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

Abstract

Despite the worldwide importance of disease complexes involving root-feeding nematodes and soilborne fungi, there have been few in-depth studies on how these organisms interact at the molecular level. Previous studies of guava decline have shown that root exudates from Meloidogyne enterolobii-parasitized guava plants (NP plants), but not from nematode-free plants (NF plants), enable the fungus Neocosmospora falciformis to rot guava roots, leading to plant death. To further characterize this interaction, NP and NF root exudates were lyophilized; extracted with distinct solvents; quantified regarding amino acids, soluble carbohydrates, sucrose, phenols, and alkaloids; and submitted to a bioassay to determine their ability to enable N. falciformis to rot the guava seedlings' roots. NP root exudates were richer than NF root exudates in amino acids, carbohydrates, and sucrose. Only the fractions NP-03 and NP-04 enabled fungal root rotting. NP-03 was then sequentially fractionated through chromatographic silica columns. At each step, the main fractions were reassessed in bioassay. The final fraction that enabled fungal root rotting was submitted to analysis using high performance liquid chromatography, nuclear magnetic resonance, mass spectrometry, energy-dispersive X-ray fluorescence, and computational calculations, leading to the identification of 1,5-dinitrobiuret as the predominant substance. In conclusion, parasitism by M. enterolobii causes an enrichment of guava root exudates that likely favors microorganisms capable of producing 1,5-dinitrobiuret in the rhizosphere. The accumulation of biuret, a known phytotoxic substance, possibly hampers root physiology and the innate immunity of guava to N. falciformis.

Keywords: 1,5-dinitrobiuret; Meloidogyne enterolobii; Neocosmospora falciformis; Psidium guajava; disease complex; guava; guava decline; nematode-fungus interaction; root exudate; root-knot nematode.

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).

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.

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.

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.

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.

Supplementary Figure 2:

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.

Supplementary Figure 4:

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.

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.

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.

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.

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.