Diagnóstico y dinámica de enfermedades en tres genotipos de fresa (Fragaria spp) en la comunidad El Castillito, Las Sabanas, departamento de Madriz

Los Pequeños productores del municipio Las Sabanas, departamento de Madriz, han adoptad o el cultivo de fresa ( Fragaria spp.) como una alternativa para mejorar su nivel de vida. Una dificultad que enfrentan es la presencia y manejo de enfermedades que afectan negativamente el desarrollo del cultivo. El estudio se estableció con el fin de iden tificar las principales enfermedades en el cultivo de fresa en el municipio Las Sabanas, así como su dinámica de desarrollo, lo cual sería la base para estudios posteriores sobre manejo agronómico. El experimento fue establecido en la finca del Sr. Cristia n Hernández, en la comunidad El Castillito, Municipio Las Sabanas, departamento de Madriz, en el periodo julio del 2005 a marzo del 2006. La zona se ubica a 1400 msnm, con precipitaciones anuales de 1200 mm. Los genotipos Festival, Britget y Chandler fuero n plantados en un diseño de Bloques Completos al Azar con cuatro réplicas. Las parcelas experimentales estuvieron constituidas por bancales de 1.0 m de ancho por 5.0 m de largo (5.0 m 2 ) evaluándose la severidad de enfermedades identificadas y graficadas en curvas de desarrollo. Nueve enfermedades fueron identificadas, 3 de ellas foliares, 1 radicular y 5 de frutos. La principal enfermedad observada fue la mancha café ( Marssonina fragariae ) la cual se presentó con mayor severidad en las variedades Festival y Britget. En relación a viruela ( Mycosphaerella fragariae ) y tizón foliar ( Phomopsis obscurans ), los genotipos mostraron similar comportamiento. Las principales enfermedades observadas en los frutos en los diferentes estados de desarrollo fueron el moho gris ( Botrytis cinerea ) y antracnosis ( Colletotrichum spp.) Se puede concluir que los materiales evaluados son susceptibles a la mayoría de las enfermedades; sin embargo, la variedad Chandler mostró menor grado de infección por mancha café y por enfermeda des en los frutos.

Diagnósis y comportamiento de enfermedades en tres genotipos de fresa (Fragaria spp.) en Las Sabanas, Madriz

Los Pequeños productores del municipio Las Sabanas departamento de Madríz han adoptado el cultivo de fresa ( Fragaria spp.) como una alternativa para mejorar su nivel de vida. Una dificultad que enfrentan es la presencia y manejo de enfermedades que afectan negativamente el desarrollo del cultivo. El presente estudio se estableció con el fin de identificar las principales enfermedades en el cultivo de fresa en el municipio Las Sabanas, así como su dinámica de desarrollo, lo cual podría considerarse como el primer paso para iniciar estudios posteriores de manejo. El experimento fue establecido en la finca del Sr. Cristian Hernández, en la comunidad El Castillito, Munici- pio Las Sabanas, departamento de Madriz, en el periodo julio del 2005 a marzo del 2006. La zona se ubica a 1400 msnm, con preci pitaciones anuales de 1200 mm. Los genotipos Festival, Britget y Chandler fueron plantados en un diseño de bloques completos al azar con cuatro réplicas. Las parcelas experimentales estuvieron consti tuidas por bancales de 1.0 m de ancho por 5.0 m de largo (5.0 m 2 ) evaluándose la severidad de enfermedades identificadas y graficadas en curvas de desarrollo. Nueve enfermedades fueron identificadas, tres de ellas foliares, una radicular y cinco de frutos. La principal enfermedad observada fue la mancha café ( Marssonina fragariae ) la cual se presentó con mayor severidad en las variedades Festival y Britget. En relación a viruela ( Mycosphaerella fragariae ) y tizón foliar ( Phomopsis obscurans ), los genotipos mostraron similar com- portamiento. Las principales enfermedades observadas en los frutos en los diferentes estados de desarrollo fueron el moho gris ( Botrytis cinerea ) y antracnosis ( Colletotrichum spp.) Se puede concluir que los materiales evaluados son susceptibles a la mayoría de las enfer medades; sin embargo, la variedad Chandler mostró menor grado de infección por mancha café y por enfermedades en los frutos.

果实采后病害生物防治及其机理研究

　　分离和筛选了5种能有效防治采后果实病害的拮抗菌。其中，季也蒙假丝酵母(Candida guiliermondii(Cast) Langeroret Guerra)从引种拮抗菌中筛选获得，枯草芽孢杆菌（Bacillus subtilis）B-912从土壤中分离筛选获得，膜醭毕赤酵母（Pichia membranefaciens hansen）从桃果实伤口上分离获得，隐球酵母(Cryptococcus albidus (Saito) Skinner)和丝孢酵母（Trichosporon sp.）从桃果实表面分离获得。本文主要研究了这些拮抗菌对桃、油桃、苹果、梨和柑桔等我国主要水果采后病害的防治效果，并对其可能的抑菌机理进行了初步研究。结果如下： 1. Sx108 CFU/mL的C．guiliermondii和P．membranefaciens悬浮液可完全抑制病菌孢子浓度为5x104个/mL时桃和油桃果实软腐病(Rhizopus stolonifer(Ehrenb.ex Fr.) Vuill.)在25℃，15℃和3℃下的发生。lx108 CFU/mL的C．albidus和Trichosporon sp.悬浮液可完全抑制孢子浓度分别为lx105个/mL和5x104个/mL时苹果灰霉病(Botrytis cinerea)和青霉病（Penicillum expansum）在23℃-25℃和1℃下的发生。C．albidus和Trichosporon sp.对梨灰、青霉病也有一定抑制效果。B-912对柑桔果实青霉病(Penicillium italicum)、绿霉病(Penicillium digitatum)和核果类果实褐腐病(Monilinia fructicola)也有极好的抑制效果。生物防治效果与拮抗菌的浓度成正比，与病菌孢子浓度成反比。 2．拮抗酵母菌在室温和冷藏条件下都能迅速在果实伤口定殖，接种酵母菌48 h后，数量可增加20倍以上。拮抗菌和病菌孢子的接种时间与生物防治效果有关，先接种拈抗菌的抑菌效果显著地好于同时或后于病菌接种的效果。 3．温度对拮抗酵母菌的抑菌活力没有明显影响，无论是在室温还是在冷藏条件下，拮抗酵母菌都有同样的抑菌效果。但拮抗细菌B-912的抑菌效果与温度有一定关系。较高的温度有利于细菌拮抗作用的发挥。 4．拮抗菌能与常规的果实采后处理措施如钙处理、化学杀菌剂、冷藏和气调贮藏相结合。酵母菌与2% CaC12配合能明显地增强其抑菌能力;拮抗菌与低浓度的杀菌剂如扑海因混合使用，可达到高浓度杀菌剂的抑病效果;C．albidus和Trichosporon sp.对果实采后气调贮藏环境有良好的适应性，它们在气调下对采后苹果、梨的灰霉病和青霉病的抑制效果比冷藏条件下的好。 5．细菌B-912的抑菌机理与其产生抗菌素有关，B-912的滤液在in vitro上能有效地抑制病菌孢子的萌发，在invivo上也能明显地抑制果实采后病害的发生。拮抗酵母菌的抑菌机理则较复杂，但主要与病菌竞争营养有关，同时，C．guilliermondii和P．membranefaciens对软腐菌的抑制还通过产生水解酶如β-1，3一葡聚糖酶和几丁酶与病原菌直接作用，并参与诱导寄主产生抗性等

Antifungal properties of Schiff bases of chitosan, N-substituted chitosan and quaternized chitosan

Schiff bases of chitosan, N-substituted chitosan, and quaternized chitosan were synthesized and their antifungal properties were analyzed against Botrytis cinerea Pers. (B. cinerea pers.) and Colletotrichum lagenarium (Pass) Ell.et halst (C. lagenarium (Pass) Ell.et halst) based on the method of D. Jasso de Rodriguez and co-workers. The results showed that quaternized chitosan had better inhibitory properties than chitosan, Schiff bases of chitosan, and N-substituted chitosan. (c) 2007 Elsevier Ltd. All rights reserved.

The influence of molecular weight of quaternized chitosan on antifungal activity

Quaternized chitosan derivatives with different molecular weights were synthesized in the laboratory. Subsequent experiments were conducted to test their antifungal activities against Botrytis cinerea Pers. (B. cinerea pers.) and Colletotrichum lagenarium (Pass) Ell.et halst (C. lagenarium (Pass) EII.et halst). Our results indicate that quaternized chitosan derivatives have stronger antifungal activities than chitosan. Furthermore, quaternized chitosan derivatives with high molecular weight are shown to have even stronger antifungal activities than those with low molecular weight. (c) 2007 Published by Elsevier Ltd.

The influence of the cationic of quaternized chitosan on antifungal activity

Quatemized chitosan: N-(2-hydroxyl-phenyl)-NN-dimethyl chitosan (NHPDCS), N-(5-chloro-2-hydroxyl-phenyl)-NN-dimethyl chitosan (NCHPDCS), N-(2-hydroxyl-5-nitro-phenyl)-NN-dimethyl chitosan (NHNPDCS) and N-(5-bromic-2-hydroxyl-phenyl)-NN-dimethyI chitosan (NBHPDCS) were synthesized and their antifungal activities against Botrytis cinerea Pers. (B. cinerea Pers.) and Colletotrichum lagenarium (Pass) Ell.et halst (C. lagenarium (Pass) Ellet halst) were investigated. The results indicated that the quaternized chitosan derivatives had better inhibitory effects than chitosan, and the antifungal activities should be affected by the cation in these compounds. (C) 2007 Elsevier B.V. All rights reserved.

Sintomatología comparada de los tizones de la flor de azalea (Rhododendron spp.)

p.81-84

First report of a novel plant lysozyme with both antifungal and antibacterial activities.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4 kDa in SDS–polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355 U/mg at pH 5.5 and 30 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 °C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.

Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchidae) associated with Hylobius sp. from Pinus pinaster in Portugal

Bursaphelenchus antoniae sp. n. is described and illustrated. Dauer juveniles were isolated from the body of the large pine weevil, Hylobius sp., collected from maritime pine (Pinus pinaster) stumps, in Portugal. Bursaphelenchus antoniae sp. n. was reared and maintained in P. pinaster wood segments and on Petri dish cultures of the fungi Botrytis cinerea and Monilinia fructicola. The new species is characterised by a relatively small body length of ca 583 μm (females) and 578 μm (males), a lateral field with two incisures, presence of a small vulval flap and a conoid female tail with a rounded or pointed terminus. Males have stout spicules with a disc-like cucullus and seven caudal papillae arranged as a single midventral precloacal papilla, one precloacal pair and two postcloacal pairs. In the character of the lateral field, B. antoniae sp. n. comes close to B. abietinus, B. rainulfi and B. hylobianum, whilst spicule characters place it within the piniperdae-group sensu Ryss et al. Morphologically, B. antoniae sp. n. is closest to B. hylobianum; the spicules of these two species having flattened, wing-like, alae on the distal third of the lamina. Bursaphelenchus antoniae sp. n. is distinguished from B. hylobianum on the arrangement of the caudal papillae (two vs three pairs). ITS-RFLP profiles and the failure to hybridise support the separation of the two species. Phylogenetic analysis of the new species, based on the 18S rDNA sequence, supports the inclusion of this new species in the B. hylobianum-group sensu Braasch. Sequence analysis of the 28S rDNA D2/D3 domain did not place the new species in a definite group.

Bursaphelenchus pinophilus Brzeski & Baujard, 1997 (Nematoda: Parasitaphelenchinae) associated with nematangia on Pityogenes bidentatus (Herbst, 1783) (Coleoptera: Scolytinae), from the Czech Republic

The occurrence of Bursaphelenchus species in the Czech Republic is poorly known, the first report of the genus being made by Kubátová et al. (2000) who reported the association of B. eremus with the hyphomycetous microfungus, Esteya vermicola, and the bark beetle, Scolytus intricatus, collected from Quercus robur, in central Bohemia. To date, four other species have been reported from the country, namely B. fungivorus (Braasch et al., 2002), B. hofmanni (see Braasch, 2001), B. mucronatus (see Braasch, 2001) and B. vallesianus (Gaar et al., 2006). More recently, a survey for Bursaphelenchus species associated with bark- and wood-boring insects in the Czech Republic identified B. pinophilus Brzeski & Baujard, 1997 from the Moravia region. Although this represents a new country record, it was also associated with nematangia on the hind wings of a new insect vector. A total of 404 bark- and wood-boring insects were collected from declining or symptomatic trees and screened for the presence of Bursaphelenchus. Bark and longhorn beetles were captured manually after debarking parts of the trunk displaying symptoms of insect attacks. Longhorn beetle larvae were also collected together with logs cut from the trunk. Logs were kept at room temperature in the laboratory until insect emergence. Each adult insect was individually dissected in water and examined for nematodes. All nematodes resembling dauer juveniles of Bursaphelenchus were collected and identified by molecular characterisation using a region of ribosomal DNA (rDNA) containing the internal transcribed spacer regions ITS1 and ITS2. ITS-RFLP analyses using five restriction enzymes (AluI, HaeIII, HinfI, MspI, RsaI) were performed to generate the species-specific profile according to Burgermeister et al. (2009). Species identification was also confirmed by morphological data after culture of the dauers on Botrytis cinerea Pers. ex Ft., growing in 5% malt extract agar. During this survey, only species belonging to the Curculionidae, subfamily Scolytinae, revealed the presence of nematodes belonging to Bursaphelenchus. Dauers of this genus were found aggregated under the elytra in nematangia formed at the root of the hind wings (Fig. 1). The dauers were identified from 12 individuals of Pityogenes bidentatus (Herbst, 1783) (Coleoptera: Scolytinae) collected under the bark of Pinus sylvestris trunks. Each insect carried ca 10-100 dauers. The ITS-RFLP patterns of the dauers so obtained confirmed the identification of B. pinophilus associated with this insect species. Bursaphelenchus pinophilus has been found mainly in Europe and has been reported from various countries such as Poland (Brzeski & Baujard, 1997), Germany (Braasch, 2001), and Portugal (Penas et al., 2007). The recent detection of this species associated with dead P. koraiensis in Korea (Han et al., 2009) expands its geographical distribution and potential importance. It has been found associated only with Pinus species, but very little is known about the insect vector. The bark beetle, Hylurgus ligniperda, was initially suggested as the insect vector by Pe-nas et al. (2006), although the nematode associated with this insect was later reclassified as B. sexdentati by morphological and molecular analysis (Penas et al., 2007). According to the literature, P. bidentatus has been cited as a vector of Ektaphelenchus sp. (Kakuliya, 1966) in Georgia, and an unidentified nematode species in Spain (Roberston et al., 2008). Interestingly, B. pinophilus was found in the nematangia formed at the root of the hind wings of P. bidentatus. Although this phenomenon is not so common in other Bursaphelenchus species, B. rufipennis has been found recently in such a structure on the hind wings of the insect Dendroctonus rufipennis (Kanzaki et al., 2008). Although other nematode species (e.g., Ektaphelenchus spp.) are frequently found associated within the same nematangia (see Kanzaki et al., 2008), in this particular case, only dauers of B. pinophilus were identified. The association between B. pinophilus and P. bidentatus represents the first report of this biological association and the association with the Scolytinae strengthens the tight and specific links between this group of Bursaphelenchus species and members of the Scolytinae (Ryss et al., 2005).

Molecular and biotechnological approaches to essential oils production in thymus caespititius

Tese de doutoramento, Biologia (Biotecnologia), Universidade de Lisboa, Faculdade de Ciências, 2014

The significance of volatile antifungal metabolites produced by trichomerma harzianum biotype Th4, in green-mould disease of commercial mushroom crops /

The aggressive mushroom competitor, Trichoderma harzianum biotype Th4, produces volatile antifungal secondary metabolites both in culture and during the disease cycle in compost. Th4 cultures produced one such compound only when cultured in the presence of Agaricus bisporus mycelium or liquid medium made from compost colonised with A. bisporus. This compound has TLC and UVabsorption and characteristics indicating that it belongs to a class of pyrone antibiotics characterised from other T. harzianum biotypes. UV absorption spectra indicated this compound was not 6-pentyl-2H-pyran-one (6PAP), the volatile antifungal metabolite widely described in Th1. Furthermore, this compound was not produced by Th1 under any culture conditions. Mycelial growth of A. bisporus, Botrytis cinerea and Sclerotium cepivorum was inhibited in the presence of this compound through volatility , diffusion and direct application. This indicates that Th4 produces novel, volatile, antifungal metabolites in the presence of A. bisporus that are likely involved in green mould disease of mushroom crops.

Contrasting effects of necrotrophic and biotrophic plant pathogens on the aphid Aphis fabae

Phytophagous insects have to contend with a wide variation in food quality brought about by a variety of factors intrinsic and extrinsic to the plant. One of the most important factors is infection by plant pathogenic fungi. Necrotrophic and biotrophic plant pathogenic fungi may have contrasting effects on insect herbivores due to their different infection mechanisms and induction of different resistance pathways, although this has been little studied and there has been no study of their combined effect. We studied the effect of the biotrophic rust fungus Uromyces viciae-fabae (Pers.) Schroet (Basidiomycota: Uredinales: Pucciniaceae) and the necrotrophic fungus Botrytis cinerea Pers. (Ascomycota: Helotiales: Sclerotiniaceae) singly and together on the performance of the aphid Aphis fabae Scop. (Hemiptera: Aphididae) on Vicia faba (L.) (Fabaceae). Alone, botrytis had an inhibitory effect on individual A. fabae development, survival and fecundity, while rust infection consistently enhanced individual aphids’ performance. These effects varied in linear relation to lesion or pustule density. However, whole-plant infection by either pathogen resulted in a smaller aphid population of smaller aphids than on uninfected plants, indicating a lowering of aphid carrying capacity with infection. When both fungi were applied simultaneously to a leaf they generally cancelled the effect of each other out, resulting in most performance parameters being similar to the controls, although fecundity was reduced. However, sequential plant infection (pathogens applied five days apart) led to a 70% decrease in fecundity and 50% reduction in intrinsic rate of increase. The application of rust before botrytis had a greater inhibitory effect on aphids than applying botrytis before rust. Rust infection increased leaf total nitrogen concentration by 30% while infection by botrytis with or without rust led to a 38% decrease. The aphids’ responses to the two plant pathogens individually is consistent with the alteration in plant nutrient content by infection and also the induction of different plant defence pathways and the possible cross-talk between them. This is the first demonstration of the complex effects of the dual infection of a plant by contrasting pathogens on insect herbivores. Key words: Vicia faba, Botrytis cinerea, Uromyces viciae-fabae, tripartite interactions, induced resistance

Asymmetric microbial reduction of ketones: absolute configuration of trans-4-ethyl-1-(1S-hydroxyethyl)cyclohexanol

A set of five fungal species, Botrytis cinerea, Trichoderma viride and Eutypa lata, and the endophytic fungi Colletotrichum crassipes and Xylaria sp., was used in screening for microbial biocatalysts to detect monooxygenase and alcohol dehydrogenase activities (for the stereoselective reduction of carbonyl compounds). 4-Ethylcyclohexanone and acetophenone were biotransformed by the fungal set. The main reaction pathways involved reduction and hydroxylations at several positions including tertiary carbons. B. cinerea was very effective in the bioreduction of both substrates leading to the chiral alcohol (S)-1-phenylethanol in up to 90% enantiomeric excess, and the cis-trans ratio for 4-ethylcyclohexanol was 0:100. trans-4-Ethyl-1-(1S-hydroxyethyl)cyclohexanol, obtained from biotransformation by means of an acyloin-type reaction, is reported here for the first time. The absolute configurations of the compounds trans-4-ethyl-1-(1S-hydroxyethyl)cyclohexanol and 4-(1S- and 4-(1R-hydroxyethyl)cyclohexanone were determined by NMR analysis of the corresponding Mosher's esters. (C) 2009 Elsevier Ltd. All rights reserved.

Efeito de biofertilizantes sobre o crescimento micelial e a germinação de esporos de alguns fungos fitopatogênicos

The biofertilizer was produced through anaerobic fermentation of cow manure adding milk, sugar, salts, cow liver parts and bone powder. After 73 days of fermentation it was evaluated the effect on micelial growth of Pythium aphanidermatum, Alternaria solani, Stemphylium solani, Septoria licopersici, Sclerotinia sclerotiorum, Botrytis cinerea, Rhizoctonia solani, Fusarium oxysporum f. sp. phaseoli and spores germination of B. cinerea, A. solani, Hemileia vastatrix and Coleosporium plumierae. In relation to micelial growth inhibition, the growth rate was calculated and it was found that, in general, concentrations over 10% caused a total inhibition of growth for the majority of fungi assayed. In case of spores germination, biofertilizer concentration over 20% has inhibited completely the germination of B. cinerea, over 10% inhibited A. solani, 5 and 1% of C. plumierae and H. vastatrix, respectively. Three different biofertilizers were also tested and one of them was less effective, which was the one produced with manure from confined cows opposed to the others produced with grazing cows.