Genotypic and Pathogenic Diversity of Colletotrichum sublineola Isolates from Sorghum (Sorghum bicolor) and Johnsongrass (S. halepense) in the Southeastern United States.

Anthracnose caused by Colletotrichum sublineola is an important disease of cultivated sorghum (Sorghum bicolor) worldwide. Anthracnose is also common on the ubiquitous wild sorghum relative Johnsongrass (S. halepense). Analysis of repetitive molecular fingerprinting markers revealed that isolates of C. sublineola from both hosts in the southeastern United States were genotypically diverse, with relatively few haplotypes found in more than one location. With few exceptions, isolates recovered from S. bicolor belonged to a population that was genetically distinct from the population recovered from S. halepense. Twenty-three isolates from cultivated sorghum were all pathogenic to at least one of 13 heritage inbred lines of S. bicolor. In all, 4 of 10 isolates from S. halepense were also pathogenic to one or more of the lines, while the rest caused no disease in greenhouse assays. The four pathogenic isolates from S. halepense were less aggressive, on average, than isolates from S. bicolor, although the ranges overlapped. Pathogenicity tests involving 15 representative pathogenic isolates from S. bicolor and S. halepense on eight heritage inbred lines of S. bicolor identified 12 races. The combined results of this study demonstrated that C. sublineola comprises two separate host-associated subpopulations in the field, even though some isolates from S. halepense were able to cause disease on S. bicolor under ideal greenhouse conditions. Nonetheless, the apparent existence of infrequent cross-infection events in the field, indicated by molecular fingerprinting, suggests that Johnsongrass has the potential to serve as a refuge and an incubator for genetic diversity in C. sublineola, which can complicate efforts to develop and deploy resistant sweet sorghum varieties in the region.

Similar Aggressiveness of Phenotypically and Genotypically Distinct Isolates of Sclerotinia sclerotiorum.

Understanding how Sclerotinia sclerotiorum aggressiveness varies among isolates may be useful for breeding programs aimed at developing common bean cultivars resistant to white mold. The aggressiveness of 20 S. sclerotiorum isolates collected in common bean fields from four Brazilian states was tested against two common bean genotypes (Pérola and A195) using two inoculation methods. The isolates were characterized using 10 microsatellite (SSR) loci, mycelial compatibility groups (MCGs), partial sequences of the oxaloacetate acetylhydrolase (OAH) gene, and morphological traits. Twenty SSR and seven OAH haplotypes, 10 MCGs, and high variability in colony morphology were found. One isolate was more aggressive when inoculated on plants of the genotype A195, but all other isolates had similar aggressiveness. Aggressiveness was not related with MCGs, SSR, OAH haplotypes, mycelial pigmentation, growth rate, or sclerotia production.

Fungicide Sensitivity of Sclerotinia sclerotiorum: A Thorough Assessment Using Discriminatory Dose, EC50, High-Resolution Melting analysis, and Description of New Point Mutation Associated with Thiophanate-Methyl Resistance.

Thiophanate-methyl (TM), fluazinam, and procymidone are fungicides extensively used for white mold control of common bean in Brazil. We assessed the sensitivity of Brazilian isolates of Sclerotinia sclerotiorum to these three fungicides using discriminatory doses and concentration that results in 50% mycelial growth inhibition (EC50) values. In total, 282 isolates from the most important production areas were screened and none was resistant to fluazinam or procymidone. The EC50 values varied from 0.003 to 0.007 and from 0.11 to 0.72 µg/ml for fluazinam and procymidone, respectively. One isolate was resistant to TM. The EC50 of the TM-resistant isolate was greater than 100 µg/ml, whereas the EC50 of the sensitive isolates varied from 0.38 to 2.23 µg/ml. The TM-resistant isolate had a L240F mutation in the ß-tubulin gene. This is the first report of mutation at codon 240 causing resistance to a benzimidazole fungicide in S. sclerotiorum. The high-resolution melting analysis allowed the distinction of TM-sensitive and -resistant isolates by specific melting peaks and curves. The TM-resistant isolate had mycelial growth, sclerotia production, and aggressiveness comparable with that of the sensitive isolates, indicating that this genotype will likely compete well against sensitive isolates in the field. This study demonstrates that resistance to TM, fluazinam, and procymidone is nonexistent or rare. Resistance management practices should be implemented, however, to delay the spread of TM-resistant genotypes.

Genetic diversity analysis of isolates of the fungal bean pathogen Pseudocercospora griseola from central and southern Brazil.

Planting resistant varieties is the most effective control measure against the angular leaf spot of dry beans, a fungal disease caused by Pseudocercospora griseola. However, dry bean varieties with durable resistance are not easily obtained. Knowledge about the genetic variability of the pathogen population is key for the success of dry bean breeding programs aimed at developing resistant materials, but finding suitable operationally simple and genetically accurate markers is not an easy task. The aim of this study was to assess the suitability of the ISSR-PCR technique to quantify the genetic variability of P. griseola isolates. Total DNA of 27 P. griseola isolates from Goiás, Minas Gerais, Espírito Santo, and Paraná States was extracted and amplified using specific primers for ISSR. Using cluster analysis, 27 genotypes were identified. The ISSR-PCR technique was suitable for assessing intraspecific variability of P. griseola. The ISSR-PCR marker was found to be highly sensitive to genetic variation and can aid in elucidating the genetic structure of the population of this plant pathogen as a support tool for the dry bean breeding programs.

First Report of Papaya Fruit Rot Caused by Colletotrichum magna in Brazil.

Species of the genus Colletotrichum are commonly reported as pathogens of fruits in tropical regions. Papaya fruits (Carica papaya L.), cv. Golden, with typical lesions of anthracnose, chocolate spot, and/or stem-end rot were collected from 18 papaya-producing areas of northeast Brazil in 2007. One hundred and fifty-five isolates of Colletotrichum spp. were obtained from the fruit lesions and cultured on potato dextrose agar. Pathogenicity tests were conducted by placing a 20-µl drop of 105 conidia ml-1 suspension on a wounded area of two healthy fruits of cv Golden at the climacteric stage. Inoculated fruits were placed in a moist chamber at 26°C (±2) for 48 h. After this period, the plastic covers of the trays used to form the moist chamber were removed and the trays were kept at 26°C (±2) for 98 h when symptoms were assessed. The causal agents of fruit rot were recovered from inoculated fruits showing symptoms of anthracnose and chocolate spot. Conidia from fresh lesions were collected and measured. Conidia dimensions were 13.49 × 3.80 µm, length/width ratio = 3.55 µm. Conidia were predominantly cylindrical to bluntly rounded ends and slightly flattened. All isolates were morphologically similar to Colletotrichum gloeosporioides Penz (1). Molecular analyses of the isolates were carried out with taxon-specific primers for C. acutatum J.H Simmonds and C. gloeosporioides (3). Only one amplicon was detected for eight isolates with the C. gloeosporioides primer. All isolates were genotyped using inter-simple sequence repeat (ISSR) primers. Three groups of isolates were found, one containing the eight C. gloeosporioides isolates, a second group comprised of 141 isolates, and a third contained six isolates. The second and third groups were more similar to each other than to the first C. gloeosporioides group. Thirty two representative isolates of the three ISSR groups were sequenced for the internal transcribed spacer (ITS) and glutamine synthetase (GS) (GenBank Nos. HM163181 and HM015847) regions. With molecular phylogenetic analyses, two well-supported clades were formed, one with the C. gloeosporioides isolates and the other with sequences highly similar (99% similarity) to the two ITS sequences available in GenBank (DQ003310 and GU358453) and the GS region of G. magna Jenkins & Winstead (DQ792873). The latter was reported in the United States and Taiwan (2,4). Isolates of C. magna and C. gloeosporioides are morphologically similar and identification needs to be based on molecular analyses. To our knowledge, this is the first report of C. magna causing rot of papaya fruit in Brazil. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) M. Z. Du et al. Mycologia 97:641, 2005. (3) P. Talhinhas et al. Appl. Environ. Microbiol. 71:2987, 2005. (4) J. G. Tsay et al. Plant Dis. 94:787, 2010.

Genetic variability of Cercospora coffeicola from organic and conventional coffee plantings, characterized by vegetative compatibility.

Cercospora leaf spot is a destructive fungal disease that has become a threat to the coffee industry in Brazil. Nevertheless, little is known about populations of its causal agent, Cercospora coffeicola. We evaluated the potential of using nitrogen-nonutilizing (nit) mutants and vegetative compatibility groups (VCGs) to characterize the genetic variability of the C. coffeicola population associated with coffee plantings in Minas Gerais state (MG), Brazil. A total of 90 monosporic isolates were obtained from samples collected according to a hierarchical sampling scheme: (i) state geographical regions (Sul, Mata, and Triângulo), and (ii) production systems (conventional and organic). Nit mutants were obtained and 28 VCGs were identified. The 10 largest VCGs included 72.31% of all isolates, whereas each of the remaining 18 VCGs included 1.54% of the isolates. Isolates of the largest VCGs were found in the three regions sampled. Based on the frequencies of VCGs at each sampled level, we estimated the Shannon diversity index, as well as its richness and evenness components. Genetic variability was high at all hierarchical levels, and a high number of VCGs was found in populations of C. coffeicola associated with both conventional and organic coffee plantings.

Brazilian isolates of Clonostachys rosea: colonization under different temperature and moisture conditions and temporal dynamics on strawberry leaves.

AIMS: In a research programme for managing diseases caused by Botryis cinerea, four isolates of the antagonistic fungus Clonostachys rosea (Cr) were obtained from different ecosystems in Brazil. We studied ecological requirements for the colonization of strawberry leaves by these isolates. METHODS AND RESULTS: Temperature effects on both mycelial growth in vitro and leaf colonization by Cr were studied. At 10 degrees C, growth on potato dextrose agar and colonization of leaf discs were poor. Optimum temperature for mycelial growth and leaf colonization was around 25 degrees C. The isolates were applied to leaves which were exposed to 0-48 h intervals of moisture. They were also applied to leaves which remained from 0 to 36 h without wetness. All isolates efficiently colonized leaves, regardless of moisture interval or the delay to begin wetness. Although all isolates survived in green leaves of whole plants, colonization decreased throughout a 49-day period. CONCLUSIONS: Brazilian isolates of Cr can establish and colonize strawberry leaves under a wide range of temperature and moisture conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: It is expected that the Brazilian isolates of Cr will establish efficiently in strawberry leaves where they can compete with B. cinerea.

The Roles of Three Fungicides in the Epidemiology of Potato Late Blight.

Three fungicides were tested in the field for efficacy on late blight caused by Phytophthora infestans. The effects of these fungicides on epidemic development, lesion growth rate and sporulation were measured. No fungicide completely arrested epidemic development under the environmental conditions of these experiments. However, the fungicide mixture, propamocarb hydrochloride plus chlorothalonil, had the most suppressive effect of the fungicides tested. The mechanism of effect included suppression of disease progress and lesion expansion. Growth chamber studies demonstrated that 24°C compared to 10 or 16°C limited cymoxanil efficacy.

Oospore Survival and Pathogenicity of Single Oospore Recombinant Progeny from a Cross Involving US-17 and US-8 Genotypes of Phytophthora infestans.

Oospores of Phytophthora infestans produced in vitro and in planta, from a cross between US-17 and US-8 genotypes, were exposed to a variety of environments and their survival was assessed. Additionally, the pathogenic characteristics of some resultant progeny isolates were assessed. Viability of oospores as measured by plasmolysis declined slightly over a period of 18 months whether they were stored in water at 4°C, in soil at 18°C, or in soil under natural field conditions. In comparison, viability as measured by germination was lower overall but appeared to increase after storage in soil. Oospores produced in planta were buried in the field in the fall of 1998, and were capable of infecting both tomato and potato leaflets when recovered in May 1999. Single oospore progeny (n = 53) from the in vitro cross were analyzed individually for genetic and pathogenicity characteristics. All 53 progeny tested for restriction fragment length polymorphisms with probe RG57 were hybrids. All but one progeny produced sporulating lesions on detached potato or tomato leaflets in growth chamber tests, but most lesions were smaller and developed more slowly than those produced by either parental isolate. In a further test of pathogenicity, under field conditions, none of a subset of 10 A2 progeny was capable of initiating a detectable epidemic in small plots of either potatoes or tomatoes.