Elaine Aparecida de Souza

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.

Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum.

Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer.

Sexual recombination in Colletotrichum lindemuthianum occurs on a fine scale

Glomerella cingulata f. sp phaseoli is the sexual phase of the fungus Colletotrichum lindemuthianum, the causal agent of common bean anthracnose. This fungus is of great concern, because it causes large economic losses in common bean crops. RAPD markers of five populations of G. cingulata f. sp phaseoli from two Brazilian states were analyzed to determine if this population possesses the sexual reproductive potential to generate the genetic variation that is observed in this phytopathogen. We identified 128 polymorphic bands, amplified by 28 random primers. The estimates of genetic similarity in this analysis ranged from 0.43 to 1.00, and the dendrogram generated from analysis of all genotypes displayed five principal groups, coinciding with the five populations. Genetic differentiation was observed between the populations (GST=0.6455); 69% of the overall observed genetic variation was between individual populations and 31% of the variance was within the sub-populations. We identified significant levels of linkage disequilibrium in all populations. However, the values of the disequilibrium ranged from low to moderate, indicating that this pathogen maintains a genetic structure consistent with sexual reproduction. The mean contribution of sexual reproduction was determined by comparison of the amplitudes of genetic similarity of isolates from sexual and asexual phases. These results support the hypothesis that recombination plays an important role in determining the amplitude of variability in this pathogen population and that this determination occurs on a fine scale.

Experimental alternatives for evaluation of progenies and clones in eucalyptus breeding programs

The feasibility of using augmented block designs and spatial analysis methods for early stage selection in eucalyptus breeding programs was tested. A total of 113 half-sib progenies of Eucalyptus urophylla and eight clones were evaluated in an 11 x 11 triple lattice experiment at two locations: Posto da Mata (Bahia, Brazil) and Sao Mateus (Minas Gerais, Brazil). Four checks were randomly allocated within each block. Plots consisted of 15 m long rows containing 6 plants spaced 3 m apart. The girth at breast height (cm/plant) was evaluated at 19 and 26 months of age. Variance analyses were performed according to the following methods: lattice design, randomized complete block design, augmented block design, Papadakis method, moving means method, and check plots. Comparisons among different methods were based on the magnitude of experimental errors and precision of the estimates of genetic and phenotypic parameters. General results indicated that augmented block design is useful to evaluate progenies and clones in early selection in eucalyptus breeding programs using moderate and low selection intensities. However, this design is not suitable for estimating genetic and phenotypic parameters due to its low precision. Check plots, nearest neighbour, Papadakis (1937), and moving means methods were efficient in removing the heterogeneity within blocks. These efficiencies were compared to that in lattice analysis for estimation of genetic and phenotypic parameters.

Determinação da variabilidade em isolados de Colletotrichum lindemuthianum por meio de marcadores morfológicos e culturais

Colletotrichum lindemuthianum (teleomorfo Glomerella cingulata f. sp. phaseoli) apresenta ampla variabilidade genetica, demonstrada por suas caracteristicas morfologicas. Com este trabalho, objetivou-se caracterizar, por meio de marcadores morfologicos, diferentes isolados de C. lindemuthianum e identificar marcadores morfologicos com uso potencial em analises geneticas. Foram avaliados os seguintes caracteres morfologicos e culturais: cor e textura das colonias, compatibilidade vegetativa e sexual, indice de velocidade de crescimento micelial (IVCM), diâmetro colonial (DC), capacidade de esporulacao (CE), dimensoes e formas conidiais, dimensoes dos ascosporos, formacao de estruturas reprodutivas e formacao de anastomoses entre hifas e conidios. Os resultados obtidos demonstraram que os isolados de C. lindemuthianum possuem ampla variabilidade genetica para todas as caracteristicas avaliadas e que a forma do conidio pode ser usada como marcador morfologico em analises geneticas.

Genetic divergence among and within Colletotrichum lindemuthianum races assessed by RAPD

A divergencia genetica entre racas de Colletotrichum lindemuthianum foi obtida utilizando marcadores RAPD. Alem das diferentes racas deste fungo, foram incluidos neste estudo tres isolados da fase sexuada de Colletotrichum lindemuthianum (Glomerella cingulata f.sp. phaseoli). A informacao do padrao de bandas geradas utilizando 11 primers, permitiu a identificacao de 133 bandas polimorficas. As bandas polimorficas foram utilizadas para determinar a divergencia genetica entre e dentro de racas do patogeno. Os isolados analisados foram distribuidos em seis grupos com similaridade relativa de 0,75. O grupo VI, formado pelos tres isolados da fase sexual do Colletotrichum lindemuthianum, foi o mais divergente. As racas agrupadas previamente pelo uso de cultivares diferenciadoras nao apresentou qualquer relacao com a classificacao obtida pelos marcadores RAPD.

Characterization of Glomerella Strains Recovered from Anthracnose Lesions on Common Bean Plants in Brazil

Anthracnose caused by Colletotrichum lindemuthianum is an important disease of common bean, resulting in major economic losses worldwide. Genetic diversity of the C. lindemuthianum population contributes to its ability to adapt rapidly to new sources of host resistance. The origin of this diversity is unknown, but sexual recombination, via the Glomerella teleomorph, is one possibility. This study tested the hypothesis that Glomerella strains that are frequently recovered from bean anthracnose lesions represent the teleomorph of C. lindemuthianum. A large collection of Glomerella isolates could be separated into two groups based on phylogenetic analysis, morphology, and pathogenicity to beans. Both groups were unrelated to C. lindemuthianum. One group clustered with the C. gloeosporioides species complex and produced mild symptoms on bean tissues. The other group, which belonged to a clade that included the cucurbit anthracnose pathogen C. magna, caused no symptoms. Individual ascospores recovered from Glomerella perithecia gave rise to either fertile (perithecial) or infertile (conidial) colonies. Some pairings of perithecial and conidial strains resulted in induced homothallism in the conidial partner, while others led to apparent heterothallic matings. Pairings involving two perithecial, or two conidial, colonies produced neither outcome. Conidia efficiently formed conidial anastomosis tubes (CATs), but ascospores never formed CATs. The Glomerella strains formed appressoria and hyphae on the plant surface, but did not penetrate or form infection structures within the tissues. Their behavior was similar whether the beans were susceptible or resistant to anthracnose. These same Glomerella strains produced thick intracellular hyphae, and eventually acervuli, if host cell death was induced. When Glomerella was co-inoculated with C. lindemuthianum, it readily invaded anthracnose lesions. Thus, the hypothesis was not supported: Glomerella strains from anthracnose lesions do not represent the teleomorphic phase of C. lindemuthianum, and instead appear to be bean epiphytes that opportunistically invade and sporulate in the lesions.

Antifungal activity and ultrastructural alterations in Pseudocercospora griseola treated with essential oils

Pseudocercospora griseola, the etiologic agent of angular leaf spot of common bean (Phaseolus vulgaris), is an important disease in all bean-producing regions worldwide and may cause extremely high yield losses. The control of this disease is made more difficult by the pathogens genetic variability and the inefficiency of fungicides. In this study, of 26 essential oils tested at different concentrations, 25 demonstrated efficiency in affecting the germination of strains 63-31 and 63-63 of the pathogen, reaching inhibition levels of between 80% and 100%. Cymbopogon citratus and Cymbopogon martinii inhibited conidia germination at all concentrations; Eugenia caryophyllata, Cinnamomum sp., Thymus vulgaris, Matricaria recutita, Cordia verbenacea, Origanum vulgare, Cymbopogon nardus, at 0.1 and 0.5%; and Zingiber officinale, Mentha arvensis, Chamaecyparis pisifera, Lavandula officinalis, Ocimum basilicum, Pimpinella anisum, Ocimum selloi, Baccharis dracunculifolia, Laurus nobilis, Citrus sinensis, Melaleuca alternifolia and Eucalyptus globulus, at 0.5%. The main constituents identified were cinnamaldehyde in Cinnamomum sp.; eugenol in E. caryophyllata; trans-β-farnesene in M. recutita; pulegone in C. verbenacea; thymol in T. vulgaris; geranial and neral in C. citratus, and geraniol in C. martini. Through transmission electron microscopy (TEM), it was verified that C. citratus, C. martini and E. caryophyllata presented direct fungitoxic action on P. griseola, causing severe damage to the cellular ultrastructure of the conidia, invalidating germination. These results indicated that essential oils are a promising alternative strategy for the control of angular leaf spot in bean, representing less risk to human health and the environment.

Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.

Procedimento para escolha de populações de milho promissoras para extração de linhagens

The identification of the population with greatest potential for inbred line extraction is directly linked to the success of a maize hybrid breeding program. This study was carried out to identify procedures for selecting these populations. Segregant S0, S1 populations and 196 S0:1 families were obtained from each of four commercial cultivars assessed: single hybrids (C 333B and Z8392), double hybrid (AG 1051) and variety (BR-105). The experiments were carried out during the 1998/99 season, in two locations in the southern region of Minas Gerais State, Brazil: Lavras and Ijaci. A 14 x 14 simple lattice design was used in the assessment of the 196 S0:1 families from each population. A randomized complete blocks design with four replications experiment was also set up to assess, simultaneously, the F1, S0 and S1 generations. The husked ear yield (kg/plot) of the F1, S0 and S1 generations was obtained and the contribution of the homozygous locos (m + a) and heterozygous locos (d) were estimated. The genetic and phenotypic parameters were also estimated using the S0:1 family experiments. A high correlation (r = 0.81) between the (m + a) estimate and the S0:1 family mean was observed. The AG 1051 population showed the greatest potential for line extraction, i.e., greatest (m + a) value. The correlation between the d and h2 estimates was low, indicating that the estimate of the contribution of the heterozygous locos was not a good indicator of the potential variability of the population.