Causal agents of Stemphylium-induced foliar diseases of tomatoes and other Solanaceae hosts in Brazil.

AIM: An extensive survey was done to clarify the prevalent Stemphylium species on Solanaceae plants across Brazil, and their host ranges. METHODS AND RESULTS: Eighty nine (89) Stemphylium isolates were obtained from naturally infected tomatoes as well as S. paniculatum, potato, eggplant, scarlet eggplant (Solanum aethiopicum var. gilo), Physalis angulata, and Capsicum species. Phylogenetic analyses encompassing the ITS-5.8S rDNA and glyceraldehyde-3-phosphate dehydrogenase genomic regions placed the isolates into two distinct groupings with either Stemphylium lycopersici or S. solani. Isolates of S. lycopersici (n = 81) were obtained infecting tomato, potato, eggplant, S. paniculatum, and P. angulata. Isolates of S. solani (n = 8) were detected in natural association with scarlet eggplant and tomato. Two isolates of S. lycopersici displayed a wide experimental host range in greenhouse bioassays, infecting accessions of 12 out of 18 species. Ocimum basilicum (Lamiaceae) was the only experimental host outside the Solanaceae family.

Phytophthora theobromicola sp. nov.: A New Species Causing Black Pod Disease on Cacao in Brazil.

Black pod disease, caused by Phytophthora species, is among the main limiting factors of cacao (Theobroma cacao L.) production. High incidence levels of black pod disease have been reported in Brazil, being induced by Phytophthora capsici, Phytophthora citrophthora, Phytophthora heveae, and Phytophthora palmivora. To assess the diversity of Phytophthora species affecting cacao in Brazil, 40 new isolates were obtained from cacao pods exhibiting symptoms of black pod disease collected in different smallholder farms in 2017. Further, ten cacao-infecting isolates morphologically identified as P. citrophthora and P. palmivora were molecularly characterized. The genomic regions beta-tubulin, elongation factor 1 alpha, heat shock protein 90, and internal transcribed spacer, and the mitochondrially encoded cytochrome c oxidase I and II genes were PCR-amplified and Sanger-sequenced from the cacao-infecting Phytophthora isolates. The morphological characterization and evaluation of the mycelial growth rates for the Phytophthora isolates were performed in vitro. Based on the molecular analysis and morphological comparisons, 19 isolates were identified as P. palmivora (clade 4). Interestingly, 31 isolates grouped together in the phylogenetic tree and were placed apart from previously known species in Phytophthora clade 2. Therefore, these isolates are considered as a new species herein referred to as Phytophthora theobromicola sp. nov., which produced papillate, semipapillate, and persistent sporangia on simple sporangiophores. The P. palmivora isolates were identified as A1 mating type by pairing each isolate with known A1 and A2 tester strains of P. capsici, but no oogonia/antheridia were observed when P. theobromicola was paired with the different tester strains. The P. theobromicola and P. citrophthora isolates showed higher mycelial growth rates, when compared to P. palmivora, on different media at 10, 15, and 20°C, but similar values were observed when grown on clarified CA media at 25 and 30°C. The pathogenicity tests carried out on pods of four cacao clones (CCN51, PS1319, Cepec2004, and CP49) showed significant variability among the isolates of both Phytophthora species, with P. theobromicola inducing higher rates of necrotic lesion expansion, when compared to P. palmivora. Here, two Phytophthora species were found associated with black pod disease in the state of Bahia, Brazil, and the previously undescribed P. theobromicola seems to be prevalent in field conditions. This is the first report of P. theobromicola on T. cacao. Also, these findings are crucial to improve the disease control strategies, and for the development of cacao materials genetically resistant to Phytophthora.

Diversity of tomato-infecting begomoviruses and spatiotemporal dynamics of an endemic viral species of the Brazilian Atlantic rain forest biome.

Yield losses induced by a complex of begomoviruses are observed across all major tomato-producing areas in Brazil. Tomato severe rugose virus (ToSRV) is the most widespread begomovirus in the country. Conversely, tomato common mosaic virus (ToCmMV) displays a more restricted geographical distribution to areas associated with the Atlantic Rain Forest (ARF) biome, encompassing the States of Espírito Santo-ES, Minas Gerais-MG, and Rio de Janeiro-RJ. Here, we characterized 277 tomato-infecting isolates collected in fields located within the ARF biome from 2006 to 2018. ToSRV displayed the highest prevalence (n = 157), followed by ToCmMV (n = 95) and tomato interveinal chlorosis virus (n = 14). Four other begomoviruses were also detected, but with very low incidences. ToCmMV was the predominant begomovirus in the ARF biome up to 2014-2015 with very low ToSRV incidence. Subsequently, ToSRV became the most prevalent species in ES and RJ, but ToCmMV was still predominating in the "Zona da Mata" meso-region in MG. Due to the remarkable endemic distribution of ToCmMV, we carried out phylogeographical studies of this virus using information from all 28 available isolates with complete DNA-A sequences. The closest common ancestor of ToCmMV was more likely originated around Coimbra-MG area ≈ 25 years before the formal report of this viral species. So far, all surveys indicated tomatoes as the only natural hosts of ToCmMV with outbreaks occurring mainly (but not exclusively) in highland areas. ToSRV shows a more widespread incidence across both highland and lowland areas of the ARF biome.

Three new species of Gliocephalotrichum causing fruit rot on different hosts from Brazil.

The genus Gliocephalotrichum (Nectriaceae), originally described as a soil-borne fungus, has been associated with postharvest diseases, especially of tropical fruits. Taxonomic studies using both morphological and molecular phylogenetic analyses have contributed to recognition of novel species in several countries. However, in Brazil, only three isolates of Gliocephalotrichum have been collected from soil samples and roots since the late 1970s. Our study expands the sample range using many Gliocephalotrichum isolates obtained from rotting fruits of tropical plant species in different states of Brazil. Polyphasic taxonomy was assessed with phylogenetic analyses of DNA sequences from four nuclear loci, morphological comparisons, and pathogenicity tests. As a result, three known species (G. bulbilium, G. longibrachium, and G. simplex) were identified from new hosts and locations in Brazil. In addition, three new species are described-G. abrachium, G. brasiliense, and G. caryocaris. A key to all Gliocephalotrichum species worldwide is provided. Although species of Gliocephalotrichum have not been considered to be important plant pathogens, this study shows they may cause postharvest fruit rot in tropical fruits and therefore have an impact in communities that depend economically on the harvest and sale of these fruits.

Morphologic, molecular, and pathogenic characterization of Phytophthora palmivora isolates causing flower rot on azalea.

The objective of this work was to characterize two Phytophthora palmivora isolates causing floral blight and rot in azalea plants and to evaluate the pathogenicity of this oomycete pathogen on several plant species. Azalea plants with symptoms of flower blight and rot were obtained in the municipality of Holambra-SP. After an attempt of isolation, colonies with Phytophthora characteristics grown only on selective V8 medium. Molecular identification of the isolates was done by amplification and sequencing of ITS and COX2 regions. In the phylogenetic analysis, the azalea isolates clustered with reference isolates of P. palmivora. Morphological characteristics were similar to those described for P. palmivora. Isolates were inoculated in healthy azalea plants and caused leaf blight and floral rot. The pathogen was re-isolated from symptomatic plants completing Koch's postulates. In a host range test, the azalea isolates were able to cause lesions on leaves of vinca, snapdragon, basil, and tomato, and affected both leaves and flowers of geranium. Fruit rot was observed on tomato, potato, sweet pepper, scarlet eggplant, zucchini, cucumber, maroon cucumber, onion, apple, papaya, guava, and carrot. This is the first report of the species P. palmivora causing flower blight and rot in azalea plants in Brazil and probably in the world.

Thiophanate-Methyl Resistance and Fitness Components of Colletotrichum musae Isolates from Banana in Brazil.

Anthracnose, caused by Colletotrichum musae, is the most important postharvest disease of banana and is widely distributed among the banana production regions in Brazil. Although thiophanate-methyl is a fungicide frequently used in Brazilian banana orchards to control Sigatoka leaf spot, Collettotrichum populations are also exposed, resulting in the evolution of fungicide resistance and the inability to manage banana anthracnose. We investigated 139 Brazilian isolates of C. musae for thiophanate-methyl sensitivity in vitro. The 50% mycelial growth inhibition (EC50) values varied between 0.003 and 48.73 µg/ml. One-hundred and thirty isolates were classified as sensitive, with EC50 values ranging from 0.003 to 4.84 µg/ml, while the remaining nine isolates were considered moderately resistant, with EC50 values ranging between 10.43 and 48.73 µg/ml. Resistant or highly resistant isolates (EC50 > 100 µg/ml) were not found. A substitution of TAC for TTC at codon 200 in a coding region of the ß-tubulin gene was associated with the moderately resistant phenotype. Applications of thiophanate-methyl formulation to detached banana fruit at the label rate (500 µg/ml) showed low efficacy in controlling the moderately resistant isolates on banana fruits. However, there is no indication of a reduction in fitness associated with fungicide resistance as sensitive and moderately resistant isolates do not differ with respect to mycelial growth rate (P = 0.098), spore production (P = 0.066), spore germination (P = 0.366), osmotic sensitivity (P = 0.051), and virulence (P = 0.057). Our results revealed absence of adaptability cost for the moderately resistant isolates, suggesting that they can be dominant in population if the fungicide continue to be applied.

Sensitivity of Brazilian Isolates of Phytophthora infestans to Commonly Used Fungicides in Tomato and Potato Crops.

Isolates of the US-1 and BR-1 clonal lineages of Phytophthora infestans, collected from tomato and potato fields of two main producing regions of Brazil (south and southeast), were tested for sensitivity to the systemic fungicide metalaxyl, plus the three protectant fungicides mancozeb, chlorothalonil, and cymoxanil. For metalaxyl, one agar test and two leaf-disc tests were carried out. For all tests, metalaxyl insensitive (I), intermediately insensitive (II), and sensitive (S) isolates were detected. There was no association among metalaxyl sensitivity and region, lineage, or host. In the agar test, 35.0% of 210 isolates were classified as I, 36.0% as II, and 29.0% as S. US-1 isolates were classified as 36.1% I, 30.6% II, and 33.3% S. BR-1 isolates were 33.3% I, 23.8% II, and 42.9% S. In leaf-disc test 1 (240 isolates tested), 24.3% were classified as I, 35.0% as II, and 40.7% as S. Isolates of US-1 were 21.0% I, 39.8% II, and 39.2% S, whereas BR-1 isolates were 36.0% I, 20.0% II, and 44.0% S. In leaf-disc test 2 (96 isolates tested), which was based on an effective dose for 50% sporulation inhibition (ED50), most isolates were either I (44.8%) or II (51.0%), and only three (4.2%) were S. The US-1 isolates were 46.0% I, 51.0% II, and 3.0% S. Isolates of the BR-1 lineage were 45.0% I, 52.0% II, and 3.0% S. For mancozeb, the ED50 for 53 of 59 isolates was below 1.0 µg/ml. No isolate grew on medium amended with more than 100 µg of chlorothalonil/ml and the ED50 for 38 of 50 isolates was below 1.0 µg/ml. For cymoxanil, the ED50 for all 47 isolates tested was below 1 µg/ml. There is no evidence of resistance of resistance of P. infestans to protectant fungicides commonly used in Brazil.

Characterization of Isolates of Phytophthora infestans from Southern and Southeastern Brazil from 1998 to 2000.

The population of Phytophthora infestans in Brazil was first characterized 12 years ago. In this research, isolates of P. infestans from potato (n = 184) and tomato (n = 267) collected in southern and southeastern Brazil were characterized to provide more detailed analysis of the current structure of the population. All 451 isolates were analyzed for mating type, and subsets of the isolates were analyzed for allozymes, restriction fragment length polymorphism fingerprint, mtDNA haplotypes, and metalaxyl resistance. Tomato isolates were all of A1 mating type, mtDNA Ib, and US-1 genotype or some variant within this clonal lineage. Of the potato isolates, 82% were A2 mating type, mtDNA IIa, BR-1 genotype, which is a new lineage of P. infestans. All A2 isolates were found on potato, whereas 91% of the A1 isolates were from tomato. A1 and A2 isolates were never found in the same field. The frequency of resistance to metalaxyl was higher in isolates from tomato (55%) than in isolates from potato (38%). After more than a decade of coexistence of isolates of the A1 and A2 mating types, the population was highly clonal, dominated by the BR-1 and US-1 clonal lineages.

Bacillus subtilis as a potential biocontrol agent of the northern leaf blight of corn

Quatro isolados de Bacillus subtilis (AP-3, AP-420, IF-36 e IF-82) foram estudados como agentes potenciais para o biocotrole da queima das folhas do milho, causada por Exserohilum turcicum. Em condiçöes de casa-de-vegetaçäo os isolados bacterianos demonstraram boa eficiência para o biocontrole. Considerando a média de 3 períodos de aplicaçäo, o isolado AP-420 foi o mais eficiente. A aplicaçäo dos antagonistas simultaneamente com a inoculaçäo de E. turcicum apresentou os melhores resultados. Em condiçöes de laboratório as bactérias produziram metabólitos extracelulares, näo-voláteis e difusíveis que inibiram o crescimento micelial e a germinaçäo de conídios de E. turcicum