Effect of 4-Ethylbenzaldehyde from the Volatilome of Annona muricata on Meloidogyne incognita.

The demand for new soil fumigants has increased as a result of more restrictive legislation regarding the use of pesticides. In the present study, the potent nematicidal activity of volatile organic compounds released by the Annona muricata leaf macerate was demonstrated. In addition, we searched in the A. muricata volatilome for a molecule with potential to be developed as a new fumigant nematicide. In the greenhouse, even the lowest concentration of soursop leaf macerate tested (1.0%) as a biofumigant caused a significant (P < 0.05) reduction in Meloidogyne incognita infectivity and reproduction when compared with the nontreated control (0%). Forty-one compounds were identified through gas chromatography-mass spectrometry analysis, of which three (sabinene, caryophyllene oxide, and 4-ethylbenzaldehyde) were selected for studies against the nematode. Among these compounds, in in vitro trails, only 4-ethylbenzaldehyde showed nematicidal activity at 250 µg ml-1. The effective doses of 4-ethylbenzaldehyde predicted to kill 50 and 95% of the M. incognita second-stage juvenile population after 48 h of exposure were 35 and 88 µg ml-1, respectively. In in vitro tests, 4-ethylbenzaldehyde at 150 µg ml-1 reduced M. incognita egg hatching to values similar (P > 0.05) to those of the commercial nematicide fluensulfone at a concentration of 200 µg ml-1. In plant experiments, as a soil fumigant, 4-ethylbenzaldehyde at a dose of 1 ml/liter of substrate had an effect similar (P > 0.05) to that of the commercial fumigant Dazomet (250 µg ml-1). Therefore, 4-ethylbenzaldehyde shows potential for development as a new nematicide.

(R)-Carvone is a potential soil fumigant against Meloidogyne incognita whose likely enzymatic target in the nematode is acetylcholinesterase.

To contribute to the development of new fumigant nematicides for the control of the plant-parasitic nematode Meloidogyne incognita, this study started with 31 volatile organic compounds reported as toxic to nematodes. At 500 µg/mL, α-ionone, (S)-carvone, (R)-carvone, 2-methylpropyl acetate, undecan-2-one, decan-2-one, and dodecan-2-one caused mortalities to M. incognita second-stage juveniles (J2) that were similar to those obtained with the commercial nematicides carbofuran (170 µg/mL) and fluensulfone (42.2 µg/mL). (R)-carvone, with a lethal concentration to 50% J2 (LC50) equal to 524 µg/mL, was selected for subsequent studies. When J2 were exposed to the (R)-carvone solution, the infectivity and reproduction on tomato were reduced. In the M. incognita egg hatching assay, (R)-carvone behaved like a true ovicide. When employed as a fumigant, (R)-carvone (3.9 g/L) was as efficient as the soil fumigant dazomet (0.245 g/L) in eliminating eggs of the nematode in a substrate to be used for tomato planting. According to in silico studies employing pharmacophoric searches and molecular docking, acetylcholinesterases are the target of (R)-carvone in the nematode.

Temperature Effects on Development of Meloidogyne Enterolobii and M. Floridensis.

Meloidogyne enterolobii and M. floridensis are virulent species that can overcome root-knot nematode resistance in economically important crops. Our objectives were to determine the effects of temperature on the infectivity of second-stage juveniles (J2) of these two species and determine differences in duration and thermal-time requirements (degree-days [DD]) to complete their developmental cycle. Florida isolates of M. enterolobii and M. floridensis were compared to M. incognita race 3. Tomato cv. BHN 589 seedlings following inoculation were placed in growth chambers set at constant temperatures of 25°C, and 30°C, and alternating temperatures of 30°C to 25°C (day-night). Root infection by the three nematode species was higher at 30°C than at 25°C, and intermediate at 30°C to 25°C, with 33%, 15%, and 24% infection rates, respectively. There was no difference, however, in the percentages of J2 that infected roots among species at each temperature. Developmental time from infective J2 to reproductive stage for the three species was shorter at 30°C than at 25°C, and 30°C to 25°C. The shortest time and DD to egg production for the three species were 13 days after inoculation (DAI) and 285.7 DD, respectively. During the experimental timeframe of 29 d, a single generation was completed at 30°C for all three species, whereas only M. floridensis completed a generation at 30°C to 25°C. The number of days and accumulated DD for completing the life cycle (from J2 to J2) were 23 d and 506.9 DD for M. enterolobii, and 25 d and 552.3 DD for M. floridensis and M. incognita, respectively. Exposure to lower (25°C) and intermediate temperatures (30°C to 25°C) decreased root penetration and slowed the developmental cycle of M. enterolobii and M. floridensis compared with 30°C.

Methyl Esters of (E)-Cinnamic Acid: Activity against the Plant-Parasitic Nematode Meloidogyne incognita and In Silico Interaction with Histone Deacetylase.

(E)-Cinnamaldehyde is very active against Meloidogyne incognita but has low persistence in soil. To circumvent this problem, esters of cinnamic acid were evaluated as a substitute for (E)-cinnamaldehyde. The best results under assays with M. incognita second-stage juveniles (J2) were obtained for the methyl esters of (E)-p-fluoro- (13), (E)-p-chloro- (14), and (E)-p-bromocinnamic acid (15), which showed lethal concentrations to 50% (LC50) J2 of 168, 95, and 216 µg/mL, respectively. Under the same conditions, the LC50 values for the nematicides carbofuran and fluensulfone were 160 and 34 µg/mL, respectively. Substances 13-15 were also active against nematode eggs, which account for most of the M. incognita population in the field. According to an in silico study, substances 13-15 can act against the nematode through inhibition of histone deacetylase. Therefore, esters 13-15 and histone deacetylase are potentially useful for the rational design of new nematicides for the control of M. incognita.

The combination of two Bacillus strains suppresses Meloidogyne incognita and fungal pathogens, but does not enhance plant growth.

BACKGROUND: The combination of biocontrol agents is a desirable strategy to improve control efficacy against the root-knot nematode (RKN) Meloidogyne incognita under field conditions. However, strains compatibility is generally tested in vitro and incompatible combinations are normally not further examined in experiments in planta. Therefore, there is virtually no information on the performance of incompatible strains. In this study, we evaluated two Bacillus strains previously described as incompatible in vitro for effects on plant growth and suppression of M. incognita, pathogenic fungi and nematophagous fungi. RESULTS: Strains BMH and INV were shown to be closely related to Bacillus velezensis. These strains, when applied individually, reduced the number of galls and eggs of M. incognita by more than 90% in tomato roots. When BMH and INV were combined (BMH + INV), RKN suppression and tomato shoot weight were lower compared to single-strain applications. Additionally, metabolites in cell-free supernatants and volatile organic compounds (VOCs) from strains BMH and INV had strong effects against the plant pathogens M. incognita, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsiii, but not against three species of nematophagous fungi. Although strain INV and the combination BMH + INV emitted fewer VOCs than strain BMH, they were still capable of killing second-stage juveniles of M. incognita. CONCLUSIONS: Bacillus strains BMH and INV inhibited M. incognita and fungal pathogens, and promoted tomato growth. However, strain INV emitted fewer VOCs and the combination BMH + INV did not enhance the activity of the biocontrol strains against the RKN or their capacity to promote plant growth. © 2021 Society of Chemical Industry.

Slight induction and strong inhibition of Heterodera glycines hatching by short-chain molecules released by different plant species.

New management tools are necessary to reduce the damage caused by the soybean cyst nematode (SCN), Heterodera glycines. Identification of molecules that can stimulate second-stage juveniles (J2) hatching in an environment without food may contribute to that. In in vitro experiments, we evaluate the effect of volatile organic compounds (VOCs) released by soybean (Glycine max), bean (Phaseolus vulgaris), ryegrass (Lolium multiflorum), and alfalfa (Medicago sativa) on H. glycines egg hatching. VOCs released by all plant species significantly (p < 0.05) increased egg hatching. Short-chain molecules released by leaves and roots of soybean and bean increased the hatching up to 71.4%. The analysis of the volatilome done by gas chromatography coupled with mass spectrometry revealed 44 compounds in the plant emissions. Four of them, namely 3-octanol, 1-hexanol, hexanal and linalool were tested individually as hatching inductors. Under concentrations of 200, 600, and 1,000 µg/ml there was no hatching induction of H. glycines J2 by these compounds. On the other hand, in these concentrations, the compounds 3-octanol and 1-hexanol caused hatching reduction with values similar to the commercial nematicide carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methyl carbamate). In subsequent tests, the compounds 1-hexanol and 3-octanol showed lethal concentration values required to kill 50% of thenematode population (LC50) of 210 and 228 µg/ml, respectively, in the first experiment and, 230 and 124 µg/mlin the second one. Although we have not identified any molecules acting as hatching factor (HF), here we present a list (44 candidate molecules) that can be explored in future studies to find an efficient HF.

Toxicities of 4,5-Dihydroisoxazoles Against Root-Knot Nematodes and in Silico Studies of Their Modes of Action.

The present work sought to contribute to the development of new nematicides. Benzaldehydes were initially converted to nitrile oxides that underwent 1,3-dipolar cycloaddition reactions with methyl acrylate to generate 4,5-dihydroisoxazoles. In in vitro tests, methyl 3-phenyl-4,5-dihydroisoxazole-5-carboxylate (1) and methyl 3-(4-chlorophenyl)-4,5-dihydroisoxazole-5-carboxylate (4) increased the mortality of Meloidogyne exigua and Meloidogyne incognita second-stage juveniles (J2). Compounds 1 and 4 presented necessary concentrations of 398 and 501 µg mL-1, respectively, to kill 50% of M. incognita J2 (LC50 values), while the value for carbofuran (positive control) was 168 µg mL-1. In in vivo tests, compounds 1 and 4 reduced the number of M. incognita galls in tomato roots by 70 and 40%, respectively, and the number of eggs by 89 and 44%. Using an in silico approach, we showed that compounds 1 and 4 were toxic to the nematodes by binding to the allosteric binding sites of the agonist-binding domains of the nematode nicotinic acetylcholine receptors. These results opened up possibilities for further investigations aimed at developing novel commercial nematicides.

Volatile emissions of watercress (Nasturtium officinale) leaves and passion fruit (Passiflora edulis) seeds against Meloidogyne incognita.

Watercress leaf and passion fruit seed macerates produce volatile organic compounds toxic to Meloidogyne incognita and 1-octanol, found in volatile emissions of watercress leaves, shows nematicidal activity against M. incognita.

BACKGROUND: Plants emit volatile organic compounds (VOCs) with several functions, including toxicity to plant-parasitic nematodes (PPNs). However, the toxicity of VOCs from watercress leaves (Nasturtium officinale) and passion fruit seeds (Passiflora edulis) against PPNs has not yet been studied. RESULTS: Biofumigation with watercress leaves and passion fruit seeds reduced the infectivity and reproduction of Meloidogyne incognita in tomato plants. The VOCs emitted by watercress leaves and passion fruit seeds caused immobility of M. incognita second-stage juveniles (J2 ). The reduction in infectivity and reproduction of M. incognita reached 89% and 99%, respectively, when J2 were exposed to watercress VOCs. Additionally, water exposed to VOCs emitted by watercress caused 79% M. incognita J2 mortality. The volatilome of the toxic water contained 12 compounds, mainly alcohols. The emissions from watercress leaves and passion fruit seeds contained 26 and 12 compounds, respectively, according to gas chromatography-mass spectrometry analysis. The 1-octanol occurring in watercress emissions demonstrated in vitro and in vivo nematicidal activity against M. incognita, with a lethal dose necessary to cause 50% mortality (LC50 ) of 382.5 µg mL−1 . CONCLUSIONS: Watercress leaf and passion fruit seed macerates emitted VOCs with nematicidal activity against M. incognita. The compound 1-octanol identified in watercress emissions may be useful for the nematicide-producing industry.

Impact of phenolic compounds on Meloidogyne incognita in vitro and in tomato plants.

Exposing second-stage juveniles (J2) of Meloidogyne incognita in vitro to a phenolic compound sometimes fails to cause J2 mortality, but in tests in vivo the same compound may reduce the infectivity and population of the nematode. This work aimed to study the effect of phenolic compounds on M. incognita through in vitro and in vivo assays. In the in vitro assay 49 phenolic compounds were screened for their toxicity to M. incognita J2. As a result, D-(-)-4-hydroxyphenylglycine, t-butylhydroquinone, L-3-(3,4-dihydroxyphenyl)alanine, sesamol, 2,4-dihydroxyacetophenone, and p-anisaldehyde increased the J2 mortality. These compounds presented, respectively, the following lethal concentrations to 50% of J2 (LC50): 365, 352, 251, 218, 210, and 85 µg/mL, while Carbofuran (positive control) had 150 µg/mL. However, none of these compounds were efficient in controlling the nematode in inoculated tomato plants, even when 2.77-fold of their LC50 were used. Although inactive in the in vitro test at 500 µg/mL, hydroquinone (3.5 mg per plant) reduced M. incognita population and galls by up to 99% to levels similar to the nematicide Carbofuran (1.2 mg per plant). Additionally, hydroquinone increased the root weight when compared to the negative and positive controls, water/NaOH and Carbofuran, respectively. In this study, we showed that some phenolic compounds, hydroquinone in particular, revealed a potential new option for the control of M. incognita.

Volatile organic compounds produced by castor bean cake incorporated into the soil exhibit toxic activity against Meloidogyne incognita.

BACKGROUND: This study showed, for the first time, the effect of volatile organic compounds (VOCs) emitted by castor bean cake added to soil on the plant-parasitic nematode Meloidogyne incognita. Identification of nematotoxic volatiles increases the chance of identifying molecules for use in commercial nematicides. RESULTS: VOCs produced after the incorporation of castor bean cake in the soil resulted in immobility (>97.3%) and death (>96.9%) of M. incognita second-stage juveniles (J2) and reduction in M. incognita egg-hatch (>74.3%) at all concentrations of cake tested (1.5-6.0%). A similar reduction in M. incognita infectivity and reproduction was observed when nematodes were exposed to these VOCs and inoculated into tomato plants or exposed directly upon contact with the soil. We identified 32 VOCs produced by castor bean cake when incorporated into soil by using solid phase micro-extraction gas chromatography coupled with mass spectrometry (SPME-GC-MS). Four of the most intense peaks in the chromatogram represented the compounds phenol, 4-methylphenol, γ-decalactone, and skatole. When M. incognita J2 were exposed to these compounds, all of the compounds demonstrated nematicidal activity with low median lethal concentration (LC50 ) values. CONCLUSION: Castor bean cake incorporated into the soil produces volatile compounds which are toxic to M. incognita. These substances show potential for application in the nematicide-producing industry. © 2018 Society of Chemical Industry.

Plant Volatiles Reduce the Viability of the Root-Knot Nematode Meloidogyne incognita Either Directly or When Retained in Water.

Volatile organic compounds (VOC) produced by green residues for the management of plant-parasitic nematodes are poorly studied for oilseed plants and some Brassica spp. To investigate the activity of VOC in vitro and as biofumigants, dry and aqueous macerates of broccoli (Brassica oleracea var. italica) shoots and sunflower (Helianthus annuus) seed were used against the root-knot nematode Meloidogyne incognita. VOC produced by sunflower seed caused higher mortality of M. incognita second-stage juveniles (J2) than VOC produced by broccoli shoots but both plant species were equally effective in decreasing the infectivity and reproduction of this nematode. The number of galls and eggs produced by the nematode in tomato roots was reduced by 89 and 95%, respectively, on average, at the highest concentrations of broccoli and sunflower seed macerates tested as biofumigants. When nematodes were placed in water exposed to broccoli VOC, J2 immobility increased and the number of galls and eggs produced by the nematode in tomato roots decreased 80 and 96%, respectively. Water exposed to sunflower seed VOC had no effect on the viability of the nematode. Gas chromatography was used to identify five and six chemical groups in broccoli and in sunflower seed macerates, respectively, but only alcohols, sulfurated VOC, and terpenes were detected in the water exposed to these plant macerates. Sulfurated VOC from the water exposed to broccoli macerates were found to be involved in its activity against M. incognita. The purified VOC dimethyl disulfide (DMDS) and 3-pentanol were tested directly against J2 and showed a lethal concentration of 176 and 918 µg/ml (ppm), respectively, whereas dimethyl sulfide had no effect against M. incognita. Furthermore, DMDS and 3-pentanol retained in water killed J2 and reduced gall formation and the number of eggs of M. incognita on tomato roots. Both these plant species produced toxic VOC to M. incognita, whereas only VOC retained in water exposed to broccoli had activity against M. incognita.

Purification and identification of metabolites produced by Bacillus cereus and B. subtilis active against Meloidogyne exigua, and their in silico interaction with a putative phosphoribosyltransferase from M. incognita.

To contribute to the development of products to control Meloidogyne exigua, the bacteria Bacillus cereus and B. subtilis were cultivated in liquid medium to produce metabolites active against this plant-parasitic nematode. Fractionation of the crude dichloromethane extracts obtained from the cultures afforded uracil, 9H-purine and dihydrouracil. All compounds were active against M. exigua, the latter being the most efficient. This substance presented a LC50 of 204 µg/mL against the nematode, while a LC50 of 260 µg/mL was observed for the commercial nematicide carbofuran. A search for protein-ligand complexes in which the ligands were structurally similar to dihydrouracil resulted in the selection of phosphoribosyltransferases, the sequences of which were used in an in silico search in the genome of M. incognita for a similar sequence of amino acids. The resulting sequence was modelled and dihydrouracil and 9H-purine were inserted in the active site of this putative phosphoribosyltransferase resulting in protein-ligand complexes that underwent molecular dynamics simulations. Calculation of the binding free-energies of these complexes revealed that the dissociation constant of dihydrouracil and 9H-purine to this protein is around 8.3 x 10-7 and 1.6 x 10-6 M, respectively. Consequently, these substances and the putative phosphoribosyltransferase are promising for the development of new products to control M. exigua.

Selection of phytotoxin producing rhizobacteria

In order to select phytotoxin producing rhizobacteria to control weed plants, twenty five bacterial strains previously isolated from the rhizospheres of various plants were grown in a liquid medium and, after cell removal by centrifugation, the liquid phases were freeze-dried and the products were extracted with ethyl acetate/methanol. The extracts were concentrated to dryness under vacuum and dissolved in water and sucrose solution to be submitted to in vitro assays of lettuce (Lactuca sativa L.) seed germination and wheat (Triticum aestivum L.) coleoptile growth. Although most samples affected coleoptile growth, only those from four strains reduced lettuce seed germination. Two strains of Bacillus cereus, one strain of B. pumilus and one of Stenotrophoonas altophilia were the most promising microorganisms for producing phytotoxin and, consequently, for the development of new weed control products.

Com o objetivo de selecionar rizobactérias produtoras de fitotoxinas para uso no controle de plantas daninhas, vinte e cinco isolados bacterianos previamente obtidos das rizosferas de diferentes plantas foram cultivados em meio líquido e, após remoção das células por centrifugação, as fases líquidas foram liofilizadas e os resíduos obtidos foram submetidos à extração com acetato de etila/metanol. Os extratos foram concentrados sob vácuo até secura e dissolvidos em água e solução de sacarose para serem submetidos a testes in vitro de germinação de sementes de alface (Lactuca sativa L.) e de crescimento de coleóptilos de trigo (Triticum aestivum L.). Embora a maior parte das amostras tenha desfavorecido o crescimento dos coleóptilos de trigo, somente as provenientes de quatro isolados reduziram a germinação das sementes de alface. Dois isolados de Bacillus cereus, um isolado de B. pumilus e um de Stenotrophomonas maltophilia foram os microrganismos mais promissores para a produção de fitotoxinas, com possibilidade de uso no desenvolvimento de novos produtos para o controle de plantas daninhas.

Selection of phytotoxin producing rhizobacteria.

In order to select phytotoxin producing rhizobacteria to control weed plants, twenty five bacterial strains previously isolated from the rhizospheres of various plants were grown in a liquid medium and, after cell removal by centrifugation, the liquid phases were freeze-dried and the products were extracted with ethyl acetate/methanol. The extracts were concentrated to dryness under vacuum and dissolved in water and sucrose solution to be submitted to in vitro assays of lettuce (Lactuca sativa L.) seed germination and wheat (Triticum aestivum L.) coleoptile growth. Although most samples affected coleoptile growth, only those from four strains reduced lettuce seed germination. Two strains of Bacillus cereus, one strain of B. pumilus and one of Stenotrophoonas altophilia were the most promising microorganisms for producing phytotoxin and, consequently, for the development of new weed control products.

Rhizobacteria able to produce phytotoxic metabolites

To contribute for the development of environmental friendly methods for weed control, a selection of rhizobacteria able to produce phytotoxic substances was carried out. Initially, 35 strains previously isolated from plants in the south of Minas Gerais State (Brazil) were grown in tryptic soy broth. After removal of bacterial cells, the resulting liquids were freeze-dried and extracted with methanol/ethyl acetate (1:1). The extracts were concentrated under vacuum and dissolved in water to be submitted to a lettuce (Lactuca sativa L.) seed assay. Metabolites produced by five strains reduced the number of normal seedlings to values statistically bellow the one observed for the negative control, being the most expressive results obtained with Bacillus cereus Frankland and Frankland, isolated from Ricinus communis L., which was able to cause rotted rootlets to 82.4 percent of seedlings. The bacterium metabolites also avoided germination of 52 percent Brachiaria decumbens Stapf seeds and the remaining 48 percent resulted in abnormal seedlings. Metabolites from B. cereus were submitted to a purification process guided by the lettuce seed assay. As a consequence, one substance causing rotted rootlets to all lettuce seedlings during the seed assay at 0.057 g/L was isolated and will be identified in future studies.

Com vistas a contribuir para o desenvolvimento de métodos não agressivos ao meio ambiente, para o controle de plantas invasoras, buscou-se selecionar rizobactérias produtoras de substâncias fitotóxicas. Inicialmente, 35 culturas previamente isoladas de plantas da região sul do Estado de Minas Gerais (Brasil) foram cultivadas em caldo soja tripticaseína. Após remoção das células bacterianas, os líquidos resultantes foram liofilizados e extraídos com metanol/acetato de etila (1:1). Os extratos foram concentrados sob vácuo e submetidos a testes com sementes de alface (Lactuca sativa L.). Os metabólitos produzidos por cinco isolados bacterianos reduziram o número de plântulas normais para valores estatisticamente inferiores aos observados para o controle negativo, sendo os mais expressivos resultados obtidos com Bacillus cereus Frankland and Frankland, isolado de Ricinus communis L., que causou necroses nas radículas de 82,4 por cento das plântulas. Os metabólitos dessa bactéria também impediram a germinação de 52 por cento das sementes de Brachiaria decumbens Stapf e fizeram com que as 48 por cento restantes dessem origem a plântulas anormais. Os metabólitos de B. cereus foram submetidos a processos de purificação direcionados por testes com sementes de alface. Em decorrência, isolou-se uma substância, que será identificada em estudos futuros. Na concentração de 0,057 g/L, causou necrose nas radículas de todas as plântulas de alface provenientes do teste com sementes.