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Soybean is the main oilseed cultivated worldwide. Even though Brazil is the world's largest producer and exporter of soybean, its production is severely limited by biotic factors. Soil borne diseases are the most damaging biotic stressors since they significantly reduce yield and are challenging to manage. In this context, the present study aimed to evaluate the potential of a bacterial strain (Ag109) as a biocontrol agent for different soil pathogens (nematodes and fungi) of soybean. In addition, the genome of Ag109 was wholly sequenced and genes related to secondary metabolite production and plant growth promotion were mined. Ag109 showed nematode control in soybean and controlled 69 and 45% of the populations of Meloidogyne javanica and Pratylenchus brachyurus, respectively. Regarding antifungal activity, these strains showed activity against Macrophomia phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. For S. sclerotiorum, this strain increased the number of healthy plants and root dry mass compared to the control (with inoculation). Based on the average nucleotide identity and digital DNA-DNA hybridization, this strain was identified as Bacillus velezensis. Diverse clusters of specific genes related to secondary metabolite biosynthesis and root growth promotion were identified, highlighting the potential of this strain to be used as a multifunctional microbial inoculant that acts as a biological control agent while promoting plant growth in soybean.

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Infective second-stage juveniles (J2) of Meloidogyne spp. migrate towards host roots, which depends on several factors, including root exudates and soil temperature. Although Meloidogyne enterolobii is a highly virulent nematode that affects major agricultural crops worldwide, there is limited ecological data about it. The objective of this study was to determine the J2 migration pattern vertically in 14-cm long segmented soil columns towards tomato (Solanum lycopersicum) and marigold (Tagetes patula) roots, each grown at two soil temperatures (20 or 26ºC). Bottomless cups with tomatoes or marigolds were attached to the top of each column; cups with no plants were used as untreated controls. Juveniles (1,000/column) were injected into a hole located 1 cm from the bottom of each column. The apparatuses were placed in growth chambers at 20 or 26ºC, and J2 were allowed to migrate for 3, 6, 9, or 12 days after injection (DAI). At each harvest, J2 were extracted from each ring of the columns and counted to compare their distribution, and root systems were stained to observe root penetration. M. enterolobii migrated over 13 cm vertically 3 DAI regardless of temperature, even without plant stimuli. The vertical migration was greater at 26ºC, where 60% of active J2 were found at distances >13 cm at 12 DAI. Temperature did not affect root penetration. Overall, a greater number of J2 was observed in tomato roots, and root penetration increased over time.

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Commercial fig tree cultivation in Brazil involves a single cultivar, 'Roxo-de-Valinhos'. The use of a single cultivar results in serious diseases and related problems. The aim of this study was to characterize fig accessions by analyzing the natural root-knot nematode and leaf rust incidence in relation to the epigenomic profile of the plant, since epigenetic variations affect plant-pathogen interactions. All plants were attacked by nematodes, indicating susceptibility; Meloidogyne incognita was the root-knot nematode species involved. Joint analysis of data showed that methylation and leaf rust incidence were correlated when observed in the same phenological phase, presenting initial evidence of the same factorial pressure loads in genotypes, suggesting similar behavior within these genotypes.

O Brasil é o maior produtor de figos da América do Sul, porém o cultivo comercial brasileiro da figueira baseia-se na plantação de uma única cultivar, o Roxo-de-Valinhos, resultando em sérios problemas relativos a pragas e doenças. Uma vez que há variações epigenéticas na interação planta-patógeno, principalmente por meio da regulação gênica, o presente trabalho objetiva realizar a caracterização in vivo de acessos de figo, por meio da análise de incidência natural de nematoides formadores de galha e de incidência natural de ferrugem, correlacionada ao seu perfil epigenômico, a fim de subsidiar trabalhos de conservação, melhoramento genético e produção da cultura. A análise dos componentes principais dos dados da caracterização dos acessos foi realizada por meio da matriz de correlação residual obtida pela análise de variância conjunta utilizando o programa GENES. Pôde-se constatar que todas as plantas foram atacadas por este patógeno, evidenciando que são suscetíveis ao mesmo. Já a análise conjunta dos dados demonstrou que a metilação e a incidência de ferrugem em folhas, quando observadas na mesma fase fenológica da planta, se correlacionam, apresentando evidências iniciais de mesmas cargas fatoriais de pressão nos genótipos, com a premissa de comportamento semelhante nos mesmos, indicando que, além do fator genético, fatores abióticos também são responsáveis pelas alterações no fenótipo das plantas, evidenciando a plasticidade fenotípica das mesmas.

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Abstract Root-knot nematode Meloidogyne incognita is among the biotic factors which has greatly affected both the yield and the quality of the tomato crop. The egg parasitic nematode, Purpureocillium lilacinum (Pl) is considered as one of the most promising agents in controlling and overcoming this plant pathogen. The nematicidal effect of the native isolate Pl AUMC 10149 on second stage juvenile's survival and egg hatching of M. incognita at different times of exposure was tested in vitro. The obtained data showed that Pl gave a maximum percentage of J2 mortality (97.6%) and egg hatching inhibition (79.8%) after 72 hours of exposure. The potentiality of Pl as well as Bio-Nematon to control M. incognita infecting tomato was conducted using different times of application in vivo. Nine treatments with five replicates were used for such bioagents compared with the nematicide Oxamyl. Each seedling was inoculated with 1000 J2s of nematode/pot and 10 mL of Pl (1x1010 CFU/mL) or Bio-Nematon spore suspension (1x108 CFU/mL) 10mL/pot. The results indicated that the most effective treatments in reducing nematode population, number of galls and egg masses of M. incognita in plant roots was performed with treatment by Pl pre-planting and post-infection with Pl (Rf 1.9) giving a significant enhancement in plant length (64.9%), fresh weight (72.52%) and shoot dry weight (163.41%) without negatively impacting environment. Therefore, the present study confirmed that using P. lilacinum AUMC 10149 can be used as a practical supplement to environmentally friendly disease management of root-knot nematodes in Egypt.

Resumo O nematoide-das-galhas Meloidogyne incognita está entre os fatores bióticos que afetaram enormemente a produção e a qualidade da cultura do tomate. O nematoide parasita de ovos, Purpureocillium lilacinum (Pl), é considerado um dos mais promissores agentes no controle e superação desse fitopatógeno. O efeito nematicida do isolado nativo Pl AUMC 10149 na sobrevivência de juvenis de segundo estágio e na eclosão dos ovos de M. incognita em diferentes momentos de exposição foi testado in vitro. Os dados obtidos mostraram que o Pl deu um percentual máximo de mortalidade de J2 (97.6%) e inibição da eclosão dos ovos (79.8%) após 72 horas de exposição. A potencialidade de Pl e de Bio-Nematon para controlar M. incognita infectando tomate foi conduzida em diferentes tempos de aplicação in vivo. Nove tratamentos com cinco repetições foram usados ​​para tais bioagentes em comparação com o nematicida Oxamyl. Cada muda foi inoculada com 1.000 J2s de nematoide / vaso e 10 mL de Pl (1×1010 CFU/mL). Ou suspensão de esporos Bio-Nematon (1×108 CFU/mL) 10mL/pot. Os resultados indicaram que os tratamentos mais eficazes na redução da população de nematoides, número de galhas e desovas de M. incognita nas raízes das plantas foram realizados com Pl pré-plantio e pós-infecção com Pl (Rf 1.9), dando um aumento significativo no comprimento da planta (64.9%), massa fresca (72.52%) e massa seca da parte aérea (163.41%) sem impactar negativamente o meio ambiente. Portanto, o presente estudo confirmou que o uso de P. lilacinum AUMC 10149 pode ser usado como um suplemento prático para o manejo ecologicamente correto de nematoides-das-galhas no Egito.

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Abstract Root-knot nematode Meloidogyne incognita is among the biotic factors which has greatly affected both the yield and the quality of the tomato crop. The egg parasitic nematode, Purpureocillium lilacinum (Pl) is considered as one of the most promising agents in controlling and overcoming this plant pathogen. The nematicidal effect of the native isolate Pl AUMC 10149 on second stage juveniles survival and egg hatching of M. incognita at different times of exposure was tested in vitro. The obtained data showed that Pl gave a maximum percentage of J2 mortality (97.6%) and egg hatching inhibition (79.8%) after 72 hours of exposure. The potentiality of Pl as well as Bio-Nematon to control M. incognita infecting tomato was conducted using different times of application in vivo. Nine treatments with five replicates were used for such bioagents compared with the nematicide Oxamyl. Each seedling was inoculated with 1000 J2s of nematode/pot and 10 mL of Pl (1x1010 CFU/mL) or Bio-Nematon spore suspension (1x108 CFU/mL) 10mL/pot. The results indicated that the most effective treatments in reducing nematode population, number of galls and egg masses of M. incognita in plant roots was performed with treatment by Pl pre-planting and post-infection with Pl (Rf 1.9) giving a significant enhancement in plant length (64.9%), fresh weight (72.52%) and shoot dry weight (163.41%) without negatively impacting environment. Therefore, the present study confirmed that using P. lilacinum AUMC 10149 can be used as a practical supplement to environmentally friendly disease management of root-knot nematodes in Egypt.

Resumo O nematoide-das-galhas Meloidogyne incognita está entre os fatores bióticos que afetaram enormemente a produção e a qualidade da cultura do tomate. O nematoide parasita de ovos, Purpureocillium lilacinum (Pl), é considerado um dos mais promissores agentes no controle e superação desse fitopatógeno. O efeito nematicida do isolado nativo Pl AUMC 10149 na sobrevivência de juvenis de segundo estágio e na eclosão dos ovos de M. incognita em diferentes momentos de exposição foi testado in vitro. Os dados obtidos mostraram que o Pl deu um percentual máximo de mortalidade de J2 (97.6%) e inibição da eclosão dos ovos (79.8%) após 72 horas de exposição. A potencialidade de Pl e de Bio-Nematon para controlar M. incognita infectando tomate foi conduzida em diferentes tempos de aplicação in vivo. Nove tratamentos com cinco repetições foram usados para tais bioagentes em comparação com o nematicida Oxamyl. Cada muda foi inoculada com 1.000 J2s de nematoide / vaso e 10 mL de Pl (1×1010 CFU/mL). Ou suspensão de esporos Bio-Nematon (1×108 CFU/mL) 10mL/pot. Os resultados indicaram que os tratamentos mais eficazes na redução da população de nematoides, número de galhas e desovas de M. incognita nas raízes das plantas foram realizados com Pl pré-plantio e pós-infecção com Pl (Rf 1.9), dando um aumento significativo no comprimento da planta (64.9%), massa fresca (72.52%) e massa seca da parte aérea (163.41%) sem impactar negativamente o meio ambiente. Portanto, o presente estudo confirmou que o uso de P. lilacinum AUMC 10149 pode ser usado como um suplemento prático para o manejo ecologicamente correto de nematoides-das-galhas no Egito.

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This research was designed to investigate the metabolite profiling, phenolics, and flavonoids content as well as the potential nematicidal properties of decoction (ZpDe), orange-yellow resin (ZpRe) and essential oil (ZpEO) from Argentinean medicinal plant Zuccagnia punctata Cav. Additionally, the antioxidant and antibacterial properties of ZpDe and ZpEO were determined. Metabolite profiling was obtained by an ultrahigh-resolution liquid chromatography MS analysis (UHPLC-ESI-QTOF/OT-MS-MS) and GCMS. The nematicidal activity was assayed by a standardized method against Meloidogyne incognita. The antioxidant properties were screened by four methods: (2,2-diphenyl-1-picrylhydrazyl assay (DPPH), Trolox equivalent antioxidant activity assay (TEAC), ferric-reducing antioxidant power assay (FRAP), and lipid peroxidation in erythrocytes (ILP). The antibacterial activity was evaluated according to the Clinical and Laboratory Standards Institute (CLSI) rules. The ZpDe, ZpRe and ZpEO displayed a strong nematicidal activity with an LC50 of 0.208, 0.017 and 0.142 mg/mL, respectively. On the other hand, the ZpDe showed a strong DPPH scavenging activity (IC50 = 28.54 µg/mL); ILP of 87.75% at 250 µg ZpDe/mL and moderated antimicrobial activity. The ZpEO showed promising activity against a panel of yeasts Candida albicans and non-albicans (ATCC and clinically isolated) with MIC values from 750 to 1500 µg/mL. The ZpDe showed a content of phenolics and flavonoid compounds of 241 mg GAE/g and 10 mg EQ/g, respectively. Fifty phenolic compounds were identified in ZpDe by ultrahigh-resolution liquid chromatography (UHPLC-PDA- Q-TOF-MS) analysis, while forty-six phenolic compounds were identified in ZpRe by UHPLC-ESI-Q-OT-MS-MS and twenty-nine in ZpEO using a GC-MS analysis, updating the knowledge on the chemical profile of this species. The results support and standardize this medicinal plant mainly as a potential environmentally friendly and sustainable bionematicide for the control of Argentinean horticultural crops including tomatoes and peppers and as a source of antimicrobial and antioxidant compounds which could be further explored and exploited for potential applications.

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Amazon chicory (Eryngium foetidum L. [Apiaceae]), also known as culantro, is native to Tropical America and the West Indies. It belongs to the unconventional food plants (UFPs) group, and in addition to be consumed as a spice herb, it possesses a wide range of ethnomedicinal uses (Paul et al. 2011). In 2019, in the eastern Amazon region of Brazil, state of Pará, producers of E. foetidum in the municipality of Castanhal (01°15'363" S 047°10'232" W) reported the occurrence of underdeveloped plants with leaf yellowing and a large number of galls in the root system, which are typical symptoms of root-knotting nematode. Soil and root samples were collected and sent to the Nematology Laboratory (LabNema) located at the Faculty of Agrarian and Veterinary Sciences, UNESP, Jaboticabal, São Paulo, Brazil. A total of 46 second-stage juveniles (J2s) were extracted per 100 cm3 of soil, and a total of 460 eggs and J2s Meloidogyne spp. were found per gram of root. Morphological and molecular techniques were used to identify the species. The analysis of the perineal patter of ten females revealed thin striations in an oval shape with a high and semi-trapezoidal dorsal arch. No striations were observed in the perivulvar region. The labial region of the ten males analyzed exhibited a non-prominent labial disc, fused and slightly recessed submedian lips, with no apparent annulations. The morphological characteristics observed in the adults were consistent with those originally described for Meloidogyne enterolobii (Yang; Eisenback, 1983), confirming the species purity of the recovered population. Three individual nematodes had their 18S rDNA region sequenced (Holterman et al. 2006) which showed an average identity of 99.7% with other sequences of M. enterolobii available in the GenBank database. A Bayesian phylogenetic tree was constructed, providing insights into the specific relationship of M. enterolobii recovered from E. foetidum with other related nematodes. Each of the three sequenced nematodes represented a unique haplotype, resulting in their separation into distinct clades. Moreover, the obtained sequences presented polymorphisms that differed from the M. enterolobii sequences already available in the database, highlighting the genetic diversity of this species in relation to its original host (Silva et al. 2021). The species M. enterolobii was also confirmed using species-specific primers for M. incognita, M. javanica, and M. enterolobii (Zijlstra et al. 2000; Tigano et al. 2010). To confirm the pathogenicity of M. enterolobii on E. foetidum, a modified Koch Postulate was conducted. Six seedlings of E. foetidum were transplanted individually to 10-liter pots containing autoclaved soil. Each pot was then inoculated with 5 mL of a suspension containing 3,000 eggs and J2s from the original population of M. enterolobii obtained from E. foetidum. After 90 days, the inoculated plants exhibited root galls with a plentiful egg mass, in contrast to the healthy non-inoculated plants. The average number of M. enterolobii nematodes recovered from the roots of the inoculated plants was 42,040 eggs and J2s, resulting in a reproduction factor (RF) of 14.0. The importance of reporting the occurrence of M. enterolobii in E. foetidum is due to the fact that this plant species is cultivated in a crop rotation system with other vegetables such as lettuce and coriander, which are also hosts of M. enterolobii. Consequently, different crop rotation strategies and control alternatives need to be considered in areas where E. foetidum is grown. This is the first report of E. foetidum serving as a host for the root-knot nematode M. enterolobii worldwide.

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MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

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Stachys byzantina belongs to the Labiatae and is known by the names "peixinho-da-horta" (Brazil) and "lamb's ear" (USA). Its importance is associated with its medicinal properties (Bahadori et al. 2020) and nutritional aspects (Milião et al. 2022). Root-knot nematodes cause severe damage to plants and suppress production. In January 2021, plants of S. byzantina in the municipality of Jaboticabal (21°14'38.7"S, 48°17'10.6"W) showed symptoms of reduced growth, yellowed leaves and the presence of galls in the roots. Initially, samples of roots from a S. byzantina were analyzed at the Nematology Laboratory (LabNema/UNESP), Jaboticabal, Brazil, estimating 20,000 eggs and juveniles of Meloidogyne sp. in 10 g of roots. To confirm the host ability of the species, a pathogenicity test was performed using Koch's postulate. For this purpose, the test was conducted in a greenhouse where 3,000 eggs and second-stage juveniles (J2) were inoculated onto three plants (n=3) of S. byzantina. After 90 days, the inoculated plants showed the same symptoms as those observed in the field. No symptom or nematode was detected in the uninoculated plant (control). Nematodes were extracted from the roots of inoculated plants and quantified. The perineal pattern of females (n=10) (Netscher and Taylor, 1974) and the labial region of males (n=10) (Eisenback and Hirschmann, 1981) were analyzed and compared with the morphological characteristics of the original description of the species (Chitwood, 1949). For analysis based on esterase isozyme phenotype, the α-method of Esbenshade and Triantaphyllou (1990) was used, and females (n=7) were examined. To confirm identification, whole genomic DNA from an adult female (n=1) was extracted using the Qiagen DNeasy® Blood & Tissue Kit and this sample was used for both genetic sequencing and the sequence-characterized amplified region techniques (SCAR). PCR amplifications were performed for the 18s rRNA gene using primers 988F and 1912R from Holterman et al (2006). Our sequence was deposited in GenBank (NCBI) under the identifier OP422209. Finally, species-specific SCAR primers (Fjav/Rjav, Me-F/Me-R, and Finc-F/Finc-R) designed by Zijlstra (2000) were used to identify Meloidogyne spp. Koch's postulate analysis yielded the following results: (n=1) 9,280 eggs and J2 (Reproduction factor, RF = 33.09); (n=2) 111,720 eggs and J2 (RF = 37.24); (n=3) 59,700 eggs and J2 (RF = 19.9) (RF mean = 30.08). The following characteristics were observed in the perineal region of females: Low and rounded trapezoidal dorsal arch with two distinct lateral lines clearly separating the dorsal and ventral arch regions, similar to the morphological features of the species description by Chitwood (1949). Males had a convex labial plate with a non-raised labial disk joining the submedial labia, a non-rugged labial region, the basal tubercles were usually wider than high, and a rounded tail tip (Eisenback and Hirschmann 1981). The α-esterase enzyme profile showed the J3 phenotype typical of M. javanica (Rm [×100] = 46.0, 54.5, and 58.9). The 18s rRNA sequences grouped Meloidogyne sp. with species such as M. enterolobii, M. incognita, and M. javanica. A DNA fragment of about 700 bp was amplified with Mj (Fjav/Rjav) primers, but not with Me (Me-F/Me-R) and Mi (Finc-F/Finc-R) primers, which confirmed the identification of M. javanica. Accurate identification and characterization of the occurrence of new hosts of M. javanica will allow us to determine the range and geographic distribution of the species. This is the first report on the occurrence of M. javanica on S. byzantina in Brazil. This report is important so that management strategies can be applied to prevent the spread of the pest to other areas.

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The effect of Radopholus similis, Pratylenchus araucensis, Meloidogyne spp., and their interaction was evaluated in seedlings of Musa AAB 'Dominico Hartón'. The study was conducted in a nursery in Palestina, Caldas department, Colombia. Forty-day-old plantain seedlings were infected separately with 750, 1,500, 2,250 and 3,000 of each species of nematodes/plant. Two experiments were conducted to evaluate the damage of R. similis, P. araucensis, Meloidogyne spp. and the mixture of 750 R. similis + 750 P. araucensis + 750 Meloidogyne spp. compared with the mixture of different proportions (1,500, 2,250 and 3,000 of each species of nematodes). Noninfected plants were included as a control treatment, for a total of 17 treatments in a randomized complete block design with ten replications. Twelve weeks after inoculation, all nematodes, both alone and in combination, reduced (p < 0.05) plantain dry root and shoot weight. In two experiments, R. similis, P. araucensis, and Meloidogyne spp. alone, each with a population density of 3,000, reduced (p < 0.05) root dry weight by 32.5%, 9.5% and 49%, respectively, and decreased (p < 0.05) shoot dry weight by 21.5%, 23%, and 31.5%, respectively, compared to the control. The interaction of nematodes with the lowest population decreased root (33%) and shoot (21%) weight. We conclude that the growth of 'Dominico Hartón' seedlings was affected by plant-parasitic nematodes, but the greatest damage occurred with concomitant nematode infection.

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A new root-knot nematode (RKN) species, Meloidogyne karsseni n. sp., associated with sweet pepper from Mexico, and a population of M. paranaensis from Guatemala, are described using data from morphological, biochemical (isozyme enzymes), molecular, and phylogenetic analyses. Meloidogyne karsseni n. sp. can be morphologically diagnosed using the combined features of the second-stage juveniles, viz. body length (345 to 422 µm), a conical rounded head region, a post-labial annule lacking transverse striation, a thin stylet 11 to 12 µm long, rounded to oval and backwardly sloping knobs, dorsal gland orifice (DGO) at 5.2 to 6.0 µm from the knobs, a hemizonid just above the secretory-excretory (SE) pore, a tapering tail with finely rounded terminus and one or two very weak constrictions at hyaline tail tip; the female characters viz. oval-to-rounded perineal pattern with coarse striation on lateral sides around the anus, low dorsal arch with finer striations, and distinctly visible lateral lines; and the male characteristics viz. a rounded and continuous head, a post-labial annule without transverse striations, a robust stylet 20 to 24 µm long, rounded-to-oval and slightly backwardly sloping knobs, and a DGO at 2.4 to 2.9 µm from the knobs. In all the studied males of M. paranaensis, a characteristic sclerotization around the duct of SE-pore was also observed for the first time. Sequences of 18S, D2-D3 of 28S, and ITS of rDNA, and cox1 of mtDNA were generated for the two species, and in the phylogenetic trees based on these genes, both species appeared in the tropical RKN species complex clade.

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Infection by the root-knot nematode (RKN), Meloidogyne incognita, impacts crop productivity worldwide, including parsley cultures (Petroselinum crispum). Meloidogyne infection involves a complex relationship between the pathogen and the host plant tissues, leading to the formation of galls and feeding sites that disorganize the vascular system, affecting the development of cultures. Herein, we sought to evaluate the impact of RKN on the agronomic traits, histology, and cell wall components of parsley, with emphasis on giant cell formation. The study consisted of two treatments: (i) control, where 50 individuals of parsley grew without M. incognita inoculation; and (ii) inoculated plants, where 50 individuals were exposed to juveniles (J2) of M. incognita. Meloidogyne incognita infection affected the development of parsley, reducing the growth of some agronomical characteristics such as root weight and shoot weight and height. Giant cell formation was noticed at 18 days after inoculation, promoting disorganization of the vascular system. Epitopes of HGs detected in giant cells reveal the continuous capacity of giant cells to elongate under the stimulus of RKN, essential processes for feeding site establishment. In addition, the detection of epitopes of HGs with low and high methyl-esterified groups indicates the PMEs activity despite biotic stress.

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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.

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The Mi-gene is widely used in different tomato cultivars to resist several Meloidogyne spp. (root-kot nematode; RKN), including M. incognita, M. javanica, and M. arenaria. Tomato cultivars with the Mi-gene are widely used in fields. However, factors such as temperatures, high initial population densities, and gene dosage can interfere with the expression of this gene. In addition, the presence of virulent species of RKN can limit the usefulness of the gene. One of the virulent species is M. haplanaria, which was identified infecting RKN-resistant tomato in Florida in 2015. The objectives of this study were to determine the initial damage threshold of M. haplanaria on tomato under greenhouse conditions and to analyze the impact of temperature and genetic background on virulence in tomato cultivars. The results showed a preliminary damage threshold of three eggs and J2/cm3 of soil. In addition, it was observed that M. haplanaria has a shorter life cycle than the virulent M. enterolobii and can infect, reproduce, and damage homozygous or heterozygous RKN-resistant tomato plants. This research demonstrated that M. haplanaria should be considered highly virulent on RKN-resistant tomato and is an important threat to agriculture in Florida.

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As a result of more restrictive legislation regarding the use of pesticides, over the last two decades, the demand for new soil fumigants has increased. These compounds can come from a variety of sources, including organic residues. In this study, we identified in the cassava wastewater volatiloma a molecule with potential to be developed as a new fumigant nematicide. Cassava wastewater (20 mL) releases volatile compounds toxic to Meloidogyne javanica second-stage juveniles (J2), causing J2 mortality up to 89%. Through gas chromatography-mass spectrometry, eight compounds were identified in the cassava wastewater volatiloma, with alcohols being the major class. The compounds ethyl butanoate and butyric acid identified in cassava wastewater volatiloma were selected for in vitro nematicidal activities and substrate fumigation tests. The lethal concentration predicted LC50-48 hr values (effective doses to kill 50% of M. javanica J2 population after 48 h of exposure) were 172.6 µg ∙ mL - 1 and 301.2 µg ∙ mL - 1 for butyric acid and ethyl butanoate, respectively. In a pot assay, the application of butyric acid and ethyl butanoate as a soil fumigant, at a dose of 0.5 mL ∙ kg - 1 substrate, significantly (P < 0.05) decreased M. javanica infectivity and reproduction compared to the negative control (water). However, ethyl butanoate proved to be a more efficient soil fumigant (P < 0.05) than butyric acid, as its application reduced the number of galls and eggs to the level of the commercial fumigant Dazomet. This study is the first to demonstrate the potential of ethyl butanoate as a soil fumigant against M. javanica.

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MAIN CONCLUSION: The pUceS8.3 is a constitutive gene promoter with potential for ectopic and strong genes overexpression or active biomolecules in plant tissues attacked by pests, including nematode-induced giant cells or galls. Soybean (Glycine max) is one of the most important agricultural commodities worldwide and a major protein and oil source. Herein, we identified the soybean ubiquitin-conjugating (E2) enzyme gene (GmUBC4; Glyma.18G216000), which is significantly upregulated in response to Anticarsia gemmatalis attack and Meloidogyne incognita-induced galls during plant parasitism by plant nematode. The GmUBC4 promoter sequence and its different modules were functionally characterized in silico and in planta using transgenic Arabidopsis thaliana and G. max lines. Its full-length transcriptional regulatory region (promoter and 5´-UTR sequences, named pUceS8.3 promoter) was able to drive higher levels of uidA (ß-glucuronidase) gene expression in different tissues of transgenic A. thaliana lines compared to its three shortened modules and the p35SdAMV promoter. Notably, higher ß-glucuronidase (GUS) enzymatic activity was shown in M. incognita-induced giant cells when the full pUceS8.3 promoter drove the expression of this reporter gene. Furthermore, nematode-specific dsRNA molecules were successfully overexpressed under the control of the pUceS8.3 promoter in transgenic soybean lines. The RNAi gene construct used here was designed to post-transcriptionally downregulate the previously characterized pre-mRNA splicing factor genes from Heterodera glycines and M. incognita. A total of six transgenic soybean lines containing RNAi gene construct were selected for molecular characterization after infection with M. incognita pre-parasitic second-stage (ppJ2) nematodes. A strong reduction in the egg number produced by M. incognita after parasitism was observed in those transgenic soybean lines, ranging from 71 to 92% compared to wild-type control plants. The present data demonstrated that pUceS8.3 is a gene promoter capable of effectively driving dsRNA overexpression in nematode-induced giant cells of transgenic soybean lines and can be successfully applied as an important biotechnological asset to generate transgenic crops with improved resistance to root-knot nematodes as well as other pests.

Assuntos

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Plant-parasitic nematodes are an important group of pests causing economic losses in agriculture worldwide. Among the plant-parasitic nematodes, the root-knot (Meloidogyne spp.) and root-lesion nematodes (Pratylenchus spp.) are considered the two most important ones affecting soybeans. In general, they damage soybean roots, causing a reduction of about one-third in productivity. The soil microbial community can exert a suppressive effect on the parasitism of plant-parasitic nematodes. Here, we investigated the effects of soil bacterial diversity on Meloidogyne javanica (Meloidogyne-assay) and Pratylenchus brachyurus (Pratylenchus-assay) suppression by manipulating microbial diversity using the dilution-to-extinction approach in two independent experiments under controlled conditions. Furthermore, we recorded the changes in the soil microbial community induced by plant-parasitic nematode infection. In Meloidogyne-assay, microbial diversity reduced the population density of M. javanica and improved plant performance. In Pratylenchus-assay, microbial diversity sustained the performance of soybean plants even at high levels of P. brachyurus parasitism. Each nematode population affected the relative abundance of different bacterial genera and altered the core microbiome of key groups within the bacterial community. Our findings provide fundamental insights into the interactions between soil bacterial diversity and plant-parasitic nematodes in soybean plants. IMPORTANCE Root-knot and root-lesion nematodes cause losses of billions of dollars every year to agriculture worldwide. Traditionally, they are controlled by using chemical nematicides, which in general have a negative impact on the environment and human health. Fortunately, the soil microbial community may suppress these pests, acting as an environmentally friendly alternative to control nematodes. However, the effects of soil microbial diversity on the parasitism of plant-parasitic nematodes still poorly understood. In this study, we provide fundamental insight into the interactions between soil bacterial diversity and plant-parasitic nematodes in soybean plants, which may be useful for the development of new strategies to control these phytopathogens.

Assuntos

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Active metabolites from plants are considered safer than synthetic chemicals for the control of plant-parasitic nematodes of the genus Meloidogyne. In the present work, 75 aqueous extracts (AEs) from different vegetative parts of 34 native plant species of the Yucatan Peninsula were evaluated against second-stage juveniles (J2s) of Meloidogyne incognita and M. javanica in microdilution assays. The highest mortality (M) against both Meloidogyne species was produced by the foliar AE from Alseis yucatanensis (M ≥ 94%) and Helicteres baruensis (M ≥ 77%) at 3% w/v after 72 h. Other active AEs at 3% were from the leaves of Croton itzaeus and stems of H. baruensis (M: 87-90%) on M. javanica and the stems of Annona primigenia and the leaves of Morella cerifera on M. incognita (M: 92-97%). The AEs from A. yucatanensis had the lowest LD50 against M. incognita (0.36% w/v), and against M. javanica (3.80% w/v). In an acute ecotoxicity assay of the most promising AEs using non-target earthworms (Eisenia fetida), the AE of A. yucatanensis had slight acute toxicity (LD50: 2.80% w/v), and the rest of the most active AEs were not ecotoxic. These tropical plants are potential candidates for further studies as biorational agents for controlling Meloidogyne species.

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For decades there have been anecdotal claims of synergistic interactions between plant-parasitic nematodes and soil-borne fungi causing decline of productivity of passion fruit (Passiflora edulis) orchards. An empirical confirmation of these disease complexes would impact disease management and plant breeding for resistance. To test those claims, we subjected passion fruit plants to single or concomitant parasitism by Meloidogyne javanica or M. incognita and Fusarium nirenbergiae or Neocosmospora sp. under controlled conditions. Non-inoculated plants served as control for the assays. The severity of shoot symptoms and variables related to plant growth, the extent of fungal lesions, and nematode reproduction were assessed to characterize the interactions. The shoot symptoms and effect on plant growth induced by the pathogens varied, but no synergy between the pathogens was observed. Moreover, the volume of tissue lesioned by the fungi was not affected by co-parasitism of the nematodes. Conversely, plant resistance to the nematodes was not affected by co-parasitism of the fungi. The interactions M. incognita-F. nirenbergiae, M. incognita-Neocosmospora sp., M. javanica-F. nirenbergiae, and M. javanica-Neocosmospora sp. were not synergistic as previously claimed, but instead neutral.

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Buckwheat (Fagopyrum esculentum Moench) belongs to the Polygonaceae family and has been widely cultivated due to its high nutritional, nutraceutical, and medicinal properties. Brazil ranks seventh-largest producer, with 66,000 tons produced in 2018. Buckwheat is also valued for its adaptability as a cover crop, in grain fields of soybean (Glycine max (L.) Merr., maize (Zea mays L.), and sorghum (Sorghum bicolor (L.) Moench) (Görgen et al. 2016, Babu et al. 2018) especially in fields highly infested with plant-parasitic nematodes (PPN). PPN cause severe root damage, suppressing plant development and yield production. In October 2018, six samples of roots and soil were collected in symptomatic patches of buckwheat, in Guaíra SP (20° 19' 32"S 48° 13' 15.4"W). Samples were analyzed in the Nematology Laboratory (LabNema), UNESP, Jaboticabal, SP, BR. Plants presented symptoms of yellow leaves and galled and volume-reduced roots. Meloidogyne sp. was found, comprising 6,320 eggs and second-stage juveniles (J2s) from 10 g of root and 1,628 J2s in 100 cm³ of soil. Adult morphological characteristics, isoenzyme phenotype of esterase, and molecular analysis were performed to identify the Meloidogyne species. The perineal patterns presented high and trapezoidal dorsal arch (n=15), and the males showed a trapezoidal labial region, including a high head cap formed by a large round labial disc that is raised above the medial lips and centrally concave (n=15) (Eisenback and Hirscmann 1981). These characteristics are typical in Meloidogyne incognita (Kofoid and White, 1912) Chitwood, 1949 (Nascimento et al., 2020; Eisenback and Hirschmann 1981; Netscher and Taylor 1974). The enzymatic phenotype was performed with females (n=8), and the phenotype I1 was verified, described by Esbenshade and Triantaphyllou (1985) as typical for M. incognita. To confirm the species DNA samples were extracted from individual females (n=6) and PCR with specific primers for M. incognita (Mi-F 5'- GTGAGGATTCAGCTCCCCAG-3' and Mi-R 5'-ACGAGGAA CATACTTCTCCGTCC-3') and M. javanica (Treub) Chitwood 1949 (Fjav 5'-GGTGCGCGATTGAACTGAGC-3' and Rjav 5'-CAG GCCCTTCAGTGGAACTATAC-3') that amplify SCAR markers described by Meng et al. (2004) and Zijlstra et al. (2000), respectively, and specific primers for M. enterolobii Yang & Eisenback 1983 that amplify rDNA-IGS2 region (Me-F 5'-AACTTTTG TGAAAGTGCCGCTG-3' and Me-R 5'-TCAGTTCAGGCAGG ATCAACC-3') described by Long et al. (2006) were tested. A fragment of 955 pb DNA size was amplified in Mi-F/R primer, which confirmed the M. incognita identification (Meng et. al., 2004). The original population was used to execute pathogenicity test. In a greenhouse, single buckwheat seeds (cv. IPR 91 Baili) were sown in six 5L pots filled with autoclaved-soil and inoculated with 3,000 eggs and J2s per pot (n=6) and control (n=6). After 60 days, the nematodes were extracted from roots and the M. incognita was confirmed. An average of 15,738 eggs and J2s were recovered, (reproductive factor = 5.24), which confirmed buckwheat as a host to M. incognita. The inoculated plants showed symptoms as those observed in the field. No symptom or nematode was noted on the control. Meloidogyne incognita has been reported causing high damage to the F. esculentum in California (Gardner and Caswell-Chen 1994) plus several crops in Brazil (Nascimento et al., 2020). However, this is the first report of this nematode infecting buckwheat in Brazil. Given the importance of buckwheat in Brazil, with extensive use as forage, cover crop, and its nutritional properties, this report is essential to specific management measures are adopted to avoid further losses.