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Persimmon (Diospyros kaki) anthracnose is a major threat in production areas worldwide. Most of the studies are focused on Colletotrichum horii, but other species have been reported as well. The association of distinct Colletotrichum species present in Brazilian persimmon production regions as well as their host ranges are yet elusive. The aims of this work were to identify and characterize Colletotrichum species associated with the persimmon anthracnose. In a survey performed in four production regions of Brazil, 88.7% and 11.3% out of 231 isolates were identified as members of Colletotrichum gloeosporioides species complex (Cgc) or Colletotrichum acutatum species complex (Cac), respectively. A subset of 18 isolates were identified through multilocus phylogenetic analysis, using ITS-rDNA region and two loci, namely GAPDH and TUB2. This study revealed the presence of four species: C. horii (38.8%) and Colletotrichum fructicola (27.7%) from the Cgc and Colletotrichum nymphaeae (27.7%) and Colletotrichum melonis (5.8%), from the Cac. Additionally, 13 isolates were selected for morphological, physiological, and pathogenic analyses. Contrasting characteristics were observed among species of the Cgc and Cac complexes. The optimal temperature for mycelial growth and germination were higher for Cgc species. The percentage of appressoria melanisation also varied across complexes. All the identified species were able to cause anthracnose-like symptoms on persimmon fruit, leaves, shoots, and sepals. Colletotrichum species from persimmon were also able to infect apple and pear. The findings will support decisions to manage anthracnose of persimmon under high infection risk due to multiple host susceptibility.

Neonectria ditissima, the causal agent of European Canker and Neonectria fruit rot, is a quarantine apple pathogen in Brazil, restricted to some production areas of Rio Grande do Sul, Santa Catarina, and Parana states. The quarantine rests on a product certification based on the absence of symptoms but it is not known whether the pathogen could be moving around in quiescent infections. In this work, we detected N. ditissima after incubation of asymptomatic commercial fruit. The identity of the pathogen was confirmed using morphological and molecular tools, and Koch’s postulates were fulfilled on “Gala” apples. We demonstrated that fruits are weak links in quarantine efforts. Fruit stored at low temperatures present quiescent N. ditissima infections, which may develop rapidly with abundant sporulation when exposed to more suitable conditions, as those of commercialization. Revised management strategies for flower and fruit treatment, as well as studies on efficient detection methods are strongly recommended.

Colletotrichum abscissum and C. gloeosporioides are the causal agents of citrus Postbloom Fruit Drop (PFD), a major disease in several countries of the American continent. These pathogens infect only floral structures; however, in the absence of floral tissue, they survive asymptomatically on the leaf tissue. Thus, detecting and quantifying pathogens on leaves are critical for epidemiological studies. Colletotrichum species are detected through isolation, serological methods and conventional PCR, but these techniques are not effective for accurate detection of PFD agents. This study aimed to develop specific, more sensitive, faster and less laborious techniques than the traditional ones routinely used. We developed, standardized and validated a multiplex real-time PCR with high sensitivity (98–99.3%) and specificity (94.3–97.1%) for the detection and quantification of both pathogens. This technique was 1000 and 10,000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. abscissum. Similarly, the multiplex qPCR was 100 and 1000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. gloeosporioides. The diagnostic parameters used to validate the multiplex qPCR showed a high positive (17–35) and low negative (0.006–0.95) likelihood ratios, low percentage of false positives (2.8–5.6%) and false negatives (0.6–1.9%), good agreement between the results generated between qPCR and multiplex qPCR, revealed by the Kappa index (0.91–0.98) and coincidental results (95–99%). All parameters indicated that the multiplex qPCR was effectively validated to detect and quantify the causal agents of PFD.

The dicarboximide fungicide iprodione has been used in Brazilian peach orchards for more than three decades to control brown rot and there are no studies on sensitivity to Monilinia fructicola in the country. In addition, isolates with reduced sensitivity to other important groups of fungicides have already been reported in Brazilian isolates. The sensitivity of M. fructicola to iprodione was evaluated using discriminatory doses and concentration resulting in 50% inhibition of mycelial growth (EC50) values. In total, 174 isolates collected from the main peach production areas were characterized for fungicide sensitivity. Disease control efficacy (DCE) was determined by comparing lesion inhibition on iprodione treated fruits with those without fungicide treatment. No resistant phenotypes were found in the M. fructicola population and 71.8% of the isolates were extremely sensitive to iprodione. The EC50 values of the isolates ranged from 0.13 to 1.01 μg/mL. DCE in wounded and non-wounded fruit was greater than 85% providing an efficient control in controlled conditions. These results suggest that iprodione could be integrated into disease management programs to prevent the establishment of resistant populations as already reported to other fungicides.

Pleoroma fotherghillae, also known as "princess flower", is an ornamental species native to Brazil and naturalized in several countries (Faravani et al. 2007). P. fotherghillae has a high economic value, with an ornamental and landscape application (Nienow et al. 2010). In September 2018, leaf spots were observed in approximately 80% of the 50 P. fotherghillae plants grown in a nursery in the municipality of Curitiba-Parana, Brazil. The spots were round-shaped, with a necrotic brown center and a reddish-brown halo, ranging from 1 to 4 mm in diameter. High leaf fall was observed among plants presenting a higher severity. Symptomatic leaves fragments were collected and disinfected as described by (Pereira et al. 2019). The fragments were transferred to a potato dextrose agar medium supplemented with streptomycin sulfate and incubated at 24 ± 1oC with a photoperiod of 12 h for 7 days. Four monosporic cultures were obtained from colonies isolated. The isolates had a grayish-white cottony aerial mycelium and reverse olive-yellow with black dots. The colonies reached approximately 60 mm in diameter, forming globular and conical pycnidia, brown to black in color with white or cream globular conidial mass. Beta conidia were hyaline, smooth, curved to the size of 19 - 25 x 1 - 1.5 μm (n = 50). No alpha nor gamma conidia were observed. The characteristics are similar to the description of Diaporthe terebinthifolli (Gomes et al. 2013). The total genomic DNA of a representative isolate, LEMIDPRPf-19-02, was extracted for amplification and sequencing of the internal transcribed spacer (ITS) region and partial of the Tubby (TUB) and thyrotroph embryonic factor (TEF) genes. The sequences of the ITS (No MN415990.1), TUB (No MW505549), and TEF (No MW505550) genes were deposited in GenBank. BLAST analysis showed similarity above 99% with D. terebinthifolli sequences (KC343219.1, KC344187.1, and KC343945.1). The multigene phylogenetic analysis, based on Bayesian Inference, grouped the isolate in a clade with other sequences of Diaporthe terebinthifolii. Four healthy plants of P. fotherghillae about 5 months old, were used for pathogenicity testing. A suspension containing 105 conidia/ml was sprayed on the surface of the leaves of four plants to the point of runoff. The plants were covered with a transparent plastic bag for 24 hours. The leaves of four other plants received sterile distilled water and served as the control treatment. The plants were kept in a greenhouse at 20±5oC. Necrotic lesions appeared 10 to 15 days after inoculation. No symptoms were observed in the control plants. The pathogen was reisolated from symptomatic leaves and had the same characteristics as the isolate LEMIDPRPf-19-02. A representative sample (MBM 331603) was deposited at the Museu do Jardim Botânico (Botanical Garden Museum) - Curitiba, Brazil. Diaporthe terebinthifolii was previously reported as endophytic in Brazil and Uruguay, isolated from Schinus terebinthifolius and Pyrus communis, respectively (Gomes et al. 2013; Sessa et al. 2017). To our knowledge, this is the first report of D. terebinthifolii causing leaf spot on P. fotherghillae in Brazil and worldwide.

Impatiens walleriana (Balsaminaceae), popularly known as Impatiens, is an African succulent and a popular ornamental plant worldwide (GBIF, 2019). In Brazil it is broadly grown indoors and outdoors, including in public parks of Curitiba, State of Parana (Viezzer et al. 2018). In September 2018, I. walleriana plants showing typical downy mildew symptoms were observed in wastelands and gardens in Curitiba. The symptoms included adaxial chlorotic leaf spots with abundant white sporulation on abaxial side (Supplementary figure 1). The disease led to severe defoliation of the plants and the incidence of the plant disease varied from 20 to 80% of plants in an area ranging from 400 to 40,000 m2. A representative sample was deposited in herbarium of the Museu Botânico Municipal de Curitiba (MBM 331601). The following morphology was observed: Sporangiophores (n = 30), hyaline, thin walled, emerging through stomata, 407.3 to 551.1 μm long, slightly swollen base, first branch at 165.8 to 324.7 μm from base, end branches 5.1 to 13.1 μm long, sporangia (n = 50) hyaline, thin-walled subglobose to ovoid, from 12.8 to 21.9 μm x 12.5 to 17.9 μm, slightly papillate. Due to morphological and genetic variations within the species Plasmopara obducens, Gorg et al. (2017) proposed the new species P. velutina and P. destructor. The morphology of the Curitiba specimen was equivalent to that described for P. destructor (Gorg et al. 2017). DNA was extracted from LEMIDPRTf-19-02 isolate and the ITS1 and cox2 regions were PCR amplified as described in Gorg et al. (2017). The resulting sequences were deposited in GenBank (ITS1, MT680628; cox2, MT952335). A BLASTn analysis of the sequences revealed 100% homology with ITS (MF372742) and cox2 (MF372728) sequences of type strain of P. destructor (GLM-F107554). A Bayesian phylogenetic analysis was performed to compare the sequences from this study with reference sequences for P. obducens, P. destructor and P. velutina (Gorg et al. 2017; Salgado-Salazar et al. 2018). The oomycete from Curitiba grouped in a reliable clade with P. destructor (Supplementary figure 2). Pathogenicity was carried out by ex vivo and in vivo tests. For ex vivo, stems with approximately four healthy leaves of I. walleriana (n = 10) were embedded in aluminum grid inside of gerbox with the stem bases immersed in distilled water. The inoculation of five stems was carried out by spraying a suspension with 6 x 104 sporangia mL-1 on the abaxial side of the leaves. Five stems with leaves inoculated with sterile water were used as controls. They were incubated in a growth chamber in the dark for 48 h at 20 °C and another 12 days in a 12 h light photoperiod. The confirmation of pathogenicity in plants (in vivo) was obtained with the inoculation of I. walleriana seedlings (one-month old) grown in 2 dm3 aluminum pots. The inoculation methodology and number of plants were the same as the stems test. After the inoculation, plants were incubated in a growth chamber for 48 h in the dark at 20 °C with 100% RH with nebulization, and another 10 days at a photoperiod of 12 hours of light. For both tests, abundant sporulation was observedwith morphology equivalent to Plasmopara destructor described by Gorg et al. (2017). No disease developed on control plants. To our knowledge, this is the first report of P. destructor on I. walleriana in Brazil (Farr and Rossman 2019, Silva et al. 2019) representing a potential loss to flower production and a reduction in flowering period in public gardens and parks.

The objectives of this work were to investigate the Botrytis species that cause gray mold in strawberries in Parana and to study their sensitivity to fungicides: procymidone (P), iprodione (I), boscalid (B), thiophanate-methyl (T), fluazinam (F), azoxystrobin (A) and difenoconazole (D). The identification of 150 isolates of Botrytis sp. was carried out by amplifying the DNA by PCR using specific primers. The sensitivity to fungicides was determined by in vitro assays with discriminatory doses and EC50 (effective concentration to inhibit 50% of the growth of the fungus), molecular characterization of mutations and ex vivo assays for disease control efficacy. Thirty phenotypes were selected from combinations (S-sensitive and R-resistant) for the EC50 studies. Molecular characterization was performed using specific primers for mutations in the cyt b, bos1 and β-tubulin genes to confirm the resistance genotype. Tests were carried out with fruit treated with the fungicides (P), (B), (F) and (A) x the different phenotypes. All isolates were confirmed as Botrytis cinerea. Forty-one isolates were classified as fungicide sensitive phenotypes and of these, six isolates showed resistance to all seven tested fungicides. The percentage of isolates resistant to the fungicides azoxystrobin, boscalid, difenoconazole, fluazinam, iprodione and procymidone was 90.7; 45.3; 33.3; 32.7; 44.0 and 43.3%, respectively. A total of 94.6% isolates were classified as highly resistant to thiophanate-methyl. The EC50 for the fungicides procymidone, fluazinam and boscalid ranged from less than 0.1 to greater than 100 μg/ml. For azoxystrobin, most isolates had an EC50 greater than 100 μg/ml. Isolates with more than one mutation for different fungicides were found. For the assay in fruits with infections from the field, only the fungicide fluazinam differed from the control treatment, showing lower final incidence of 33.3% and control efficiency of 55.6%. For the assay with inoculated fruits, the fungicide fluazinam best controlled the disease for the three isolates presenting an average final incidence of 33.3% and an average control efficiency of 61.9%. Therefore, disease management in the state of Parana should be reviewed in terms of registering new fungicides to gray mold and the use of integrated management measures to reduce the reliance on fungicides.

Quinone-outside inhibitor (QoI) fungicides are effective tools for preharvest control of brown rot of stone fruit. These fungicides have a very specific site of action so the risk of resistance selection is high. The sensitivity of Monilinia fructicola (G. Winter) Honey isolates to azoxystrobin (QoI) was investigated in 143 isolates collected between 2002 and 2011 from four Brazilian states in orchards with different frequencies of fungicide use (0 to 6 fungicides sprays/season). Sensitivity of the isolates to azoxystrobin was determined in vitro, by inhibition of mycelial growth and spore germination on fungicide-amended media or ex vivo by pathogen inoculation in untreated or treated fruit with azoxystrobin. Potential mutations in codons 143, 137, and 129 of the cytochrome b (Cyt b) gene and the occurrence of an intron immediately after codon 143 were analyzed in a subpopulation of the isolates. The M. fructicola population of São Paulo State was less sensitive to the fungicide than the population from the states of Paraná, Santa Catarina, and Rio Grande do Sul. The low sensitivity of the isolates was confirmed also by comparing to the sensitivity of the baseline isolates. Mutations in G143A, F129L, and G137R in Cyt b gene were not found. In addition, 58 isolates tested showed an intron after codon 143 in Cyt b gene. Our results indicate that other mechanisms of selection for low sensitivity to QoI fungicides should be investigated.

Bidens pilosa L. (Asteraceae) is an aggressive weed in annual and perennial crops with high potential to reduce crop yields in several countries (Dias et al. 2017). It also may host pathogens such as Alternaria dauci (J.G. Kuhn) J.W. Groves & Skolko (Lin et al. 2015) and Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Cho et al. 2013). In Brazil, this weed is distributed across all grape-producing areas. Leaf blight on grape, caused by Pseudocercospora vitis (Lev.) Speg., is an important disease that causes premature defoliation. In March 2018, plants of B. pilosa showing leaf spots were found in a 1-ha organic orchard of fox grape cultivar Bordo in the metropolitan region of Curitiba in Parana state, Brazil. The orchard had high leaf blight incidence (above 80%). The disease normally started in early summer; in December, and around March, complete defoliation occurred caused by leaf blight. B. pilosa was distributed throughout the orchard, and around 50% of leaves were infected. Leaf spots initially appeared slightly chlorotic, becoming pale brown to dark brown. Lesions were circular or irregular, 2 to 8 mm in diameter, and often coalesced. Microscopic examination revealed that conidia were hyaline to light brown, cylindrical, straight or slightly curved, with a rounded apex. They had 4 to 10 transverse septa, 24 to 82 μm in length and 5 to 8 μm in width, with conidiophores grouped in synnema. Isolates were recovered from lesions on tissue surfaces that were disinfested by immersion in 70% alcohol for 30 s and then in 0.5% NaClO for 30 s. Pieces were washed three times with sterile distilled water and cultured on potato dextrose agar (PDA) at room temperature for 9 days under continuous fluorescent light. Hyphal tips of the obtained colony were transferred to V8 agar medium and cultured at room temperature for 4 weeks under continuous fluorescent light. Individual spores were collected and deposited in V8 agar medium to obtain one monosporic isolate (PCLEMID02). The colony presented slow growth, with 15-mm diameter after 1 week of PDA cultivation at 25°C under a 12-h photoperiod, and was dark gray on top, black on reverse. The morphological and cultural characteristics of the isolate were consistent with those described for Pseudocercospora sp. (Ellis 1971). To confirm the species, the nuclear gene regions ITS, ACT, and EF-1α of isolate PCLEMID02 were amplified and sequenced for multigene phylogenetic analysis. The DNA sequences generated were deposited in GenBank (MH330685, MH891492, and MH891491). Phylogenetic analysis was performed with the concatenated sequence alignment of three genes using MEGA 6.0 software. The neighbor-joining phylogenetic tree showed that isolate PCLEMID02 from B. pilosa clustered in one separate clade with 100% bootstrap support with sequences of P. vitis (GenBank GU269829.1, GU320533.1, and GU384541.1). To perform the pathogenicity test, a suspension containing 1 × 10⁴ conidia/ml was prepared from one isolate (PCLEMID02) with 3-week-old cultures grown on V8 agar. The conidial suspension was sprayed on the leaf surface of B. pilosa and grapes. It used 20 leaves ranging in age from 30 to 45 days. Another 20 leaves received water instead of inoculum and served as controls. All leaves were left in a moist chamber for 24 h and subsequently stored in a greenhouse at 28 ± 2°C with a 12-h photoperiod. Only the inoculated leaves showed symptoms. Leaf blight on grape and leaf spots on B. pilosa were observed 12 and 25 days after inoculation, respectively. Both the inoculated leaves and the controls were submitted to the reisolation process. The pathogen was effectively reisolated only on symptomatic leaves and presented the same cultural, morphological, and molecular characteristics as the colony used for inoculation. Thus, B. pilosa is a host of P. vitis and may allow the survival of the pathogen in the dormancy period of the grapevine and, consequently, contribute primary inoculum for the development of leaf blight on grape.

Os grupos 3 e 4 de anastomose (AG-3 e AG-4) do fungo Rhizoctonia solani são importantes grupos associados à batata no mundo. No Brasil, o AG-3 é relatado afetando principalmente batata e fumo. Já o AG-4 causa perdas consideráveis em culturas de importância econômica, como a soja, o feijão e o amendoim, podendo ocorrer também em hortaliças como o espinafre, o pimentão, o brócolis, o tomate, a batata e frutíferas como o melão. Recentemente foi constatada, em Brasília-DF, a associação de R. solani a plantas invasoras em áreas de cultivo de batata. Entretanto, não há informação a respeito da etiologia do patógeno bem como do papel de espécies invasoras como outras hospedeiras no ciclo do patógeno. Objetivou-se com esse estudo caracterizar isolados de R. solani obtidos de batata e de outras três espécies de plantas invasoras associadas a áreas de cultivo da cultura: juá-de-capote [Nicandra physaloides (L.) Pers., Solanaceae], beldroega (Portulaca oleracea L., Portulacaceae), e caruru (Amaranthus deflexus L., Amaranthaceae). Foi confirmada a hipótese de que os isolados obtidos de R. solani de beldroega, caruru e juá-de-capote pertencem ao grupo 4 de anastomose e são patogênicos à batata, exceto o isolado de beldroega. Estes isolados apresentaram patogenicidade cruzada às três espécies e também patogênicos à maria-pretinha (Solanum americanum Mill.), uma outra espécie de Solanaceae invasora. A classificação dos isolados no grupo AG-4 HGI ou no grupo AG-4 HGIII (isolado de caruru) foi confirmada através de características culturais e moleculares (seqüenciamento da região ITS-5.8S do rDNA). Os resultados deste trabalho trazem implicações importantes para o manejo das podridões radiculares de Rhizoctonia em batata.The anastomosis groups 3 and 4 (AG-3 and AG-4) of the fungus Rhizoctonia solani are important groups associated with potatoes worldwide. In Brazil, the AG-3 is reported affecting mainly potatoes and tobacco. The AG-4 cause considerable losses in crops of economic importance, such as soybean, beans and peanuts and may also occur in vegetables such as spinach, pepper, broccoli, tomatoes, potatoes and fruit such as melons. The association of R. solani with invasive plants was recently established in potato production areas from Brasília, DF. However, there is no information about the etiology of the pathogen as well as the role of invasive species as alternative hosts in the life cycle of the pathogen. The objective of this study was to characterize isolates of R. solani obtained from potatoes and three other invasive plant species associated with areas of potato production: Shoo-fly plant [Nicandra physaloides (L.) Pers., Solanaceae], pigweed (Portulaca oleracea L., Portulacaceae), and low-amaranth (Amaranthus deflexus L., Amaranthaceae). It was confirmed the hypothesis that the R. solani isolates obtained from pigweed, low-amaranth and Shoo-fly plant belong to the anastomosis group 4 and, except for the isolate from pigweed, are pathogenic to potatoes. These isolates were cross pathogencic to all the three weed species tested and also to American nightshade (Solanum americanum Mill.), another Solanaceae invasive of potato fields. The placement of the isolates in the group AG-4 HGI or in the group AG-4 HGIII (isolate from caruru) was confirmed by cultural and molecular characteristics (sequencing of the ITS-5.8S region of rDNA). The results of this study provide important implications for the management of the Rhizoctonia root rot in potatoes.

Made available in DSpace on 2018-11-12T17:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2015. Added 1 bitstream(s) on 2021-07-14T17:51:34Z : No. of bitstreams: 1 S1808-16572015000100225.pdf: 843757 bytes, checksum: b15a68db35270397d28f44f2c6d0f0da (MD5) RESUMO O presente estudo teve como objetivos: (i) determinar, in vitro, o efeito da temperatura sobre o crescimento micelial (10 a 35oC) e a germinação de conídios (10 a 45oC) de Corynespora cassiicola ; (ii) avaliar, in vitro , o efeito do regime de luz (luz contínua, fotoperíodo de 12 h e escuro) sobre o crescimento micelial, a esporulação e a viabilidade dos esporos de C. cassiicola ; e (iii) verificar, in vivo , a influência da temperatura (20 a 30oC) e da duração do período de molhamento foliar (4 a 48 h) no desenvolvimento da mancha-alvo em folhas de acerola. As temperaturas ótimas, estimadas a partir das equações, para o crescimento micelial e a germinação de esporos foram de 30 e 29oC, respectivamente. O regime de luz não afetou o crescimento micelial e a germinação dos esporos, entretanto, luz contínua favoreceu a esporulação, e a ausência de luz durante a produção dos esporos diminuiu sua viabilidade. Houve um incremento na área das lesões da mancha-alvo em folhas de acerola conforme a temperatura aumentou até 30oC. Foram necessárias 12 h de molhamento foliar para que a infecção e as lesões ocorressem. ABSTRACT The objective of this study were: (i) to assess, in vitro , the effect of temperature on mycelial growth (10-35oC) and spores germination (10-45oC); (ii) to evaluate, in vitro¸the effect of the light regime (continuous light, 12 h photoperiod and continuous darkness) on mycelial growth, sporulation and viability of the spores of C. cassiicola ;and (iii) to assess, in vivo,the effect of temperature (20-30oC) and the duration of leaf wetness (4-48 h) in the development of target spot in leaves of barbados cherry. The estimated maximum temperatures for mycelia growth and spore germination were 30 and 29oC, respectively. The light regime did not affect the mycelial growth and spores germination. However, continuous light regime favored the sporulation, and the absence of light during spore production decreased the spore viability. The target spot lesions area in barbados cherry leaves increased with the increment in the temperature up to 30oC. Twelve hours of leaf wetness were necessary for the infection and lesion development of target spot. Universidade Estadual Paulista Faculdade de Engenharia Departamento de Fitossanidade, Engenharia Rural e Solos Centro Estadual de Educação Tecnológica Paula Souza ETEC Sebastiana Augusta de Moraes Universidade de São Paulo Escola Superior de Agronomia Luiz de Queiroz Departamento de Fitopatologia e Nematologia Universidade Estadual Paulista Faculdade de Engenharia Departamento de Fitossanidade, Engenharia Rural e Solos

A migração da citricultura paulista das regiões norte e central para a região sudoeste, tem submetido a cultura à clima mais chuvoso e, consequentemente, à maior ocorrência da podridão floral dos citros (PFC). A PFC, cujo agente causal é C. acutatum, induz a abscisão de frutos jovens e pode causar perdas de 100%. Alguns componentes do monociclo dessa doença ainda encontram-se indefinidos. Os processos de sobrevivência e colonização têm sido sustentados por evidências oriundas de experimentos não conclusivos. Até o momento, os métodos de detecção não têm sido eficazes para detectar o patógeno em folhas assintomáticas de citros, além de não serem capazes de quantificá-lo. Diante dessas lacunas, esse trabalho buscou avaliar o período de sobrevivência do patógeno na superfície de folhas assintomáticas entre as floradas; verificar se o patógeno coloniza os tecidos das folhas de citros; estabelecer um método sensível para detecção e quantificação de C. acutatum e C. gloeosporioides na superfície foliar e; monitorar quantitativamente os patógenos em campos de produção de citros no Estado de São Paulo. A sobrevivência do patógeno foi avaliada em condições controladas, casa de vegetação e condições ambientais. Foi notado que o patógeno sobreviveu até sete meses em condições controladas, até dez meses em casa de vegetação e até seis meses sob condições ambientais. Chuvas regulares associadas ao molhamento foliar prolongado, auxiliaram na manutenção e sobrevivência do inóculo na superfície foliar. O processo de colonização do patógeno foi investigado por microscopia de luz, transmissão e confocal. Não foi observada colonização do patógeno em tecidos foliares. O peg de penetração proveniente dos apressórios penetrou a cutícula e ficou restrito numa camada péctica, acima da parede periclinal da epiderme. Diferentes métodos foram avaliados para a detecção e quantificação de C. acutatum e C. gloeosporioides em folhas de citros. Foi desenvolvida uma PCR em tempo real (qPCR) e uma PCR multiplex em tempo real (qPCR multiplex), ambas específicas e altamente sensíveis à detecção dos patógenos. O DNA da planta não influenciou na amplificação da qPCR. As qPCR foram muito mais sensíveis que a PCR convencional e Nested-PCR. Distintos métodos para processamento das amostras e para obtenção do DNA foram avaliados. O método do congelamento e a maceração do tecido foliar, foram os mais eficazes para o processamento da amostras e, a extração do DNA (CTAB ou Qiagen) foi melhor método de obtenção do DNA para quantificação dos patógenos. O spot e extração do DNA foram validados para a detecção dos patógenos tanto para a qPCR quanto para a qPCR multiplex. O monitoramento dos patógenos foi realizado em áreas localizadas em duas distintas regiões do Estado de São Paulo. Foi notado considerável aumento na quantidade de inóculo quando ocorreram chuvas regulares, com muitos dias com chuvas. O inóculo se concentrou no interior da copa em épocas com baixo volume de chuvas. A quantidade de inóculo de C. acutatum na área localizada em Santa Cruz do Rio Pardo foi consideravelmente maior que na área de Barretos e ambas apresentaram quantidades similares de C. gloeosporioides. The migration of the citrus culture in São Paulo State from the northern and central regions to the south-western region submitted the culture to a wetter climate and, consequently, a higher incidence of postbloom fruit drop (PFD). PFD, whose causal agents are C. acutatum and C. gloeosporioides, induces the abscission of young fruits and can causes loss of 100% of the culture. Some monocycle components of this disease are still undetermined. The survival and colonization processes are supported by evidences from inconclusive experiments. To date, the detection methods have not been effective in detecting and quantifying the pathogen in asymptomatic citrus leaves. This study aimed to assess the survival period of C. acutatum on the surface of asymptomatic leaves between the flowering seasons, to verify whether the pathogen colonizes the tissues of citrus leaves of citrus, to establish a sensitive method for detection and quantification of C. acutatum and C. gloeosporioides on leaves surface and to monitor quantitatively the pathogens in citrus orchards in the State of São Paulo in Brazil. The survival of the pathogen was evaluated under controlled conditions, in a greenhouse and in environmental conditions. The pathogen survived for seven months under controlled conditions, ten months in a greenhouse and six months under environmental conditions. Regular rainfall associated with the prolonged leaf wetness favored the maintenance and survival of the inoculum on the leaf surface. The colonization process of the pathogen was investigated by light, confocal and transmission microscopy. Pathogen colonization was not observed on leaf tissues. The penetration peg from appressoria penetrated the cuticle and was restricted in a pectic layer on the periclinal epidermal wall. Different methods were evaluated for the detection and quantification of C. acutatum and C. gloeosporioides on leaves of citrus plants. It was developed a realtime PCR (qPCR) and a real-time multiplex PCR (qPCR multiplex), both specific and highly sensitive in the detection of pathogens. The plant DNA did not influence the qPCR amplification. The qPCRs were much more sensitive than the conventional PCR and Nested-PCR. Different methods for sample processing and for DNA extraction were evaluated. The freezing and maceration methods of foliar tissue were the most effective for sample processing and the DNA extraction (CTAB or Qiagen) was best method for extracting the DNA for pathogen quantification. The Spot and DNA extraction were validated for the detection of pathogens for both qPCR and multiplex qPCR. The monitoring of pathogens was carried out in two separate regions of the State of São Paulo in Brazil. It was observed a considerable increase in the number of inoculum when regular rainfall occurred, with many rainy days. The inoculum was concentrated within the tree canopy in seasons with low volume of rainfall. The number of inoculum of C. acutatum the at region of Santa Cruz do Rio Pardo was considerably higher than in the region of Barretos and both showed similar amounts of C. gloeosporioides.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:49:36Z No. of bitstreams: 1 S0100-54052010000200007.pdf: 1055426 bytes, checksum: 3c7c5a8a14f443daa7fc900a45edb477 (MD5) Made available in DSpace on 2013-08-22T18:49:36Z (GMT). No. of bitstreams: 1 S0100-54052010000200007.pdf: 1055426 bytes, checksum: 3c7c5a8a14f443daa7fc900a45edb477 (MD5) Previous issue date: 2010-06-01 Made available in DSpace on 2013-09-30T19:39:58Z (GMT). No. of bitstreams: 2 S0100-54052010000200007.pdf: 1055426 bytes, checksum: 3c7c5a8a14f443daa7fc900a45edb477 (MD5) S0100-54052010000200007.pdf.txt: 47292 bytes, checksum: 94171fb68a32afcdf69afc1bde3bda46 (MD5) Previous issue date: 2010-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:10:37Z No. of bitstreams: 2 S0100-54052010000200007.pdf: 1055426 bytes, checksum: 3c7c5a8a14f443daa7fc900a45edb477 (MD5) S0100-54052010000200007.pdf.txt: 47292 bytes, checksum: 94171fb68a32afcdf69afc1bde3bda46 (MD5) Made available in DSpace on 2014-05-20T15:10:37Z (GMT). No. of bitstreams: 2 S0100-54052010000200007.pdf: 1055426 bytes, checksum: 3c7c5a8a14f443daa7fc900a45edb477 (MD5) S0100-54052010000200007.pdf.txt: 47292 bytes, checksum: 94171fb68a32afcdf69afc1bde3bda46 (MD5) Previous issue date: 2010-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Os grupos 3 e 4 de anastomose (AG-3 e AG-4) do fungo Rhizoctonia solani são importantes grupos associados à batata no mundo. No Brasil, o AG-3 é relatado afetando principalmente batata e fumo. Já o AG-4 causa perdas consideráveis em culturas de importância econômica, como a soja, o feijão e o amendoim, podendo ocorrer também em hortaliças como o espinafre, o pimentão, o brócolis, o tomate, a batata e frutíferas como o melão. Recentemente foi constatada, em Brasília-DF, a associação de R. solani a plantas invasoras em áreas de cultivo de batata. Entretanto, não há informação a respeito da etiologia do patógeno bem como do papel de espécies invasoras como outras hospedeiras no ciclo do patógeno. Objetivou-se com esse estudo caracterizar isolados de R. solani obtidos de batata e de outras três espécies de plantas invasoras associadas a áreas de cultivo da cultura: juá-de-capote [Nicandra physaloides (L.) Pers., Solanaceae], beldroega (Portulaca oleracea L., Portulacaceae), e caruru (Amaranthus deflexus L., Amaranthaceae). Foi confirmada a hipótese de que os isolados obtidos de R. solani de beldroega, caruru e juá-de-capote pertencem ao grupo 4 de anastomose e são patogênicos à batata, exceto o isolado de beldroega. Estes isolados apresentaram patogenicidade cruzada às três espécies e também patogênicos à maria-pretinha (Solanum americanum Mill.), uma outra espécie de Solanaceae invasora. A classificação dos isolados no grupo AG-4 HGI ou no grupo AG-4 HGIII (isolado de caruru) foi confirmada através de características culturais e moleculares (seqüenciamento da região ITS-5.8S do rDNA). Os resultados deste trabalho trazem implicações importantes para o manejo das podridões radiculares de Rhizoctonia em batata. The anastomosis groups 3 and 4 (AG-3 and AG-4) of the fungus Rhizoctonia solani are important groups associated with potatoes worldwide. In Brazil, the AG-3 is reported affecting mainly potatoes and tobacco. The AG-4 cause considerable losses in crops of economic importance, such as soybean, beans and peanuts and may also occur in vegetables such as spinach, pepper, broccoli, tomatoes, potatoes and fruit such as melons. The association of R. solani with invasive plants was recently established in potato production areas from Brasília, DF. However, there is no information about the etiology of the pathogen as well as the role of invasive species as alternative hosts in the life cycle of the pathogen. The objective of this study was to characterize isolates of R. solani obtained from potatoes and three other invasive plant species associated with areas of potato production: Shoo-fly plant [Nicandra physaloides (L.) Pers., Solanaceae], pigweed (Portulaca oleracea L., Portulacaceae), and low-amaranth (Amaranthus deflexus L., Amaranthaceae). It was confirmed the hypothesis that the R. solani isolates obtained from pigweed, low-amaranth and Shoo-fly plant belong to the anastomosis group 4 and, except for the isolate from pigweed, are pathogenic to potatoes. These isolates were cross pathogencic to all the three weed species tested and also to American nightshade (Solanum americanum Mill.), another Solanaceae invasive of potato fields. The placement of the isolates in the group AG-4 HGI or in the group AG-4 HGIII (isolate from caruru) was confirmed by cultural and molecular characteristics (sequencing of the ITS-5.8S region of rDNA). The results of this study provide important implications for the management of the Rhizoctonia root rot in potatoes. Universidade Estadual Paulista Campus de Ilha Solteira FEIS USP Campus de Piracicaba ESALQ UNESP Campus de Jaboticabal FCAV Universidade Federal Rural de Pernambuco (UFRPE) Dept. Agronomia Swiss Federal Institute of Technology ETHZ Institute of Integrative Biology Universidade Estadual Paulista Campus de Ilha Solteira FEIS UNESP Campus de Jaboticabal FCAV

A antracnose é uma das principais doenças que afetam a goiaba no Estado de São Paulo. Tanto Colletotrichum gloeosporioides quanto Colletotrichum acutatum, são relatados como sendo os agentes causais da doença. Os objetivos do trabalho foram caracterizar e identificar 54 isolados de Colletotrichum oriundos de lesões de goiaba, baseados nos aspectos culturais, morfológicos, moleculares e enzimáticos, além da caracterização patogênica de isolados representativos de cada espécie de Colletotrichum identificadas. A caracterização cultural foi avaliada mediante a mensuração do crescimento micelial dos isolados a 25 ºC, além dos aspectos culturais, como a coloração e a topografia das colônias. Na caracterização morfológica foram mensurados comprimento e largura de conídios, bem como avaliados os seus formatos. Oligonucleotídeos específicos foram utilizados na caracterização molecular, visando identificar as espécies dos isolados de Colletotrichum. A caracterização enzimática envolveu a mensuração, in vitro, do halo de degradação dos substratos da amilase, proteinase, celulase, pectinase e lipase. Por fim, alguns isolados representativos e identificados foram utilizados na caracterização patogênica, sendo avaliados os períodos de latência e incubação, a área lesionadas dos frutos e a esporulação. Baseados na coloração das colônias, os isolados foram reunidos em 9 grupos diferentes. Os mesmos puderam se reunidos em dois grupos distintos de acordo com a taxa do crescimento micelial. Os conídios apresentaram os formatos: (i) reto, fusiforme, com ápices afilados, (ii) reto, oblongo, com ápices arredondados, (iii) reto, clavado, afilado em uma extremidade e (iv) reto, com constrição. As dimensões variaram de 11,4 a 16,8 µm de comprimento por 2,6 a 4,9 µm de largura. O uso de oligonucleotídeos específicos permitiu identificar C. acutatum e C. gloeosporioides entre os isolados avaliados. Grande parte dos isolados, 94%, foram identificados como pertencendo à espécie C. gloeosporioides, enquanto que apenas 4% foram identificados como C. acutatum. Em relação à caracterização enzimática, apenas a atividade celulolítica proporcionou diferenças significativas entre C. gloeosporioides e C. acutatum. A patogenicidade dos isolados avaliados mostrou alta variabilidade na severidade da doença nos frutos, contudo não foi possível evidenciar diferenças significativas que distinguissem C. acutatum de C. gloeosporioides. Os períodos de incubação e latência foram menores para os isolados de C. acutatum em relação aos isolados de C. gloeosporioides. C. acutatum produziu quantidade superior de esporos nos frutos inoculados quando comparados a C. gloeosporioides. Observou-se, ainda, correlação positiva entre a área do halo de degradação de pectina, lipídio e amido e a área lesionada dos frutos afetados pelos isolados avaliados. Anthracnose is one of the major diseases affecting guava in the State of São Paulo. Both Colletotrichum gloeosporioides and Colletotrichum acutatum are reported as the causal agents of the disease. The objectives of this work were to characterize and to identify 54 Colletotrichum isolates from guava, based on cultural, morphological, molecular, enzymatic, and pathogenic aspects. Cultural characterization was achieved by measuring the mycelial growth at 25 ° C, as well as reporting cultural aspects, such as color and topography of the colonies. In the morphological characterization it was measured length and width of conidia, and rated their shapes. CaInt2 and CgInt specific primers were used in the molecular identification of the Colletotrichum isolates. The enzymatic characterization was performed by measuring, in vitro degradation of starch, protein, cellulose, pectin and lipid. Finally some representative and identified isolates were used in the pathogenic characterization, evaluated by latency and incubation periods, diseased area and sporulation. Based on the color of the colonies, the isolates were grouped in 9 different groups. These same isolates showed two distinct growth paterns according to the mycelial growth rates. Conidia showed shapes: (i) straight, fusiform, with acute ends, (ii) straight, oblong, with round ends, (iii) straight, clavate, tapered at one end and (iv) straight, with a constriction in the middle. Conidia size ranged from 11.4 to 16.8 µm in length by 2.6 to 4.9 µm in width. The use of specific primers identified C. acutatum and C. gloeosporioides among the isolates. Most of the isolates (94%) were identified as C. gloeosporioides, while only (6%) were identified as C. acutatum. In the enzymatic characterization, only cellulolytic activity revealed significant differences between C. gloeosporioides and C. acutatum. Pathogenicity of the isolates was highly variable, but could not help to distinguish between C. acutatum and C. gloeosporioides. The incubation and latency periods were shorter for C. acutatum in relation to C. gloeosporioides. C. acutatum produced higher amounts of spores on inoculated fruits compared to C. gloeosporioides. There was also a positive correlation between in vitro degradation of pectin, lipid and starch, and the diseased area for tested isolates.

O Brasil é o único produtor, em escala comercial, de guaraná do mundo e o Estado do Amazonas destaca-se como o segundo maior produtor do Brasil, sendo superado apenas pelo Estado da Bahia. O principal fator limitante à expansão da cultura do guaranazeiro na região amazônica é a antracnose, causada por Colletotrichum guaranicola Alburq. Este trabalho teve como objetivo caracterizar isolados do fungo visando sua correta identificação. Foram realizadas as caracterizações cultural, morfológica, fisiológica, enzimática, patogênica e molecular (filogenia multilocus). Na caracterização morfo-cultural houve variação no tamanho de conídios e apressórios entre os isolados, porém todos ficaram dentro da faixa de amplitude descrita para a espécie C. guaranicola e para duas outras espéciess (C. gloeosporioides e C. boninense). Predominaram conídios de formato cilindro a oblongo e apressórios arredondados. Com exceção de um grupo de isolados de Maués (série C), todos os demais exibiram hilo na base do conídio. Comprimento e largura de conídios e apressórios não foram características importantes na diferenciação dos isolados de C. guaranicola devido à sobreposição no tamanho. A taxa de crescimento micelial foi determinada em meio BDA e permitiu diferenciar dois grupos de isolados: com taxa de crescimento acima de 10 mm/dia e abaixo dessa taxa. Com exceção de C-12 e C-14, todos os isolados da série C cresceram acima dos demais, comparando-se aos isolados padrões das espécies C. gloeosporioides, C. boninense e C. acutatum. Todos os demais cresceram abaixo desse valor. O maior crescimento foi registrado para C-09 (12,25 mm/dia) e o menor para 2601 (2,71 mm/dia). Os demais ficaram na faixa intermediária. Diferença na coloração da colônia também permitiu separar os isolados em duas categorias: colônia branca a creme amarelada e colônias cinza a cinza escuro, semelhante aos padrões de C. boninense e C. gloesoporioides, respectivamente. Todos os isolados cresceram melhor na faixa de 25 ºC, porém os isolados C-18 e C-19 foram capazes de crescer em temperaturas mais altas, em relação aos demais isolados. A caracterização enzimática revelou que C. guaranicola produz várias enzimas, destacando-se a pectinase. Por outro lado, houve baixa produção de amilase. Somente dois isolados de C. guaranicola (C-18 e C-19) e os padrões de C. gloeosporioides e C. boninense foram capazes de utilizar o ácido cítrico como fonte de carbono. Com exceção dos isolados 2361, 2419 e 2554, os demais isolados testados foram capazes de utilizar o tartarato de amônio. A caracterização filogenética foi realizada com base na amplificação e sequenciamento parcial dos genes Gliceraldeído-3-Fosfato Desidrogenase (GAPDH), Actina, Quitina Sintase, Calmodulina e região transcrita interna (ITS). Os isolados testados formaram dois clados distintos, o primeiro (25 isolados) agrupou no complexo C. boninense próximo a C. petchii e o outro no complexo C. gloeosporioides próximo a C. siamense. A caracterização patogênica foi realizada com base no diâmetro da lesão produzida por oito isolados de C. guaranicola em dois genótipos de guaranazeiro (suscetível e resistente). O isolado C-18 expressou maior severidade quando inoculado nos dois genótipos, enquanto o isolado 2512 demonstrou a menor severidade. Brazil is the world\'s sole producer of guaraná on a commercial scale, and the Amazonas State stands out as Brazil\'s the second largest producer, only after the Bahia State. The main limiting factor for the expansion of guaraná culture in the Amazon region is anthracnose caused by Colletotrichum guaranicola Alburq. This study aimed to characterize isolates of the fungus for its correct identification. Cultural, morphological, physiological, enzymatic, pathogenic, and molecular characterizations were performed (multilocus phylogeny). In the morpho-cultural characterization, there was variation in the size of conidia and appressoria among the isolates, but all remained within the range of amplitude described for the species C. guaranicola and for two other species (C. gloeosporioides and C. boninense). The cylinder-shaped conidia were oblong and the appressoria were round, except for a group of isolates of Maués (C series), all other isolates exhibited a hilo at the base of the conidium. Length and width of conidia and appressoria were not important features in the differentiation of isolates of C. guaranicola due to the overlap in size. The mycelial growth rate was determined in BDA medium and allowed to differentiate two groups of isolates: growth rate above 10 mm/day and growth rate below 10 mm/day. With the exception of C-12 and C-14, all C-series isolates grew above the rest, comparing to standard isolates of species C. gloeosporioides, C. boninense and C. acutatum. All others grew below the standard values. The highest growth was registered for C-09 (12.25 mm/day) and the lowest for 2601 (2.71 mm/day). The others remained within an intermediate range. Color difference of the colony also allowed to separate the isolates into two categories: white to yellowish cream colony and grey to dark grey colonies, similar to standards of C. boninense and C. gloesoporioides, respectively. All isolates grew best at the range of 25°C, but the isolates C-18 and C-19 were able to grow at higher temperatures, compared to other isolates. The enzymatic characterization showed that C. guaranicola produces various enzymes, mainly pectinase. On the other hand, there was low production of amylase. Only two isolates of C. guaranicola (C-18 and C-19) and the standards of C. gloeosporioides and C. boninense were able to use citric acid as a carbon source. With the exception of isolates 2361, 2419 and 2554, the other isolates tested were able to use the ammonium tartrate. The phylogenetic characterization was performed based on amplification and partial sequencing of genes Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Actin, Chitin Synthase, Calmodulin and internal transcribed section (ITS). The isolates tested formed two distinct clads. The first (25 isolates) grouped in the complex C. boninense near C. petchii and the other in the complex C. gloeosporioides near C. siamense. The pathogenic characterization was performed based on the lesion diameter caused by eight isolates of C. guaranicola in two guaraná genotypes (susceptible and resistant). The isolate C-18 expressed greater severity when inoculated into both genotypes, while isolate 2512 showed least severity.