Photosynthetic and antioxidative alterations in coffee leaves caused by epoxiconazole and pyraclostrobin sprays and Hemileia vastatrix infection.

Coffee leaf rust (CLR), caused by Hemileia vastatrix, is a major disease affecting coffee production worldwide. In this study, an in-depth analysis of the photosynthetic performance of coffee leaves challenged or not with H. vastatrix and sprayed with either epoxiconazole (EPO) or pyraclostrobin (PYR) was performed by combining chlorophyll a fluorescence images, photosynthetic pigment pools and the activities of chitinase (CHI), ß-1,3-glucanase (GLU), peroxidase (POX) and catalase (CAT). The CLR severity was higher in the control plants, but reduced in plants sprayed with both PYR and EPO. Also, the CLR severity was reduced in plants sprayed with PYR compared with plants sprayed with EPO. Plants sprayed with either EPO or PYR showed maximal photosystem II quantum efficiency (Fv/Fm) values ranging from 0.78 to 0.80, which were quite similar to those obtained with inoculated plants (values ranging from 0.74 to 0.77). The decreases in the Fv/Fm ratio values and parallel increases in the F0 values in the inoculated plants, which were not observed in the control plants (sprayed with water) and were confirmed by images of the initial fluorescence (F0) and Fv/Fm parameters in the regions of the leaf tissue containing pustules and in the asymptomatic leaf tissue, indicated that photosynthesis was negatively impacted. When effective photosystem II quantum yield (Y(II)) values approached zero with a high photosynthetic photon flux density, high values of quantum yield of regulated energy dissipation (Y(NPQ)) in association with a high carotenoid concentration were noted in the inoculated plants sprayed either with PYR or EPO. The increased CLR severity in inoculated plants in contrast to inoculated plants sprayed with either PYR or EPO was associated with greater POX activity and a reduced photosynthetic pigment concentration. POX and CAT activities were increased in inoculated plants sprayed with either EPO or PYR when compared with control plants. CHI and GLU activities were maintained at high levels in the leaves of inoculated plants, regardless of the fungicide sprayed, indicating that CHI and GLU are less important for coffee resistance against CLR. The results of the present study clearly demonstrated that plants sprayed with either EPO or PYR showed milder CLR symptoms with adequate photosynthetic performance and optimal conditioning of their antioxidant systems.

Colletotrichum boninense Causing Anthracnose on Coffee Trees in Brazil.

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.

Genetic characterization of an elite coffee germplasm assessed by gSSR and EST-SSR markers.

Coffee is one of the main agrifood commodities traded worldwide. In 2009, coffee accounted for 6.1% of the value of Brazilian agricultural production, generating a revenue of US$6 billion. Despite the importance of coffee production in Brazil, it is supported by a narrow genetic base, with few accessions. Molecular differentiation and diversity of a coffee breeding program were assessed with gSSR and EST-SSR markers. The study comprised 24 coffee accessions according to their genetic origin: arabica accessions (six traditional genotypes of C. arabica), resistant arabica (six leaf rust-resistant C. arabica genotypes with introgression of Híbrido de Timor), robusta (five C. canephora genotypes), Híbrido de Timor (three C. arabica x C. canephora), triploids (three C. arabica x C. racemosa), and racemosa (one C. racemosa). Allele and polymorphism analysis, AMOVA, the Student t-test, Jaccard's dissimilarity coefficient, cluster analysis, correlation of genetic distances, and discriminant analysis, were performed. EST-SSR markers gave 25 exclusive alleles per genetic group, while gSSR showed 47, which will be useful for differentiating accessions and for fingerprinting varieties. The gSSR markers detected a higher percentage of polymorphism among (35% higher on average) and within (42.9% higher on average) the genetic groups, compared to EST-SSR markers. The highest percentage of polymorphism within the genetic groups was found with gSSR markers for robusta (89.2%) and for resistant arabica (39.5%). It was possible to differentiate all genotypes including the arabica-related accessions. Nevertheless, combined use of gSSR and EST-SSR markers is recommended for coffee molecular characterization, because EST-SSRs can provide complementary information.

First Report of Cercospora apii Leaf Spot on Capsicum chinense in Brazil.

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.

First Report of Leaf Blight on Rubus brasiliensis Caused by Colletotrichum acutatum in Brazil.

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.

Insecticide use and organophosphate resistance in the coffee leaf miner Leucoptera coffeella (Lepidoptera: Lyonetiidae).

Increasing rates of insecticide use against the coffee leaf minerLeucoptera coffeella(Guérin-Méneville) and field reports on insecticide resistance led to an investigation of the possible occurrence of resistance of this species to some of the oldest insecticides used against it in Brazil: chlorpyrifos, disulfoton, ethion and methyl parathion. Insect populations were collected from ten sites in the state of Minas Gerais, Brazil and these populations were subjected to discriminating concentrations established from insecticide LC99s estimated for a susceptible standard population. Eight of the field-collected populations showed resistance to disulfoton, five showed resistance to ethion, four showed resistance to methyl parathion, and one showed resistance to chlorpyrifos. The frequency of resistant individuals in each population ranged from 10 to 93% for disulfoton, 53 to 75% for ethion, 23 to 76% for methyl parathion, and the frequency of resistant individuals in the chlorpyrifos resistant population was 35%. A higher frequency of individuals resistant to chlorpyrifos, disulfoton and ethion was associated with greater use of insecticides, especially other organophosphates. This finding suggests that cross-selection, mainly between organophosphates, played a major role in the evolution of insecticide resistance in Brazilian populations of L. coffeella. Results from insecticide bioassays with synergists (diethyl maleate, piperonyl butoxide and triphenyl phosphate) suggested that cytochrome P450-dependent monooxygenases may play a major role in resistance with minor involvement of esterases and glutathione S-transferases.

Effects of Angular Leaf Spot and Rust on Yield Loss of Phaseolus vulgaris.

ABSTRACT Three field experiments were conducted in 1997, 1998, and 1999 to investigate the effects of angular leaf spot and rust, separately or combined, on host growth and yield of individual bean plants (Phaseolus vulgaris). In each experiment, three treatments were established by inoculating cv. Carioca with Phaeoisariopsis griseola, Uromyces appendiculatus, or with both pathogens. An additional control treatment was not inoculated, but was sprayed with a fungicide. In the 1997 and 1999 experiments, angular leaf spot reached higher disease levels than rust, whereas in 1998, rust was more severe than angular leaf spot. Host growth, expressed as healthy leaf area duration (HAD), and yield were the highest in 1997 and lowest in 1998. In each experiment, the treatments did not differ significantly to the area under leaf area progress curve, HAD, and healthy leaf area absorption (HAA). All inoculated treatments had significantly more severe disease and less yield than the control treatment. Based on the analysis of 60 plants in each experiment, yield was not related to the areas under disease progress curve for either or both diseases. In 1997 and 1999, yield was related to HAD (R(2) = 0.57 and 0.43) and HAA(R(2) = 0.60 and 0.55). Based on the combined analysis of all 36 plots, angular leaf spot reduced the leaf area because of defoliation, whereas rust did not affect the leaf area. Rust reduced yield more than four times that of angular leaf spot, although the decrease in photosynthesis to angular leaf spot was twice that of rust.

Incidence-Severity Relationships in the Pathosystem Coffea arabica-Hemileia vastatrix.

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.