Note SOURCES OF RESISTANCE TO Crinipellis perniciosa IN PROGENIES OF CACAO ACCESSIONS COLLECTED IN THE BRAZILIAN AMAZON

The witches’ broom disease caused by the fungus Crinipellis perniciosa is the main phytossanitary constraint for cacao production in Brazil. The integrated management of the disease involves resistance as one of the components. The breeding program conducted by the Brazilian Institution, CEPLAC is directed toward the pyramidation of resistance genes from different sources to achieve a more durable resistance. This study aimed to identify sources of resistance in progenies of cacao accessions collected in the basins of ten Amazonian rivers and compared to progenies from the Peruvian clones ‘Scavina 6’ and ‘Sacavina 12’. Progenies from 40 Amazonian accessions and ‘Scavina’ were evaluated in the field for six years for witches’ broom resistance through multivariate and repeated measurement analyses evaluating the effect of progeny, area, block, year, and their interactions. There were differences in the mean number of vegetative brooms on some Amazonian progenies and ‘Scavina’ descendants. There was an increase in the number of vegetative brooms in the last year for ‘Scavina’ progenies, but that was not observed for the Amazonian progenies 64, 66, 156, 194, 195, 269 and 274. There were different gene/alleles for resistance in the Amazonian progenies in comparison to the traditional ‘Scavina’ accessions. These new sources of resistance will be important for pyramiding resistance genes and consequently increasing the stability and durability of the resistance to witches’ broom.


INTRODUCTION
Cacao (Theobroma cacao) is a perennial species that grows spontaneously in the tropical plains of South America (Cuatrecasas, 1964); it is grown mainly in the Amazonian region and in Bahia State in Brazil, one of the world's five largest producers of cacao.Diseases are responsible for the largest losses in production in the last two decades (Bowers et al., 2001).
The basidiomycete Crinipellis perniciosa, causal agent of the witches' broom disease, is one of the most damaging cacao pathogens.The fungus infects all meristematic tissues, including flower cushions, pods, and growing parts of the stem (Silva et al., 2002).Infected stems produce the vegetative brooms, which are hypertrophyc and hyperplasic branches possibly caused by hormonal imbalances induced by the pathogen.This disease was detected in the main Brazilian cacao-growing region, Bahia State, in 1989 (Pereira et al., 1990).Witches' broom disease caused up to 90% losses in some cacao crops (Luz et al., 2005), leading to unemployment of rural workers and the destruction of what remains of the Atlantic forest (Trevizan & Marques, 2002).The disease control should ideally involve the use of resistant genotypes.Progress in cacao breeding programs around the world are considered poor in comparison to other crops, possibly because of the small number of studies on sources of resistance from wild cacao accessions (Rios-Ruiz, 2001).
The search for resistance to witches' broom in South America began in the 1930's, through collecting-expeditions to the Amazonian region, including areas in Ecuador, Peru, and Colombia.During these expeditions, pods of wild cacao apparently free from the disease were collected and their progenies introduced mainly into Trinidad (Fonseca, 1988).The best sources of resistance, the accessions 'Scavina 6' and 'Scavina 12' were obtained in Peru by Pound (1938;1943).These accessions showed immunity to the disease in Trinidad (Bartley, 1994), susceptibility in Ecuador (Bartley, 1981), Peru (Rios-Ruiz, 1989), and in Rondônia State, Brazil (Anderbrhan et al., 1998).The 'Scavina 6' accession is widely used by the Comissão Executiva do Plano da Lavoura Cacaueira -CEPLAC's breeding program.However, because of the variable reactions of these accessions in other regions and to the existence of genetic variations of the pathogen population in Bahia State (Pires, 2003), the breeding programs need to incorporate new sources of resistance as a strategy to obtain extended resistance (Pinto & Pires, 1998).
The Brazilian Amazon harbors great part of the cacao genetic diversity.Accessions with a wide variation in terms of vigour, morphological characteristics, productivity, and disease resistance were collected in that region (Vello & Medeiros, 1965;Barriga et al., 1985;Almeida & Almeida, 1987;Almeida et al., 1987;1995;Bartley et al., 1988;Almeida & Dias, 2001), mainly in the States of Acre, Rondônia, and Amazonas, (Almeida, 1996;Almeida et al., 1987).These accessions were termed CAB (cacao from the Brazilian Amazon) and maintained as part of CEPLAC's germplasm.In 1997, open pollinated pods from 40 selected accessions were introduced in Bahia State to be evaluated for witches' broom resistance (Luz et al., 1997;2000).Seedlings from these accessions were selected for witches' broom resistance in greenhouse through artificial inoculation (Luz et al., 1997;2000).Greenhouse-selected plants evaluated under field conditions were the basis of the present study, targeted at evaluating resistance to witches' broom disease on cacao progenies obtained from 40 accessions collected from ten river basins in the Brazilian Amazon.

Field experiments
Seedlings from 40 cacao accessions collected in different Amazonian regions were used (Table 1).From seedlings not showing symptoms of witches' broom in the greenhouse (Luz et al., 1997;2000), 36 individuals from each progeny were transferred to the field six months after germination along with the same number of seedlings of 'Scavina 6' and 'Scavina 12' progenies, used as resistant controls, and of 'Catongo', used as susceptible control.The field experiments were installed in 1997, in three areas at CEPLAC, Ilhéus, Bahia State, Brazil (18 progenies in the first area, 10 in the second and 13 in the third, plus the three controls in all areas -Table 1).The three experiments were set up in a randomized blocks design, with three replicates of 12 plants per plot spaced 3.0 × 1.5 m, and planted under older cacao trees highly infected with C. perniciosa.Plants were evaluated individually every three months for six years (1999 to 2004) for witches' broom symptoms, yield, and other agronomical traits.The number of vegetative brooms on 30 cacao trees, used as natural inoculum sources and under which the evaluated seedlings were planted was taken once in 2005 for comparison purposes.

Statistical analysis
The number of vegetative brooms per plant per year was the variable here analyzed.To simplify presentation of the results, the three experiments were evaluated jointly, as elicited by the use of the controls in all experiments and the lack of interaction between controls and areas allowed (Pimentel-Gomes, 1990).Furthermore, the main objective of this study was to compare the Amazonian progenies with the 'Scavina' controls.Multivariate analyses were used to study the effects of area, block within the areas, progeny, and interactions between controls and areas by considering the mean value of vegetative brooms per plant per year as separate variables.The interpretation of the effect of the year and the effects of interactions between year and area and progeny was done with the analyses of repeated measurements (Danford, 1960).To better understand the interaction progeny x year, each of the six years was successively removed from the analyses, enabling the comparison of individual contributions to the interaction.All studied progenies were compared through contrasts with 'Scavina 6' and the Amazonian progeny with the lowest number of vegetative brooms.All analyses were done with the program SAS (SAS Institute, 1988).

Controls
Because only six out of 108 plants (36 plants per area) of the Catongo progeny, used as susceptible control, survived to the Crinipellis perniciosa inoculum pressure, this control was not considered.

Joint evaluation of the areas
The interaction between controls and areas was not significant (P = 0.1981; Wilk's test), allowing the combined analysis of the areas as a single experiment (Pimentel-Gomes, 1990).

Number of vegetative brooms
Effects of area and blocks within the areas were significant (P < 0.0001; Wilk's test).Therefore the average number of vegetative brooms for the progenies was corrected for these factors.The mean number of vegetative brooms per year differed among progenies (P < 0.0001; Wilk's test).The highest average number of vegetative brooms per plant per year (total of four evaluations per year) among all progenies studied was 22.6 brooms (average observed in 2004 for the Amazonian progeny 272).On the other hand, the mean number observed for thirty randomly selected plants among the ones kept in the experiment as inoculum sources was 71.8 vegetative brooms per plant in only one observation.

DISCUSSION
The Amazonian progenies included genotypes of great resistance once they passed through an initial selection at CEPLAC's ERJOH station (Fonseca & Albuquerque, 1996), followed by another one in the greenhouse (Luz et al., 1997), and were finally selected in the field by the mortality of susceptible plants *Probability of committing an error in accepting the hypothesis of difference between the progenies with six annual means of vegetative brooms.2).The differences among progenies in regard to the mean number of vegetative brooms per year indicates that different genes/alleles of resistance might be expressing in these progenies.The existence of interaction between year of evaluation and progenies shows that these progenies behaved differently along the years, 2004 being the most important year for the interaction.In this year, there was an increase in the number of vegetative brooms for 'Scavina 6' and '12' progenies; the same trend was not observed for the progenies of 64,66,156,194,195 269 and 274 (Table 2,Figure1).These progenies have different averages of vegetative brooms per year in comparison to 'Scavina 6' (P > 0.05; Wilks Test; Table 3) and a lower mean number of vegetative brooms than 'Scavina 6' progenies (data not shown) along the six years, what demonstrates, again, the presence of different genes/alleles of resistance.
Apart from the different genetic structure for the resistance in some Amazonian progenies and 'Scavina 6', apparently differences among superior progenies, as seen for 195 and 64 (Table 3 -P > 0.0406) also occur.These differences indicate a differential reaction to the disease and imply in the expression of different resistance genes/allelles.
The changes in the resistance pattern of 'Scavina' descendants were already observed in the proximity of the areas where this experiment was conducted, and also a clear distinction between isolates of C. perniciosa obtained from susceptible and resistant cacao genotypes according to RAPD markers (Pires, 2003).This adaptation of the fungus to 'Scavina' might also have occurred in the areas where the experiments were installed.
Therefore, two aspects are important to emphasize: 1) once changes in the pattern of disease resistance in 'Scavina' progenies were recorded again, the need for new resistance genes and their association aiming at the amplification of the genetic diversity in the field and the achievement of a more durable resistance is confirmed; and 2) the identification of Amazonian progenies with genes/alleles of resistance different from those of 'Scavina 6', will allow the necessary association of them.

Table 1 -
Cacao accessions evaluated for witches' broom resistance under field conditions for six years at CEPLAC/ CEPEC.

December 2006 caused
by high disease pressure.This resistance is illustrated by the low number of vegetative brooms on Amazonian progenies, the highest average being 22.6 vegetative brooms per plant per year(272, year 2004;  Table