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| Fruit of cacao infected by fungus causing black pod (copyright: CEPLAC / CEPEC) |
PETER MOON
Brazilian Science Communication Agency
The search for resistant plants
In the state of Bahia, the Brazilian state that leads cocoa production with 70% of harvest, black pod disease and broom witch disease cause a loss of 30% to 40% of the fruits annually. Black pod accounted for 14% of the losses (30,000 tons), while witch-broom accounted for the loss of 20,000 tons (9%), says agronomist and phytopathologist Edna Dora Newman Luz of the Center for Cacao Research (CEPLAC / CEPEC), in Ilhéus, Bahia. Today, black pod results in greater loss of fruit than the dreaded witch's broom.
"The losses with black pod in the cacao plantations of Bahia in recent years have been higher than the world average. Annually, the disease causes on average losses of up to 40% of the world production. Here in Bahia, they were around 14% in 2017. Worldwide, the disease is responsible for the death of 10% of the cocoa trees, annually."
"However, until the mid-1980s the losses caused by black pod were much higher, reaching 30% of production," reveals Luz. She believes that climate change is responsible for reducing losses.
Black pod is caused by seven species of the genus Phytophthora. In Brazil, there are currently three species: Phytophthora citrophthora, P. palmivora and P. capsici.
The Phytophthora spores move in the water. Therefore, it is the rainwater that falls on the contaminated leaves and fruit that carries the spores to infect other leaves, fruits and trees.
"Phytophthora is very sensitive to moisture. It needs rain to propagate its spores, which are transported by water," says Luz.
"In the past, in the 1970s and 1980s, the climate in southern Bahia was more humid. The weather was very rainy, which helped spread the disease. Today, because of climate change, the climate in the region has become drier. It rains a lot less today than it did 30 or 40 years ago. The reduction in rainfall reduced the propagation of Phytophthora spores and, consequently, the rate of infections fell 30% to 14% of production.
"Still, 14% is a very high loss level. Cacao farmers seek to keep black pod under control by regularly inspecting the trees and removing the affected fruits, preventing the disease from spreading or, where possible, applying copper-based fungicides.
The option to actually bring down the incidence of the disease is genetic and genomic breeding, selecting varieties resistant to this type of fungus.
This research began with the geneticist Mariana Barreto, at the State University of Santa Cruz (UESC). The first part of the research involved the work of phytopathology, to verify the various degrees of resistance or susceptibility of cacao to Phytophthora. For this, Barreto collected leaf samples of approximately 300 cacao trees from an experimental belonging to a large food company.
The samples of the three Phytophthora species were obtained with Luz from the Laboratory of Phytophthora (Phytolab) of CEPEC / CEPLAC, where the research was carried out.
To obtain relevant statistical results, Barreto produced, from the leaf samples collected from the cacao trees, three sets of 12 thousand Petri dishes to make the resistance and susceptibility tests with each of the three species of fungus that infest cacao trees in Bahia.
In each one of the 12,000 Petri dishes, Barreto dripped a drop with a solution in which there were spores of a single species of the pathogen. She repeated the operation three times, one for each of the three Phytophthora species separately, totaling 36,000 petri dishes.
After a few days, Barreto analyzed the dishes one by one under light, assigning a note according to the degree of resistance or susceptibility to infection by Phytophthora.
"Based on a scale of notes where the levels of resistance or susceptibility of plants, from the most resistant to the most susceptible, I checked each of the 36 thousand dishes. It was a meticulous work that lasted 9 long months," says Barreto.
It was from this research of phytopathology that Barreto was able to identify, among the 300 trees from which leaves had been collected for analysis, which were the cacao trees with the most natural resistance to each of the three Phytophthora species. "Every time I identified a sample that did not have the disease, it meant that it came from a tree that possessed genes resistant to Phytophthora," Barreto says.
The results suggested that only two or three phytopathogen-resistant genes existed in the cacao tree, not a single one, nor dozens of genes.
"This was evidence that it would be possible to select resistant plants," concludes plant geneticist Anete Pereira de Souza, leader of the Laboratory of Molecular Genetic Analysis at the Center for Molecular Biology and Genetic Engineering at the Biology Institute of the State University of Campinas (Unicamp). The results of phytopathology work were published in 2015.
Investigating Cocoa DNA
The presence of clones resistant to Phytophthora was the first stage of the work, which involved the genome research of cacao DNA, in search of Phytophthora resistance genes. This stage was carried out at the Souza laboratory in Campinas, in collaboration with researchers from the State University of Santa Cruz and a laboratory of a chocolate company located in the USA, too.
To carry out the genomic study, Barreto went to fetch samples from the same cocoa farm where she had previously collected leaves. This time, the Bahian geneticist collected leaf samples from 265 trees. Then the genotyping work of all those individuals was carried out to detect molecular markers that could indicate which regions of the genome where the resistance genes could be.
The DNA of cacao (Theobroma cacao) has ten chromosomes. To determine in which chromosomes were located the resistance regions for each of the three Phytophthora species, Barreto performed a genetic-statistical analysis as well as bioinformatics.
"We found regions of resistance to the three Phytophthora species in six cocoa chromosomes. We know this because in the same chromosomes we know other genes resistant to fungal diseases," explains Souza.
Specifically, the statistical analysis pointed to the existence of regions of resistance to the species Phytophthora capsici on chromosomes 1, 2, 3 and 4. In the case of P. citrophthora and P. palmivora, only one resistance region was identified for each species. But both regions are on the same chromosome, number 6.
"Now that we know the regions of the cacao genome where the resistance genes are, we can develop markers to individually identify which plants have the genes responsible for resistance in such regions," says Souza.
Once this is done, it will be possible through genomic breeding to cross such individuals with other plants susceptible to the disease, thereby producing new black pod resistant plants, which will be readily identifiable by molecular markers.
Among the three Phytophthora species currently infesting the cacao trees in Bahia, "P. palmivora is the most common, followed by P. citrophthora. P. palmivora is a species that infects plants all over the world, both in the cacao plantations of the Americas and in those which grow in Africa," explains specialist Edna Dora Luz.
The two largest cocoa producers are in Africa: Ivory Coast and Ghana. The third is Indonesia, in Southeast Asia. Brazil currently ranks sixth among the largest producers.
"P. citrophthora is a species restricted to the Americas, being the most aggressive of the three species that occur in Bahia," explains Luz.
According to the researcher, all species cause the same symptoms in the fruits, but each Phytophthora species reaches the plant in a different way. While one species is restricted to leaves and fruits, another also attacks the roots, for example. "When the focus of infection is on the fruit, black pod can partially or totally destroy the seeds contained inside. The fruit rot, looks very dark, chocolate-colored black, and with a strong stench of fish," says Barreto.
Because the disease attacks individual fruits, the farmers' procedure to try to keep the Phytophthora under control is to remove and destroy the infected fruits, preventing the infection from spreading to the other fruits and causing the epidemic to spread to surrounding trees. "You have to be careful also with the remnants of the fruit peels that have been broken. The disease can also spread from them," says Luz.
According to Souza, "the importance of this genomic work is mainly related to the more aggressive species of Phytophthora: P. citrophthora. Other studies abroad have already identified regions of resistance in P. palmivora and P. capsici, which occur elsewhere. Our work is the first to do the same with P. citrophthora.
In addition, it was necessary to do this long study to identify and confirm the existence of resistance genes to the three fungi in the cacao plantations belonging to the cacao breeding programs in Brazil, and specifically in Bahia.
contacts for interviews:
Anete Pereira de Souza
Unicamp, Campinas (SP)
Phone: (5519) 3521-1132
Mobile: (5519) 99111-6547
e-mail: anete@unicamp.br
Mariana Barreto
Universidade Estadual de Santa Cruz (UESC), Ilhéus (BA)
Mobile: (5571) 98862-8672
e-mail: maribiologista@gmail.com
Edna Dora Luz - cacao specialist
Centro de Pesquisa do Cacau (CEPLAC/CEPEC), Ilhéus (BA)
Mobile: (5573) 98831-1595
email: ednadora@yahoo.com.br
Press agent: Peter Moon
Mobile: 5511 95630-9353
In 1990, the Brazilian production of cocoa was 350 thousand tons. Today, it has fallen by half. Over the past 30 years, diseases such as black pod and witch-brooms have gradually brought down national production. In 2017, Brazil produced 170,000 tons of cocoa, insufficient volume to satisfy the desire of the Brazilian people for chocolate, a business of R$ 14 billion a year.
Although Brazil is one of the centers of origin of the cocoa plant (Theobroma cacao) and has already been the largest producer in the world, since 1998 the country is an importer of cocoa. In 2017, 60,000 tons of the product were imported.
If it were not the losses caused by disease, the country could be self-sufficient. Losses of cocoa fruits due to black pod and witch's broom are estimated to have reduced the 2017 crop by 50,000 tons, which could otherwise have reached 225,000 tons.
The search for ways to reduce the incidence of black pod and witch's broom has therefore become a pressing issue for Brazilian cocoa producers. How to do it? The answer is through the genomic improvement of cocoa.
In thesis, it is possible to develop new clones from plants resistant to the agents that cause black pod disease, some species of fungi of the genus Phytophthora. In a recent genomic study, researchers from Bahia and São Paulo announce the discovery of six regions in the genome of cocoa that confer resistance to black pod. The discovery is the first concrete step for the development of new clones or varieties of cacao trees resistant to the disease.
If it were not the losses caused by disease, the country could be self-sufficient. Losses of cocoa fruits due to black pod and witch's broom are estimated to have reduced the 2017 crop by 50,000 tons, which could otherwise have reached 225,000 tons.
The search for ways to reduce the incidence of black pod and witch's broom has therefore become a pressing issue for Brazilian cocoa producers. How to do it? The answer is through the genomic improvement of cocoa.
In thesis, it is possible to develop new clones from plants resistant to the agents that cause black pod disease, some species of fungi of the genus Phytophthora. In a recent genomic study, researchers from Bahia and São Paulo announce the discovery of six regions in the genome of cocoa that confer resistance to black pod. The discovery is the first concrete step for the development of new clones or varieties of cacao trees resistant to the disease.
 |
| Fruit of cacao infected by fungus causing black pod (copyright: CEPLAC / CEPEC) |
The search for resistant plants
In the state of Bahia, the Brazilian state that leads cocoa production with 70% of harvest, black pod disease and broom witch disease cause a loss of 30% to 40% of the fruits annually. Black pod accounted for 14% of the losses (30,000 tons), while witch-broom accounted for the loss of 20,000 tons (9%), says agronomist and phytopathologist Edna Dora Newman Luz of the Center for Cacao Research (CEPLAC / CEPEC), in Ilhéus, Bahia. Today, black pod results in greater loss of fruit than the dreaded witch's broom.
"The losses with black pod in the cacao plantations of Bahia in recent years have been higher than the world average. Annually, the disease causes on average losses of up to 40% of the world production. Here in Bahia, they were around 14% in 2017. Worldwide, the disease is responsible for the death of 10% of the cocoa trees, annually."
"However, until the mid-1980s the losses caused by black pod were much higher, reaching 30% of production," reveals Luz. She believes that climate change is responsible for reducing losses.
Black pod is caused by seven species of the genus Phytophthora. In Brazil, there are currently three species: Phytophthora citrophthora, P. palmivora and P. capsici.
The Phytophthora spores move in the water. Therefore, it is the rainwater that falls on the contaminated leaves and fruit that carries the spores to infect other leaves, fruits and trees.
"Phytophthora is very sensitive to moisture. It needs rain to propagate its spores, which are transported by water," says Luz.
"In the past, in the 1970s and 1980s, the climate in southern Bahia was more humid. The weather was very rainy, which helped spread the disease. Today, because of climate change, the climate in the region has become drier. It rains a lot less today than it did 30 or 40 years ago. The reduction in rainfall reduced the propagation of Phytophthora spores and, consequently, the rate of infections fell 30% to 14% of production.
"Still, 14% is a very high loss level. Cacao farmers seek to keep black pod under control by regularly inspecting the trees and removing the affected fruits, preventing the disease from spreading or, where possible, applying copper-based fungicides.
The option to actually bring down the incidence of the disease is genetic and genomic breeding, selecting varieties resistant to this type of fungus.
This research began with the geneticist Mariana Barreto, at the State University of Santa Cruz (UESC). The first part of the research involved the work of phytopathology, to verify the various degrees of resistance or susceptibility of cacao to Phytophthora. For this, Barreto collected leaf samples of approximately 300 cacao trees from an experimental belonging to a large food company.
The samples of the three Phytophthora species were obtained with Luz from the Laboratory of Phytophthora (Phytolab) of CEPEC / CEPLAC, where the research was carried out.
To obtain relevant statistical results, Barreto produced, from the leaf samples collected from the cacao trees, three sets of 12 thousand Petri dishes to make the resistance and susceptibility tests with each of the three species of fungus that infest cacao trees in Bahia.
In each one of the 12,000 Petri dishes, Barreto dripped a drop with a solution in which there were spores of a single species of the pathogen. She repeated the operation three times, one for each of the three Phytophthora species separately, totaling 36,000 petri dishes.
After a few days, Barreto analyzed the dishes one by one under light, assigning a note according to the degree of resistance or susceptibility to infection by Phytophthora.
"Based on a scale of notes where the levels of resistance or susceptibility of plants, from the most resistant to the most susceptible, I checked each of the 36 thousand dishes. It was a meticulous work that lasted 9 long months," says Barreto.
It was from this research of phytopathology that Barreto was able to identify, among the 300 trees from which leaves had been collected for analysis, which were the cacao trees with the most natural resistance to each of the three Phytophthora species. "Every time I identified a sample that did not have the disease, it meant that it came from a tree that possessed genes resistant to Phytophthora," Barreto says.
The results suggested that only two or three phytopathogen-resistant genes existed in the cacao tree, not a single one, nor dozens of genes.
"This was evidence that it would be possible to select resistant plants," concludes plant geneticist Anete Pereira de Souza, leader of the Laboratory of Molecular Genetic Analysis at the Center for Molecular Biology and Genetic Engineering at the Biology Institute of the State University of Campinas (Unicamp). The results of phytopathology work were published in 2015.
 |
| Geneticist Anete Pereira de Souza in her laboratory in Unicamp (copyright: Mariana Vargas Cruz) |
Investigating Cocoa DNA
The presence of clones resistant to Phytophthora was the first stage of the work, which involved the genome research of cacao DNA, in search of Phytophthora resistance genes. This stage was carried out at the Souza laboratory in Campinas, in collaboration with researchers from the State University of Santa Cruz and a laboratory of a chocolate company located in the USA, too.
To carry out the genomic study, Barreto went to fetch samples from the same cocoa farm where she had previously collected leaves. This time, the Bahian geneticist collected leaf samples from 265 trees. Then the genotyping work of all those individuals was carried out to detect molecular markers that could indicate which regions of the genome where the resistance genes could be.
The DNA of cacao (Theobroma cacao) has ten chromosomes. To determine in which chromosomes were located the resistance regions for each of the three Phytophthora species, Barreto performed a genetic-statistical analysis as well as bioinformatics.
"We found regions of resistance to the three Phytophthora species in six cocoa chromosomes. We know this because in the same chromosomes we know other genes resistant to fungal diseases," explains Souza.
Specifically, the statistical analysis pointed to the existence of regions of resistance to the species Phytophthora capsici on chromosomes 1, 2, 3 and 4. In the case of P. citrophthora and P. palmivora, only one resistance region was identified for each species. But both regions are on the same chromosome, number 6.
"Now that we know the regions of the cacao genome where the resistance genes are, we can develop markers to individually identify which plants have the genes responsible for resistance in such regions," says Souza.
Once this is done, it will be possible through genomic breeding to cross such individuals with other plants susceptible to the disease, thereby producing new black pod resistant plants, which will be readily identifiable by molecular markers.
Among the three Phytophthora species currently infesting the cacao trees in Bahia, "P. palmivora is the most common, followed by P. citrophthora. P. palmivora is a species that infects plants all over the world, both in the cacao plantations of the Americas and in those which grow in Africa," explains specialist Edna Dora Luz.
The two largest cocoa producers are in Africa: Ivory Coast and Ghana. The third is Indonesia, in Southeast Asia. Brazil currently ranks sixth among the largest producers.
"P. citrophthora is a species restricted to the Americas, being the most aggressive of the three species that occur in Bahia," explains Luz.
According to the researcher, all species cause the same symptoms in the fruits, but each Phytophthora species reaches the plant in a different way. While one species is restricted to leaves and fruits, another also attacks the roots, for example. "When the focus of infection is on the fruit, black pod can partially or totally destroy the seeds contained inside. The fruit rot, looks very dark, chocolate-colored black, and with a strong stench of fish," says Barreto.
Because the disease attacks individual fruits, the farmers' procedure to try to keep the Phytophthora under control is to remove and destroy the infected fruits, preventing the infection from spreading to the other fruits and causing the epidemic to spread to surrounding trees. "You have to be careful also with the remnants of the fruit peels that have been broken. The disease can also spread from them," says Luz.
According to Souza, "the importance of this genomic work is mainly related to the more aggressive species of Phytophthora: P. citrophthora. Other studies abroad have already identified regions of resistance in P. palmivora and P. capsici, which occur elsewhere. Our work is the first to do the same with P. citrophthora.
In addition, it was necessary to do this long study to identify and confirm the existence of resistance genes to the three fungi in the cacao plantations belonging to the cacao breeding programs in Brazil, and specifically in Bahia.
contacts for interviews:
Anete Pereira de Souza
Unicamp, Campinas (SP)
Phone: (5519) 3521-1132
Mobile: (5519) 99111-6547
e-mail: anete@unicamp.br
Mariana Barreto
Universidade Estadual de Santa Cruz (UESC), Ilhéus (BA)
Mobile: (5571) 98862-8672
e-mail: maribiologista@gmail.com
Edna Dora Luz - cacao specialist
Centro de Pesquisa do Cacau (CEPLAC/CEPEC), Ilhéus (BA)
Mobile: (5573) 98831-1595
email: ednadora@yahoo.com.br
Press agent: Peter Moon
Mobile: 5511 95630-9353
Barreto, M.A., Rosa, J.R.B.F., Holanda, I.S.A. et al. 2018. QTL mapping and identification of corresponding genomic regions for black pod disease resistance to three Phytophthora species in Theobroma cacao L. Euphytica (2018) 214:188
https://doi.org/10.1007/s10681-018-2273-5
Barreto, M.A., Santos, J.C.S., Corrêa, R.X. et al. 2015. Detection of genetic resistance to cocoa black pod disease caused by three Phytophthora species. Euphytica (2015) 206: 677.
https://doi.org/10.1007/s10681-015-1490-4
https://doi.org/10.1007/s10681-015-1490-4
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