Root-knot nematodes associated with carrot crop in Federal District region, and reaction of cultivars

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Programa de Pós-Graduação em Fitopatologia, 2017.A cenoura é uma das hortaliças mais importantes no mercado brasileiro, principalmente, para a comercialização in natura. A produção dessa hortaliça pode ser severamente afetada pela infecção de nematoides parasitas de plantas, sendo que mais de 90 espécies deste grupo de patógenos já foram associadas à referida cultura. O nematoide das galhas (Meloidogyne spp.) é o grupo que causa maiores perdas na cultura da cenoura. Os objetivos desse trabalho foram: (1) identificar as espécies de Meloidogyne presentes em áreas de cultivo de cenoura no Distrito Federal e no Município de Cristalina (GO); (2) avaliar a resposta de cultivares de cenoura quanto aos níveis naturais de infecção por espécies de Meloidogyne nas safras de 2013 e 2015, e (3) avaliar a reação de cultivares de cenoura em condições de campo („Brasília-CNPH‟, „Brasília-Agrocinco‟ e „Nantes‟) e em condições de casa de vegetação („Brasília-CNPH‟, „Brasília-Agrocinco‟ e „Kuronan‟) aos nematoides M. incognita, M. javanica e M. enterolobii (avaliada somente em condições controladas em casa de vegetação). Amostras de solo foram coletadas em campos comerciais de cenoura do Distrito Federal (DF) e de Cristalina, Goiás (GO). Ao todo foram coletadas 32 amostras compostas (22 amostras no DF e dez amostras em Cristalina, GO). Foi possível detectar nematoides das galhas em 22 amostras e identificar as espécies dos mesmos em 19 amostras. Por meio do fenótipo de esterase, foram identificadas as espécies M. javanica e M. incognita, ambas ocorreram isoladamente ou em mistura populacional. Meloidogyne javanica foi a espécie predominante nas áreas amostradas. Em relação à avaliação de cultivares comerciais quanto à produtividade, verificou-se diferença estatística significativa tanto entre as cultivares de cenoura dentro de cada safra, como entre as safras. As cultivares mais produtivas foram: „Brasília-CNPH‟, „Brasília-Horticeres‟, „BRS Planalto-CNPH‟, „BRS Planalto-Agrocinco‟, „Brasília-Agrocinco‟ e „Brasília-Tecnoseed‟. A cultivar Brasília-CNPH obteve produtividade semelhante nas duas safras. Quanto à reação ao nematoide das galhas, foi observada diferença estatística para o fator de aumento/redução da população de juvenis do nematoide (J2) no solo (FA), entre as cultivares avaliadas nas duas safras. A população „CNPH 1212554‟ foi a que apresentou maior FA na safra 2013 e a cultivar „Brasília-Tecnoseed‟ na safra 2015. Nas duas safras, as cultivares que apresentaram menor quantidade de nematoides por grama de casca de raiz foram: „Brasília-CNPH‟ e „Brasília-Agrocinco‟. Quanto à reação das cultivares ao nematoide das galhas verificou-se que para as variáveis: índice de massa de ovos (IMO), índice de galhas (IG) e fator de reprodução (FR), as cultivares „Kuronan‟, „Brasília-Agrocinco‟ e „Brasília-CNPH‟ comportaram-se como suscetiveis ao ataque de M. incognita, M. incognita + M. javanica e resistentes à espécie M. enterolobii em condições de casa de vegetação. No campo, somente a cultivar „Nantes‟ mostrou suscetibilidade frente aos nematoides M. incognita e M. incognita + M. javanica e as cultivares „Brasília- Agrocinco‟ e „Brasília-CNPH‟ comportaram-se como resistentes, de acordo com os valores de IMO e IG.Carrot is among the most important vegetable crops in Brazil, mainly, for the fresh root market. The production of this vegetable can be severely affected by the infection of plant parasitic-nematodes, with more than 90 species being associated with this crop.The root-knot nematodes (Meloidogyne spp.) is the main group of these soil-borne pathogens that causes yield and quality losses in the carrot crop. The main objectives of this study were: (1) to identify Meloidogyne species with natural occurrence in carrot growing areas in the Federal District and in Cristalina, State of Goiás (GO); (2) to evaluate reaction of carrot cultivars to Meloidogyne species under field conditions in 2013 and 2015 crop seasons; (3) to evaluate a subset of carrot cultivars under field („Brasília-CNPH‟, „Brasília-Agrocinco‟ and „Nantes‟) and under greenhouse conditions („Brasília-CNPH‟, „Brasília-Agrocinco‟ and „Kuronan‟) to M. incognita, M. javanica and M. enterolobii (evaluated only in controlled assays under greenhouse conditions). Soil samples were collected in carrot fields from the Federal District (DF) and from Cristalina (GO). A total of 32 samples were collected (22 samples in the Federal District and ten samples in Cristalina, GO). It was possible to detect root-knot nematodes in 22 samples and to identify the species in 19 samples. Analyses using the esterase phenotype, indicated the presence of the species M. javanica and M. incognita, which appeared either isolated or in a population mix. Meloidogyne javanica was the predominant species in the sampled areas. The cultivars with the highest yields were: „Brasília-CNPH‟, „Brasília-Horticeres‟, „BRS Planalto-CNPH‟, „BRS Planalto-Agrocinco‟, „Brasília-Agrocinco‟ and „Brasília-Tecnoseed‟. The cultivar „Brasília CNPH‟ displayed similar root yields in both crop seasons. It was observed statistical difference for the factor of increase/decrease of juveniles in soil (FA) between cultivars evaluated in the two crop seasons. The population „CNPH 1212554‟ was the one with the highest FA in the 2013 crop season and cv. Brasília-Tecnoseed in the 2015 crop season. The cultivars with the lowest number of nematodes per gram of root skin in the two crop seasons were: „Brasília-CNPH‟ and „Brasília-Agrocinco‟. As to the reaction of the carrot cultivars to the root-knot nematode, it was verified that for the variables: egg mass index (IMO), gall index (GI) and reproduction factor (FR): cultivars „Kuronan‟, „Brasília-Agrocinco‟ and „Brasília-CNPH‟ displayed a susceptible reaction to the attack of M. incognita, M. incognita + M. javanica and showed resistant reaction to M. enterolobii under greenhouse. Under field conditions, only cultivar „Nantes‟ showed susceptibility to M. incognita and to M. incognita + M. javanica, whereas „Brasília-Agrocinco‟ and „Brasília-CNPH‟ cultivars displayed a resistant reaction, according to IMO and GI values

Identification of the Control Region of Pancreatic Expression of Bmp4 In Vitro and In Vivo

<div><p>Bone morphogenetic protein 4 (Bmp4) was recently shown to be related to glucose homeostasis in mouse adult pancreas through the regulation of insulin production. We previously revealed the predominant expression of <i>Bmp4</i> in adult pancreas by <i>in vivo</i> imaging of transgenic mice. However, the control regions for predominant <i>Bmp4</i> expression in the adult pancreas are unclear. In this study, we established transgenic (Tg) mice that allow real time <i>in vivo</i> bioluminescence imaging of the enhancer/promoter activity of the <i>Bmp4</i> gene. Tg mice expressing firefly luciferase with a 7 kb upstream region and 5′-non-coding sequence (three exons and two introns) of the <i>Bmp4</i> gene showed pancreatic expression of bioluminescence, while the Tg mice bearing luciferase with the 7 kb upstream region alone did not show pancreatic expression of the reporter gene. Interestingly, pancreatic expression of bioluminescence was also present in Tg mice harboring the truncated promoter without exon IA and IB, indicating the presence of a cryptic promoter in front of exon II. Furthermore, the bioluminescence signal was not detected in embryonic pancreas, but increasing signals were observed in neonatal and infantile Tg mice depending on the genotypes observed. These results suggested that a novel mechanism of transcription is involved in pancreatic expression of the <i>Bmp4</i> gene.</p></div

Luciferase activity of Bmp4Luc reporter constructs <i>in vitro</i>.

<p>(A) Mouse <i>Bmp4</i> is located on the long arm of mouse chromosome 14, and consists of four exons (IA, IB, II, III and IV) shown by boxes with two transcription start sites (TSS1, TSS2). Dark boxes show the coding sequence. The position of each element is counted from TSS1 as +1. Triangles denote splicing spans. (B) Luciferase activity of Bmp4Luc reporter constructs. NIT1 and NIH3T3 cells were transiently cotransfected with reporter plasmids and <i>Renilla</i> luciferase vector (pRL-TK) as a transfection control. The <i>x</i> axes show relative luciferase activities: firefly luciferase (FL) from the reporter plasmid was normalized to <i>Renilla</i> (RL) luciferase activity from the control vector. All data are the mean ± SE from three independent experiments. **Represents a significant difference (p<0.01) by Student’s <i>t</i>-test.</p

Developmental expression of bioluminescence in Bmp4Luc Tg mice.

<p>(A) Bioluminescence signals in −6815/+4513 Bmp4Luc (line #17), (B) −6815/+618 Bmp4Luc (line #19) and (C) +1532/+4513 Bmp4Luc (Line #8) were determined at embryonic stage (E17.5), lactation stages (P5 and P16) and Weaning stage (P28 and beyond) using Xenogen IVIS Imaging System and the same reporter assay <i>in vivo</i> and <i>ex vivo</i> as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061821#pone-0061821-g002" target="_blank">Figure 2</a>. Embryonic bioluminescent images were obtained from laparotomy-treated embryonic bodies with the abdomen immersed in sterile PBS containing 0.3 mg/ml D-luciferin.</p

Number of Tg mice expressing bioluminescence signals in pancreas and skin during pancreatic maturation against the number of tested mice.

<p>Luciferase activity in the pancreas was detected by <i>ex vivo</i> imaging of upper 10⁵ (p/s/cm²/sr) and in skin by <i>in vivo</i> imaging of upper 10⁵ (p/s/cm²/sr) using the Xenogen IVIS Imaging System. The numbers of bioluminescence positive lines observed from the total tested are shown. Colored boxes represent a positive bioluminescence signal.</p

<i>In vivo</i> bioluminescent signals in Bmp4Luc Tg mice.

<p>(A–D) <i>In vivo</i> imaging assays of three representative lines of Bmp4Luc Tg mice [−6815/+4513 Bmp4Luc (line #17), −6815/+618 Bmp4Luc (line #19) and +1532/+4513 Bmp4Luc (Line #8)], and age matched (12–15-weeks-old). Transgenic mice were anesthetized with 2% isoflurane gas and subcutaneously injected with D-luciferin. Bioluminescent images were obtained by 1 min exposure in the imaging system. The minimum and maximum photons/second values for each figure are indicated in each rainbow bar scale. <i>Ex vivo</i> imaging assays were performed using dissected pancreas from transgenic mice immersed in sterile PBS containing 0.3 mg/ml D-luciferin, and subjected to imaging analysis. (E) The bioluminescence signals in each pancreas lines were quantified using Living Image software, and the intensity of luminescence is expressed as photons/second/cm²/steradian (p/s/cm²/sr) in individual transgenic mice and is indicated by individual color bars. The data are the mean ± SE from three independent experiments. **Represents a significant difference (p<0.01) by Student’s <i>t</i>-test.</p

Number of Tg mice lines expressing bioluminescence signals against the number of tested lines.

<p>Bioluminescence signals in the pancreas were detected by <i>ex vivo</i> imaging of upper 10⁵ (p/s/cm²/sr) and in the skin by <i>in vivo</i> imaging of upper 10⁵ (p/s/cm²/sr) using the Xenogen IVIS Imaging System. The numbers of bioluminescence positive lines observed from the total tested are shown.</p

In vivo bioluminescent signals in p7 kb-Bmp4Luc transgenic mice.

<p>(A) In vivo imaging assays of the three lines of transgenic mice (#1, #8 and #17). Transgenic mice were anesthetized with 2% isoflurane gas and intraperitoneally injected with D-luciferin. Bioluminescent images were obtained by 1 min exposure in the imaging system. The minimum and maximum photons/second values for each figure are indicated in each rainbow-colored bar scale. Strong bioluminescent signals were detected in the upper abdomen (black arrows) in all three lines in vivo. (B) The strong signal in the upper abdomen was confirmed to be localized to the pancreas (black arrow), by abdominal exploration in a transgenic mouse injected with D-luciferin. (C) Ex vivo imaging assays. Dissected tissues, spleen (S), liver (Li), pancreas (P), lung (Lu) and kidney (K) from wild-type (upper part of the dish) and transgenic mice (bottom part of the dish) were immersed in sterile PBS containing 0.3 mg/ml D-luciferin, and subjected to imaging analysis. (D) Western blot analysis of various tissues from transgenic mice, using antibodies to murine Bmp4, luciferase and α-tubulin.</p

Luciferase activity of truncated Bmp4Luc reporter constructs.

<p>(A) The truncated Bmp4Luc reporter was generated by restriction enzyme digestion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061821#s4" target="_blank">Materials and Methods</a> to determine luciferase activity as described in Fig. 1B. (B) Bioluminescence signals were detected in +2.9 kb-Bmp4Luc Tg mice (#30) using the same reporter assay <i>in vivo</i> and <i>ex vivo</i> as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061821#pone-0061821-g002" target="_blank">Figure 2</a>. **Represents a significant difference (p<0.01) by Student’s <i>t</i>-test.</p

Establishment Bmp4Luc Tg mice.

*<p>Representative lines used for <i>in vivo</i> imaging. (n), number.</p