Oliveiro Guerreiro-Filho

Phenol Contents, Oxidase Activities, and the Resistance of Coffee to the Leaf Miner Leucoptera coffeella

We examined the role of phenolic compounds, and the enzymes peroxidase and polyphenol oxidase, in the expression of resistance of coffee plants to Leucoptera coffeella (Lepidoptera: Lyonetiidae). The concentrations of total soluble phenols and chlorogenic acid (5-caffeoylquinic acid), and the activities of the oxidative enzymes peroxidase (POD) and polyphenol oxidase (PPO), were estimated in leaves of Coffea arabica, C. racemosa, and progenies of crosses between these species, which have different levels of resistance, before and after attack by this insect. The results indicate that phenols do not play a central role in resistance to the coffee leaf miner. Differences were detected between the parental species in terms of total soluble phenol concentrations and activities of the oxidative enzymes. However, resistant and susceptible hybrid plants did not differ in any of these characteristics. Significant induction of chlorogenic acid and PPO was only found in C. racemosa, the parental donator of the resistance genes against L. coffeella. High-performance liquid chromatography (HPLC) analysis also showed qualitative similarity between hybrids and the susceptible C. arabica. These results suggest that the phenolic content and activities of POD and PPO in response to the attack by the leaf miner may not be a strong evidence of their participation in direct defensive mechanisms.

Genetic diversity and structure of Ethiopian, Yemen and Brazilian Coffea arabica L. accessions using microsatellites markers

Genetic diversity among 115 coffee accessions from the Coffea Germplasm Collection of IAC was assessed using SSR markers. The germplasm represents 73 accessions of Coffea arabica derived from spontaneous and subspontaneous plants in Ethiopia and Eritrea, species center of origin and diversity, 13 commercial cultivars of C. arabica developed by the Breeding Program of IAC, 1 accession of C. arabica cv. ‘Geisha’, 13 accessions of C. arabica from Yemen, 5 accessions of C. eugenioides, 4 accessions of C. racemosa and 6 accessions of C. canephora. Genetic analysis was performed using average number of alleles per locus (A), proportion of polymorphic loci (P), Shannon’s genetic index (H′ and G′ST) and clustering analysis. All evaluated species were distinguished by a cluster analysis based on Jaccard’s coefficient. Differentiation between the cultivated plants of C. arabica and accessions derived from spontaneous and subspontaneous plants was observed. Spontaneous and subspontaneous accessions from Ethiopia were separated according to the geographical origin: east and west of the Great Rift Valley. Cultivated plants showed a low genetic diversity with a division in two groups: accessions from Yemen (H′=0,028) and Brazilian commercial cultivars (H′=0,030). The results agreed with previously reported narrow genetic basis of cultivated plants of C. arabica and supported the hypotheses about domestication of the species. This study also showed a significant genetic diversity among accessions from Ethiopia and Eritrea present in the Germplasm Collection of IAC. This diversity is specially observed in accessions from Sidamo (H′=0,143), Kaffa (H′=0,142) and Illubabor (H′=0,147) indicating their importance as source of genetic variability for coffee breeding programs.

CARACTERIZAÇÃO ANATÔMICA DE FOLHAS DE CAFEEIROS RESISTENTES E SUSCETÍVEIS AO BICHO-MINEIRO (1)

The leaf miner Leucoptera coffeella (Guerin-Meneville, 1842) (Lepidoptera: Lyonetiidae) is the major pest of coffee culture, being responsible for significant production losses as result of premature leaf fall, and consequent reduction of the photosynthetic area. The Coffee Breeding Program of the Agronomic Institute (IAC) has transferred through crossing genes that confer resistance to leaf miner from Coffea racemosa to the susceptible species C. arabica. The main objective of this study was to characterize leaf tissues, at histological level, from the parental species C. racemosa and C. arabica, and also from hybrids exhibiting different resistance levels. Histological analyses were performed in leaf transversal sections, and included measurements of cuticles and epidermis thickness, total palisade and spongy parenchyma, total leaf thickness, and percentage of the palisade parenchyma from total mesophyll. Results revealed that there were significant differences in leaf thickness between parental species C. arabica and C. racemosa. However, in hybrids no such difference could be observed between resistant and susceptible progenies, suggesting that the anatomical differences of parental genotypes are not related to resistance mechanisms to L. coffeella. When leaf lesions caused by insect attack were measured one and four days after larvae eclosion a slow insect development was observed in resistant plants, which could be related to a presence of specific chemicals in the palisade parenchyma.

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation.

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

Caracterização de cultivares de Coffea arabica mediante utilização de descritores mínimos

Cerca de 70% do cafe produzido e comercializado mundialmente e oriundo de Coffea arabica. A especie apresenta base genetica estreita sendo as cultivares bastante aparentadas e originarias em sua maioria das tradicionais cultivares Tipica e Bourbon. Este trabalho foi desenvolvido com o objetivo de identificar a eficiencia de descritores minimos na caracterizacao de cultivares de cafeeiros e como diferenciadores entre cultivares a serem submetidas ao processo de protecao de cultivares no Brasil. Foram avaliadas trinta e oito caracteristicas botânicas ou tecnologicas das plantas, folhas, flores, frutos, sementes, assim como tres caracteristicas agronomicas. Utilizaram-se vinte e nove cultivares de cafeeiros selecionadas pelo Instituto Agronomico, sendo avaliadas trinta plantas de cada cultivar. Os resultados evidenciaram que apenas com a utilizacao das caracteristicas porte, cor do fruto, resistencia ao agente da ferrugem, Hemileia vastatrix e ciclo de maturacao e possivel obter uma discriminacao eficiente dos diferentes grupos de cultivares avaliadas. A cor das folhas jovens e o diâmetro da copa revelaram-se importantes descritores na discriminacao de cultivares do grupo Mundo Novo. Nao foi possivel, porem, identificar descritores eficientes na discriminacao das cultivares dos grupos Catuai Vermelho, Catuai Amarelo e Icatu Vermelho.

Large-scale analysis of differential gene expression in coffee genotypes resistant and susceptible to leaf miner–toward the identification of candidate genes for marker assisted-selection

BackgroundA successful development of herbivorous insects into plant tissues depends on coordination of metabolic processes. Plants have evolved complex mechanisms to recognize such attacks, and to trigger a defense response. To understand the transcriptional basis of this response, we compare gene expression profiles of two coffee genotypes, susceptible and resistant to leaf miner (Leucoptera coffella). A total of 22000 EST sequences from the Coffee Genome Database were selected for a microarray analysis. Fluorescence probes were synthesized using mRNA from the infested and non-infested coffee plants. Array hybridization, scanning and data normalization were performed using Nimble Scan® e ArrayStar® platforms. Genes with foldchange values +/-2 were considered differentially expressed. A validation of 18 differentially expressed genes was performed in infected plants using qRT-PCR approach.ResultsThe microarray analysis indicated that resistant plants differ in gene expression profile. We identified relevant transcriptional changes in defense strategies before insect attack. Expression changes (>2.00-fold) were found in resistant plants for 2137 genes (1266 up-regulated and 873 down-regulated). Up-regulated genes include those responsible for defense mechanisms, hypersensitive response and genes involved with cellular function and maintenance. Also, our analyses indicated that differential expression profiles between resistant and susceptible genotypes are observed in the absence of leaf-miner, indicating that defense is already build up in resistant plants, as a priming mechanism. Validation of selected genes pointed to four selected genes as suitable candidates for markers in assisted-selection of novel cultivars.ConclusionsOur results show evidences that coffee defense responses against leaf-miner attack are balanced with other cellular functions. Also analyses suggest a major metabolic reconfiguration that highlights the complexity of this response.

Flutuação populacional do bicho-mineiro em cultivares de café arábica resistentes à ferrugem

The intensity of infestation of leaf-miner, Leucoptera coffeella (Guerin-Meneville) was investigated in coffee cultivars Obata IAC 1669-20 and Tupi IAC 1669-33, both resistant to the leaf rust agent, Hemileia vastatrix Berk. et Br., and Ouro Verde Amarelo IAC 4397, susceptible to this coffee disease, at field assays in Campinas, SP, Brazil. The incidence of coffee rust and presence of natural enemies, as well as the plant leafiness, were also observed. In Obata IAC 1669-20 and Tupi IAC 1669-33 cultivars the curves of population fluctuation revealed high occurrence of the coffee leaf miner between April and November. However analysis in the Ouro Verde Amarelo IAC 4397 cultivar demonstrated the occurrence of two peaks of infestation, the first in April-May and the second in August-September. Also, a higher index of damaged leaves was observed in Tupi IAC 1669-33 and Obata IAC 1669-20 cultivars in comparison to Ouro Verde Amarelo IAC 4397. This result may not indicate a higher susceptibility to L. coffeella of those cultivars, but otherwise could be the result of a better leaf retention, as a consequence of the leaf rust resistance exhibited by both cultivars. On the other hand, the damage observed in leaves from Ouro Verde Amarelo IAC 4397 cultivar is less frequent, probably in consequence of the high rate of leaf falling. Based on these observed differences, distinct strategies for this pest management are suggested.

Antixenosis resistance to leaf miner Leucoptera coffeella in Coffea species

The coffee leaf miner, Leucoptera coffeella, is the most important pest of Coffea arabica plantations in Brazil, and all cultivars are susceptible to the insect. However, distinct reactions have been observed in other species, such as C. congensis and C. canephora. In this work, the occurrence of antixenotic response was surveyed in individuals of Coffea species, selected for resistance to the leaf miner at both field and laboratory conditions. Tests performed on young plants from C. arabica cultivars Icatu Precoce IAC 3282, Mundo Novo IAC 515-20, Tupi IAC 1669-33 and Catuaí Amarelo IAC 62 demonstrated that these were preferred for oviposition when pooled with seedlings from C. canephora cv. Apoatã IAC 2258. Apoatã was also preferred over C. racemosa for oviposition. Similar results were observed in tests using detached leaves, and higher amount of eggs was counted in the cv Obatã IAC 1669-20 and Catuaí Vermelho IAC 81, and lower oviposition frequency was observed in C. canephora cv. Guarini IAC 1598. C. congensis showed an oviposition frequency between C. arabica and C. canephora. Also, the results suggest that the frequency of oviposition is co-related with the plant resistance level, during larvae infection.