Complete genome sequence of Bacillus velezensis strain Ag109, a biocontrol agent against plant-parasitic nematodes and Sclerotinia sclerotiorum.

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.

First report of root-knot nematode, Meloidogyne javaniva, infecting Stachys byzantina on São Paulo, Brazil.

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.

Characterizing the Interaction between Root-knot Nematodes and Soil-borne Fungi which are Pathogenic to Passion Fruit (Passiflora Edulis).

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.

Nematicidal Screening of Aqueous Extracts from Plants of the Yucatan Peninsula and Ecotoxicity.

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.

Interactions between Soil Bacterial Diversity and Plant-Parasitic Nematodes in Soybean Plants.

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.

Screening of the hatching control of root-knot nematodes using extract concentrations from oat genotypes

Even though oats (Avena spp.) have the capacity to produce numerous compounds with the potential to act antagonistically against plant pathogens, studies on the genotypic effect of this crop focusing on their nematicidal activity are limited. The objective of this study was to verify the effect of the aqueous extracts prepared with the biomass of oat genotypes, on the hatching of second-stage juveniles of root-knot nematodes (Meloidogyne javanica and Meloidogyne incognita). The bioassays were carried out in a completely randomized design with four replications. Eighteen extracts were evaluated, consisting of a combination of six oat genotypes Agro Quaraí, Agro Esteio, Embrapa 139, AF 12202, UPFPS Farroupilha, and AF 1345 Ucraniana, and three extract concentrations (5 %, 10 %, and 20 % w/v). Three additional treatments were added to the study (distilled water, chemical nematicides abamectin, and imidacloprid + thiodicarb). The treatments and the suspension containing nematode eggs were placed in Petri dishes and incubated in a growth chamber for ten days. At the 5 % and 10 % w/v concentration levels, a genotype effect was observed in the hatching of juveniles for both nematode species. For the control of M. javanica the extracts of Embrapa 139 had a better performance, while the extracts of Agro Quaraí and AF1345 Ucraniana performed better when under the control of M. incognita. Thus, oat biomass formation might have the ability to suppress the nematode population in the soil, and could therefore, be used for the management of root-knot nematodes.

Physiological changes in Solanum lycopersicum L. in the presence of root-knot nematodes and salicylic acid / Alterações fisiológicas em Solanum lycopersicum L. na presença de nematóides das galhas e ácido salicílico

Nematodes of the genus Meloidogyne spp. are one of the tomato culture's main pathogens. According to their soil infestation level, physiological changes may occur. Consequently, it is extremely important to employ given products to minimize their effect. A feasible option is the salicylic acid, a phytohormone that can induce into resistance due to its ability to increase the production level of some pathogenicity proteins. Therefore, the purpose hereof was to assess the effect of salicylic acid on the physiology of tomato plants subject to different populational densities of Meloidogyne javanica. The design employed was one of randomized blocks, in an incomplete factorial scheme, using the Box Central Compound (BCC) matrix with five nematode populational densities (PD) (0; 5815; 20000; 34184; and 40000 eggs per plant) and five salicylic acid doses (0.0; 0.29; 1.0; 1.71; and 2.0 mM), with four repetitions and two plants per experimental patch. The gas exchanges, fluorescence, and chlorophyll levels were evaluated 45 days after the transplant and soil infestation. The data were submitted for analysis of variance through the F test and, in the significance cases, the polynomial regression analysis was performed. For stomatal conductance, a greater reduction was seen at the PD of 21755 eggs per plant, a fact that might have decreased the liquid assimilation of CO2 and the efficiency of carboxylation. The salicylic acid (SA) affected the CO2 liquid assimilation and the efficiency of carboxylation. The infestation of M. javanica in tomato plants negatively affected the gas exchange and the chlorophyll levels because the SA application did not mitigate the negative effect thereof.

Os nematoides do gênero Meloidogyne spp. são um dos principais patógenos na cultura do tomate, podendo causar alterações fisiológicas em função do nível de infestação do solo. O uso de produtos que minimize seus efeitos é de suma importância, nesse sentido, o ácido salicílico pode ser uma alternativa viável, visto que, esse fitormônio pode induzir a resistência devido à capacidade de proporcionar um aumento na produção de algumas proteínas de patogenicidade. Com isso, objetivou-se avaliar o efeito do ácido salicílico na fisiologia do tomateiro submetido a diferentes densidades populacionais de Meloidogyne javanica. O delineamento utilizado foi o de blocos casualizados, em esquema fatorial incompleto, utilizando-se a matriz Composto Central de Box (CCB) com cinco densidades populacionais (DP) de nematoides (0; 5815; 20000; 34184 e 40000 ovos por planta) e cinco doses de ácido salicílico (0,0; 0,29; 1,0; 1,71 e 2,0 mM), com quatro repetições e duas plantas por parcela experimental. Foram avaliadas as trocas gasosas, fluorescências e índices de clorofilas aos 45 dias após o transplantio e infestação do solo. Os dados foram submetidos à análise de variância pelo teste F e nos casos de significância foi realizada análise de regressão polinomial. Para a condutância estomática, observou-se uma maior redução na DP de 21755 ovos por planta, fato que pode ter ocasionado reduções na assimilação líquida de CO2 e na eficiência de carboxilação. O ácido salicílico (AS) influenciou na assimilação líquida de CO2 e eficiência de carboxilação. A infestação por M. javanica em plantas de tomateiro influenciou negativamente nas trocas gasosas e nos teores de clorofila, sendo que a aplicação do AS não atenuou os efeitos negativos desses patógenos.

Nematicidal potential of essential oils of Ageratum fastigiatum, Callistemon viminalis and Schinus terebinthifolius / Potencial nematicida de óleos essenciais de Ageratum fastigiatum, Callistemon viminalis e Schinus terebinthifolius

Some essential oils are rich in nematicidal compounds and can be used for the management of plant-parasitic nematodes. Laboratory and field studies aiming to evaluate the efficiency of this compounds are necessary. The objective of this research was to assess the nematicidal potential of essential oils from leaves of Ageratum fastigiatum and Callistemon viminalis, and green and mature fruits of Schinus terebinthifolius on the root-knot nematode Meloidogyne javanica. In laboratory, the essential oil of S. terebinthifolius green fruits reduced hatching by more than 80% and increased juvenile mortality by 300% when compared to Tween 20 + water. In the field, none of the essential oils controlled M. javanica in lettuce. In conclusion, the essential oil of S. terebinthifolius green fruits reduces the egg hatching and kills J2 of M. javanica in laboratory. In an infested field with an average of 555 J2/100 cm3 of soil, the application of the essential oils of S. terebinthifolius, C. viminalis and A. fastigiatum does not control M. javanica in lettuce.

Alguns óleos essenciais são ricos em compostos nematicidas e poderiam ser usados no manejo de fitonematoides. Estudos laboratoriais e de campo que visam avaliar a eficiência desses compostos são necessários. O objetivo desta pesquisa foi avaliar o potencial nematicida de óleos essenciais de folhas de Ageratum fastigiatum e Callistemon viminalis e frutos verdes e maduros de Schinus terebinthifolius sobre o nematoide de galhas Meloidogyne javanica. Em laboratório, o óleo essencial de frutos verdes de S. terebinthifolius reduziu mais de 80% a eclosão e aumentou em 300% a mortalidade de juvenis em comparação com Tween 20 + água. No campo, nenhum óleo essencial controlou M. javanica em alface. Em conclusão, o óleo de frutos verdes de S. terebinthifolius reduz a eclosão e mata J2 de M. javanica em laboratório. Em campo com infestação média de 555 J2/100 cm3 de solo, a aplicação dos óleos essenciais de S. terebinthifolius, C. viminalis e A. fastigiatum não controla M. javanica em alface.

Identifying resistance to root-knot nematodes in Capsicum genotypes / Identificação de genótipos de Capsicum resistentes a nematoides de galha

The present study aimed to evaluate Capsicum accessions for resistance to Meloidogyne incognita race 3, Meloidogyne javanica and Meloidogyne enterolobii. Two experiments with different genotypes of hot and sweet peppers were carried out in a completely randomized design. The first experiment was conducted in a 31 x 3 factorial scheme with 27 genotypes of Capsicum annuum, two cultivars of hot pepper, one line of Capsicum frutescens and tomato 'Santa Cruz Kada', and three species of nematodes (M. incognita race 3, M. javanica and M. enterolobii). In the second experiment, we used a factorial scheme 39 x 3 with 36 accessions of C. annuum, two hot pepper cultivars and the 'Santa Cruz Kada' tomato and three nematodes species mentioned earlier. The total number of eggs and second-stage juveniles (TNEJ), number of eggs and second-stage juveniles per gram of root (NEJGR), reproduction index (RI) and reproduction factor (RF) were evaluated. Based on RI and RF, the genotypes CNPH 185, CNPH 187 and CNPH 680 were resistant and very resistant to M. incognita race 3 and M. javanica, simultaneously. The C. frutescens line presented resistance to the three root-knot nematode species.

O presente trabalho teve por objetivo avaliar acessos de Capsicum quanto à resistência a Meloidogyne incognita raça 3, Meloidogyne javanica e Meloidogyne enterolobii. Foram realizados dois experimentos, com diferentes genótipos de pimentas e pimentões, em delineamento inteiramente casualizado sendo o primeiro em esquema fatorial 31 x 3 com 27 genótipos de Capsicum annuum, duas cultivares de pimenta, uma linhagem de Capsicum frutescens, o tomateiro 'Santa Cruz Kada' e três espécies de nematoides (M. incognita raça 3, M. javanica e M. enterolobii). No segundo experimento foi utilizado esquema fatorial 39 x 3 com 36 acessos de C. annuum, duas cultivares de pimenta, o tomateiro 'Santa Cruz Kada' e três espécies de nematoides mencionadas anteriormente. Avaliou-se o número total de ovos e juvenis de segundo estádio (NTOJ), número de ovos e juvenis de segundo estádio por grama de raízes (NOJGR), índice de reprodução (IR) e fator de reprodução (FR). Com base no FR e IR os genótipos CNPH 185, CNPH 187 e CNPH 680 foram resistentes e muito resistentes a M. incognita raça 3 e M. javanica, simultaneamente. A linhagem de C. frutescens apresentou resistência às três espécies de nematoides de galha.

Earthworms and root-knot nematodes: effect on soil biological activity and tomato growth / Minhocas e nematoides das galhas: efeitos na atividade biológica do solo e crescimento do tomate

Earthworms are a representative soil invertebrate, and their living habits are known to influence a large diversity of organisms. The objective of this study was to evaluate the ability of Amynthas spp. to change the biological attributes of soil, and its potential to reduce infection by root-knot nematodes on tomato crop. The study was conducted in the greenhouse of the Diagnostic Center Marcos Enrietti, Federal University of Paraná, Brazil. The treatments earthworms at the following densities: control (absence of earthworms), two, four, six, and eight, which were inoculated into different pots, with five replicates per group. In each pot, a single tomato plant (Solanum lycopersicum) was used, and a suspension of Meloidogyne javanica containing 3000 eggs and/or juveniles was added 14 days after seeding. During the experiment, edaphic respiration was evaluated at 96-h intervals. After 91 days, soil microbial biomass carbon (MBC), microbial soil respiration (MSR), the metabolic quotient (qCO2), dry mass of roots (DMR), dry mass of plants (DMP), and the number of root galls were determined per plant. We observed that inoculation with higher earthworm densities increased the MBC. Furthermore, the lowest earthworm density (two animals) resulted in a MBC that was 75% higher than that of the control treatment (earthworms absent). There was a positive correlation between MBC and DMP, anda negative correlation between MBC and qCO2. The DMR was not influenced by inoculation withearthworms. A linear increase in DMP was observed with earthworms; however, gall formations on thetomato root were not suppressed.(AU)

As minhocas são um dos mais representativos invertebrados do solo e sabe-se que seus hábitos de vida influenciam uma grande diversidade de outros organismos. O objetivo deste estudo foi avaliar a capacidade de Amynthas spp. em alterar alguns atributos biológicos do solo e seu potencial em reduzir a infecção de nematoides formadores de galhas na cultura do tomate. O estudo foi conduzido em casa de vegetação no Centro Diagnóstico Marcos Enrietti, Universidade Federal do Paraná, Brasil. Os tratamentos foram diferentes densidades de minhocas: Controle (ausência de minhocas), dois, quatro, seis e oito indivíduos inoculados por vaso, com cinco repetições. Em cada vaso foi utilizada uma única plântula de tomate (Solanum lycopersicum), onde, aos 14 dias após a semeadura foi adicionada uma suspensão contendo 3000 ovos e/ou juvenis de Meloidogyne javanica. Durante o experimento, a respiração edáfica foi avaliada em intervalos de 96 horas. Após 91 dias, o carbono da biomassa microbiana (MBC), respiração microbiana (MSR), quociente metabólico (qCO2), massa seca das raízes (DMR), massa seca da planta (DMP) e o número de galhas por planta foram determinados. Como resultados, observou-se que a inoculação de altas densidades de minhocas aumentou o MBC. Além disso, baixas densidades de minhocas (dois indivíduos) mostraram valores de MBC 75% maiores, comparados ao tratamento controle (ausência de minhocas). Houve uma correlação positiva entre MBCe DMP, negativa entre MBC e qCO2. A DMR não foi influenciada pela inoculação de minhocas. Umaumento linear da DMP foi observado com o aumento da densidade de minhocas, sem ocorrer supressãoda formação de galhas nas raízes.(AU)

Earthworms and root-knot nematodes: effect on soil biological activity and tomato growth / Minhocas e nematoides das galhas: efeitos na atividade biológica do solo e crescimento do tomate

Earthworms are a representative soil invertebrate, and their living habits are known to influence a large diversity of organisms. The objective of this study was to evaluate the ability of Amynthas spp. to change the biological attributes of soil, and its potential to reduce infection by root-knot nematodes on tomato crop. The study was conducted in the greenhouse of the Diagnostic Center Marcos Enrietti, Federal University of Paraná, Brazil. The treatments earthworms at the following densities: control (absence of earthworms), two, four, six, and eight, which were inoculated into different pots, with five replicates per group. In each pot, a single tomato plant (Solanum lycopersicum) was used, and a suspension of Meloidogyne javanica containing 3000 eggs and/or juveniles was added 14 days after seeding. During the experiment, edaphic respiration was evaluated at 96-h intervals. After 91 days, soil microbial biomass carbon (MBC), microbial soil respiration (MSR), the metabolic quotient (qCO2), dry mass of roots (DMR), dry mass of plants (DMP), and the number of root galls were determined per plant. We observed that inoculation with higher earthworm densities increased the MBC. Furthermore, the lowest earthworm density (two animals) resulted in a MBC that was 75% higher than that of the control treatment (earthworms absent). There was a positive correlation between MBC and DMP, anda negative correlation between MBC and qCO2. The DMR was not influenced by inoculation withearthworms. A linear increase in DMP was observed with earthworms; however, gall formations on thetomato root were not suppressed.

As minhocas são um dos mais representativos invertebrados do solo e sabe-se que seus hábitos de vida influenciam uma grande diversidade de outros organismos. O objetivo deste estudo foi avaliar a capacidade de Amynthas spp. em alterar alguns atributos biológicos do solo e seu potencial em reduzir a infecção de nematoides formadores de galhas na cultura do tomate. O estudo foi conduzido em casa de vegetação no Centro Diagnóstico Marcos Enrietti, Universidade Federal do Paraná, Brasil. Os tratamentos foram diferentes densidades de minhocas: Controle (ausência de minhocas), dois, quatro, seis e oito indivíduos inoculados por vaso, com cinco repetições. Em cada vaso foi utilizada uma única plântula de tomate (Solanum lycopersicum), onde, aos 14 dias após a semeadura foi adicionada uma suspensão contendo 3000 ovos e/ou juvenis de Meloidogyne javanica. Durante o experimento, a respiração edáfica foi avaliada em intervalos de 96 horas. Após 91 dias, o carbono da biomassa microbiana (MBC), respiração microbiana (MSR), quociente metabólico (qCO2), massa seca das raízes (DMR), massa seca da planta (DMP) e o número de galhas por planta foram determinados. Como resultados, observou-se que a inoculação de altas densidades de minhocas aumentou o MBC. Além disso, baixas densidades de minhocas (dois indivíduos) mostraram valores de MBC 75% maiores, comparados ao tratamento controle (ausência de minhocas). Houve uma correlação positiva entre MBCe DMP, negativa entre MBC e qCO2. A DMR não foi influenciada pela inoculação de minhocas. Umaumento linear da DMP foi observado com o aumento da densidade de minhocas, sem ocorrer supressãoda formação de galhas nas raízes.

Earthworms and root-knot nematodes: effect on soil biological activity and tomato growth / Minhocas e nematoides das galhas: efeitos na atividade biológica do solo e crescimento do tomate

Earthworms are a representative soil invertebrate, and their living habits are known to influence a large diversity of organisms. The objective of this study was to evaluate the ability of Amynthas spp. to change the biological attributes of soil, and its potential to reduce infection by root-knot nematodes on tomato crop. The study was conducted in the greenhouse of the Diagnostic Center Marcos Enrietti, Federal University of Paraná, Brazil. The treatments earthworms at the following densities: control (absence of earthworms), two, four, six, and eight, which were inoculated into different pots, with five replicates per group. In each pot, a single tomato plant (Solanum lycopersicum) was used, and a suspension of Meloidogyne javanica containing 3000 eggs and/or juveniles was added 14 days after seeding. During the experiment, edaphic respiration was evaluated at 96-h intervals. After 91 days, soil microbial biomass carbon (MBC), microbial soil respiration (MSR), the metabolic quotient (qCO2), dry mass of roots (DMR), dry mass of plants (DMP), and the number of root galls were determined per plant. We observed that inoculation with higher earthworm densities increased the MBC. Furthermore, the lowest earthworm density (two animals) resulted in a MBC that was 75% higher than that of the control treatment (earthworms absent). There was a positive correlation between MBC and DMP,

As minhocas são um dos mais representativos invertebrados do solo e sabe-se que seus hábitos de vida influenciam uma grande diversidade de outros organismos. O objetivo deste estudo foi avaliar a capacidade de Amynthas spp. em alterar alguns atributos biológicos do solo e seu potencial em reduzir a infecção de nematoides formadores de galhas na cultura do tomate. O estudo foi conduzido em casa de vegetação no Centro Diagnóstico Marcos Enrietti, Universidade Federal do Paraná, Brasil. Os tratamentos foram diferentes densidades de minhocas: Controle (ausência de minhocas), dois, quatro, seis e oito indivíduos inoculados por vaso, com cinco repetições. Em cada vaso foi utilizada uma única plântula de tomate (Solanum lycopersicum), onde, aos 14 dias após a semeadura foi adicionada uma suspensão contendo 3000 ovos e/ou juvenis de Meloidogyne javanica. Durante o experimento, a respiração edáfica foi avaliada em intervalos de 96 horas. Após 91 dias, o carbono da biomassa microbiana (MBC), respiração microbiana (MSR), quociente metabólico (qCO2), massa seca das raízes (DMR), massa seca da planta (DMP) e o número de galhas por planta foram determinados. Como resultados, observou-se que a inoculação de altas densidades de minhocas aumentou o MBC. Além disso, baixas densidades de minhocas (dois indivíduos) mostraram valores de MBC 75% maiores, comparados ao tratamento controle (ausência d

Earthworms and root-knot nematodes: effect on soil biological activity and tomato growth / Minhocas e nematoides das galhas: efeitos na atividade biológica do solo e crescimento do tomate

Earthworms are a representative soil invertebrate, and their living habits are known to influence a large diversity of organisms. The objective of this study was to evaluate the ability of Amynthas spp. to change the biological attributes of soil, and its potential to reduce infection by root-knot nematodes on tomato crop. The study was conducted in the greenhouse of the Diagnostic Center Marcos Enrietti, Federal University of Paraná, Brazil. The treatments earthworms at the following densities: control (absence of earthworms), two, four, six, and eight, which were inoculated into different pots, with five replicates per group. In each pot, a single tomato plant (Solanum lycopersicum) was used, and a suspension of Meloidogyne javanica containing 3000 eggs and/or juveniles was added 14 days after seeding. During the experiment, edaphic respiration was evaluated at 96-h intervals. After 91 days, soil microbial biomass carbon (MBC), microbial soil respiration (MSR), the metabolic quotient (qCO2), dry mass of roots (DMR), dry mass of plants (DMP), and the number of root galls were determined per plant. We observed that inoculation with higher earthworm densities increased the MBC. Furthermore, the lowest earthworm density (two animals) resulted in a MBC that was 75% higher than that of the control treatment (earthworms absent). There was a positive correlation between MBC and DMP,

As minhocas são um dos mais representativos invertebrados do solo e sabe-se que seus hábitos de vida influenciam uma grande diversidade de outros organismos. O objetivo deste estudo foi avaliar a capacidade de Amynthas spp. em alterar alguns atributos biológicos do solo e seu potencial em reduzir a infecção de nematoides formadores de galhas na cultura do tomate. O estudo foi conduzido em casa de vegetação no Centro Diagnóstico Marcos Enrietti, Universidade Federal do Paraná, Brasil. Os tratamentos foram diferentes densidades de minhocas: Controle (ausência de minhocas), dois, quatro, seis e oito indivíduos inoculados por vaso, com cinco repetições. Em cada vaso foi utilizada uma única plântula de tomate (Solanum lycopersicum), onde, aos 14 dias após a semeadura foi adicionada uma suspensão contendo 3000 ovos e/ou juvenis de Meloidogyne javanica. Durante o experimento, a respiração edáfica foi avaliada em intervalos de 96 horas. Após 91 dias, o carbono da biomassa microbiana (MBC), respiração microbiana (MSR), quociente metabólico (qCO2), massa seca das raízes (DMR), massa seca da planta (DMP) e o número de galhas por planta foram determinados. Como resultados, observou-se que a inoculação de altas densidades de minhocas aumentou o MBC. Além disso, baixas densidades de minhocas (dois indivíduos) mostraram valores de MBC 75% maiores, comparados ao tratamento controle (ausência d

Nematicidal activity of crambe extracts on Meloidogyne spp. / Atividade nematicida de extratos de crambe sobre Meloidogyne spp.

Alternative methods for the control of nematodes, such as the use of plant secondary metabolites, can be explored for integrated pest management systems. The objective of this work was to assess the best solvent for obtaining allyl isothiocyanate from Crambe abyssinica leaves, and the effects of this extract on Meloidogyne incognita and M. javanica. Dry leaves of C. abyssinica at 200 mg L-1 were used to prepare extracts by using water (by infusion and grinding), acetone, water + ethanol (hydroalcoholic extraction), methanol, hexane, and chloroform as solvents. Following the evaporation of the solvents, the residue was resuspended in water for use in the experiments. Distilled water and chemical nematicide were used as control treatments. Once the most effective extracts were defined, the following dosages of dried crambe leaves were used: 0, 200, 300, 400, and 500 mg L-1. High performance liquid chromatography (HPLC) was used to quantify the allyl isothiocyanate present in the extracts. After the solvents evaporated, the residues were eluted with water and used in assays with 200 eggs for the hatching test or 200 second stage juveniles (J2) for mobility and mortality tests. The hydroalcoholic extract was the most effective in reducing the hatching of M. incognita and M. javanica juveniles, by 71.6 and 74.4 percentage points, respectively. The mortality of M. incognita and M. ja

Medidas alternativas de controle de fitonematoides, como o uso de metabólitos secundários produzidos pelas plantas, podem ser exploradas no contexto do manejo integrado em fitossanidade. O objetivo deste trabalho foi verificar quais os melhores solventes para obtenção de alil isotiocianato de folhas de Crambe abyssinica, e o efeito dos mesmos sobre Meloidogyne incognita e Meloidogyne javanica. Folhas secas de crambe na concentração de 200 mg L-1 foram utilizadas para o preparo dos extratos com os solventes água (para os extratos obtidos por infusão e trituração), acetona (cetônico), água + álcool etílico (hidroalcoólico), álcool metílico (metanólico), hexano (hexânico) e clorofórmio (clorofórmico). Após a rotoevaporação dos solventes o resíduo foi ressuspenso em água para utilização nos ensaios. Água destilada e nematicida foram usados como tratamentos controle. Após a seleção dos melhores extratos, os selecionados foram avaliados nas seguintes doses: 0, 200, 300, 400 e 500 mg L-1 de folhas secas de crambe. Para quantificar o alil isotiocianato presente nos extratos foi utilizada cromatografia líquida de alta pressão (HPLC). Para avaliar a eclosão foram utilizados 200 ovos e para motilidade e mortalidade, 200 juvenis de segundo estádio (J2). O extrato hidroalcoólico foi o mais efetivo em reduzir a eclosão de juvenis de M. incognita e M. javanica, com redução de 71.6 e 74.4 pont

Nematicidal activity of crambe extracts on Meloidogyne spp / Atividade nematicida de extratos de crambe sobre Meloidogyne spp

Alternative methods for the control of nematodes, such as the use of plant secondary metabolites, can be explored for integrated pest management systems. The objective of this work was to assess the best solvent for obtaining allyl isothiocyanate from Crambe abyssinica leaves, and the effects of this extract on Meloidogyne incognita and M. javanica. Dry leaves of C. abyssinica at 200 mg L-1 were used to prepare extracts by using water (by infusion and grinding), acetone, water + ethanol (hydroalcoholic extraction), methanol, hexane, and chloroform as solvents. Following the evaporation of the solvents, the residue was resuspended in water for use in the experiments. Distilled water and chemical nematicide were used as control treatments. Once the most effective extracts were defined, the following dosages of dried crambe leaves were used: 0, 200, 300, 400, and 500 mg L-1. High performance liquid chromatography (HPLC) was used to quantify the allyl isothiocyanate present in the extracts. After the solvents evaporated, the residues were eluted with water and used in assays with 200 eggs for the hatching test or 200 second stage juveniles (J2) for mobility and mortality tests. The hydroalcoholic extract was the most effective in reducing the hatching of M. incognita and M. javanica juveniles, by 71.6 and 74.4 percentage points, respectively. The mortality of M. incognita and M. ja

Medidas alternativas de controle de fitonematoides, como o uso de metabólitos secundários produzidos pelas plantas, podem ser exploradas no contexto do manejo integrado em fitossanidade. O objetivo deste trabalho foi verificar quais os melhores solventes para obtenção de alil isotiocianato de folhas de Crambe abyssinica, e o efeito dos mesmos sobre Meloidogyne incognita e Meloidogyne javanica. Folhas secas de crambe na concentração de 200 mg L-1 foram utilizadas para o preparo dos extratos com os solventes água (para os extratos obtidos por infusão e trituração), acetona (cetônico), água + álcool etílico (hidroalcoólico), álcool metílico (metanólico), hexano (hexânico) e clorofórmio (clorofórmico). Após a rotoevaporação dos solventes o resíduo foi ressuspenso em água para utilização nos ensaios. Água destilada e nematicida foram usados como tratamentos controle. Após a seleção dos melhores extratos, os selecionados foram avaliados nas seguintes doses: 0, 200, 300, 400 e 500 mg L-1 de folhas secas de crambe. Para quantificar o alil isotiocianato presente nos extratos foi utilizada cromatografia líquida de alta pressão (HPLC). Para avaliar a eclosão foram utilizados 200 ovos e para motilidade e mortalidade, 200 juvenis de segundo estádio (J2). O extrato hidroalcoólico foi o mais efetivo em reduzir a eclosão de juvenis de M. incognita e M. javanica, com redução de 71.6 e 74.4 pont

AFLP and SRAP markers linked to the mj gene for root-knot nematode resistance in cucumber

Root-knot nematodes (Meloidogyne spp.) are an important worldwide pest of cucumber (Cucumis sativus L.). Molecular markers linked to the Javanese root-knot nematode (M. javanica) resistance gene mj in cucumber may aid marker assisted selection. One-hundred AFLP (EcoRI-MseI) and 112 SRAP were used to screen resistant and susceptible parents for polymorphisms to develop molecular markers linked to the mj gene. Of the 100 AFLP primers, 92 produced bands and two yielded candidate markers (E-ATT/M-CAA and E-AAC/M-CTG). These two bands were cut off from polyacrylamide gel, cloned and sequenced. Primers designed from the sequences did not yield polymorphic bands between the parents. In addition, the sequences did not contain any restriction site or indel to be used to convert them to CAPS or SCAR markers. The two sequences obtained from polymorphic AFLP markers were used primarily to design D1F, D1R, D17F and D17R primers. SRAP forward and reverse primers were used in combination with these four specific primers to search for polymorphisms between parents. Of the 112 primer combinations 11 yielded polymorphisms between parents. MapMaker Exp 3.0 software was used to analyze the 11 markers. Two markers were identified that flanked the mj gene at distance of 16.3 and 19.3 cM. The results indicated that these markers should be useful to develop molecular markers flanking the mj gene.

Nematóides causadores de galhas (Meloidogyne spp.) em raízes de pepino Cucumis sativus L.) são de ocorrência mundial. Marcadores moleculares ligados ao gene mj que confere resistência a M. javanica em pepino podem auxiliar na seleção de plantas em programas de melhoramento genético. Cem AFLP (EcoRI-MseI) e 112 SRAP foram usados para a seleção de parentais resistentes e susceptíveis, por meio de polimorfismos, para o desenvolvimento de marcadores moleculares ligados ao gene mj. Entre 100 oligonucleotídeos iniciadores para AFLP, 92 geraram fragmentos amplificados de DNA e dois produziram candidatos a marcadores (E-ATT/M-CAA e E-AAC/M-CTG). Os dois fragmentos amplificados foram clonados e seqüenciados. Oligonucleotídeos iniciadores sintetizados a partir das seqüências de nucleotídeos obtidas não produziram fragmentos polimórficos entre os parentais. Além disso, as seqüências de nucleotídeos não contêm sítios de restrições ou deleções que possam ser convertidos em marcadores CAPS ou SCAR. As duas seqüências de nucleotídeos obtidas para os marcadores polimórficos AFLP foram usadas primeiramente para sintetizar os oligonucleotídeos iniciadores específicos D1F, D1R, D17F e D17R. Oligonucleotídeos iniciadores senso e antisenso para SRAP foram usados em combinações com os quatro oligonucleotídeos iniciadores específicos para identificação de polimorfismos entre os parentais. Dentre 112 combinações testadas, 11 geraram polimorfismos entre indivíduos parentais. O programa MapMaker Exp 3.0 foi usado para análise desses 11 marcadores. Foram identificados dois marcadores inseridos no gene mj nas distancias de 16.3 e 19.3 cM. Os resultados indicam que esses marcadores podem ser usados para o desenvolvimento de marcadores moleculares para o gene mj.

AFLP and SRAP markers linked to the mj gene for root-knot nematode resistance in cucumber

Root-knot nematodes (Meloidogyne spp.) are an important worldwide pest of cucumber (Cucumis sativus L.). Molecular markers linked to the Javanese root-knot nematode (M. javanica) resistance gene mj in cucumber may aid marker assisted selection. One-hundred AFLP (EcoRI-MseI) and 112 SRAP were used to screen resistant and susceptible parents for polymorphisms to develop molecular markers linked to the mj gene. Of the 100 AFLP primers, 92 produced bands and two yielded candidate markers (E-ATT/M-CAA and E-AAC/M-CTG). These two bands were cut off from polyacrylamide gel, cloned and sequenced. Primers designed from the sequences did not yield polymorphic bands between the parents. In addition, the sequences did not contain any restriction site or indel to be used to convert them to CAPS or SCAR markers. The two sequences obtained from polymorphic AFLP markers were used primarily to design D1F, D1R, D17F and D17R primers. SRAP forward and reverse primers were used in combination with these four specific primers to search for polymorphisms between parents. Of the 112 primer combinations 11 yielded polymorphisms between parents. MapMaker Exp 3.0 software was used to analyze the 11 markers. Two markers were identified that flanked the mj gene at distance of 16.3 and 19.3 cM. The results indicated that these markers should be useful to develop molecular markers flanking the mj gene.

Nematóides causadores de galhas (Meloidogyne spp.) em raízes de pepino Cucumis sativus L.) são de ocorrência mundial. Marcadores moleculares ligados ao gene mj que confere resistência a M. javanica em pepino podem auxiliar na seleção de plantas em programas de melhoramento genético. Cem AFLP (EcoRI-MseI) e 112 SRAP foram usados para a seleção de parentais resistentes e susceptíveis, por meio de polimorfismos, para o desenvolvimento de marcadores moleculares ligados ao gene mj. Entre 100 oligonucleotídeos iniciadores para AFLP, 92 geraram fragmentos amplificados de DNA e dois produziram candidatos a marcadores (E-ATT/M-CAA e E-AAC/M-CTG). Os dois fragmentos amplificados foram clonados e seqüenciados. Oligonucleotídeos iniciadores sintetizados a partir das seqüências de nucleotídeos obtidas não produziram fragmentos polimórficos entre os parentais. Além disso, as seqüências de nucleotídeos não contêm sítios de restrições ou deleções que possam ser convertidos em marcadores CAPS ou SCAR. As duas seqüências de nucleotídeos obtidas para os marcadores polimórficos AFLP foram usadas primeiramente para sintetizar os oligonucleotídeos iniciadores específicos D1F, D1R, D17F e D17R. Oligonucleotídeos iniciadores senso e antisenso para SRAP foram usados em combinações com os quatro oligonucleotídeos iniciadores específicos para identificação de polimorfismos entre os parentais. Dentre 112 combinações testadas, 11 geraram polimorfismos entre indivíduos parentais. O programa MapMaker Exp 3.0 foi usado para análise desses 11 marcadores. Foram identificados dois marcadores inseridos no gene mj nas distancias de 16.3 e 19.3 cM. Os resultados indicam que esses marcadores podem ser usados para o desenvolvimento de marcadores moleculares para o gene mj.

Efeito de Inseticidas no Controle de Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) e de Nematóides Fitoparasitos na Qualidade Tecnológica e na Produtividade da Cana-de-Açúcar / Effect of Insecticides on Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) and Fitoparasitic Nematodes Control on Technological Quality and Sugarcane Yield

The objective of this work was to study the effect of insecticides on sugarcane root froghopper and fitoparasitic nematodes populations and on technological quality and sugarcane yield. Two experiments were carried out, under field conditions, and different insecticides were applied once or twice during the froghopper infestation period. The most efficient compounds on root froghopper control were aldicarb 150G 12 kg/ha, thiamethoxan 10GR 30 kg/ha, carbofuran 100G 40 kg/ha, applied only in the begining of pest infestation period (October) and aldicarb 150G 6+6 kg/ha and thiamethoxan 10GR 15+15 kg/ha, in which a half dose was applied in the begining (October) and the other half was applied in the middle of pest infestation period (January). In experiment 1, Pratylenchus zeae Grahan was detected in high populations and aldicarb 150G at 12 and at 6+6 kg/ha and carbofuran 100G at 40 kg/ha were the most efficient nematicides, decreasing nematode populations at least untill three months after application. Low populations of P. zeae and Meloidogyne javanica (Treub) Chitwood were detected in experiment 2. The insecticides application resulted in significative increase of sugar content in the stalks, juice purity, and stalks and sugar yields (until 6,12 ton of recoverable sugar per ha), but the split dose applied in two periods did not result in significative increase of yield when compared to only one application.

Com o objetivo de avaliar a eficiência de inseticidas no controle da cigarrinha das raízes e de nematóides fitoparasitos e seus reflexos sobre a qualidade tecnológica e a produtividade da cana-de-açúcar, conduziram-se dois experimentos, na região canavieira de Piracicaba, SP, aplicando-se diversos inseticidas em uma ou em duas ocasiões, ao longo do período de ataque da cigarrinha. Aldicarbe 150G 12 kg/ha, tiametoxam 10GR 30 kg/ha e carbofuram 100G 40 kg/ha, aplicados no início do período de infestação da cigarrinha (outubro) e aldicarbe 150G 6+6 kg/ha e tiametoxam 10GR 15+15 kg/ha, com a primeira metade da dose aplicada no início do período de infestação (outubro) e a outra no meio do período (janeiro), foram os produtos mais eficientes no controle da cigarrinha-das-raízes, contribuindo para incrementos na pol por centocana, na pureza do caldo e na produtividade de açúcar (até 6,12 t ATR/ha). No experimento 1 foram observadas populações elevadas de Pratylenchus zeae Grahan, sendo o aldicarbe 150G a 12 e 6+6 kg/ha e o carbofuram 100G a 40 kg/ha os nematicidas mais eficientes, reduzindo as populações do nematóide pelo menos até três meses após aplicação. No experimento 2 foram detectadas populações baixas de P. zeae e Meloidogyne javanica Grahan foi encontrada nas áreas do ensaio 1 e P. zeae e Meloidogyne javanica (Treub) Chitwood. O parcelamento da dose dos inseticidas para aplicação em duas épocas não resultou em incrementos significativos de produtividade, quando comparada com os resultados obtidos com uma só aplicação.

Interações entre Pratylenchus brachyurus e Meloidogynejavanica em soja

The interactive effects between Pratylenchus brachyurus and Meloidogynejavanica on soybean cv. Crista-Una were evaluated. Ten treatments (noninoculated check; single inoculations of P. brachyurus at three inoculum levels; single inoculations of M. javanica at two inoculum levels; and mixed inoculations of both species at four different combinations) were stablished, with five replications, in a randomized block design. Sixty days after the inoculations, fresh root weight and top dry weight values were determined, as well as the final population of the nematodes. Antagonism between the species was observed in the mixed infestations, being generally P. brachyurus more adversely affected. Significant reduction hi plant growth only occurred with single P. brachyurus inoculations of 5,000 or 10,000 nematodes per plant.

Avaliaram-se os efeitos de interações entre Pratylenchus brachyurus e Meloidogynejavanica na soja 'Cristalina', sob condição de telado. Dez tratamentos (testemunha não inoculada; infestações simples de P. brachyurus em três níveis de inóculo; infestações simples de M. javanica em dois níveis de inóculo; e infestações conjuntas das duas espécies em quatro combinações de níveis de inóculo) foram estabelecidos, repetidos cinco vezes e arranjados em blocos casualizados. Após 60 dias das inoculações, determinaram-se os pesos de matéria fresca de raízes e matéria seca da parte aérea das plantas, bem como estimaram-se as populações finais dos nematóides. Observou-se antagonismo entre as duas espécies nas infestações conjuntas, sendo P. brachyurus geralmente a mais afetada. Reduções significativas nos pesos só ocorreram nas infestações simples de P. brachyurus nos níveis de 5000 e 10000 nematóides por planta.

Interações entre Pratylenchus brachyurus e Meloidogynejavanica em soja

The interactive effects between Pratylenchus brachyurus and Meloidogynejavanica on soybean cv. Crista-Una were evaluated. Ten treatments (noninoculated check; single inoculations of P. brachyurus at three inoculum levels; single inoculations of M. javanica at two inoculum levels; and mixed inoculations of both species at four different combinations) were stablished, with five replications, in a randomized block design. Sixty days after the inoculations, fresh root weight and top dry weight values were determined, as well as the final population of the nematodes. Antagonism between the species was observed in the mixed infestations, being generally P. brachyurus more adversely affected. Significant reduction hi plant growth only occurred with single P. brachyurus inoculations of 5,000 or 10,000 nematodes per plant.

Avaliaram-se os efeitos de interações entre Pratylenchus brachyurus e Meloidogynejavanica na soja 'Cristalina', sob condição de telado. Dez tratamentos (testemunha não inoculada; infestações simples de P. brachyurus em três níveis de inóculo; infestações simples de M. javanica em dois níveis de inóculo; e infestações conjuntas das duas espécies em quatro combinações de níveis de inóculo) foram estabelecidos, repetidos cinco vezes e arranjados em blocos casualizados. Após 60 dias das inoculações, determinaram-se os pesos de matéria fresca de raízes e matéria seca da parte aérea das plantas, bem como estimaram-se as populações finais dos nematóides. Observou-se antagonismo entre as duas espécies nas infestações conjuntas, sendo P. brachyurus geralmente a mais afetada. Reduções significativas nos pesos só ocorreram nas infestações simples de P. brachyurus nos níveis de 5000 e 10000 nematóides por planta.