlings watered with NF-03 65.7a 184.2a
Inoculated seedlings watered with NF-04 67.2a 189.7a
CV% 12.1 7.7

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.

Treatments Rootlets’ unrotten, fresh mass Fresh shoot mass
Uninoculated seedlings watered with sterile, distilled water (control) 64.4*a 185.2a
Inoculated seedlings watered with sterile, distilled water (control) 64.7a 198.8a
Inoculated seedlings watered with NP-03-F1 70.1a 159.7a
Inoculated seedlings watered with NP-03-F2 36.2b 87.7b
Inoculated seedlings watered with NP-03-F3 65.7a 184.2a
Inoculated seedlings watered with NP-03-F4 67.2a 189.7a
Inoculated seedlings watered with NP-03-F5 63.5a 198.6a
CV% 15.1 9.7

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 code Solvent used Dry mass (mg) Relative mass (%)a
NP-03-F2-3+4-DI Diethyl ether 4.9 1.32
NP-03-F2-3+4-ET Ethanol 291.6 78.42
NP-03-F2-3+4-ME Methanol 72 19.37
NP-03-F2-3+4-IN Insoluble 3.3 0.89

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 code Column fractions Dry mass (mg) Relative mass (%)a
NP-03-F2-3+4-ET-01 1–10 40.7 13.6
NP-03-F2-3+4-ET-02 11–14 36.2 12.1
NP-03-F2-3+4-ET-03 15–20 45.1 15
NP-03-F2-3+4-ET-04 21–25 22.6 7.5
NP-03-F2-3+4-ET-05 26–32 89.4 29.8
NP-03-F2-3+4-ET-06 33–50 16.6 5.5
NP-03-F2-3+4-ET-07 51 (methanol) 45.8 15.3
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