Identification of southern corn rust resistance QTNs in Chinese summer maize germplasm via multi-locus GWAS and post-GWAS analysis.

Southern corn rust (SCR) caused by Puccinia polysora Underw is a major disease leading to severe yield losses in China Summer Corn Belt. Using six multi-locus GWAS methods, we identified a set of SCR resistance QTNs from a diversity panel of 140 inbred lines collected from China Summer Corn Belt. Thirteen QTNs on chromosomes 1, 2, 4, 5, 6, and 8 were grouped into three types of allele effects and their associations with SCR phenotypes were verified by post-GWAS case-control sampling, allele/haplotype effect analysis. Relative resistance (RRR) and relative susceptibility (RRs) catering to its inbred carrier were estimated from single QTN and QTN-QTN combos and epistatitic effects were estimated for QTN-QTN combos. By transcriptomic annotation, a set of candidate genes were predicted to be involved in transcriptional regulation (S5_145, Zm00001d01613, transcription factor GTE4), phosphorylation (S8_123, Zm00001d010672, Pgk2- phosphoglycerate kinase 2), and temperature stress response (S6_164a/S6_164b, Zm00001d038806, hsp101, and S5_211, Zm00001d017978, cellulase25). The breeding implications of the above findings were discussed.

Clonal Expansion and Dispersal Pathways of Puccinia polysora in China.

Southern corn rust (SCR) caused by Puccinia polysora is one of the most devastating diseases in the world. In recent years, SCR has been upgraded from a minor to a major disease around the world, including in China. However, little is known about its population genetics and structure in China. In this study, we analyzed 288 isolates collected from various localities during 2017 in seven Chinese provinces: Guangxi, Guangdong, Anhui, Hunan, Shandong, Henan, and Shaanxi. The isolates were analyzed using nine microsatellite markers. The population structure, genetic diversity, and reproduction mode of P. polysora were investigated based on genotype data. Strong genotypic diversity was detected and clonal reproduction was dominant. The populations collected from the pathogen's winter-reproductive regions harbored more genotypes than those collected from the pathogen's epidemic regions. The spatial differences in genotypic richness, and evenness among the populations were significant, and showed a decreasing trend from south to north. Most isolates were clustered into two clonal groups. Two high-frequency multilocus genotypes (MLGs), MLG1 and MLG2, were widely distributed in all populations. Our analyses confirmed that P. polysora employed clone dispersal from the pathogen's winter-reproductive regions to the pathogen's epidemic regions, and in addition to the sources from the pathogen's winter-reproductive regions, the pathogen in Anhui and Hunan might also have other sources from areas such as Taiwan, China, or/and Southeast Asia, and the pathogen went through a genetic bottleneck during its dispersal. These findings provide initial insights into the reproduction mode and dispersal pathways of P. polysora in China.

How omnivory affects the survival and choices of earwig Doru luteipes (Scudder) (Dermaptera: Forficulidae)?

Abstract Doru luteipes (Scudder, 1876) is an omnivorous predator that finds different food resources in the corn plant: eggs of Spodoptera frugiperda (J.E. Smith, 1797), uredospores of Puccinia polysora (Underw, 1897), and pollen. Knowing the survival and food preferences of this predator is essential to define its relevance as a biological control agent. We hypothesize that the foraging behavior and predatory capacity of D. luteipes may be affected when several food resources, especially eggs of S. frugiperda, uredospores of P. polysora, and pollen are concurrently in the same plant. The survival of D. luteipes in the nymph stage and their preference among food resources, often available in corn plants, were determined. To verify the survival of D. luteipes, newly hatched nymphs were fed exclusively with 1- uredospores of P. polysora, 2- eggs of S. frugiperda, 3- corn pollen, 4- a combination of uredospores + eggs, and 5- artificial diet (control). In another experiment, nymphs and adults of D. luteipes with 24 and 48 hours of fasting were individually released in the center of a container with four diets: 1- uredospores of P. polysora, 2- eggs of S. frugiperda, 3- corn pollen, 4- artificial diet, and maintained for 10 minutes, to evaluate the food choice and feeding time. The exclusive feeding with S. frugiperda eggs caused low nymph survival (8%), but the combination of P. polysora uredospores + S. frugiperda eggs allowed 58.3% survival. D. luteipes preferred feeding during the nighttime and the most significant proportions of choices by nymphs and adults were for pollen and diet, with adults spending more time eating pollen. These findings indicate that the trophic choices of D. luteipes are relevant to understand its contribution as an agent to control pest insects and fungal diseases in corn.

Resumo Doru luteipes (Scudder, 1876) é um predador onívoro, que encontra na planta do milho diferentes recursos alimentares: ovos de Spodoptera frugiperda (J.E. Smith, 1797), uredósporos de Puccinia polysora (Underw, 1897) e pólen. Para definição da relevância desse predador como agente de controle biológico, conhecer a sobrevivência e preferência alimentar é essencial. Nós hipotetizamos que o comportamento de forrageamento e a capacidade de predação de D. luteipes podem ser afetados quando uma mesma planta oferece ovos, uredósporos e pólen, concomitantemente. A sobrevivência de D. luteipes na fase de ninfa e sua preferência entre os recursos alimentares, frequentemente disponíveis nas plantas de milho, foram determinados. Para verificar a sobrevivência de D. luteipes, ninfas recém eclodidas foram alimentadas exclusivamente com 1- uredósporos de P. polysora, 2- ovos de S. frugiperda, 3- pólen de milho, 4- combinação de uredósporos + ovos e 5- dieta artificial (controle). Em outro experimento, ninfas e adultos de D. luteipes com 24 e 48 horas de jejum foram liberadas individualmente, em recipientes contendo quatro dietas: 1- uredósporos de P. polysora, 2- ovos de S. frugiperda, 3- pólen de milho, 4- dieta artificial e mantidas durante 10 min, sendo avaliados a escolha pelo alimento e o tempo de alimentação. A alimentação exclusiva com ovos de S. frugiperda ocasionou baixa sobrevivência das ninfas (8%), porém a combinação de uredósporos de P. polysora + ovos de S. frugiperda possibilitou sobrevivência de 58,3%. D. luteipes preferiu se alimentar durante o período noturno e as maiores proporções de escolhas das ninfas e dos adultos ocorreram no pólen e na dieta, sendo que os adultos gastaram mais tempo se alimentando de pólen. Estas descobertas indicam que as escolhas tróficas de D. luteipes são relevantes para compreender sua contribuição como agente de controle de insetos-praga e doença fúngicas em milho.

How omnivory affects the survival and choices of earwig Doru luteipes (Scudder) (Dermaptera: Forficulidae)? / Como a onivoria afeta a sobrevivência e escolhas da tesourinha Doru luteipes (Scudder) (Dermaptera: Forficulidae)?

Doru luteipes (Scudder, 1876) is an omnivorous predator that finds different food resources in the corn plant: eggs of Spodoptera frugiperda (J.E. Smith, 1797), uredospores of Puccinia polysora (Underw, 1897), and pollen. Knowing the survival and food preferences of this predator is essential to define its relevance as a biological control agent. We hypothesize that the foraging behavior and predatory capacity of D. luteipes may be affected when several food resources, especially eggs of S. frugiperda, uredospores of P. polysora, and pollen are concurrently in the same plant. The survival of D. luteipes in the nymph stage and their preference among food resources, often available in corn plants, were determined. To verify the survival of D. luteipes, newly hatched nymphs were fed exclusively with 1- uredospores of P. polysora, 2- eggs of S. frugiperda, 3- corn pollen, 4- a combination of uredospores + eggs, and 5- artificial diet (control). In another experiment, nymphs and adults of D. luteipes with 24 and 48 hours of fasting were individually released in the center of a container with four diets: 1- uredospores of P. polysora, 2- eggs of S. frugiperda, 3- corn pollen, 4- artificial diet, and maintained for 10 minutes, to evaluate the food choice and feeding time. The exclusive feeding with S. frugiperda eggs caused low nymph survival (8%), but the combination of P. polysora uredospores + S. frugiperda eggs allowed 58.3% survival. D. luteipes preferred feeding during the nighttime and the most significant proportions of choices by nymphs and adults were for pollen and diet, with adults spending more time eating pollen. These findings indicate that the trophic choices of D. luteipes are relevant to understand its contribution as an agent to control pest insects and fungal diseases in corn.

Doru luteipes (Scudder, 1876) é um predador onívoro, que encontra na planta do milho diferentes recursos alimentares: ovos de Spodoptera frugiperda (J.E. Smith, 1797), uredósporos de Puccinia polysora (Underw, 1897) e pólen. Para definição da relevância desse predador como agente de controle biológico, conhecer a sobrevivência e preferência alimentar é essencial. Nós hipotetizamos que o comportamento de forrageamento e a capacidade de predação de D. luteipes podem ser afetados quando uma mesma planta oferece ovos, uredósporos e pólen, concomitantemente. A sobrevivência de D. luteipes na fase de ninfa e sua preferência entre os recursos alimentares, frequentemente disponíveis nas plantas de milho, foram determinados. Para verificar a sobrevivência de D. luteipes, ninfas recém eclodidas foram alimentadas exclusivamente com 1- uredósporos de P. polysora, 2- ovos de S. frugiperda, 3- pólen de milho, 4- combinação de uredósporos + ovos e 5- dieta artificial (controle). Em outro experimento, ninfas e adultos de D. luteipes com 24 e 48 horas de jejum foram liberadas individualmente, em recipientes contendo quatro dietas: 1- uredósporos de P. polysora, 2- ovos de S. frugiperda, 3- pólen de milho, 4- dieta artificial e mantidas durante 10 min, sendo avaliados a escolha pelo alimento e o tempo de alimentação. A alimentação exclusiva com ovos de S. frugiperda ocasionou baixa sobrevivência das ninfas (8%), porém a combinação de uredósporos de P. polysora + ovos de S. frugiperda possibilitou sobrevivência de 58,3%. D. luteipes preferiu se alimentar durante o período noturno e as maiores proporções de escolhas das ninfas e dos adultos ocorreram no pólen e na dieta, sendo que os adultos gastaram mais tempo se alimentando de pólen. Estas descobertas indicam que as escolhas tróficas de D. luteipes são relevantes para compreender sua contribuição como agente de controle de insetos-praga e doença fúngicas em milho.

How omnivory affects the survival and choices of earwig Doru luteipes (Scudder) (Dermaptera: Forficulidae)? / Como a onivoria afeta a sobrevivência e escolhas da tesourinha Doru luteipes (Scudder) (Dermaptera: Forficulidae)?

Abstract Doru luteipes (Scudder, 1876) is an omnivorous predator that finds different food resources in the corn plant: eggs of Spodoptera frugiperda (J.E. Smith, 1797), uredospores of Puccinia polysora (Underw, 1897), and pollen. Knowing the survival and food preferences of this predator is essential to define its relevance as a biological control agent. We hypothesize that the foraging behavior and predatory capacity of D. luteipes may be affected when several food resources, especially eggs of S. frugiperda, uredospores of P. polysora, and pollen are concurrently in the same plant. The survival of D. luteipes in the nymph stage and their preference among food resources, often available in corn plants, were determined. To verify the survival of D. luteipes, newly hatched nymphs were fed exclusively with 1- uredospores of P. polysora, 2- eggs of S. frugiperda, 3- corn pollen, 4- a combination of uredospores + eggs, and 5- artificial diet (control). In another experiment, nymphs and adults of D. luteipes with 24 and 48 hours of fasting were individually released in the center of a container with four diets: 1- uredospores of P. polysora, 2- eggs of S. frugiperda, 3- corn pollen, 4- artificial diet, and maintained for 10 minutes, to evaluate the food choice and feeding time. The exclusive feeding with S. frugiperda eggs caused low nymph survival (8%), but the combination of P. polysora uredospores + S. frugiperda eggs allowed 58.3% survival. D. luteipes preferred feeding during the nighttime and the most significant proportions of choices by nymphs and adults were for pollen and diet, with adults spending more time eating pollen. These findings indicate that the trophic choices of D. luteipes are relevant to understand its contribution as an agent to control pest insects and fungal diseases in corn.

Resumo Doru luteipes (Scudder, 1876) é um predador onívoro, que encontra na planta do milho diferentes recursos alimentares: ovos de Spodoptera frugiperda (J.E. Smith, 1797), uredósporos de Puccinia polysora (Underw, 1897) e pólen. Para definição da relevância desse predador como agente de controle biológico, conhecer a sobrevivência e preferência alimentar é essencial. Nós hipotetizamos que o comportamento de forrageamento e a capacidade de predação de D. luteipes podem ser afetados quando uma mesma planta oferece ovos, uredósporos e pólen, concomitantemente. A sobrevivência de D. luteipes na fase de ninfa e sua preferência entre os recursos alimentares, frequentemente disponíveis nas plantas de milho, foram determinados. Para verificar a sobrevivência de D. luteipes, ninfas recém eclodidas foram alimentadas exclusivamente com 1- uredósporos de P. polysora, 2- ovos de S. frugiperda, 3- pólen de milho, 4- combinação de uredósporos + ovos e 5- dieta artificial (controle). Em outro experimento, ninfas e adultos de D. luteipes com 24 e 48 horas de jejum foram liberadas individualmente, em recipientes contendo quatro dietas: 1- uredósporos de P. polysora, 2- ovos de S. frugiperda, 3- pólen de milho, 4- dieta artificial e mantidas durante 10 min, sendo avaliados a escolha pelo alimento e o tempo de alimentação. A alimentação exclusiva com ovos de S. frugiperda ocasionou baixa sobrevivência das ninfas (8%), porém a combinação de uredósporos de P. polysora + ovos de S. frugiperda possibilitou sobrevivência de 58,3%. D. luteipes preferiu se alimentar durante o período noturno e as maiores proporções de escolhas das ninfas e dos adultos ocorreram no pólen e na dieta, sendo que os adultos gastaram mais tempo se alimentando de pólen. Estas descobertas indicam que as escolhas tróficas de D. luteipes são relevantes para compreender sua contribuição como agente de controle de insetos-praga e doença fúngicas em milho.

A comprehensive dynamic immune acetylproteomics profiling induced by Puccinia polysora in maize.

Lysine-ε-acetylation (Kac) is a reversible post-translational modification that plays important roles during plant-pathogen interactions. Some pathogens can deliver secreted effectors encoding acetyltransferases or deacetylases into host cell to directly modify acetylation of host proteins. However, the function of these acetylated host proteins in plant-pathogen defense remains to be determined. Employing high-resolution tandem mass spectrometry, we analyzed protein abundance and lysine acetylation changes in maize infected with Puccinia polysora (P. polysora) at 0 h, 12 h, 24 h, 48 h and 72 h. A total of 7412 Kac sites from 4697 proteins were identified, and 1732 Kac sites from 1006 proteins were quantified. Analyzed the features of lysine acetylation, we found that Kac is ubiquitous in cellular compartments and preferentially targets lysine residues in the -F/W/Y-X-X-K (ac)-N/S/T/P/Y/G- motif of the protein, this Kac motif contained proteins enriched in basic metabolism and defense-associated pathways during fungal infection. Further analysis of acetylproteomics data indicated that maize regulates cellular processes in response to P. polysora infection by altering Kac levels of histones and non-histones. In addition, acetylation of pathogen defense-related proteins presented converse patterns in signaling transduction, defense response, cell wall fortification, ROS scavenging, redox reaction and proteostasis. Our results provide informative resources for studying protein acetylation in plant-pathogen interactions, not only greatly extending the understanding on the roles of acetylation in vivo, but also providing a comprehensive dynamic pattern of Kac modifications in the process of plant immune response.

First Report of Southern Rust (Puccinia polysora) on Corn (Zea mays) in Michigan.

In Michigan, corn (Zea mays) is grown on 2.35 million acres with an annual production valued at $1.36 billion dollars (USDA-NASS). Southern rust is caused by the obligate biotrophic fungus Puccinia polysora Underw. and is often ranked in the top five most destructive corn diseases in the southern U.S. (Mueller et al. 2020). Yield losses due to southern rust in the northern U.S. have been considerable and estimated to be 231 million bushels from 2016 to 2019. In 2020 and 2021, corn leaf samples exhibiting signs typical of infection by P. polysora were collected from commercial production fields across Michigan. In 2020, samples were collected from two counties, Branch and Hillsdale. In 2021, samples were collected from 10 additional counties, Allegan, Barry, Calhoun, Eaton, Ingham, Kent, Montcalm, Shiawassee, Tuscola, and Van Buren. Uredinia of P. polysora were observed aggregated primarily on the upper leaf surface, light cinnamon brown to bright orange, ovular, and surrounded by yellow halos. Urediniospores were yellow to gold in the center, hyaline along the outer membrane, ellipsoid to obovoid, with short echinulations on the surface when viewed at 40x magnification under a light microscope. Urediniospores (n=180) measured 17 to 28 x 26 to 38 µm. No telia were observed. To confirm identification, the large subunit (28S) rDNA was amplified and sequenced with rust specific primers as described in Aime (2006) and Aime et al. (2018) on a sample collected from Shiawassee County in 2021. The resulting DNA sequence (GenBank Accession No. OL468037) shared 99.79% identity (932/934 bp) with P. polysora voucher BPI 863756 (GenBank Accession No. GU058024; Dixon et al. 2010). This report serves as the first documentation of southern rust in Michigan and an initial survey of its distribution throughout the state. Because of the ability of P. polysora spores to travel long distances via wind (Cammack 1959), it is likely that disease was present but not detected in additional counties not included in this report. Although widespread yield losses due to southern rust have not been documented in Michigan, there have been anecdotal reports of 30 bushels per acre losses in grain corn and 30% loss of tonnage from silage fields. Understanding the distribution of southern rust can help inform future disease management and corn breeding research. Furthermore, the distribution of southern rust is projected to move poleward by 15° by 2100 due to increasing global temperatures (Ramirez-Cabral et al. 2017), and the movement of southern rust into northern corn growing regions should be documented. Additional Michigan counties with confirmations of southern rust will continue to be reported via the corn IPMpipe https://corn.ipmpipe.org/.

Quantitative Resistance Loci to Southern Rust Mapped in a Temperate Maize Diversity Panel.

Southern rust is a severe foliar disease of maize (Zea mays) resulting from infection with the obligate biotrophic fungus Puccinia polysora. This disease reduces photosynthetic productivity, which in turn reduces yields, with the greatest yield losses (up to 50%) associated with earlier onset infections. P. polysora urediniospores overwinter only in tropical and subtropical regions but cause outbreaks when environmental conditions favor initial infection. Increased temperatures and humidity during the growing season combined with an increased frequency of moderate winters are likely to increase the frequency of severe southern rust outbreaks in the U.S. Corn Belt. In summer 2020, a severe outbreak of southern rust was observed in eastern Nebraska, United States. We scored a replicated maize association panel planted in Lincoln, NE for disease severity and found that disease incidence and severity showed significant variation among maize genotypes. Genome-wide association studies identified four loci associated with significant quantitative variation in disease severity. These loci were associated with candidate genes with plausible links to quantitative disease resistance. A transcriptome-wide association study identified additional genes associated with disease severity. Together, these results indicate that substantial diversity in resistance to southern rust exists among current temperate-adapted maize germplasm, including several candidate loci that may explain the observed variation in resistance to southern rust.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Multiyear Evaluation of Fungicide Efficacy and Application Timing for Control of Southern Rust in Hybrid Corn in Arkansas.

The efficacy and timing of eight foliar fungicides to manage southern rust of corn (caused by Puccinia polysora Underwood) was investigated over 4 years in three field experiments. Each experiment consisted of one-, two-, or three-fungicide application timings at tassel, milk, or dent growth stages with quinone outside inhibitor (QoI), demethylation inhibitor (DMI), or QoI + DMI fungicides. Each year trace amounts of southern rust were observed in the field at tassel, except in 2018, when rust was not observed until physiological maturity. Southern rust severity on ear leaf and two leaves above the ear leaf was approximately 50, 35, 75, and 0% at dent in 2015, 2016, 2017, and 2018, respectively. Applications that contained a QoI or QoI + DMI fungicide provided greater southern rust control than DMI fungicides, with little variation within fungicide classes. Applications of QoI or QoI + DMI fungicides applied at tassel provided greater disease control (52.5%) than those applied at milk (5.8%) or dent (1.4%), and greater yield protection (40.4%) than those applied at milk (23.7%) or dent (2.6%) when final rust development was severe (>40%). When rust development increased later in the season, after milk growth stage, a trend of better disease control was observed with fungicides applied at milk (57.8%) compared with tassel (35.2%), but grain yield protection was similar, with an average yield protection of 7.4%. There was no yield benefit with fungicides applied in the absence of disease or at the dent growth stage. Southern rust was most effectively managed with QoI or QoI + DMI fungicides applied at tassel when southern rust was present and environmental conditions favored rust development.

Identification and Fine Mapping of RppM, a Southern Corn Rust Resistance Gene in Maize.

Southern corn rust (SCR) caused by Puccinia polysora Underw. is a major disease causing severe yield losses during maize production. Here, we identified and mapped the SCR resistance gene RppM from the near-isogenic line Kangxiujing2416 (Jing2416K), which harbors RppM in the genetic background of the susceptible inbred line Jing2416. In this study, the inheritance of SCR resistance was investigated in F2 and F3 populations derived from a cross between Jing2416K and Jing2416. The observed 3:1 segregation ratio of resistant to susceptible plants indicated that the SCR resistance is controlled by a single dominant gene. Using an F2 population, we performed bulked segregant analysis (BSA) sequencing and mapped RppM to a 3.69-Mb region on chromosome arm 10S. To further narrow down the region harboring RppM, we developed 13 insertion/deletion (InDel) markers based on the sequencing data. Finally, RppM was mapped to a region spanning 110-kb using susceptible individuals from a large F2 population. Two genes (Zm00001d023265 and Zm00001d023267) encoding putative CC-NBS-LRR (coiled-coiled, nucleotide-binding site, and leucine-rich repeat) proteins, a common characteristic of R genes, were located in this region (B73 RefGen_v4 reference genome). Sequencing and comparison of the two genes cloned from Jing2416K and Jing2416 revealed sequence variations in their coding regions. The relative expression levels of these two genes in Jing2416K were found to be significantly higher than those in Jing2416. Zm00001d023265 and Zm00001d023267 are thus potential RppM candidates.

Severity evaluation methods for southern polysora corn rust / Métodos de avaliação da severidade da ferrugem polysora do milho

Among the biotrophic organisms that attack the corn crop the most important for Central Brazil is the Puccinia polysora Underw, which is the causal agent of polysora corn rust. Due to the need to study the methods used in the assessments better, this work was performed. An experiment to identify the best methodology for assessing polysora rust was set up in Itumbiara ­ Goiás - Brazil, with 22 hybrids and an experimental randomized block design with three replications. So as to evaluate disease severity in relation to the leaf area affected, the Area Under the Disease Progress Curve (AUDPC) and a single evaluation were used, while also considering the severity of the disease on the leaf area affected, 30 days after flowering (30 d.a.f.) Both based on a proposal diagrammatic scale from Agroceres with grades ranging from one (highly resistant) to nine (highly susceptible), which were employed considering the plot as a whole. So as to calculate the AUDPC five evaluations of disease severity at intervals of 10 days which begun 50 days after sowing were carried out. It was found that it was possible to determine the level of genotype resistance by using the AUDPC or by using the single assessment at 30 d.a.f., with the assessment of disease severity by means of the AUDPC calculation allowing for better understanding of the evolution of the disease in time and that the one only or single assessment at 30 d.a.f. allows us to work with a greater number of genotypes, due to the practicality of the methodology.

Dentre os organismos biotróficos que atacam a cultura do milho o mais importante para o Brasil Central é a Puccinia polysora Underw, agente causal da ferrugem polysora do milho. Devido à necessidade de se estudar melhor os métodos utilizados nas avaliações, realizou-se este trabalho. Um experimento para identificar a melhor metodologia para avaliação da ferrugem polysora foi montado em Itumbiara ­ Goiás, com 22 híbridos e o delineamento experimental de blocos casualizados com três repetições. Utilizou-se para as avaliações da severidade da doença em relação à área foliar afetada, a Área Abaixo da Curva de Progresso da Doença (AACPD) e a avaliação única, considerando também a severidade da doença em relação à área foliar afetada, aos 30 dias após florescimento (30 d.a.f.). Ambas baseadas na escala diagramática da Agroceres, com notas variando de um (altamente resistente) a nove (altamente suscetível), que foi empregada considerando a parcela com um todo. Para o cálculo da AACPD foram realizadas cinco avaliações da severidade da doença a intervalos de 10 dias e iniciadas aos 50 dias após semeadura. Verificou-se que tanto pela AACPD ou pela avaliação única aos 30 d.a.f. foi possível a determinação do nível de resistência dos genótipos, sendo que a avaliação da severidade da doença através do cálculo da AACPD permite entender melhor a evolução da doença no tempo e a avaliação única aos 30 d.a.f. nos permite trabalhar com um maior número de genótipos, devido à praticidade da metodologia.