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The use of resistant cultivars is the most effective strategy for controlling coffee leaf rust caused by the fungus Hemileia vastatrix. To assist the development of such cultivars, amplified fragment-length polymorphism (AFLP) markers linked to two loci of coffee resistance to races I and II as well as pathotype 001 of H. vastatrix were converted to sequence-characterized amplified region (SCAR) and cleaved amplified polymorphic site (CAPS) markers. In total, 2 SCAR markers and 1 CAPS marker were validated in resistant and susceptible parents as well as in 247 individuals from the F2 population. The efficiency of these markers for marker-assisted selection (MAS) was evaluated in F2:3 and backcross (BCrs2) populations genotyped with the developed markers and phenotyped with race II of H. vastatrix. The markers showed 90% efficiency in MAS. Therefore, the developed markers, together with molecular markers associated with other rust resistance genes, were used for F3:4 and BCrs3 coffee selection. The selected plants were analyzed using two markers associated with coffee berry disease (CBD) resistance, aiming for preventive breeding. MAS of F3:4 and BCrs3 individuals with all resistance loci was feasible. Our phenotypic and genotypic approaches are useful for the development of coffee genotypes with multiple genes conferring resistance to coffee leaf rust and CBD. Made available in DSpace on 2022-01-27T01:59:16Z (GMT). No. of bitstreams: 1 marker-assisted-pyramiding.pdf: 22779626 bytes, checksum: 7a76b7c036947b04bf1229d56a0e8c4e (MD5) Previous issue date: 2021

Soil borne fungi can be very importan on coffee plantations and in the nursery. One of the key measure to get vigorous plants in the field is the quality of the seedling and the site of planting. The great majority of the diseases on seedlings came from the nursery. Another source of pathogens is to plant in soil that has suffered clearing of forests. The soil's organic matter contains soil pathogens that attack coffee seedlings after planting. Sometimes plants can die. The main soil borne disease of coffee are: Rhizoctoniosis, Rosellinia root rot, Ceratocystis canker stain and Coffee Wilt Disease. For these reasons the main measures of integrated control is exclusion of the pathogen and avoidance.

One of the objectives of integrated production (IP) are rationalizing the use of pesticides. For conilon coffee (Coffea canephora), a coffee leaf rust (Hemileia vastatrix) management system based on IP principles needs to be proposed and validated. Thus, this study aimed to quantify the economic and environmental effects of rust management based on disease monitoring for decision-making regarding fungicide use in conilon coffee (integrated coffee production, ICP-Conilon). The ICP-Conilon system was compared with the conventional rust management system (CS) based on a fixed schedule of fungicide application by delineating two plots of C. canephora clonal fields. The incidence of leaf rust in the plants from each clone was quantified monthly and, in the ICP-Conilon plots, systemic fungicide was applied only to the clones/planting rows with monthly disease incidence of ≥5%. The ICP-Conilon system resulted in an average reduction of 71.3% in the fungicide volume applied/ha/year, compared to that in the CS plots. Thus, it was possible to reduce, on average, 21% of the rust management cost in clonal conilon coffee plantations.

Genomic selection (GS) emphasizes the simultaneous prediction of the genetic effects of thousands of scattered markers over the genome. Several statistical methodologies have been used in GS for the prediction of genetic merit. In general, such methodologies require certain assumptions about the data, such as the normality of the distribution of phenotypic values. To circumvent the non-normality of phenotypic values, the literature suggests the use of Bayesian Generalized Linear Regression (GBLASSO). Another alternative is the models based on machine learning, represented by methodologies such as Artificial Neural Networks (ANN), Decision Trees (DT) and related possible refinements such as Bagging, Random Forest and Boosting. This study aimed to use DT and its refinements for predicting resistance to orange rust in Arabica coffee. Additionally, DT and its refinements were used to identify the importance of markers related to the characteristic of interest. The results were compared with those from GBLASSO and ANN. Data on coffee rust resistance of 245 Arabica coffee plants genotyped for 137 markers were used. The DT refinements presented equal or inferior values of Apparent Error Rate compared to those obtained by DT, GBLASSO, and ANN. Moreover, DT refinements were able to identify important markers for the characteristic of interest. Out of 14 of the most important markers analyzed in each methodology, 9.3 markers on average were in regions of quantitative trait loci (QTLs) related to resistance to disease listed in the literature. Made available in DSpace on 2020-10-16T09:14:16Z (GMT). No. of bitstreams: 1 Sousa-et-al-2020.pdf: 564113 bytes, checksum: 4574b3c97e267472a1844b5733671891 (MD5) Previous issue date: 2021

The biotrophic fungus Hemileia vastatrix causes coffee leaf rust (CLR), one of the most devastating diseases in Coffea arabica . Coffee, like other plants, has developed effective mechanisms to recognize and respond to infections caused by pathogens. Plant resistance gene analogs (RGAs) have been identified in certain plants as candidates for resistance ( R ) genes or membrane receptors that activate the R genes. The RGAs identified in different plants possess conserved domains that play specific roles in the fight against pathogens. Despite the importance of RGAs, in coffee plants these genes and other molecular mechanisms of disease resistance are still unknown. This study aimed to sequence and characterize candidate genes from coffee plants with the potential for involvement in resistance to H. vastatrix . Sequencing was performed based on a library of bacterial artificial chromosomes (BAC) of the coffee clone ?Híbrido de Timor? (HdT) CIFC 832/2 and screened using a functional marker. Two RGAs, HdT_LRR_RLK1 and HdT_LRR_RLK2, containing the motif of leucine-rich repeat-like kinase (LRR-RLK) were identified. Based on the presence or absence of the HdT_LRR_RLK2 RGA in a number of differential coffee clones containing different combinations of the rust resistance gene, these RGAs did not correspond to any resistance gene already characterized (SH1-9). These genes were also analyzed using qPCR and demonstrated a major expression peak at 24 h after inoculation in both the compatible and incompatible interactions between coffee and H. vastatrix . These results are valuable information for breeding programs aimed at developing CLR-resistant cultivars, in addition to enabling a better understanding of the interactions between coffee and H. vastatrix. Made available in DSpace on 2022-01-26T14:00:41Z (GMT). No. of bitstreams: 1 receptor-like-kinase.pdf: 797260 bytes, checksum: 18841c8c3f9b08408ff2d898ecc48880 (MD5) Previous issue date: 2021

Genomic Selection (GS) has allowed the maximization of genetic gains per unit time in several annual and perennial plant species. However, no GS studies have addressed Coffea arabica, the most economically important species of the genus Coffea. Therefore, this study aimed (i) to evaluate the applicability and accuracy of GS in the prediction of the genomic estimated breeding value (GEBV); (ii) to estimate the genetic parameters; and (iii) to evaluate the time reduction of the selection cycle by GS in Arabica coffee breeding. A total of 195 Arabica coffee individuals, belonging to 13 families in generation of F2, susceptible backcross and resistant backcross, were phenotyped for 18 agronomic traits, and genotyped with 21,211 SNP molecular markers. Phenotypic data, measured in 2014, 2015, and 2016, were analyzed by mixed models. GS analyses were performed by the G-BLUPmethod, using the RKHS (Reproducing Kernel Hilbert Spaces) procedure, with a Bayesian algorithm. Heritabilities and selective accuracies were estimated, revealing moderate to high magnitude for most of the traits evaluated. Results of GS analyses showed the possibility of reducing the cycle time by 50%, maximizing selection gains per unit time. The effect of marker density on GS analyses was evaluated. Genomic selection proved to be promising for C. arabica breeding. The agronomic traits presented high complexity for they are controlled by several QTL and showed low genomic heritabilities, evidencing the need to incorporate genomic selection methodologies to the breeding programs of this species. Made available in DSpace on 2019-03-09T00:34:09Z (GMT). No. of bitstreams: 1 Earlyselectionenabledbytheimplementation.pdf: 585900 bytes, checksum: fde1543a047334f2e1fa8511738aba4e (MD5) Previous issue date: 2019-03-08

Híbrido de Timor is the principal source for disease and pest resistance genes in C. arabica breeding program worldwide. The part of the chromosome responsible for resistance introgressed from C. canephora to Híbrido de Timor are claimed to affect the cup quality of the C. arabica cultivars derived from the crossing program of Híbrido de Timor. Therefore, this work was done to study the genome introgression of Híbrido de Timor and its impact on the cup quality on the C. arabica cultivars. For genome introgression and genetic relationship analysis, seventy-six accessions from C. arabica, C. canephora and Híbrido de Timor were analyzed using AFLP and SSR molecular markers. To understand the effect of genome introgressed from Híbrido de Timor on cup quality, three C. arabica, seven Híbrido de Timor and six cultivars derived from Híbrido de Timor × C. arabica were genotyped using SSR molecular markers and sensorial analysis was performed. The genetic diversity analysis among the tested genotypes showed high genetic similarity between Híbrido de Timor with C. arabica and clear differentiation among coffee species. The analysis of genome introgression of C. arabica and C. canephora var Robusta into Híbrido de Timor not reach 30% of C. canephora genome. The sensorial analysis of coffee genotypes demonstrated non-significant difference on cup quality parameters among C. arabica cv Bourbon and cultivars derived from Híbrido de Timor that showed the possibility of developing C. arabica cultivars without affecting the cup quality. Similarly, the SSR marker diversity showed high genetic similarity between the Bourbon and the C. arabica cultivars derived from Híbrido de Timor. Made available in DSpace on 2020-10-08T09:14:01Z (GMT). No. of bitstreams: 1 Setotaw-et-al-2020-1.pdf: 713983 bytes, checksum: 13e69a86eb54c40482aa1060a44dbf0f (MD5) Previous issue date: 2020

The biotrophic fungus Hemileia vastatrix Berk & Broome is the most destructive coffee pathogen in Brazil. Better understanding of the population genetics of H. vastatrix would provide important insights into its biology, epidemiology, and evolutionary potential. The aim of the present study was to assess the genetic diversity and population structure of H. vastatrix in Minas Gerais (Brazil) using ribosomal DNA (rDNA) sequences. The analyzes were performed by sequencing the internal transcribed spacers ITS1 and ITS2, and the 5.8S gene from 15 H. vastatrix populations. Of the 82 sequences obtained, 68 ribotypes were found, as defined by 108 nucleotide substitutions and five indels. Of the 68 ribotypes, 64 were exclusively found in one population. Analysis of molecular variance (AMOVA) and FST fixation index indicated moderate genetic differentiation among field populations, which were divided according to geographic origin. In conclusion, analysis of the nuclear ITS1?5.8S-ITS2 rDNA sequence diversity in the H. vastatrix population of Minas Gerais revealed that most ribotypes are restricted to a single population and that there exists greater genetic diversity within than among field populations. Made available in DSpace on 2020-10-08T09:13:15Z (GMT). No. of bitstreams: 1 Santana-et-al-2018-1.pdf: 367751 bytes, checksum: 884fd5bf84adea9f3d7d4764589ab727 (MD5) Previous issue date: 2018

Clubroot of crucifers is a root disease caused by the fungus Plasmodiophora brassicae, responsible for causing more than 40% economic damage to brassica crops. This disease has led to a gradual decline in brassica production in several countries, including Brazil, where an approximate volume of 50 thousand tons are marketed every year. There are few studies involving the control of this disease, which led to the development of an experiment with four treatments: T1. negative control, T2. biostimulant (phosphorus + auxin + fulvic acid), T3. biostimulant + fungicide (phosphorus + auxin + fulvic acid + cyazofamid) and T4. fungicide (cyazofamid). The lowest incidence value obtained in the last evaluation was 21% (79% control) with the mixture of biostimulant and fungicide; the biostimulant alone did not significantly differ from the negative control, considering the condition of high inoculum density, which serves as a basis for future studies with a mixture of new compounds and biocontrol agents compatible with the fungicide.

Over the years, breeding programs have sought efficient strategies to select genotypes with superior performance. Genome-wide selection (GWS) emerged in 2001, aiming to increase efficiency and accelerate selection gain. This technique is considered essential in the breeding of perennial species, such as Coffea canephora, mainly due to the potential to increase the gain per unit of time. Thus, this study aimed to apply the GWS principle, evaluate the efficiency of this technique in C. canephora population using SNP molecular markers, and evaluate eight main phenotypic traits. A total of 165 genotypes were evaluated, being 51 of varietal group of Conilon, 32 of Robusta, and 82 intervarietal hybrids. Through the sequencing of the RAPiD Genomics company, 18,111 SNP markers were identified, of which 14,429 were used after quality analysis. All traits showed good predictive capacity, except for fruit maturation time, fruit size, and yield per plant. The lower values of genomic heritability found for these traits may justify the low values of predictive capacity obtained. The accuracy values estimated were considered as moderate to high, ranging from 67 to 82%. By shortening the cycle time from 6 to 3 years, GWS provided selective efficiency ranging from 22 to 146%. Results revealed that GWS provides higher gains per unit of time. Therefore, GWS proved to be a useful and promising tool for the breeding of C. canephora for accurately predicting the individuals’ genotypes, shortening the time required to complete the selection cycle and providing gains in selective efficiency per unit of time.

Twelve tetraphenylphosphonium allyldithiocarbimates were synthesized and fully characterized by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), infrared and nuclear magnetic resonance (NMR) spectroscopies. The spectroscopic data indicated that the allyldithiocarbimate anions present Z configuration, as confirmed by X-ray crystallography. These new compounds inhibited the germination of Phakopsora pachyrhizi and Hemileia vastatrix at very low doses, with IC50 (concentration to achieve 50% of inhibition of spore germination) values ranging from 0.028 to 0.166 mmol L-1, and IC90 (concentration to achieve 90% of inhibition of spore germination) values varying from 0.156 to 0.528 mmol L-1. Allyldithiocarbimate salts are lead compounds for the development of new antifungals for coffee leaf rust and Asian soybean rust, devastating plant diseases with limited control options.

Oligonychus ilicis (McGregor) (Acari: Tetranychidae), is one of the most important mite pests found in coffee plants. One way to reduce intensive pest attacks is management via resistant plants. The purpose of this study was to evaluate the resistance of 14 C. canephora genotypes to O. ilicis by antixenosis and antibiosis mechanisms and to determine the preimaginal conditioning effect on resistant genotypes using O. ilicis reared on C. canephora. Resistance by antixenosis was not found; however, nine genotypes showed resistance to O. ilicis via an antibiosis mechanism. Two of these resistant genotypes showed a more persistent resistance than the O. ilicis reared on C. canephora, which indicated that a previous host can affect the selection of a genotype by O. ilicis. The study may provide a model to evaluate the preimaginal conditioning effect on plant resistance, which can be useful for plant breeders.

A major problem in the strawberry production chain is the occurrence of fruit rot caused by fungi, thus, accurate identification of the pathogens associated with these rots is important for suitable management of diseases. During an assessment of postharvest diseases of strawberry plants, a rot caused by Calonectria was found. The surface of the infected fruits showed a slight leakage of liquid and the presence of mycelium with clearly defined hyphae containing a mass of conidiophores and conidia. Morphological examination was performed on isolates derived from single germinated conidium cultures, and phylogenetic analyses of four DNA loci (β-tubulin, histone H3, elongation factor and calmodulin) was undertaken. In order to confirm the pathogenicity, strawberry fruits and plants were dipped or sprayed with a conidial suspension. Strawberry fruits showed rot symptoms similar to those initially observed, and the plants developed leaf spot symptoms. The fungus was successfully reisolated from the infected tissues. Based on morphological and phylogenetic analysis, the fungus was shown to be distinct, and is described here as Calonectria fragariae sp. nov. This finding provides additional information for the better understanding of strawberry diseases in Brazil, enabling the development of more effective strategies of management of these diseases.

Coffee leaf rust (Hemileia vastatrix) and Asian soybean rust (Phakopsora pachyrhizi) are diseases that cause great losses in the productivity of these crops, not only in Brazil but on a global scale. Coffee and soybean varieties grown are susceptible to these diseases. Thus, it is necessary to search for efficient compounds for their chemical control, mainly from the group of protectors or residuals so that they can be formulated with systemic fungicides to control the diseases. This allows not only the efficient management of diseases but also the prevention of the emergence of resistant mutants in the populations of these pathogens. In this context, the present study aimed to evaluate the sensitivity and effect of bis(N-R-sulfonyldithiocarbimato)zincate(II) salts fungicides on the epidemiological components of pathosystems coffee × H. vastatrix and soybean × P. pachyrhizi. Initially, four zinc(II) complexes salts (1A, 2A, 1B, and 2B) with N-R-sulfonyldithiocarbimates were synthesized. In the first experiment, the in vitro sensitivity of H. vastatrix and P. pachyrhizi was studied for the four compounds synthesized and mancozeb at 0.5, 5.0, 50.0, 100.0 and 200.0 µmol L-1. All the compounds synthetized in this study had inhibitory effects on H. vastatrix and P. pachyrhizi. In the greenhouse it was studied the effect of bis(N-R-sulfonyldithiocarbimato)zincate(II) salts on the epidemiological components of coffee leaf rust and Asian soybean rust. For the pathosystem coffee × H. vastatrix, there were no differences in the values obtained for the bis(N-R-sulfonyldithiocarbimato)zincate(II) salts and mancozeb for the latent period. For the sporulated lesion variable, the control treatment had a mean value of 149.0 lesions/leaf, differing significantly from the other treatments. The mean value of compound 2B was estimated as 25.0 lesions/leaf, differing significantly from treatments 1A, 1B, 2B, and mancozeb. Treatments 1A, 1B, 2B, and mancozeb did not differ significantly from each other. For the Asian soybean rust, the area under the disease progress curve had a mean value of 75.8 for the control, while for the 2A treatment the value was 4.1, differing from the other compounds. The treatments 1A, 1B, 2A, and mancozeb did not differ significantly from each other. In conclusion, compounds 1A, 2A, and 1B were more efficient in the control of the coffee leaf rust, while compound 2A was efficient in the control of the Asian soybean rust.

Physiology-based differentiation of SH genes and Hemileia vastatrix races is the principal method employed for the characterization of coffee leaf rust resistance. Based on the gene-for-gene theory, nine major rust resistance genes (SH1-9) have been proposed. However, these genes have not been characterized at the molecular level. Consequently, the lack of molecular data regarding rust resistance genes or candidates is a major bottleneck in coffee breeding. To address this issue, we screened a BAC library with resistance gene analogs (RGAs), identified RGAs, characterized and explored for any SH related candidate genes. Herein, we report the identification and characterization of a gene (gene 11), which shares conserved sequences with other SH genes and displays a characteristic polymorphic allele conferring different resistance phenotypes. Furthermore, comparative analysis of the two RGAs belonging to CC-NBS-LRR revealed more intense diversifying selection in tomato and grape genomes than in coffee. For the first time, the present study has unveiled novel insights into the molecular nature of the SH genes, thereby opening new avenues for coffee rust resistance molecular breeding. The characterized candidate RGA is of particular importance for further biological function analysis in coffee. Made available in DSpace on 2020-07-15T11:11:10Z (GMT). No. of bitstreams: 1 in-silico-guided-structural.pdf: 2828975 bytes, checksum: 122284f694e73c0616a4956d5037e108 (MD5) Previous issue date: 2020

The use of molecular markers to detect polymorphism at DNA level is one of the most significant developments in molecular biology techniques. With the development of new next-generation sequencing technologies, the discovery of SNP became easier and faster, and the costs of data point were reduced. The development and use of SNP markers for coffee have provided new perspectives for the evaluation of genetic diversity and population structure via different statistical approaches. In this study, 72 Coffea canephora genotypes were analyzed to identify the SNP markers and apply them to genetic studies and selection of parents/hybrids in genetic breeding. As many as 117,450 SNP were identified using the RAPiD Genomics platform. After quality analyses, 33,485 SNP were validated for analyses of genetic diversity and population structure. Genotypes were separated based on their varietal groups, and Hybrids were differentiated using the clustering and Bayesian approach. Coffee accessions mistakenly identified in the germplasm and breeding program were detected. The Conilon varietal group presented the lowest genetic dissimilarity values, suggesting the introduction of new accessions in the germplasm bank. The highest genetic distances values were observed among genotypes of the heterotic groups (Conilon and Robusta). The markers were efficient in evaluating the genetic diversity and population structure of C. canephora. Promising crosses were selected within and between the varietal groups. Hybrids with greater genetic distances were selected, which were important for C. canephora breeding programs. Made available in DSpace on 2020-09-26T09:11:15Z (GMT). No. of bitstreams: 1 Alkimim-et-al-2018-1.pdf: 2838689 bytes, checksum: bc5a94f08d2d4dbe59d11b083d54f31e (MD5) Previous issue date: 2018

We provide a transcriptional profile of coffee rust interaction and identified putative up regulated resistant genes Coffee rust disease, caused by the fungus Hemileia vastatrix, is one of the major diseases in coffee throughout the world. The use of resistant cultivars is considered to be the most effective control strategy for this disease. To identify candidate genes related to different mechanism defense in coffee, we present a time-course comparative gene expression profile of Caturra (susceptible) and Híbrido de Timor (HdT, resistant) in response to H. vastatrix race XXXIII infection. The main objectives were to obtain a global overview of transcriptome in both interaction, compatible and incompatible, and, specially, analyze up-regulated HdT specific genes with inducible resistant and defense signaling pathways. Using both Coffea canephora as a reference genome and de novo assembly, we obtained 43,159 transcripts. At early infection events (12 and 24 h after infection), HdT responded to the attack of H. vastatrix with a larger number of up-regulated genes than Caturra, which was related to prehaustorial resistance. The genes found in HdT at early hours were involved in receptor-like kinases, response ion fluxes, production of reactive oxygen species, protein phosphorylation, ethylene biosynthesis and callose deposition. We selected 13 up-regulated HdT-exclusive genes to validate by real-time qPCR, which most of them confirmed their higher expression in HdT than in Caturra at early stage of infection. These genes have the potential to assist the development of new coffee rust control strategies. Collectively, our results provide understanding of expression profiles in coffee-H. vastatrix interaction over a time course in susceptible and resistant coffee plants.

The aim of this study was to evaluate the resistance of Botrytis cinerea to the fungicides currently used for its control in Brazil. Isolates of the fungus were collected from different strawberry-producing fields in the states of Espírito Santo, Minas Gerais, and São Paulo, Brazil. First, a total of 183 isolates were identified at the species level using specific primers for the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene. The isolates were grown on potato dextrose agar (PDA) containing the fungicides procymidone, iprodione, and thiophanate-methyl in different concentrations: 0.0 (control), 0.1; 1.0; 10.0; 100.0 and 1,000.0 μg∙mL−1. The percentage of mycelial growth inhibition was used to determine the effective concentration of the fungicide that was able to inhibit colony growth by 50% (EC50). Approximately 25.7% of the isolates were resistant to iprodione, 53.0% were resistant to procymidone, and 93.0% were resistant to thiophanate-methyl. Moreover, crossresistance and multiple resistance were verified, with 19.7% of the isolates showing resistance to 3 fungicides simultaneously. This finding explains the ineffectiveness of fungicides application to control gray mold in strawberry fields in Brazil and highlights the need for new strategies to manage this disease in the culture.

Physiology-based differentiation of SH genes and Hemileia vastatrix races is the principal method employed for the characterization of coffee leaf rust resistance. Based on the gene-for-gene theory, nine major rust resistance genes (SH1-9) have been proposed. However, these genes have not been characterized at the molecular level. Consequently, the lack of molecular data regarding rust resistance genes or candidates is a major bottleneck in coffee breeding. To address this issue, we screened a BAC library with resistance gene analogs (RGAs), identified RGAs, characterized and explored for any SH related candidate genes. Herein, we report the identification and characterization of a gene (gene 11), which shares conserved sequences with other SH genes and displays a characteristic polymorphic allele conferring different resistance phenotypes. Furthermore, comparative analysis of the two RGAs belonging to CC-NBS-LRR revealed more intense diversifying selection in tomato and grape genomes than in coffee. For the first time, the present study has unveiled novel insights into the molecular nature of the SH genes, thereby opening new avenues for coffee rust resistance molecular breeding. The characterized candidate RGA is of particular importance for further biological function analysis in coffee.

Incidence-severity relationships for coffee rust were studied to determine if the easily assessed incidence could be used to evaluate host resistance and fungicide treatment. At two locations in each of 3 years, the incidence of rust on 300 leaves was compared with two assessments of severity: (i) the average number of sporulating pustules per leaf, and (ii) the estimated leaf area with rust. For nine or 10 assessments in time at one location and pooled over 3 years, the average number of sporulating pustules per leaf (Y sp) was well related with the incidence of leaves with rust (X) as Y sp = 0.02982+ 0.017035X +0.000573X 2; R 2 = 0.87. The leaf area with rust (Y la) was also well related with incidence of leaves with rust as Y la = 0.001 − 0.01076X +0.008376X 2; R 2 = 0.92. For two independent data sets from a second location obtained over two seasons, the above models satisfactorily fit the relationships for the average sporulating pustules per leaf (R 2 = 0.97 and 0.96) and for the estimated leaf area with rust (R 2 = 0.95 and 0.98). Therefore, the readily determined incidence can be used to estimate both measures of disease severity of coffee rust.

There are more than 300 blackberry (Rubus) species worldwide. Rubus brasiliensis Mart. is a native Brazilian species found in tropical forests. In January 2009, samples of R. brasiliensis with severe leaf blight were collected from an area of rain forest in the city of São Miguel do Anta, State of Minas Gerais, Brazil. Dark spots began developing in the young leaves and progressed to necrotic spots with occasional twig dieback. From the spots, a fungus was isolated with the following morphology: acervuli that were 20 to 50.0 × 50 to 125.0 μm and hyaline amerospores that were ellipsoid and fusiform and 7.5 to 23.75 × 2.5 to 5.0 μm. On the basis of these morphological characteristics, the fungus was identified as Colletotrichum acutatum. In Brazil, C. acutatum is reported in apple, citrus, strawberry, peach, plum, nectarine, olive, medlar, and yerba-mate, but it was not reported as the causal agent of leaf blight in R. brasiliensis. A sample was deposited in the herbarium at the Universidade Federal de Viçosa, Minas Gerais, Brazil (VIC 31210). One representative isolate, OLP 571, was used for pathogenicity testing and molecular studies. Identity was confirmed by amplifying the internal transcribed spacer (ITS) regions of the ribosomal RNA with primers ITS4 (3), CaInt2 (a specific primer for C. acutatum [2]) and CgInt (a specific primer for C. gloeosporioides [1]). Isolates of C. acutatum (DAR78874 and DAR78876) and C. gloeosporioides (DAR78875) obtained from Australian olive trees were used as positive controls. The primers ITS4 and CaInt2 amplified a single DNA product of 500 bp expected for C. acutatum. OLP 571 was grown for 7 days on potato dextrose agar. Young leaves of R. brasiliensis were inoculated with a conidial suspension (106 conidia/ml) on young leaves. Inoculated plants were maintained in a moist chamber for 2 days and subsequently in a greenhouse at 25°C. Necrotic spots similar to those described were detected on young leaves 3 days after the inoculation. Control leaves, on which only water was sprayed, remained healthy. The same fungus was reisolated from the inoculated symptomatic tissues. To our knowledge, this is the first report of C. acutatum causing leaf blight in the native species of R. brasiliensis in Brazil. References: (1) P. R. Mills et al. FEMS Microbiol. Lett. 98:137, 1999. (2) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

In Brazil, Capsicum chinense Jacq. is the predominant species of commercial hot peppers because of its popular citrus-like aroma and adaptability to different soils and climates (4). In June 2010, 30 samples of C. chinense with severe leaf spot were collected from a field in the city of Viçosa, state of Minas Gerais, Brazil. Symptoms were observed on leaves, calyxes, fruits, and stems on most of the plants found in the area. On leaves, symptoms included amphigenous lesions that were initially circular to ellipsoid, 1 to 5 mm in diameter, whitish to tan in the center, and surrounded by a dark brown or reddish purple border. Lesions coalesce and turned necrotic with age. A fungus isolated from the lesions matched well with the description of Cercospora apii Fresen. It formed erumpent stromata that were dark brown and spherical to irregular; fascicule conidiophores were clear brown or pale, straight or curved, unbranched, geniculate, 22.5 to 80 × 5 to 7.5 μm, 0 to 3 septate, subtruncate apex; and conidia were solitary, hyaline to subhyaline, filiform, base truncate, tip acute, straight to curved, 12.5 to 140 × 3.5 to 5 μm, and 0 to 11 septate (1,2). A sample was deposited in the herbarium of the Universidade Federal de Viçosa, Minas Gerais, Brazil (VIC 31415). Identity was confirmed by amplifying part of the calmodulin gene with species-specific primers CercoCal-apii and CercoCal-R (3) of fungal DNA from a single-spore culture. In amplification reaction, initial denaturation step was done at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C (30 s), annealing at 56°C (30 s), and elongation at 72°C (30 s). Primers CercoCal-apii and CercoCal-R amplified a single DNA product of 176 bp, and coupled with the morphological characteristics, confirmed the identity of the fungus as Cercospora apii. To check pathogenicity, a 6-mm-diameter plug of the isolate was removed from the expanding edge of a 21-day-old culture grown on potato dextrose agar (PDA) and placed in contact with the adaxial face of the leaves of 8-week-old C. chinense grown in 2-liter plastic pots with soil substrate. Six plants, one per pot, were inoculated with the isolate and six plants were inoculated with the fungus-free PDA plug. Inoculated plants were maintained in a moist chamber for 24 h and then subsequently kept in a greenhouse at 26°C. Leaf spot was observed in all inoculated plants 15 days after inoculation and symptoms were similar to those expressed in the field. The fungus was reisolated from the inoculated plants and matched well with the description of Cercospora apii. All fungus-free PDA inoculated plants remained healthy. Cercospora apii comprises a complex of 281 morphologically indistinguishable species that can infect an extremely wide host range (2). To our knowledge, this pathogen has the potential to cause significant damage to the hot pepper industry of Brazil. References: (1) C. Chupp. A Monograph of the Fungus Cercospora. Cornell University Press, Ithaca, NY, 1954. (2) P. W. Crous and U. Braun. CBS Biodivers. Ser. 1:1, 2003. (3) M. Groenewald et al. Phytopathology 95:951, 2005. (4) S. D. Lannes et al. Sci. Hortic. 112:266, 2007.

In Brazil, dieback and necrosis of leaves and berries of coffee trees (Coffea arabica and C. canephora) are common symptoms of anthracnose disease caused by Colletotrichum gloeosporioides (Penz.) Sacc. In April 2010, these symptoms were observed in 100% of the plants from different coffee plantations in the Brazilian states of Espírito Santo and Bahia. Ten isolates were obtained from symptomatic leaves and berries from these areas. Of the 10 isolates, one had distinct conidial morphology with hyaline and ellipsoid conidia measuring 10 to 16 × 5.0 to 7.5 μm and melanized irregular or spatulated-shaped appressoria measuring 7.5 to 11.0 × 5.5 to 8.5 μm, formed either solitary or concatenated, which concurred with the conidia description of Colletotrichum boninense. In order to confirm the identity of this isolate, the internal transcribed spacer (ITS) rRNA region and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were sequenced (GenBank Accession Nos. JF683320 and JF331654, respectively) and compared to sequences from a database of C. boninense, confirming that the isolate was definitely C. boninense sensu lato, since it was exactly identical to other sequences in a large clade of isolates. To verify the pathogenicity of C. boninense in coffee and to compare the symptoms with those caused by C. gloeosporioides, leaves and berries were inoculated with the isolate of C. boninense and one representative isolate of C. gloeosporioides, both expressing the GFP (green fluorescent protein) gene. The isolates were grown for 7 days on potato dextrose agar and a conidial suspension (106 conidia × ml–1) was used to inoculate the organs, wounded and non-wounded, at different stages of development. In non-wounded organs, the conidial suspension was inoculated on the surface, and in leaves and berries used as control, the suspensions were substituted for sterile water. Leaves and berries were wounded with a sterilized needle and inoculated with 20 and 10 μl of the conidial suspension, respectively. Inoculated materials were incubated at 25°C and 100% relative humidity. The experiment was performed twice and evaluated daily for a week. No symptoms were observed on the control and non-wounded organs, while wounded organs exhibited typical anthracnose symptoms for both species. In berries, C. gloeosporioides consistently caused more severe symptoms at a faster rate than C. boninense. Both fungi caused necrosis in young but not old leaves. Typical acervuli were observed on the lesions and the fungus was successfully recovered from the inoculated tissues, which was confirmed by fluorescence microscopy, fulfilling Koch's Postulates. C. boninense has been identified as a pathogen causing anthracnose in a range of hosts worldwide. However, in Brazil, it has only been reported in pepper (Capsicum annuum) (3), passion fruit (Passiflora) (4), Hippeastrum (1) and in the medicinal plant Maytenus ilicifolia (2). To our knowledge, this is the first report of C. boninense associated with anthracnose of coffee trees in Brazil. Since the symptoms are similar to those caused by C. gloeosporioides, it can be stated that both species are associated with this disease in commercial coffee plantations in Brazil. Therefore, control strategies should consider the occurrence of C. boninense. References: (1) D. F. Farr et al. Mycol. Res. 110:1395, 2006. (2) S. A. Pileggi et al. Can. J. Microbiol. 55:1076, 2009. (3) H. J. Tozze et al. Plant Dis. 93:106, 2009. (4) H. J. Tozze et al. Australas. Plant Dis. Notes 5:70, 2010.

Nickel(II) complexes of general formula [Ni(RSO2N = CS2)(PPh3)2] (2a–h) or [Ni(RSO2N = CS2)dppe] (3a–h), where R = methyl (a), ethyl (b), butyl (c), octyl (d), phenyl (e), 4-isopropylphenyl (f), 4-tert-butylphenyl (g), 2-naphthyl (h), PPh3 = triphenylphosphine and dppe = 1,2-bis(diphenylphosphine)ethane were prepared, from which six are new substances (2f–h and 3f–h). The new compounds were characterized by elemental analysis of C, H, N and Ni, and by IR, UV–Vis and 1H, 13C and 31P NMR spectroscopies. The data were consistent with the formation of square planar nickel(II) complexes with mixed ligands, what was confirmed by single crystal X-ray diffraction studies on compounds 2f and 2h. These complexes present intramolecular Ni H C anagostic interactions. All complexes inhibited the germination of Hemileia vastatrix and Phakopsora pachyrhizi, the causal agents of devastating diseases on soybean and coffee cultures. The most active compounds presented IC90 values as low as 405 µM against H. vastatrix, and 280 μM against P. pachyrhizi. Thus, the title compounds are target molecules for the development of new agrochemicals against the Asian soybean rust and Coffee leaf-rust diseases.

This study aimed to quantify the levels of resistance of 39 hybrids “Clone 83 × Clone 02”of Coffea canephora var. conilon to the races II (v5) and XXXIII (v5,7,9) of H. vastatrix. To study the resistance of the genotypes, leaf discs were removed from the plants and spreaded on the top of a stainless steel screen inside gerbox as a moist chamber. The gerboxes were mantained at 22 ± 2 °C in the dark for 48 h and under 12 h of photoperiod, respectively. The hybrids were classified into four levels: resistant (R), moderately resistant (MR), moderately susceptible (MS) and susceptible (S). Leaf discs (%) containing sporulated lesions was the resistance component that had the greatest absolute correlation (0.95). The level of resistance of the hybrids varyied according to the race of the pathogen. The percentage of R, MR, MS and S of the hybrids to race II and XXXIII were 25 %, 25 %, 38 %, and 11 %, and 13 %, 37 %, 42 %, and 8 %, respectively. Nine (25 %) and five (13 %) hybrids were R to race II and XXXIII, respectively. Four hybrids (21, 30, 48 and 66) showed vertical resistance to races II and XXXIII. Possibly these hybrids have the resistance factor SH6, because they were resistant to race XXXIII v5,7,9. Leaf discs (%) with sporulating lesions can be used as a reference in future studyies of screening genotypes with horizontal resistance to C. canephora genotypes to leaf rust.

In Brazil, coffee leaf rust, a fungal disease caused by Hemileia vastatrix Berk. et Br., was first detected in Coffea arabica in January 1970, in the southern region of Bahia state. Today, the disease is present in virtually all arabica and conilon (Coffea canephora) coffee-growing areas of Brazil, and continues to threaten coffee production with losses that range from 30 to 50 %. The disease is usually less severe at elevations above 1,200 m, where the environment is less conducive for the rust. Disease risk is increased in arabica coffee compared to conilon, and lower production is expected in the year following an epidemic due to early defoliation and drying of branches. Several varieties were developed in the country using sources of resistance from germplasm collections in Portugal. However, very few are completely resistant, instead exhibiting various levels of partial resistance. The disease is currently managed through the use of protectant and systemic fungicides including copper, triazoles and strobilurins that should be applied following rules of decision that vary according to the risk scenario. A review of the biology and epidemiology of coffee rust in Brazil is presented and the best management practices for controlling the disease based on advances in breeding for resistance and crop protection are discussed.

Fire blight, a disease of apple trees caused by Erwinia amylovora, occurs worldwide except in South America, South-Central Africa, and most of Oceania. Ecological niche models can determine the potential distribution of species and measure the risk of pest invasion. This study aims to develop global climate suitability models using MaxEnt software for E. amylovora and to determine the regions in which apple cultivation and the bacterium are most likely to co-occur. Most occurrence data for E. amylovora (93%) are from the northern hemisphere, distributed between 63.90 to 14.56 degree days in regions of Africa, Central America, North America, Asia, and Europe. The only country in the southern hemisphere that this bacteria has been detected is New Zealand (Oceania). Apples are cultivated on every continent except Antarctica, between 61.55 to 44.41 degree days. We find that regions of South-Eastern Africa, Argentina, Australia, Southern Brazil, Bolivia, Chile, China, the United States, Madagascar, Morocco, Mexico, New Zealand, Peru, Tunisia, Uruguay, and the majority of Europe are suitable for both E. amylovora and apple cultivation. These results provide information on the potential worldwide distribution of E. amylovora in apple production area.

The main phytosanitary problems affecting global coffee production are the fungal diseases known as rust, caused by Hemileia vastatrix Berkeley and Broome, and coffee berry disease (CBD), induced by Colletotrichum kahawae Waller and Bridge. The main disease control strategy is the use of resistant coffee cultivars. Hibrido de Timor is the most important source of resistant varieties used in breeding programs worldwide. The objective of this work was to characterize the diversity and disease resistance of 152 HdT genotypes from the germplasm collection at the Universidade Federal de Vicosa (UFV). Accessions were phenotyped with H. vastatrix races II and XXXIII. Molecular analysis was carried out with 29 random microsatellite markers or single sequence repeats (SSRs), and two SSRs associated with the CBD resistance gene Ck-1. All accessions in the germplasm collection were resistant to H. vastatrix race II, and 141 were resistant to H. vastatrix race XXXIII. Based on the presence of markers, there were 106 accessions containing the CBD resistance gene Ck-1. In the diversity study, the 152 accessions clustered into 21 different groups. A unique molecular profile (fingerprint) was determined for each individual, using 52 alleles from 22 SSR markers. The HdT germplasm of UFV was highly diverse, and included 99 accessions with multiple disease resistance genes, including the CBD resistance gene Ck-1, and others conferring resistance to H. vastatrix races II and XXXIII.

The common bean (Phaseolus vulgaris L.) is widely cultivated in Brazil and is known as a very important crop for families in this country. Fusarium wilt severely harms common beans and has become a big issue for this crop. In order to assist the breeding programs that target resistance to this disease, the evaluation of genetic diversity of the pathogen and its molecular characterization are crucial. Thus, the present goal was to identify Fusarium isolates obtained from several places in Brazil using molecular tools; select molecular markers for these isolates; and analyze their diversity. All of isolates were molecularly identified as Fusarium oxysporum f. sp. phaseoli (Fop). By using seven selected SSR markers, the results of diversity obtained by the dendrogram and the Bayesian analysis formed four groups where a large diversity of this fungus was found within each state. However, the groups were more homogenous according to the collection source and the pathogenicity test. More specifically, group 2 was composed of the most virulent strains and originated from Minas Gerais State – UFV, and group 3 was mostly composed by isolates from Goias state. Group I was also more diverse in terms of location and virulence. The overall results indicated a positive correlation between Fusarium diversity and its virulence to common bean. Furthermore, the use of these markers was effective in molecular identification and in detecting polymorphism within F. oxysporum f. sp. phaseoli.

The objective of this study was to assess whether the source of Colletotrichum graminicola isolates (leaves or stalks) affects anthracnose leaf blight (ALB) and anthracnose stalk rot (ASR) development. Ten greenhouse experiments were conducted, five to evaluate ALB and five to evaluate ASR, each using a distinct commercial maize hybrid varying in reaction to the diseases. A collection of 20 isolates, 10 recovered from symptomatic leaves, and ten from symptomatic stalks, were inoculated on the five hybrids. Maize leaves were inoculated 15 days after planting and ALB was evaluated 15 days after inoculation. Stalks were inoculated at pretasseling and ASR evaluated 30 days later. Both ALB and ASR were assessed using a 0–5 ordinal scale. No differences in ALB and ASR severity were observed between the two groups of isolates regardless of hybrid based on the non-parametric test. The ability of C. graminicola to infect and cause anthracnose on any part of the maize plant seemed to not depend on the source of the isolate Made available in DSpace on 2017-06-09T00:08:59Z (GMT). No. of bitstreams: 1 Colletotrichumgraminicola1.pdf: 487050 bytes, checksum: ab301efb9fc23bbc3f95042ed0c5a4ab (MD5) Previous issue date: 2016-01-25

This study investigated the ability of different triazole and strobilurin fungicides either alone or combination when applied to the leaves and triazole to the soil for control of Coffee Leaf Rust (CLR). The experiments were performed in a greenhouse using three month-old coffee seedlings, and in the field with five years old plants using the susceptible variety ‘Catuai vermelho’, at elevation of 750 m, from 2010 to 2012, in Viçosa, Minas Gerais State, Brazil. The effects of triazole (triadimenol, cyproconazole and epoxiconazole) and strobilurin (azoxystrobin, trifloxystrobin, pyraclostrobin) was first studied in a greenhouse to evaluate the translaminar effect on CLR control. Based on the results obtained in the greenhouse, a field experiment was performed to evaluate the effect of triazole applied to the soil and triazole + strobilurin sprayed on CLR control and the effect on coffee berry yield of the plants. The different fungicides applied in the greenhouse reduced CLR severity by 8.9 % (trifloxystrobin), 79.4 % (azoxystrobin), 82.0 % (triadimenol), 93.2 % (cyproconazole), 96.6 % (epoxiconazole) and 98.5 % (pyraclostrobin) compared to that in the control plants. In the field experiment triazole + strobilurin fungicides provided effective CLR control. The co-application of epoxiconazole and pyraclostrobin or azoxystrobin sprayed twice resulted in the most effective CLR control and maintained the highest coffee berry yield. The incidence of CLR was 83.5, 54.0 and 78.5 % for the control treatments, whereas the incidence did not exceeded 24.4, 37.0 and 39.0 % on the sprayed plants. The average coffee berry yield increased by 33.9 % (lowest value) when applying triadimenol to the soil in November followed by cyproconazole + trifloxystrobin sprayed in December and February, and increased by 99.1 % (highest value) in the treatment with epoxiconazol + pyraclostrobin sprayed in December and March compared with plants that did not receive any fungicide application. The control treatment and that of triadimenol applied to the soil in November followed by cyproconazole + trifloxystrobin sprayed in December and February yielded less than 1800 (1320 to 1800) kg/ha of processed coffee, whereas the other treatments more than 2100 (2142 to 2676) kg/ha. Treatments with cyproconazole applied to the soil in November followed by cyproconazole + azoxystrobin sprayed on the leaves in December and February, epoxiconazol + pyraclostrobin sprayed on the leaves in December and March, and cyproconazole + azoxystrobin sprayed on leaves in December, February and April yielded more than 2340 (2340 to 2676) kg/ha of processed coffee. This is the first report indicating that a triazole fungicide applied to the soil and triazole + strobilurin fungicide sprayed on the leaves, efficiently controlled CLR and significantly increased coffee berry yield for three seasons.

Coffee is the most traded commodity in the world, and Brazil is its largest producer. Coffee leaf rust, caused by the biotrophic fungus Hemileia vastatrix, is the most important coffee disease, reducing coffee yield by 35–50%. This study aimed to use the ratio of variable and maximum fluorescence of dark-adapted tissue (Fv/Fm) as a parameter to differentiate presymptomatic tissue from healthy tissue during disease development in plants sprayed with pyraclostrobin and epoxiconazole after 4 days postinoculation. Visual severity was considered as an indicative of apparent disease and true severity as an indicative of both apparent and non-apparent disease. There was a significant linear relationship between the areas of true severity and visual severity, and for each additional unit in the visual severity, there was an increase of 1.53 units on the true severity. For the epoxiconazole and pyraclostrobin treatments, coffee leaf rust symptoms decreased according to both visual and Fv/Fm images. Pustules on the leaves sprayed with epoxiconazole were smaller in size than those on the leaves of non-sprayed plants but bigger than those sprayed with pyraclostrobin. The reduction in Fv/Fm values at the pustule epicentres present on the leaves of plants sprayed with epoxiconazole, and pyraclostrobin was greater than those of the non-sprayed plants. This finding was expected and reflects the importance of these fungicides in prohibiting the progress of coffee leaf rust. The photosynthetic capacity of Coffea arabica was affected by H. vastatrix infection, and the Fv/Fm parameter was able to show this effect before the visual symptoms were noticed.

Sorghum anthracnose is one of the most important and destructive diseases of sorghum. Genetic resistance has been the most efficient strategy to control the disease, but the high variability of the pathogen population in Brazil has resulted in only modest efficacy. Accordingly, we investigated the variability of Colletotrichum sublineolum in response to sorghum populations with three levels of genetic diversity: pure stand, three-way hybrids and physical mixtures of three-way hybrids. Six plots of each treatment were planted in different areas and at different dates. A total of 480 isolates, that is 40 single-conidium isolates per plot, were collected from the field experiment to characterize the variability of the pathogen in each host population. Isolates were inoculated in a greenhouse on a differential line set composed of eight sorghum inbred lines. Our results reveal that the pathogen populations derived from three-way combinations had higher pathotype diversity than did those derived from pure stand host populations. More complexly, virulent phenotypes were also developed in genetically diverse stands compared to pure stand host populations. The diversification of the host population limits pathogen adaptation, thus resulting in a significantly higher number of pathotypes. The results of this study will improve the management of sorghum anthracnose in the field by helping sorghum breeders maintain disease resistance.

Adoption of resistant cultivars is the primary measure used to control anthracnose stalk rot. The goal of this study was to identify maize-resistant genotypes to anthracnose stalk rot, which are similar to the hybrid 2B710. Experiments were performed at Embrapa Maize and Sorghum experimental fields in Brazil. The first experimental trial evaluated 234 maize lines as well as two commercials hybrids, BRS1010 (susceptible) and 2B710 (resistant). Artificial inoculations were performed with a strain at the blister (R2) phase, and evaluation of disease severity was performed after 30 days. The second experimental trial evaluated 48 maize lines and hybrids, inoculated with two Colletotrichum graminicola strains. In the first trial, eight resistance groups were formed, and the last lines were more resistant, as was the hybrid 2B710, with values between 11.50% and 23.0% of severity. In the second trial, there was an interaction between the two factors, lines and isolates, and the lines often showed the same reaction features as those obtained in the first trial. However, the disease severity was higher for most lines, even when using other isolates. These lines with effective levels of resistance could be used in future studies of inheritance, in programs to develop hybrids, and to identify molecular markers associated with resistance to anthracnose stalk rot in maize. RESUMO: O uso de cultivares resistentes é a principal medida para o manejo da antracnose do colmo em milho. Neste trabalho, objetivou-se identificar linhagens com níveis de resistência à antracnose do colmo, similar ao híbrido 2B710, considerado resistente. Dois experimentos foram conduzidos na Embrapa Milho e Sorgo. No primeiro experimento, foram avaliados 234 linhagens e os híbridos BRS1010 (suscetível) e 2B710 (resistente). Foi realizada inoculação artificial com um isolado de C. graminicola , na fase de pré-pendoamento e, após 30 dias, foi realizada a avaliação da severidade da antracnose no colmo. O segundo experimento foi conduzido com 48 linhagens e os híbridos inoculados com dois isolados de C. graminicola . No primeiro experimento, os genótipos formaram oito grupos com base na severidade da doença e as linhagens do último grupo foram consideradas as mais resistentes, incluindo o híbrido 2B710, em que os genótipos apresentaram valores de severidade entre 11,50 a 23%. No segundo experimento, houve interação entre os fatores linhagens e isolados e, de modo geral, as linhagens apresentaram a mesma tendência de reação obtida no primeiro experimento, no entanto, a severidade da doença foi maior para a maioria das linhagens, mesmo quando utilizado o outro isolado. Com isso, foi possível realizar a seleção de linhagens com bons níveis de resistência, as quais podem ser utilizadas em programas de melhoramento, em estudos de herança, desenvolvimento de híbridos e identificação de marcadores moleculares, associados com resistência à antracnose do colmo.

– Anthracnose stalk rot (ASR) disease caused by Colletotrichum graminicola can lead to major corn yield losses in Brazil. Studies on plant resistance and genetic variation in pathogen populations are required towards the development of ASR disease management strategies. However, the lack of adequate methods for C. graminicola inoculation in maize stalk has hindered these efforts. The main objective of the study was to develop a confident and feasible method for C. graminicola inoculation into maize stalk. Three inoculation methods were evaluated: TCMP (toothpick colonized by pathogen mycelium), TICS (toothpicks immersed in conidial suspension), and ICS (injection of conidial suspension). The effect of the internode position, the phenological stage, and the period of time required for maize reactions to ASR infection were also evaluated. The infection severity was assessed by a diagrammatic scale with six levels of severity from 0 (no necrotic spots) to 5 (100 % infection). The TICS was the best method, inoculation in the third internode and assessment of disease severity at tasselling stage (VT) 30 days after inoculation presented the more accurate results. The procedure was validated under field conditions. Key words: Zea mays; stalk rot; anthracnose. METODO PARA INOCULACAO DE Colletotrichum graminicola EM COLMOS DE MILHO RESUMO – A podridao do colmo do milho causada por Colletotrichum graminicola causa perdas severas em lavouras de milho. Estudos com resistencia de plantas e variabilidade genetica do patogeno sao necessarios para o desenvol-vimento de estrategias de manejo desta doenca. Por outro lado, ha ausencia de uma metodologia adequada para a inoculacao de C. graminicola em colmos. O objetivo principal do trabalho foi estabelecer uma metodologia confiavel e pratica para a inoculacao de C. graminicola. Foram avaliados tres metodos de inoculacao em colmo: palitos de dente colonizados por micelio; palitos de dente imersos em suspensao de conidios; e injecao de suspensao de conidios. Nos tambem determinamos o efeito da posicao do entreno inoculado, o estadio fenologico para a inoculacao e o periodo de tempo ideal apos a inoculacao para avaliar a severidade da doenca. O metodo de palitos de dente imersos em suspensao de conidios inoculado no terceiro entreno no estadio fenologico de pendoamento (VT) e avaliado aos 30 dias apos a inoculacao obteve o melhor resultado. A metodologia foi validada em plantas cultivadas em condicoes de campo. Palavras-chave: Zea mays; podridao de colmo, antracnose.

The most important disease of Coffea arabica is coffee leaf rust caused by the fungus Hemileia vastatrix. The purpose of this study was to characterize the inheritance of coffee resistance gene(s) to race II of this pathogen and to identify and map molecular markers linked to this trait. Different populations were used: F2 (160 plants), BCr (20), and BCs (135), derived from a cross between the resistant genotype Hı´brido de Timor UFV 427-15 and the susceptible cultivar Catuaı´ Amarelo UFV 2143-236 (IAC 30). The segregation analysis showed that the resistance of Hı´brido de Timor to race II of the H. vastatrix is conferred by a single dominant gene. The amplification of 176 AFLP (Amplified fragment length polymorphism) primer combinations using bulked segregant analysis (BSA) allowed the identification of three molecular markers linked to the resistance gene. Genetic mapping of these three markers in the F2 population indicated that they are distributed on both sides, flanking the resistance gene. The markers E.CTC/M.TTT405 and E.CGT/M.TGT 300 were found linked to the resistance gene at 8.69 cM (LOD 18.91) and 25.10 cM (LOD 5.37), respectively, while E.CCT/M.TTC230 was localized on the other side of the gene, at 20.50 cM (LOD 6.15). These markers are the first rust resistance markers identified in Hı´brido de Timor and can be useful for marker assisted selection in coffee breeding programs.

Countering the economic hurdle caused by coffee leaf rust disease is most appealing at this time as it has posed a major threat to coffee production around the world. Establishing differential expression profiles at different times following pathogen invasion in both innate and acquired immunities unlocks the molecular components of resistance and susceptibility. Suppression subtractive hybridization (SSH) was used to identify genes differentially over-expressed and repressed during incompatible and compatible interactions between Coffea arabica and Hemileia vastatrix. From 433 clones of expressed sequence tags (ESTs) sequenced, 352 were annotated and categorized of which the proportion of genes expressed during compatible interaction were relatively smaller. The result showed upregulation and downregulation of various genes at 12 and 24 h after pathogen inoculation in both interactions. The use of four different databases in searching for gene homology resulted in different number of similar sequences. BLASTx against EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics Institute) database being with the maximum (100%) hits for all the annotated sequences. RT-qPCR analysis of seven resistance-signaling genes showed similar expression patterns for most of the genes in both interactions, indicating these genes are involved in basal (non-specific) defense during which immune reactions are similar. Using SSH, we identified different types of resistance related genes that could be used for further studies towards resistant cultivar development. The potential role of some of the resistance related proteins found were also discussed.

Toxigenic fungi that infect corn grains are among the major problems in Brazil as they are associated with toxicological implications for both humans and animals. Mechanical-physical methods have been used to separate contaminated grains in lots of corn kernels based on the symptoms caused by fungi. The objective of this study was to assess whether the presence or the type of symptoms were associated with fungal incidence and total fumonisin levels in corn kernel samples from the field. Kernels of two hybrids were separated visually into three groups: 1) asymptomatic or symptomatic exhibiting 2) kernel rot or 3) kernel streak symptoms. A high frequency of Fusarium spp. was observed in asymptomatic (69.5%) and kernel rot (69.9%). Stenocarpella sp. and Penicillium sp. were found in kernel rot (16.0%) and kernel streak (44.1%), respectively. High fumonisin levels (7.24 μg.g-1), above the maximum tolerated limit, were detected only in kernel rot. No association between fumonisin levels and the frequency of Fusarium spp. in kernels was found. Made available in DSpace on 2017-09-24T19:54:16Z (GMT). No. of bitstreams: 1 Symptomalogicalaspects.pdf: 3180767 bytes, checksum: 57fd4ca890fbb919fa4c09684ecdef9e (MD5) Previous issue date: 2017-07-03

Selecting superior genotypes is facilitated by marker-assisted selection (MAS), which is particularly suitable for transferring disease resistance alleles because it nullifies environmental effects and allows selection of resistant individuals in the absence of the pathogen or race, enabling preventive breeding. Molecular markers linked to two major genes (SH3 and SH?), conferring resistance to coffee rust, and those linked to the Ck-1 gene, conferring resistance to coffee berry disease (CBD), have previously been identified. These markers were validated and used in a progeny of crosses between Indian selections with Coffea arabica cultivars. Eleven resistant individuals homozygous for SH3 were identified by MAS. Of these, seven carry SH? from Híbrido de Timor and the gene introduced from Coffea liberica (SH3). SH? was characterized as derived from Coffea canephora. Thus, it was possible to identify C. arabica genotypes carrying important genes for rust resistance introgressed from other coffee species. MAS also allowed identification of sources of CBD resistance for use in preventive breeding for resistance to this serious disease. Using two validated molecular markers, two coffee plants carrying Ck-1 were identified: the UFV 328-60 genotype (F2) was resistant and homozygous based on both molecular markers but exhibited no markers related to SH3 and SH?, and the UFV 317-12 genotype (F1) was resistant and homozygous but resistant and heterozygous based on CBD-Sat207 and CBD-Sat235, respectively. Along with possessing Ck-1, the latter carries SH?. Overall, plants carrying different genes for resistance to rust and CBD were identified. These plants are important sources for gene pyramiding in breeding programs aimed at multiple and durable resistance.

The main management strategy for coffee leaf rust (Hemileia vastatrix) in conilon coffee is based on the application of fungicides using a fixed schedule without considering the epidemiological aspects of the disease. In order to turn chemical control less subjective and more efficient, two systems of coffee leaf rust management were tested: 1) application of systemic fungicide based on the intensity of the disease present in each conilon clone of the “Conilon Vitoria” variety, according to the disease incidence; threshold of 5 % incidence of the disease was adopted to spray the fungicide, and 2) application of systemic fungicide to the soil, followed by foliar application in the beginning of the rainy season. The experiments were performed in two municipalities of the Espirito Santo State, Nova Venecia e Castelo. Monthly evaluations of disease incidence were performed between September 2010 and August 2011. The results showed that chemical control of coffee leaf rust in conilon coffee should be performed differently, for each clone, of the clonal Vitoria variety. The 5 % threshold of the disease incidence was reached at different time of the coffee grower season. The strategy to apply systemic fungicide to the leaves, to control conilon leaf rust, based on the disease incidence of 5 % was more effective than soil application system. Application of systemic fungicide to the soil did not control the disease. In conclusion, monitoring the incidence of coffee leaf rust in all the clones is an important strategy to decide the appropriate time to apply fungicides.

The objective of this study was to investigate the effect of silicon and chitosan as alternative controls for postharvest rot of strawberries (gray mold) caused by Botrytis cinerea. Chitosan and potassium silicate applications were performed at the preharvest stage using the following treatments: chitosan once a week; potassium silicate once a week; potassium silicate once a week + chitosan once a week; and without application. An additional variable was introduced at postharvest, with one-half of the total fruit harvested from each plot dipped in chitosan and the other half not dipped in chitosan. Potassium silicate alone was not effective for rot control. Fruits from plants that received the chitosan application showed 64% less area under the rot progress curve (AURPC) than fruits from plants that were not treated with chitosan. Harvested fruits that were chitosan dipped showed 48% less AURPC than fruits that were not treated at postharvest. Chitosan application in the field and at postharvest is a promising strategy for the management of postharvest strawberry rot.

Coffee leaf rust, caused by Hemileia vastatrix, is the most devastating disease of coffee. Since limited information is available in the literature on silicon (Si) affecting plant diseases in coffee, this study was designed to investigate foliar application of potassium silicate (PS), a source of soluble (Si), on infection process of coffee leaf rust at the microscopic level. The foliar Si concentration for plants sprayed with water and PS has no significant difference (0.24 and 0.30 dag kg−1, respectively). X-ray microanalysis indicated that the deposition of Si on the leaves of the plants that were sprayed with PS was greater in comparison to the leaf samples from the plants sprayed with water. Rust severity on leaves of plants sprayed with water or sprayed with PS reached 44% and 32%, respectively, at 36 days after inoculation (dai). Plates of polymerised PS were observed on the leaf surfaces of the plants sprayed with the product, in contrast to its absence on the leaf surfaces of plants sprayed with water. At 36 dai, a greater number of uredia were observed on the leaf surfaces of plants sprayed with water in comparison to the leaf surfaces of plants sprayed with PS. On fractured leaf tissues that were sprayed with PS, less fungal colonisation was observed in comparison to the leaves of plants sprayed with water. In conclusion, the results of this study suggest that the effect of foliar-applied Si on the control of the coffee leaf rust development may be attributed to the physical role of the polymerised PS, its osmotic effect against urediniospores germination, or both.

All the transcriptome sequencing data mentioned in the original article is publicly available at the National Center of Biotechnology Information (NCBI).

Potassium (K) is one of the essential nutrients for plants and is involved in many cellular processes which might influence the severity of diseases. There are few reports of the effect of increasing concentrations of K in the field on the severity of the Asian soybean rust (ASR) caused by Phakopsora pachyrhizi. In this context, the objective of this work was to verify the influence of increasing concentrations of K on ASR, in the absence and presence of chemical control, in conditions that highly favored the development of the disease. Two experiments were conducted under field conditions and two also in the field but in pots with a capacity of 100 L. Evaluated concentrations of K were 0.0, 100, 200, and 400 mg/dm³, respectively in a randomized complete block design. The application of the fungicide mixture azoxystrobin (200 g/L) + cyproconazol (80 g/L) was performed at 45, 60, and 75 days after emergence. The severity of the disease, area under the disease progress curve (AUDPC), rate of disease progression (r), soybean productivity, and contents of chlorophyll a, b and total were evaluated. The severity, AUDPC, and r decreased with the increase of the concentration of K and fungicide application, while the rate of disease control and soybean productivity increased. Contents of chlorophyll a, b and total also had higher values according to the increase in the concentration of K. Potassium fertilization reduced the severity of ASR and grain yield under high disease pressure with or without chemical control.

Three experiments were carried out in an experimental field from Viçosa city, Minas Gerais, Brazil, to compare late blight (LB) (Phytophthora infestans) and early blight (EB) (Alternaria grandis) epidemics under different environmental conditions and fungicide application programs. Each experiment consisted of two side-by-side trials that were arranged in a randomized complete block design with five treatments and five replications. At 30 days after planting the potato (‘Ágata’ cultivar) plants in plots of trials 1 and 2 were inoculated with an isolate of P. infestans (A2 mating type) and four isolates of A. grandis, respectively. Fungicide applications were initiated at seven days after inoculation and repeated at 7- or 15-day intervals. The severity of LB and EB was assessed every two days, and the area under disease progress curve (AUDPC) was calculated for both diseases. The yield was quantified by weighing the tubers of all the plants from each plot after their maturation. Yield data were converted into kg ha-1 and the percentage of loss was calculated for each treatment. Based on the AUDPC, the LB severity was greater than the EB severity under all three environmental conditions, and this was reflected in the yield. LB and EB caused yield losses as high as of 82 and 45%, respectively. The fungicide effects on both LB and EB and the associated yield losses changed according to the environmental conditions, therefore, indicating the importance of using tools such as a forecast system to help farmers to decide the best time for fungicide application.

The drying time of pesticides on the leaves and the resistance of the deposits to rain removal are essential for success in controlling plant diseases. Because the fungicide application is performed during the rainy season, it is crucial to study the resistance of chemical deposits on the leaf surface for the control of the coffee leaf rust caused by Hemileia vastatrix. Thus, the present work analyzed the effect of an simulated rain (30 min), after several drying time of the deposits, on the removal of (i) copper oxychloride alone and associated to mineral and the adjuvant polyoxyethylene alkylphenol ether and also (ii) before several drying time, on the removal of the epoxiconazol + pyraclostrobin formulation mixed with copper hydroxide and micronutrients applied on coffee leaves, for the control of leaf rust. Around 85% and 45% of the treatments had copper retention > 30% after 120 min and 480 min, respectively, of drying time before the rain. At 480 min, retention of copper oxychloride + 0.75% mineral oil was > 30%, whereas copper oxychloride + adjuvant polyoxyethylene alkylphenol ether at 0.05% and 0.1%, respectively was 40% or higher. When copper oxychloride was added to 0.5% mineral oil at 0.05% to 0.20% of adjuvant polyoxyethylene alkylphenol ether at 480 min of drying time, copper retention was > 30%. When copper oxychloride was added to 0.75% mineral oil, copper retention was > 30% at 0.05% and 0.10 % of adjuvant polyoxyethylene alkylphenol ether. Five of the treatments with copper retention > 50% with 120 min of drying time were reduced to only two (copper oxychloride + 0.75% mineral oil; copper oxychloride + (1.0% mineral oil + 0.1% adjuvant polyoxyethylene alkylphenol ether) with copper retention > 30%, at the drying time of 480 min. All the treatments differed from the control but did not differ between each other for the coffee leaf rust control. The percentage of control ranged from 94.6% to 100% and from 71.3% to 99.5% at 180 min and 480 min of drying time, respectively. Copper retention varied from 32.2% to 55.2% and from 23.7% to 51.5%, at 180 min and 480 min of drying time, respectively, for the best treatments. The treatment with the highest copper retention was copper oxychloride + 0.75% mineral oil, both at 180 min and 480 min of drying time. The best treatments for leaf rust control were not always those with the highest copper retention by the leaves. The highest copper retention by the leaf surface was achieved with copper oxychloride mixed with 0.75% mineral oil; this treatment resulted in > 97% of coffee leaf rust control. Adding adjuvant polyoxyethylene alkylphenol ether to the mixture does not necessarily favor the tenacity and efficiency of the copper oxychloride for the coffee leaf rust control. We concluded that the copper oxychloride acts differently for the coffee leaf rust control, depending on the concentration of added mineral oil and adjuvant polyoxyethylene alkylphenol ether time zero. Pyraclostrobin + epoxiconazol alone or mixed with copper hidroxide controlled 100% of the disease after 30 min, 120 min and 480 min of spraying, but when micronutrients was added to pyraclostrobin + epoxiconazol and copper hidroxide control of coffee leaf rust reached 100% at drying time of 120 and 480 min.

Coffee leaf rust caused by the fungus Hemileia vastatrix is one of the most important leaf diseases of coffee plantations worldwide. Current knowledge of the H. vastatrix genome is limited and only a small fraction of the total fungal secretome has been identified. In order to obtain a more comprehensive understanding of its secretome, we aimed to sequence and assemble the entire H. vastatrix genome using two next-generation sequencing platforms and a hybrid assembly strategy. This resulted in a 547 Mb genome of H. vastatrix race XXXIII (Hv33), with 13,364 predicted genes that encode 13,034 putative proteins with transcriptomic support. Based on this proteome, 615 proteins contain putative secretion peptides, and lack transmembrane domains. From this putative secretome, 111 proteins were identified as candidate effectors (EHv33) unique to H. vastatrix, and a subset consisting of 17 EHv33 genes was selected for a temporal gene expression analysis during infection. Five genes were significantly induced early during an incompatible interaction, indicating their potential role as pre-haustorial effectors possibly recognized by the resistant coffee genotype. Another nine genes were significantly induced after haustorium formation in the compatible interaction. Overall, we suggest that this fungus is able to selectively mount its survival strategy with effectors that depend on the host genotype involved in the infection process.

Este trabalho objetivou caracterizar acessos de C. canephora oriundos de cultivos comerciais e bancos de germoplasma brasileiros. Um total de 130 acessos das coleções do IAC (São Paulo), UFV (Minas Gerais) e coletados em plantios comerciais no Espírito Santo e Rondônia foram genotipados com 20 novos microssatélites. Métodos multivariados foram utilizados para estimar a relação entre os acessos. Foi observado alto nível de polimorfismo e dois grupos foram identificados: o primeiro foi constituído pelos genótipos conservados nas coleções de germoplasma do IAC e UFV e o segundo foi composto pelos acessos coletados em plantios comerciais. Os acessos do Espírito Santo e de Rondônia formaram dois subgrupos distintos. Apesar do grande polimorfismo encontrado nas lavouras brasileiras de café canéfora, incrementar essa diversidade é necessário, pois um novo limiar de ganhos genéticos é esperado nos programas de melhoramento com a intensificação do uso do germoplasma conservado. This work aimed to characterize accessions that represent the C. canephora germplasm conserved and cultivated in Brazil. A total of 130 accessions from germplasm banks of IAC (São Paulo), UFV (Minas Gerais) and also collected in plantations of the State of Espírito Santo and Rondônia were evaluated with a set of 20 new microsatellite primers. Multivariate methods were used to estimate the relationship among the accessions. High level of polymorphism and two major diversity clusters were identified. First cluster was composed by the accessions conserved in the IAC and UFV collections and the second was formed by accessions collected in areas under cultivation. Accessions from Espírito Santo and Rondônia were clear separated, composing two subclusters. Despite the great polymorphism found in Brazilian plantations, the diversity may be increased, because a new threshold in the genetic gains is expected on breeding programs with the intensification of the use of conserved germplasm.

A diversidade fenotípica de Magnaporthe grisea foi avaliada baseada em amostras de folhas com brusone coletadas de oito lavouras de arroz, 'Primavera' e 'Bonança', durante os anos de 2001/2002 e 2002/2003, no Estado de Goiás. O número de isolados de M. grisea, em cada lavoura, utilizado para teste de virulência variou de 28 a 47. Foram utilizados três diferentes índices baseados no tipo de reação em oito diferenciadoras internacionais e oito diferenciadoras brasileiras. A diversidade fenotípica das subpopulações de M. grisea de 'Primavera' e 'Bonança' foi semelhante pelos índices de Shannon, Simpson e Gleason. O índice de Simpson foi mais sensível do que Shannon e Gleason quanto ao número de patótipo e desvio padrão da freqüência utilizando diferenciadoras brasileiras. Entretanto, o índice de Gleason foi melhor quanto ao número de patótipos e desvio padrão da freqüência, considerando as diferenciadoras internacionais. O tamanho da amostra não influenciou significativamente os índices de diversidade. Os dois conjuntos de diferenciadoras utilizados nesse estudo evidenciaram diversidade fenotípica de diferentes maneiras em todas as oito subpopulações analisadas. A diversidade fenotípica, determinada pelas diferenciadoras brasileiras foi menor nas lavouras de 'Primavera' do que em 'Bonança', independente do índice, ano e local. Considerando as diferenciadoras brasileiras, as quatro subpopulações de 'Primavera' não mostraram distribuição uniforme, predominando apenas um patótipo nas subpopulações. A distribuição uniforme de patótipos foi observada em três subpopulações de 'Bonança'. A diversidade de patótipos de M. grisea foi determinada com maior precisão combinando as diferenciadoras brasileiras e o índice de Simpson. The phenotypic diversity of Magnaporthe grisea was evaluated based on leaf samples with blast lesions collected from eight commercial fields of the upland rice cultivars 'BRS Primavera' and 'BRS Bonança', during the growing seasons of 2001/2002 and 2002/2003, in Goias State. The number of M. grisea isolates from each field utilized for virulence testing varied from 28 to 47. Three different indices were used based on reaction type in the eight standard international differentials and eight Brazilian differentials. The M. grisea subpopulations of ´Primavera' and 'Bonança', as measured by Simpson, Shannon and Gleason indices, showed similar phenotypic diversities. The Simpson index was more sensitive relation than those of Shannon and Gleason for pathotype number and standard deviation utilizing Brazilian differentials. However, the Gleason index was sensitive to standard deviation for international differentials. The sample size did not significantly influence the diversity index. The two sets of differential cultivars used in this study distinguished phenotypic diversity in different ways in all of the eight subpopulations analyzed. The phenotypic diversity determined based on eight differential Brazilian cultivars was lower in commercial rice fields of 'Primavera' than in the fields of 'Bonança,' independent of the diversity index utilized, year and location. Considering the Brazilian differentials, the four subpopulations of 'BRS Primavera' did not show evenness in distribution and only one pathotype dominated in the populations. The even distribution of pathotype was observed in three subpopulations of 'BRS Bonança'. The pathotype diversity of M. grisea was determined with more precision using Brazilian differentials and Simpson index.

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi since it was first reported in Brazil in 2001 has been subject of studies regarding the use of fungicides to its control. Low efficiency of triazol (demethylator inhibitors-DMI’s) or stobilurin (quinone outside inhibitor-QoI’s) has been reported on the control of ASR under field conditons. For this reason experiments were conducted to study the effects of these systemic fungicides associate with protectant multi-site fungicides (MSF) on ASR control preventively and at the onset of the epidemic. All combinations of fungicides were effective to control ASR and promoted greater soybean yield gain compared to the control. The analysis of the efficacy of ASR control in the lower, medium and upper part of the plant canopy, showed that application of Epoxiconazole + Pyraclostrobin and Cyproconazole + Azoxystrobin reduced ASR and leaf fall (%), and increased soybean yield grain. The severity of the disease increased from the lower to the upper part of the plant. The application of DMI+ QoI associated with MSF applyed when disease severity reached 2%, promoted effective reduction of ASR, increased yield and reduced leaf fall. Overal the DMI’s + QoI’s associated with MSF was 68% and 70.2% more efficient on ASR control and soybean grain yield, respectively than the control. Mancozeb and Chlorothalonil associated with the lowest dose of Epoxiconazol + Pyraclostrobin (10.2 + 33.2 g.a.i./ha) or Cyproconazole + Azoxystrobin (12.5 + 30.0 g.a.i./ha), respectively increased soybean grain yield at least 45.0%; the same combinations of the fungicides Epoxiconazol + Pyraclostrobin (15.3 + 49.8 g.a.i./ha) and Cyproconazole + Azoxystrobin (25.0 + 60.0 g.a.i./ha) 50% less than the commercial dose, increased 89.5% and 109.0%, respectively. The use of high risk (DMI and QoI) fungicides combined to low risk (MSF) applyied properly, can be a new strategy for the management of ASR at short and long term, even after the epidemics has reached 2.0% severity. Furthermore, due to the residual effect, MSF can promote higher longevity of DMI and QoI molecules.