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 Renata Lopes (renatasil82@gmail.com) on 2016-09-20T12:27:34Z No. of bitstreams: 1 suzanameiraribeiro.pdf: 1332777 bytes, checksum: b4c35efe19a33f78ca2cec41c685e844 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:24:02Z (GMT) No. of bitstreams: 1 suzanameiraribeiro.pdf: 1332777 bytes, checksum: b4c35efe19a33f78ca2cec41c685e844 (MD5) / Made available in DSpace on 2016-09-26T20:24:02Z (GMT). No. of bitstreams: 1 suzanameiraribeiro.pdf: 1332777 bytes, checksum: b4c35efe19a33f78ca2cec41c685e844 (MD5) Previous issue date: 2010-06-12 / Doenças causadas por patógenos, incluindo bactérias e fungos podem gerar muitos problemas à saúde humana e à agricultura. Em cultivares economicamente importantes, tais patógenos podem contribuir para perdas significativas no rendimento da produção agrícola. Um fator agravante para esses dois cenários é o uso intensivo de compostos convencionais para combater esses patógenos, levando à seleção de microrganismos extremamente resistentes. Além disso, tais compostos podem trazer severas conseqüências ao meio ambiente bem como à saúde humana e animal. Desta forma, a busca por novos compostos capazes de controlar efetivamente bactérias e fungos patogênicos resistentes, têm sido prioritária. Por estas razões, este trabalho reporta o isolamento de uma nova proteína heterodimérica (Pa-AFP1) de sementes de Passiflora alata com propriedades antifúngicas. Pa-AFP1 foi purificada utilizando precipitação com sulfato de amônio 70-100%, subseqüente cromatografia de troca aniônica em Q-Sepharose e cromatografia de HPLC fase reversa em coluna C4. Análise da massa molecular por meio de gel Tricina-SDS-PAGE revelou uma massa molecular de aproximadamente 4.500 Da para a cadeia menor e cerca de 7.000 Da para cadeia maior totalizando um heterodímero com cerca de 11.500 Da. A massa da forma oligomérica também foi obtida por espectrometria de massa (11569. 63 Da). Análise da seqüência N-terminal mostrou alta identidade entre Pa-AFP1 e albuminas 2S, adicionando assim uma nova proteína à pequena lista de albuminas 2S com atividade antimicrobiana. Além disso, Pa-AFP1 foi capaz de inibir eficientemente o fungo filamentoso Colletotrichum gloeosporiodes, mas foi incapaz de inibir bactérias e fungos leveduriformes. Em suma, Pa-AFP1 poderá futuramente contribuir para o desenvolvimento de produtos biotecnológicos contra fungos fitopatogênicos. / Diseases caused by pathogens, including bacteria and fungi can generate many problems to human health and agriculture. In important economically cultivars, such pathogens may contribute to significant losses in yield of agricultural production. The aggravating factor for these two scenarios is the intensive use of conventional compounds to combat these pathogens, leading to selection of highly resistant microorganisms. Furthermore, these compounds can bring severe consequences to the environment, human and animal health. Thus the search for new compounds capable of effectively controlling bacteria and fungi resistant has been a priority. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptide molecular masses of approximately 4,500 Da and 7,000 Da, in the presence of β-mercapthoetanol, while by removing the reducing agent, a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimeric mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporiodes, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi.

Datura metel is a herbaceous plant found in almost all tropical parts of the world. It belongs to the family Solanaceae whose members, viz. Duboisia, Atropa, Hyoscyamus and Datura plants are known to produce tropane alkaloids- hyoscyamine and scopolamine which are most noted for their therapeutic use as anti-cholinergic agents. Since these alkaloids are produced in very low amounts in plants, alternative sources and methods of production for these alkaloids have been crucial in meeting the demands for these drugs. Endophytic fungi inhabiting a plant may have the potential to produce the same compounds as the host plants. The aim of the present study was to search for tropane alkaloid producing endophytic fungal isolates from Datura metel. Eighteen endophytic fungi were isolated from various tissues of Datura metel and screened for the presence of three tropane alkaloid biosynthetic genes- putrescine N-methyltransferase (PMT), tropinone reductase I (TRI) and hyoscyamine 6β-hydroxylase (H6H) using PCR-based screening approach. Six endophytic fungal isolates were found to possess the PMT, TR1 and H6H genes. The fungi were identified using molecular taxonomy as Col letotrichum boninense, Phomopsis sp., Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col letotrichum gloeosporioides and the identity was confirmed using colony and spore morphology. The production of tropane alkaloids hyoscyamine and scopolamine by the fungi has been ascertained using various techniques like TLC, HPLC and ESI-MS/MS by comparison with the authentic reference standards. The amount of tropane alkaloids produced by all six fungi in liquid cultures was quantified using HPLC analysis. Among the six tropane alkaloid-producing fungi Col letotrichum incarnatum gave the highest yields of hyoscyamine and scopolamine which were 3.906 mg/L and 4.13 mg/L, respectively. With an aim to characterize the tropane alkaloid biosynthetic genes in these fungi, the PMT gene was isolated from five of the endophytic fungi- Col letotrichum boni-nense, Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col-letotrichum gloeosporioides for the first time and the sequence analysis showed high ho-mology (98%) to the Datura metel PMT cDNA. The gene was found to be devoid of introns in the fungi. Further phylogenetic analysis of the full length PMT sequence from the fungi strongly supports the hypothesis of horizontal gene transfer between the host plant and endophytic fungi. For further in detail characterization of fungal PMT, the Col letotrichum boninense PMT gene was taken as a representative. CbPMT gene was cloned in pRSET A expres-sion vector and heterologously expressed in E. coli and biochemically characterized. For optimal yield of soluble protein upon heterologous expression different conditions such as IPTG concentration, temperature and time post induction were optimized. Optimal yield was obtained by inducing the culture by 0.25 mM IPTG once it had reached and O.D. of 0.6 and incubating at 37◦ C for 3 h. The recombinant CbPMT enzyme expressed as histidine tagged fusion protein was purified using Ni-NTA affinity chromatography. Gel elution studies were carried out to determine molecular weight of the protein and it was found that the protein exists as a homodimer in solution with some amount also present as a monomer. Catalytic activity of the purified recombinant enzyme was studied for its dependence on both substrates putrescine as well as S-adenosylmethionine (SAM). The Km and Vmax values for putrescine were found to be 464 µM and 18.55 nkat/mg, respectively, while those for S-adenosylmethionine were found to be 628 µM and 18.63 nkat/mg, respectively. Optimum temperature for activity was found to be 37◦ C and optimum pH range was found to be 8-9. Fluorescence spectroscopy was used to study the binding affinity of both the sub-strates to the enzyme. Fluorescence quenching data for each substrate was analysed by using a nonlinear regression curve fit and Kd values were found to be 0.309 mM for pu-trescine and 0.118 mM for SAM, respectively. Circular dichroism spectrum of the enzyme indicated a pattern typical for alpha helix in the secondary structure. Binding of either substrate led to increase in ellipticity of the protein. Fluorescence quenching studies with collisional quenchers- acrylamide, potassium iodide, and cesium chloride indicated that the native protein is folded in a conformation that allows tryptophan residues to be acces-sible for quenching. The fraction of tryptophan residues (fa ) accessible for quenching by acrylamide (1.06) was found to be higher than that for potassium iodide (0.54) while that cesium ions was the least (0.38). The neutral quencher acrylamide could access all the tryptophans meaning that none of tryptophans are completely buried inside hydrophobic cores. the differential accessibility to the charged quenchers, however, indicates that more of the tryptophans are surrounded by positively charged amino acids. The unfolding of the protein was studied with the aid of chaotropic agents guanidine-HCl and urea and thermodynamic parameters were determined. The denaturant m-values were found to be 2.313 kcal/mol/M for Gdn-HCl and 2.345 kcal/mol/M for urea respectively. The free energy of unfolding was estimated to be 2.635 kcal/mol for Gdn-HCl and 4.630 kcal/mol for urea. Since no reports are available about the thermodynamics of folding and unfolding of PMT from any plant source, this study contributes towards the understanding of protein stability. Although a lot of reports are available on the biochemical characterization of PMT from different plant sources, the crystal structure of PMT is not yet available. In the current work, homology based modelling studies on CbPMT were carried out to get some idea about the protein tertiary structure. Homology based modelling studies showed that a significant amount of protein is present as α-helices which are present on the surface while the β-sheets are present in the interior of the protein. Each monomer of the protein is capable of binding both the substrates and hence the dimerization property of the enzyme could be a purely structural one leading to more stability and solubility of the protein. In conclusion, this study has shown for the first time that endophytic fungi have significant potential to be used for tropane alkaloid production and six such fungal strains have been identified. Although the production of tropane alkaloids by endophytic fungi is not very high, it can be scaled up by over-expressing the biosynthetic gene putrescine N-methyltransferase in the highest producer- Col letotrichum incarnatum to further increase the yield. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for these drugs economically.

O crescente aumento da tolerância aos fungicidas atualmente utilizados é um dos principais problemas tanto na área clínica quanto na agrícola e tem estimulado o desenvolvimento de estratégias alternativas para o controle de fungos patogênicos, tais como o tratamento fotodinâmico antimicrobiano (TFA), que pode utilizar fotossensibilizadores (FS) de origem natural ou sintética. Alguns FS, como as cumarinas e furanocumarinas, são produzidos por espécies de Citrus como limão Tahiti (Citrus latifolia) e toranjas (Citrus paradisi). O presente estudo investigou o TFA in vitro de conídios do fungo fitopatogênico Colletotrichum acutatum com: (1) o corante fenotiazínico azul de metileno (MB), (2) uma furanocumarina comercial, 8-metoxipsoraleno (8-MOP), (3) uma cumarina comercial, (2H-chromen-2-one), (4) a cumarina 5,7-dimetoxicumarina extraída no presente trabalho a partir de óleo da casca do limão Tahiti, (5) a cumarina 7-hidroxicumarina extraída no presente trabalho a partir de óleo essencial de toranja rubi, (6) duas frações extraídas por De Menezes e colaboradores (2014a) a partir de um resíduo da indústria citrícola, uma delas constituída pela furanocumarina isopimpinelina e outra constituída por uma mistura das cumarinas 7-metoxicumarina e 5,7-dimetoxicumarina (3:1), (7) cinco extratos brutos obtidos a partir de óleos da casca e essencial de limão Tahiti e óleos essenciais de toranjas rubi e branca. Com exceção do extrato bruto obtido do óleo essencial de toranja branca, todos os demais extratos apresentaram cumarinas e/ou furanocumarinas em sua composição. As suspensões de conídios foram tratadas com os diferentes compostos (50 ?M para os compostos comerciais e as frações puras e 12,5 mg L-1 para os extratos e a mistura) e expostas à radiação solar por 1 h. O MB foi exposto à radiação solar por 30 min, 1 e 2 h. Determinou-se o efeito do tratamento fotodinâmico em folhas de mudas de laranja Valencia (Citrus sinensis) com (1) 8-MOP, (2) mistura de cumarinas, (3) 5,7-dimetoxicumarina e (4) o extrato bruto de limão Tahiti. Adicionalmente, experimentos para avaliar a estabilidade do 8-MOP exposto à radiação solar por até 12 h foram realizados. Nesses experimentos, soluções de 8-MOP (50 ?M) foram expostas à radiação solar e alíquotas foram retiradas após os diferentes tempos de exposição. Após as exposições, estudos de espectroscopia de absorção no UV e emissão de fluorescência foram realizados, além da realização de experimentos para avaliar a eficácia do TFA com os FS previamente expostos à radiação solar por 6 e 12 h. Os TFA mais eficazes foram com o MB, 8-MOP (ambos 50 ?M), a mistura de cumarinas e um dos extratos brutos obtidos de limão Tahiti (ambos 12,5 mg L-1), que mataram 99,998%, 99,378%, 99,336% e 65,049% dos conídios, respectivamente, após 1 h de exposição à radiação solar. Nenhum dano nas folhas de laranja foi observado após o TFA com os compostos. Os espectros de absorção e emissão de fluorescência das soluções de 8-MOP expostas à radiação solar mostraram fotodegradação do composto após 12 h de exposição. Entretanto, as soluções de 8-MOP expostas por 6 e por 12 h ainda foram capazes de matar 99,978% e 95,591% dos conídios de C. acutatum, respectivamente / Increasing tolerance to currently used fungicides is a major problem both in clinical and agricultural areas and has stimulated the development of alternative strategies to control pathogenic fungi such as the antimicrobial photodynamic treatment (APDT), that can use natural or synthetic photosensitizers (PS). Some PS, such as coumarins and furocoumarins are produced by Citrus species such Tahiti acid lime (Citrus latifolia) and grapefruit (Citrus paradisi). This study investigated the in vitro APDT of conidia of the plant-pathogenic fungi C. acutatum with: (1) the phenothiazinium dye methylene blue (MB), (2) commercial furocoumarin, 8-methoxypsoralen (8-MOP), (3) commercial coumarin (2H-chromen-2-one), (4) coumarin 5,7-dimethoxycoumarin, extracted in this study from Tahiti lime peel oil, (5) coumarin 7-hidroxycoumarina, extracted in this study from ruby grapefruit essential oil, (6) two fractions extracted by De Menezes et al (2014a) from a Citrus industry residue, one of them constituted by the furocoumarin isopimpinellin, and the other constituted by the mixture of coumarins 7-methoxycoumarin and 5,7-dimethoxycoumarin (3:1), (7) five crude extracts obtained from Tahiti lime peel or essential oil and ruby and white grapefruit essential oil. Except the extract from the white grapefruit, all the others presented coumarins and/or furocoumarins in their composition. Conidial suspentions were treated with the different PS (50 ?M for commercial and pure compounds and 12.5 mg L-1 for the extracts and the mixture) and exposed to solar radiation for 1 h. The MB was exposed to solar radiation for 30 min, 1 and 2 h. The APDT effect on the leaves of Valencia orange trees (Citrus sinensis) with (1) 8-MOP, (2) the mixture of coumarins, (3) 5,7-dimethoxycoumarin, and (4) the most efective extract from Tahiti lime were determined. Additionally, experiments to assess the 8-MOP stability after exposure to solar radiation for 12 h were realized. 8-MOP solutions (50 ?M) were exposed under solar radiation and rates were removed after different times of exposition. After the expositions, absorption spectroscopic studies in UV and fluorescence emission were realized, as well as holding the realization of experiments of APDT efficacy evaluation after 6 and 12 h to exposure under solar radiation. The more effective treatments were with MB, 8-MOP (both 50 ?M), the coumarins mixture and one of the extracts from Tahiti lime (both 12.5 mg L-1), killing at least 99,998%, 99,378%, 99,336% and 65,049% of the conidia, respectively after 1 h under solar radiation. No damage to orange tree leaves was observed after APDT with any of the compounds. The absorption and fluorescence intensity spectra showed the 8-MOP photodegradation after 12 h of exposition, although the 8-MOP solutions exposed under solar radiation for 6 and 12 h still killed at least 99,978% and 95,591% of the C. acutatum conidia, respectively

Fruit rot caused by Aspergillus niger and Colletotrichum sp. could cause rapid and severe damage on orange fruits. Current control method of orange fruits is mainly applied by usage of harmful pesticides, leading to chemical residues on fruits, environmental pollution and human poisoning. One of alternative methods of reducing pesticides is to use botanical extracts. This study was conducted to evaluate the in vivo antifungal efficacy of aqueous extracts from the leaves of neem and basket plants against A. niger and Colletotrichum sp. Orange fruits artificially inoculated by fruit rot pathogens were immersed into leaf extracts of 6% (w/v) neem or basket plants for 30 s, and kept for 11 days to record lesion length at room temperature. Orange fruits immersed into sterile distilled water were used as the control treatment. The results showed that at 11 days after inoculation, extracts of neem and basket plants significantly reduced the Aspergillus rot lesions by 109.08 and 124.00 mm, respectively. In addition, anthracnose lesions on orange fruits were statistically inhibited by treatments of neem and basket plants, with the average lesion diameters approximately 160.00 and 154.75 mm, respectively, at day 11 of the conducting experiment. The results of this study showed that leaf extracts of neem and basket plant at the concentration of 6% could be used as a natural alternative to control the in vivo growth of rot pathogens of orange fruits. These extracts have a bright future in modern plant protection to replace conventional synthetic pesticides in agro-ecosystem. / Thối trái bởi Aspergillus niger và Colletotrichum sp. gây ra các thiệt hại nghiêm trọng trên cam. Biện pháp phòng trừ bệnh trên trái cam hiện nay chủ yếu dựa vào thuốc hóa học, dẫn đến tồn dư thuốc trên trái cây, ô nhiễm môi trường và gây độc cho con người. Một trong các phương pháp thay thế giúp giảm sử dụng thuốc hóa học là sử dụng dịch trích thực vật. Nghiên cứu này đã được thưc hiện để đánh giá hiệu quả in vivo của dịch trích ở nồng độ 6% của neem hoặc lược vàng đối với A. niger và Colletotrichum sp. Các trái cam đã lây nhiễm nhân tạo tác nhân gây thối trái thì được nhúng vào dịch trích ở nồng độ 6% của neem hoặc lược vàng trong 30 giây, và giữ đến 11 ngày để ghi nhận chiều dài vết bệnh ở nhiệt độ phòng. Cái trái cam được nhúng vào nước cất thì dùng như nghiệm thức đối chứng. Kết quả cho thấy ở 11 ngày sau khi chủng bệnh, dịch trích neem và lược vàng làm giảm đáng kể vết thối Aspergillus lần lượt là 109,08 và 124,00 mm. Bên cạnh đó, vết bệnh thán thư trên trái cam đã bị ức chế có ý nghĩa thống kê bởi các dịch trích neem và lược vàng, với đường kính trung bình các vết bệnh lần lượt là 160,00 và 154,75 mm, ở ngày 11 của thí nghiệm. Kết quả của nghiên cứu này đã chỉ ra rằng dịch trích neem và lược vàng ở nồng độ 6% có thể sử dụng như một biện pháp thay thế tự nhiên trong việc phòng trừ sự phát triển của tác nhân gây thối trái cam. Các loại dịch trích này có tương lai trong bảo vệ thực vật hiện đại, thay thế các loại thuốc hóa học tổng hợp truyền thống trong hệ sinh thái nông nghiệp.

Fruit rot caused by Aspergillus niger or Colletotrichum musae is an important post-harvest disease on orange, chilli and Cavendish banana fruits. The use of synthetic fungicides has been a traditional strategy for the management of the fruit rot disease, but these chemicals adversely affect human health and environment. Therefore, the objective of this study was to evaluate the effects of CaCl2 on in vitro hyphal growth and in vivo lesion inhibition. First, aqueous solutions of CaCl2 at three concentrations of 20, 40 and 60 mM were assessed for their inhibitory effect against hyphal growth in vitro. Next, mature fruits were immersed into a solution of 20 mM CaCl2 for 20 - 30 s, then inoculated by a pathogen suspension at the density of 106 conidia mL-1 and observed for 12 days. The results showed that 20 mM CaCl2 was the most effective concentration in antifungal assay to Aspergillus isolated from orange rot. The treatment of CaCl2 continued to gain efficacy on limiting lesions’ development on orange fruits until 12 days after inoculation (DAI). On chilli, CaCl2 at concentrations of 20 and 40 mM inhibited well on the growth of Aspergillus hyphae isolated from chilli rot. However, calcium treatment was not effective on chilli fruits. On Cavendish banana, solutions of CaCl2 at concentrations of 20, 40 and 60 mM highly limited fungal growth of Colletotrichum in vitro conditions. The application of CaCl2 solution could inhibit anthracnose lesion length of Cavendish banana variety, but its efficacy did not prolong until 6 DAI. In general, the good results were obtained from the 20 mM CaCl2 in almost all the studied assays. Management of rot diseases on fruits by employing 20 mM CaCl2 could be suitable to replace the current hazardous agro-chemicals. / Thối trái do nấm Aspergillus niger hay nấm Colletotrichum musae là bệnh sau thu hoạch thường gặp trên cam, ớt và chuối già. Thuốc trừ nấm tổng hợp là biện pháp truyền thống quản lý bệnh thối trái nhưng lại ảnh hưởng bất lợi đến sức khỏe con người và môi trường. Vì vậy, mục tiêu của nghiên cứu là đáng giá ảnh hưởng của CaCl2 đối với sự sinh trưởng in vitro của nấm và sự ức chế vết bệnh ở điều kiện in vivo. Đầu tiên, dung dịch CaCl2 ở các nồng độ 20, 40 và 60 mM được sử dụng để đánh giá khả năng ức chế sự sinh trưởng in vitro của nấm bệnh. Tiếp theo, trái trưởng thành được nhúng vào dung dịch CaCl2 20 mM trong 20 - 30 s, rồi lây nhiễm với huyền phù mầm bệnh ở mật số 106 bào tử mL-1 và quan sát đến 12 ngày. Kết quả cho thấy CaCl2 20 mM có hiệu quả ức chế tốt đối với nấm Aspergillus phân lập từ bệnh thối trái cam. CaCl2 tiếp tục thể hiện hiệu quả ức chế bệnh trên trái cam đến 12 ngày sau lây bệnh. Trên ớt, CaCl2 20 và 40 mM cho hiệu quả ức chế sự phát triển nấm Aspergillus phân lập từ bệnh thối trái ớt. Tuy nhiên, xử lý CaCl2 không mang lại hiệu quả mong đợi trên trái ớt. Trên chuối già, dung dịch CaCl2 ở các nồng độ 20, 40 và 60 mM ức chế tốt sợi nấm Colletotrichum trong điều kiện in vitro. Dung dịch canxi có thể ức chế vết bệnh thán thư trên chuối già, nhưng hiệu quả không kéo dài đến 6 ngày sau lây bệnh. Nhìn chung, các kết quả tốt đều đạt được khi xử lý bằng CaCl2 20 mM ở hầu hết các thí nghiệm. Việc kiểm soát bệnh thối trái bằng CaCl2 20 mM có thể thay thế cho hóa chất nông nghiệp độc hại hiện nay.

Anthracnose, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. Cloning and characterization of the host resistance (R) genes against the pathogen will improve our knowledge of molecular mechanisms underlying host resistance and facilitate the development of resistant alfalfa cultivars. However, the intractable genetic system of cultivated alfalfa, owing to its tetrasomic inheritance and outcrossing nature, limits the ability to carry out genetic analysis in alfalfa. Nonetheless, the model legume Medicago truncatula, a close relative of alfalfa, provides a surrogate for cloning the counterparts of many agronomically important genes in alfalfa. In this study, we used genetic map-based approach to clone RCT1, a host resistance gene against C. trifolii race 1, in M. truncatula. The RCT1 locus was delimited within a physical interval spanning ~200 kilo-bases located on the top of M. truncatula linkage group 4. Complementation tests of three candidate genes on the susceptible alfalfa clones revealed that RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad spectrum anthracnose resistance. Thus, RCT1 offers a novel resource to develop anthracnose-resistant alfalfa cultivars. Furthermore, the cloning of RCT1 also makes a significant contribution to our understanding of host resistance against the fungal genus Colletotrichum.

A cultura do mamão exerce uma grande importância para a fruticultura nacional em função de aspectos sociais e econômicos, destacando o Brasil como um dos grandes produtores mundiais da fruta. Entre os fatores biológicos destacam-se a ação de fitopatógenos que provocam grandes prejuízos em condições de campo e também em pós-colheita. Com isso, aumenta-se a procura por novos produtos de base natural que auxiliem na redução dessas perdas. Desta forma, objetivou-se com este trabalho avaliar o efeito de diferentes óleos essenciais associados a biofilmes no controle da antracnose do mamoeiro e na melhoria da qualidade dos frutos em póscolheita. Foi utilizado o delineamento inteiramente casualizado em todos os ensaios. Dentre os tratamentos, biofilmes foram utilizados: gelatina, parafina e óleo de girassol os quais foram incorporados aos óleos essenciais de noni (Morinda citrifolia), Alecrim pimenta (Lippia sidoides) e seus respectivos compostos majoritários representados pelo ácido octanóico e timol. Após os tratamentos e decorridos 24 h, os frutos foram inoculadas com discos de 6 mm de diâmetro de C. gloeosporioides, e mantidos em câmara úmida por dez dias em temperatura ambiente de 27 ± 3ºC. Os óleos essenciais apresentara efici ncia significativa na m ê inibição da germinação de conídios de Colletotrichum gloeosporioides. O controle da doença, foi avaliado de acordo com a Área Abaixo da Curva de Progresso da Doença (AACPD), onde constatou-se que o biofilme mais eficiente foi o de parafina com noni. Verificou-se que todos os óleos essenciais reduziram a germinação de conídios de C. gloeosporioides, em função do aumento das concentrações. Com relação ao crescimento micelial observou-se que os óleos essenciais de noni e lippia obtiveram maior efeito inibitório. Na avaliação da perda de massa dos frutos foram observados menores perdas no tratamento utilizando parafina incorporado ao óleo essencial de alecrim pimenta e parafina incorporado ao óleo essencial de noni. Com relação à influência do tratamento sobre o período de amadurecimento dos frutos o óleo de girassol + noni seguido de óleo de girassol + ácido octanóico mantiveram os frutos sem amadurecer por maior tempo. Através dos resultados foi possível concluir que os biofilmes incorporados com óleos essenciais são uma opção viável para o controle de C. gloeosporioides em mamão e para manutenção de sua qualidade póscolheita. / The papaya culture has great importance for national fruit growing due to its social and economic aspects, highlighting Brazil as one of the world's great fruit producers. Among the biological factors are the action of phytopathogens that cause great damage in field conditions and also in post-harvest. This will increase the demand for new natural-based products to help reduce these losses. Thus, the objective of this work was to evaluate the effect of different essential oils associated with biofilms in the papaya anthracnose control and in the post-harvest quality of fruits. A completely randomized design was used in all trials. Among the treatments, biofilms were used: gelatin, paraffin and sunflower oil which were incorporated into the essential oils of noni (Morinda citrifolia), rosemary pepper (Lippia sidoides) and their respective major compounds represented by octanoic acid and thymol. After treatments and after 24 h, the fruits were inoculated with disks of 6 mm diameter of C. gloeosporioides, and kept in a humid chamber for ten days at room temperature of 27 ± 3ºC. The essential oils presented significant efficiency in the inhibition of the conidial germination of Colletotrichum gloeosporioides. Disease control was assessed according to the Area Under the Disease Progression Curve (AACPD), where it w u h h m biofilm was paraffin with noni. It was verified that all the essential oils reduced the germination of conidia of C. gloeosporioides, due to the increase of the concentrations. Regarding mycelial growth, it was observed that the essential oils of noni and lippia had a greater inhibitory effect. In the evaluation of the loss of mass of the fruits were observed lower losses in the treatment using paraffin incorporated into the essential oil of rosemary pepper and paraffin incorporated into essential oil of noni. Regarding the influence of the treatment on the ripening period of the fruits, the sunflower oil + noni followed by sunflower oil + octanoic acid maintained the fruits without maturing for a longer time. Through the results it was possible to conclude that biofilms incorporated with essential oils are a viable option for the control of C. gloeosporioides in papaya and for maintenance of its post-harvest quality.

Submitted by (ana.araujo@ufrpe.br) on 2017-02-07T18:23:56Z No. of bitstreams: 1 Fabiana Aparecida Cavalcante Silva.pdf: 934712 bytes, checksum: d4f03c8904e031e8ea5d8cd19ffcfece (MD5) / Made available in DSpace on 2017-02-07T18:23:56Z (GMT). No. of bitstreams: 1 Fabiana Aparecida Cavalcante Silva.pdf: 934712 bytes, checksum: d4f03c8904e031e8ea5d8cd19ffcfece (MD5) Previous issue date: 2008-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cotton is a crop of considerable importance worldwide, the production of which is affected by diverse biotic and abiotic factors. Stress caused by insect attacks and phytopathogens stand out among the biotic factors, the control of which is significantly hindered due the variability of these organisms and their consequent capacity for adaptation. Fungi are the phytopathogens that most cause damage to cotton plants, especially Colletotrichum gossypii South var. cephalosporioides A.S. Costa, a ramulosis-causing disease characterized by dwarfism and an over-budding of the branches, affecting the development of the buds and number of bolls. The main form of dissemination is through contaminated seeds and the most effect method of control is by means of chemical fungicides or resistant varieties which are obtained after exhaustive tests conducted in fieldwork and greenhouses. The use of biochemical and molecular tools that assist in the identification of resistant genotypes in improvement programs is of considerable importance, as they offer reliability and reduce selection costs in the development process of new varieties. In the present study, a biochemical/molecular study was carried out on cotton varieties submitted toinfection with the ramulosis fungus with the aim of identifying markers associated to the resistance process. In the first assay, biochemical descriptors were assessed in four infected cotton varieties. The plants were inoculated 20 days after cultivation, receiving a fungal concentration of 1 x 106 conidias/mL. Evaluations occurred at 3, 15 and 30 days following inoculation. Proline, peroxidase and catalase levels were quite expressive in the resistant BRS Antares cultivar, exhibiting a rapid response at 3 days after inoculation. Proline and catalase levels are indicated as useful tools for the identification of ramulosis-resistant varieties in cotton crop selection studies. The second assay sought to identify differentially expressed transcripts in cotton plants submitted to infection with the ramulosis fungus. Two varieties with opposing traits regarding resistance to this disease were used: BRS Antares and CNPA Precoce I, respectively resistant and susceptible. Ten RAPD oligonucleotides were used in the RT-PCR reactions at an annealing temperature of 35 ºC. Following an analysis of the band patterns in the control and inoculated plants, 12 down-regulated transcripts, 13 overregulated transcripts and ten activated transcripts were identified. A band ofapproximately 200 bp, obtained with the V10 oligonucleotide, was sequenced and exhibited 86% homology with the GmChl 3 gene, which codifies for thechlorophyllase III enzyme in soybean. This enzyme has considerable importance for vegetal cells submitted to oxidative damage, as it reduces this damage and modulates different defense pathways. The data obtained in the present study are applicable to studies on cotton plant responses to pathogen attacks and offers relevant information for crop improvement programs. / O algodão é uma cultura de grande importância mundial cuja produção é afetada por diversos fatores bióticos e abióticos. Dentre os bióticos destacam-se os estresses provocados pelo ataque de insetos e fitopatógenos, cujo controle é bastante difícil devido à variabilidade destes organismos e,conseqüente, a capacidade adaptativa. Entre os fitopatógenos, os fungos são os que causam mais danos ao algodoeiro,destacando-se o Colletotrichum gossypii South var. cephalosporioides A.S. Costa, causador da ramulose, doença caracterizada pelo nanismo e super-brotamento dos ramos, que afeta o desenvolvimento das maçãs e o número de capulhos. A principal forma de disseminação é via sementes contaminadas e o modo de controle mais efetivo é por meio de fungicidas químicos ou de cultivares resistentes que são obtidas por meio de exaustivos testes conduzidos em campo e casa de vegetação. A utilização de ferramentas bioquímicas e moleculares que auxiliem na identificação de genótipos resistentes nos programas de melhoramento são de grande relevância uma vez que, além da confiabilidade, reduz os custos de seleção nos processos de desenvolvimento de novas cultivares. No presente trabalho procedeu-se a um estudo bioquímico e molecular em cultivares de algodão submetido à infecção com o fungo da ramulose, visando identificar marcadores associados ao processo de resistência. No primeiro ensaio, avaliaram-se descritores bioquímicos em quatro cultivares de algodão infectado com o fungo causador da ramulose. As plantas foram inoculadas aos 20 dias após o cultivo, recebendo uma concentração fúngica de 1 x 106 conídios/mL. As avaliações ocorreram aos 3, 15 e 30 dias após a inoculação. Observou-se que os teores de prolina, peroxidase e catalase foram bastante expressivos na cultivar resistente BRS Antares apresentando resposta rápida logo aos 3 dias após a inoculação. Os teores de prolina e catalase foram indicados como ferramentas úteis para identificação de cultivares resistentes a ramulose nos trabalhos de seleção na cultura do algodão. No segundo ensaio buscou-se identificar transcritos diferencialmente expressos em plantas de algodão submetidas a infecção com o fungo da ramulose. Duas cultivares antagônicas em relação à resistência a esta doença foram utilizadas, BRS Antares e CNPA Precoce I, respectivamente, resistente e susceptível. Dez oligonucleotídeos RAPD foram utilizados nas reações de RT-PCR a uma temperatura de anelamento de 35 ºC. Após análise dos padrões de banda nas plantas controle e inoculada foram identificados doze transcritos sub-regulados, treze super-regulados e dez ativados. Uma banda de aproximadamente 200 pb obtida com o oligonucleotídeo V10 na cultivar resistente foi sequenciada e a apresentou homologia de 86 % com o gene GmChl 3 que codifica para enzima clorofilase III em soja. Esta enzima apresenta grande importância para as células vegetais submetidas a danos oxidativos uma vez que reduz estes estragos além de modular diferentes vias de defesa. Os dados obtidos no presente trabalho são de grande aplicabilidade em estudos de resposta do algodão ao ataque de patógenos além de oferecer informações relevantes para os programas de melhoramento da cultura.

Açaí palm (Euterpe oleracea Mart.) is native to the Amazon and presents great economic interest in Brazil and in the world. Brazil is the world's largest producer of açaí fruit, the state of Pará being the largest national producer. This production was destined for local consumption, but the conquest of new markets and the increase in exports stimulated the expansion of commercial plantations on land. However, the high demand for seedlings has limited the implantation of commercial plantations, due to the slow growth, high sensitivity to the water deficit and the occurrence of anthracnose caused by Colletotrichum. The study objective was to evaluate the growth promotion, water deficit mitigation and reduction of anthracnose by rhizobacteria in açaí palm seedlings. The experiment was carried out in a completely randomized design in the nursery conditions with açaí seedlings inoculated with rhizobacteria R-35, R-38, BRM32113, BRM32111, R-58, R-61 and R-92. All rhizobacteria promoted growth, accumulation of biomass, higher content of nutrients and chlorophylls, as well as improvements in photosynthetic performance. The rhizobacteria BRM32113, BRM32111, R-58 and R-92 were inoculated in açaí seedlings submitted to the water deficit. Depletion of soil water up to 50% of field capacity reduced water potential and photosynthetic performance. However, the higher activity of antioxidant enzymes alleviated the effects of water deficit on açaizeiro seedlings inoculated with rhizobacteria. The infection of Colletotrichum sp. in leaves of açaí seedlings altered the capture of light and the assimilation of CO2 in the mesophyll, resulting in lower photosynthetic performance. However, when the açaí seedlings were inoculated with the R-92 rhizobacteria the severity of the anthracnose was reduced and the damages in the photosynthetic apparatus were attenuated. The results show that the rhizobacteria accelerate the growth of the açaí palm seedlings, which can contribute to decrease the time of quality seedlings, greater tolerance to water deficit and greater resistance to anthracnose. The application of rhizobacteria can be used as a clean technology to aid in the sustainable management of the production of açaí palm seedlings in nurseries. / O açaizeiro (Euterpe oleracea Mart.) é uma palmeira nativa da Amazônia e de grande interesse econômico no Brasil e no mundo. O Brasil é o maior produtor mundial do fruto de açaí, sendo o estado do Pará o maior produtor nacional. Essa produção era destinada ao consumo local, porém a conquista de novos mercados e o aumento nas exportações estimulou a expansão dos plantios comerciais em terra firme. No entanto, a alta demanda por mudas tem limitado a implantação de plantios comerciais, devido ao crescimento lento, alta sensibilidade ao déficit hídrico e a ocorrência de antracnose causada por Colletotrichum sp. O objetivo do estudo foi avaliar a promoção de crescimento, mitigação do déficit hídrico e redução da antracnose por rizobactérias em mudas de açaizeiro. O experimento foi realizado em delineamento inteiramente casualizado nas condições de viveiro com mudas de açaizeiro inoculadas com as rizobactérias R-35, R-38, BRM- 32113, BRM-32111, R-58, R-61 e R-92. Todas rizobactérias promoveram crescimento, acúmulo de biomassa, maior conteúdo de nutrientes e clorofilas, além de melhoria no desempenho fotossintético. As rizobactérias BRM-32113, BRM-32111, R-58 e R-92 foram inoculadas em mudas de açaizeiro submetidas ao déficit hídrico. A diminuição do conteúdo de água no solo até 50% da capacidade de campo reduziram o potencial hídrico e o desempenho fotossintético. Porém, a maior atividade das enzimas antioxidantes aliviou os efeitos do déficit hídrico nas mudas de açaizeiro inoculadas com as rizobactérias. A infecção de Colletotrichum sp. em folhas de mudas de açaizeiro alterou a captação de luz e a assimilação de CO2 no mesofilo, resultando em menor desempenho fotossintético. Porém, quando as mudas de açaizeiro foram inoculadas com a rizobactéria R-92, a severidade da antracnose foi reduzida e os danos no aparato fotossintético foram atenuadas. Os resultados revelam que as rizobactérias aceleram o crescimento das mudas de açaizeiro, podendo contribuir para diminuir o tempo de obtenção das mudas de qualidade, maior tolerância ao déficit hídrico e maior resistência a antracnose. A aplicação das rizobactérias pode ser utilizada como uma tecnologia limpa para auxiliar no manejo sustentável da produção de mudas de açaizeiro em viveiros.