1Department of Plant Pathology, Federal University of Višosa, 36571-000, Višosa, MG, Brazil; 2Aracruz Celulose S.A. C.P. 331011, 29197-000, Aracruz-ES, Brazil

Background and objectives
Eucalyptus rust, caused by Puccinia psidii, was described in Brazil in 1944 and was practically unnoticed for almost 30 years until 1973, when more than 400,000 seedlings of Eucalyptus grandis were refused for planting due to the disease. Since then, several rust outbreaks have been registered in young plants in the field, in coppice and in clonal hedges. The disease affects shoots and juvenile leaves, causing necrosis and deformation of the attacked organs. Small erupting pustules that are slightly green initially and become yellow at a later stage are the main characteristic for the diagnosis of the disease [1]. Because Eucalyptus is generally planted in areas favorable to the disease, improved genotypes must be resistant to Puccinia psidii. Thus, the objectives of this work were to identify clones resistant to the rust among elite clones of a Brazilian forest company and to characterize their resistance reaction at the histological level.

Materials and methods
Three replicates of five plants from each of 13 elite clones were inoculated with a suspension of 2x104 urediniospores/mL and incubated in moist chamber, at 20░C, in the dark, for the first 24 h, and in a growth chamber at 23░C, with a 12 h photoperiod, in the next 11 days. The number of sori per 2.4 cm2 of leaf area were evaluated in two leaves of each plant [2]. For the histopathological studies, one susceptible and one resistant clone were selected. Drops of 2 microliters of spore suspension at 105 urediniospores / mL were inoculated on the abaxial surface of detached leaves placed in Petri dishes containing wet filter paper under a nylon screen. Inoculated leaves were incubated at 20▒1░C, in the dark during the first 24 h, and then under a 12 h photoperiod. At 6 h intervals up to 60 h after inoculation, rectangular fragments of leaves, containing one drop of inoculum suspension, were collected and cleared with chloral hydrate : distilled water (5:2 v/v) during five days, under laboratory conditions. After clarification, the fragments were treated with lactophenol containing Aniline Blue (0.25%) and Trypan Blue (0.25%) for 12 h, and then transferred to chloral hydrate, where they were kept for two days. Subsequently, they were mounted in glicerin:lactophenol (1:1) and observed under light microscope, using phase contrast. For 50 urediniospores on each replicate, the percentage of germination , formation of apressorium, mode of penetration, formation of primary and secondary hyphae and formation and development of haustorium were recorded.

Results and conclusions
Four clones showed complete resistance to P. psidii. Among these, two displayed typical hypersensitive reaction (HR) and two did not show any macroscopic HR symptoms. The resistant clone selected for the histological studies was in the later group. No differences were observed between the phases of germination, formation of apressorium and penetration in both resistant and susceptible clones. On both clones, more than 90% of urediniospore germination was observed 6 h after inoculation, more than 90% of the germinated uredinospores formed apressorium at 18 h, and more than 75% of penetration was registered at 12 h. The penetration peg penetrated directly through the anticlinal walls of the epidermal cells. Penetration through stomata was rarely observed. Primary hyphae were observed on S and R genotypes respectively at 12 and 18 h after inoculation and haustorium formation at 18 and 24 h. In the cytoplasm of cells containing haustorium and in adjacent cells of the resistant genotype a dye accumulation was observed, indicative of cellular death. At 48 h after inoculation a HR was visible with naked eye. In the susceptible genotype, the hyphae branched and colonized the tissues after the first haustorium formation. Thus, the resistance was due to a HR response immediately after haustorium formation.


1. Alfenas AC, Demuner NL, Barbosa MM, 1989. Correio AgrÝcola 1, 18-20.
2. Ruiz RAR, Alfenas AC, Ferreira FA, Vale FXR, 1989. Fitopatologia Brasileira 14, 55-61.