Sobrevivência de Fusarium tucumaniae, agente causal da podridão vermelha da raiz da soja, em condições de campo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Fontes de matéria orgânica para inibição de fitopatógenos habitantes do solo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Extração e análise eletroforética em gel de poliacrilamida (SDS-PAGE) de proteínas totais de folhas e raízes de Piper tuberculatum

O Estado do Pará é o principal produtor brasileiro de pimenta-do-reino (Piper nigrum Link), entretanto a sua produção tem sido bastante afetada pela doença conhecida como fusariose. O Fusarium solani f. sp. piperis é o agente causador desta doença que afeta o sistema radicular da planta, causando o apodrecimento das raízes e a queda das folhas levando à morte da planta. Algumas piperáceas nativas da região amazônica, entre elas a espécie Piper tuberculatum Jacq., têm se mostrado resistentes à infecção pelo F. solani f. sp. piperis, e desta forma têm sido utilizadas em estudos de interação planta-patógeno. Neste trabalho foram avaliadas cinco condições de extração de proteínas com o objetivo de selecionar tampões adequados para a extração de proteínas totais de folhas e raízes de P. tuberculatum. Os tampões utilizados para a extração de proteínas de raízes e folhas foram: tampão salino, tampão sacarose, tampão glicerol, tampão uréia e tampão fosfato de sódio. As análises quantitativas mostraram que os tampões sacarose, glicerol e uréia foram mais eficientes na extração de proteínas de folhas e raízes. Análises de SDS-PAGE mostraram padrões diferenciados de bandas em extratos protéicos de folhas e raízes obtidos com os diferentes tampões. Os resultados obtidos neste trabalho contribuem para a identificação de tampões de extração adequados para a obtenção de amostras de proteínas totais em estudos de interação P. tuberculatum - F. solani f. sp. piperis.

Isolamento, caracterização e expressão em sistema bacteriano de um gene que codifica uma proteína transportadora de lipídeos de Piper nigrum

A pimenteira-do-reino (Piper nigrum L.) constitui uma das espécies de pimenta mais amplamente utilizadas no mundo, pertencendo à família Piperaceae, a qual compreende cerca de 1400 espécies distribuídas principalmente no continente americano e sudeste da Ásia, onde esta cultura originou. A pimenteira-do-reino foi introduzida no Brasil no século XVII, e tornou-se uma cultura de importância econômica desde 1933. O Estado do Pará é o principal produto brasileiro de pimenta-do-reino, contudo sua produção vem sendo afetada pela doença fusariose causada pelo fungo Fusarium solani f. sp. piperis. Estudos prévios revelaram a identificação de sequencias de cDNA diferencialmente expressas durante a interação da pimenteira-do-reino com o F. solani f. sp. piperis. Entre elas, uma sequencia de cDNA parcial que codifica para uma proteína transportadora de lipídeos (LTP), a qual é conhecida por seu importante papel na defesa de plantas contra patógenos e insetos. Desta forma, o objetivo principal deste trabalho foi isolar e caracterizar as sequencias de cDNA e genômica de uma LTP de pimenteira-do-reino, denominada PnLTP. O cDNA completo da PnLTP isolado por meio de experimentos de RACE apresentou 621 bp com 32 pb and 235 bp nas regiões não traduzidas 5‘ e 3‘, respectivamente. Este cDNA contem uma ORF de 354 bp codificando uma proteína deduzida de 117 resíduos de aminoácidos que apresentou alta identidade com LTPs de outras espécies vegetais. Análises das sequencias revelou que a PnLTP contem um potencial peptídeo sinal na extremidade amino-terminal e oito resíduos de cisteína preditos por formar quatro pontes de dissulfeto, as quais poderiam contribuir para a estabilidade desta proteína. O alinhamento entre as sequencias de cDNA e genômica revelou a ausência de introns na região codificante do gene PnLTP, o que está de acordo ao encontrado em outros genes de LTPs de plantas. Por último, a PnLTP madura foi expressa em sistema bacteriano. Experimentos adicionais serão realizados com o objetivo de avaliar a habilidade da PnLTP recombinante em inibir o crescimento do F. solani f. sp. piperis.

Controle biológico da podridão radicular (Pythium aphanidermatum) em cultivos hidropônicos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

Fungal microbiota from rain water and pathogenicity of Fusarium species isolated from atmospheric dust and rainfall dust

In order to determine the presence of Fusarium spp. in atmospheric dust and rainfall dust, samples were collected during September 2007, and July, August, and October 2008. The results reveal the prevalence of airborne Fusarium species coming from the atmosphere of the South East coast of Spain. Five different Fusarium species were isolated from the settling dust: Fusarium oxysporum, F. solani, F. equiseti, F. dimerum, and F. proliferatum. Moreover, rainwater samples were obtained during significant rainfall events in January and February 2009. Using the dilution-plate method, 12 fungal genera were identified from these rainwater samples. Specific analyses of the rainwater revealed the presence of three species of Fusarium: F. oxysporum, F. proliferatum and F. equiseti. A total of 57 isolates of Fusarium spp. obtained from both rainwater and atmospheric rainfall dust sampling were inoculated onto melon (Cucumis melo L.) cv. Piñonet and tomato (Lycopersicon esculentum Mill.) cv. San Pedro. These species were chosen because they are the main herbaceous crops in Almeria province. The results presented in this work indicate strongly that spores or propagules of Fusarium are able to cross the continental barrier carried by winds from the Sahara (Africa) to crop or coastal lands in Europe. Results show differences in the pathogenicity of the isolates tested. Both hosts showed root rot when inoculated with different species of Fusarium, although fresh weight measurements did not bring any information about the pathogenicity. The findings presented above are strong indications that long-distance transmission of Fusarium propagules may occur. Diseases caused by species of Fusarium are common in these areas. They were in the past, and are still today, a problem for greenhouses crops in Almería, and many species have been listed as pathogens on agricultural crops in this region. Saharan air masses dominate the Mediterranean regions. The evidence of long distance dispersal of Fusarium spp. by atmospheric dust and rainwater together with their proved pathogenicity must be taken into account in epidemiological studies.

A role for jasmonate in pathogen defense of Arabidopsis

To investigate the role of jasmonate in the defense of plants against fungal pathogens, we have studied a mutant of Arabidopsis, fad3–2 fad7–2 fad8, that cannot accumulate jasmonate. Mutant plants were extremely susceptible to root rot caused by the fungal root pathogen Pythium mastophorum (Drechs.), even though neighboring wild-type plants were largely unaffected by this fungus. Application of exogenous methyl jasmonate substantially protected mutant plants, reducing the incidence of disease to a level close to that of wild-type controls. A similar treatment with methyl jasmonate did not protect the jasmonate-insensitive mutant coi1 from infection, showing that protective action of applied jasmonate against P. mastophorum was mediated by the induction of plant defense mechanisms rather than by a direct antifungal action. Transcripts of three jasmonate-responsive defense genes are induced by Pythium challenge in the wild-type but not in the jasmonate-deficient mutant. Pythium species are ubiquitous in soil and root habitats world-wide, but most (including P. mastophorum) are considered to be minor pathogens. Our results indicate that jasmonate is essential for plant defense against Pythium and, because of the high exposure of plant roots to Pythium inoculum in soil, may well be fundamental to survival of plants in nature. Our results further indicate that the fad3–2 fad7–2 fad8 mutant is an appropriate genetic model for studying the role of this important signaling molecule in pathogen defense.

Characterization of Two Novel Type I Ribosome-Inactivating Proteins from the Storage Roots of the Andean Crop Mirabilis expansa1

Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.

Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site.

The tobacco N and Arabidopsis RPS2 genes, among several recently cloned disease-resistance genes, share highly conserved structure, a nucleotide-binding site (NBS). Using degenerate oligonucleotide primers for the NBS region of N and RPS2, we have amplified and cloned the NBS sequences from soybean. Each of these PCR-derived NBS clones detected low-or moderate-copy soybean DNA sequences and belongs to 1 of 11 different classes. Sequence analysis showed that all PCR clones encode three motifs (P-loop, kinase-2, and kinase-3a) of NBS nearly identical to those in N and RPS2. The intervening region between P-loop and kinase-3a of the 11 classes has high (26% average) amino acid sequence similarity to the N gene although not as high (19% average) to RPS2. These 11 classes represent a superfamily of NBS-containing soybean genes that are homologous to N and RPS2. Each class or subfamily was assessed for its positional association with known soybean disease-resistance genes through near-isogenic line assays, followed by linkage analysis in F2 populations using restriction fragment length polymorphisms. Five of the 11 subfamilies have thus far been mapped to the vicinity of known soybean genes for resistance to potyviruses (Rsv1 and Rpv), Phytophthora root rot (Rps1, Rps2, and Rps3), and powdery mildew (rmd). The conserved N- or RPS2-homologous NBS sequences and their positional associations with mapped soybean-resistance genes suggest that a number of the soybean disease-resistance genes may belong to this superfamily. The candidate subfamilies of NBS-containing genes identified by genetic mapping should greatly facilitate the molecular cloning of disease-resistance genes.

Incidence of Stagonospora meliloti and Acrocalymma medicaginis in Lucerne crowns and roots in eastern Australia, and their comparative aggressiveness to Lucerne and inheritance of reaction to S. meliloti in lucerne

The research presented indicates that lucerne crown and root rot caused by Stagonospora meliloti is prevalent in southern New South Wales, whereas Acrocalymma medicaginis is the more commonly observed pathogen in Queensland. Although both pathogens cause reddening of internal root and crown tissue of lucerne, they can be distinguished by symptomatology. S. meliloti causes a diffuse red blotching of the internal tissue accompanied by the presence of an external lesion, whereas A. medicaginis causes red streaking at the extremity of wedge-shaped, dry-rotted tissue. Inoculation of propagules of a susceptible lucerne clone indicated that S. meliloti was the more aggressive pathogen. Although A. medicaginis does not cause leaf disease, there was a strong relationship between the leaf and root reaction of clones to S. meliloti. Inheritance of resistance to S. meliloti in lucerne appeared to be conditioned by a single dominant gene, based on segregations observed in S-1 and F-1 populations, but not in a backcross population from the same family where an excess of susceptible individuals (74% v. expected of 50%) was obtained in a cross of a resistant F-1 individual to the susceptible parent. Resistance appears to be highly heritable, however, and amenable to population improvement by breeding. A conclusion of the research is that breeding for resistance to S. meliloti for lucernes to be grown in southern Australia would appear to be a worthwhile objective. Presently, no highly resistant cultivars exist anywhere in the world.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

What influences fungal communities in cotton soils

Soilborne diseases such as Fusarium wilt, Black root rot and Verticillium wilt have significant impact on cotton production. Fungi are an important component of soil biota with capacity to affect pathogen inoculum levels and their disease causing potential. Very little is known about the soil fungal community structure and management effects in Australian cotton soils. We analysed surface soils from ongoing field experiments monitoring cotton performance and disease incidence in three cotton growing regions, collected prior to 2013 planting, for the genetic diversity and abundance as influenced by soil type, environment and management practices and link it with disease incidence and suppression. Results from the 28S LSU rRNA sequencing based analysis indicated a total of 370 fungal genera in all the cotton soils and the top 25 genera in abundance accounted for the major portion of total fungal community. There were significant differences in the composition and genetic diversity of soil fungi between the different field sites from the three cotton growing regions. Results for diversity indices showed significantly greater diversity in the long-term crop rotation experiment at Narrabri (F6E) and experiments at Cowan and Goondiwindi compared to the Biofumigation and D1 field experiments at ACRI, Narrabri. Diversity was lowest in the soils under brassica crop rotation in Biofumigation experiment. Overall, the diversity and abundance of soil fungal community varied significantly in the three cotton growing regions indicating soil type and environmental effects. These results suggest that changes in soil fungal community may play a notable role in soilborne disease incidence in cotton.

Controle da podridão de raiz e promoção de crescimento em hidroponia com bactérias.

2009

Phosphonate applied as a pre-plant dip controls Phytophthora cinnamomi root and heart rot in susceptible pineapple hybrids.

The effectiveness of pre-plant dips of crowns in potassium phosphonate and phosphorous acid was investigated in a systematic manner to develop an effective strategy for the control of root and heart rot diseases caused by Phytophthora cinnamomi in the pineapple hybrids 'MD2' and '73-50' and cultivar Smooth Cayenne. Our results clearly indicate that a high volume spray at planting was much less effective when compared to a pre-plant dip. 'Smooth Cayenne' was found to be more resistant to heart rot than 'MD2' and '73-50', and 'Smooth Cayenne' to be more responsive to treatment with potassium phosphonate. Based on cumulative heart rot incidence over time 'MD2' was more susceptible to heart rot than '73-50' and was more responsive to an application of phosphorous acid. The highest levels of phosphonate in roots were reached one month after planting and levels declined during the next two months. Pre-plant dipping of crowns prior to planting is highly effective to control root and heart rot in the first few months but is not sufficient to maintain health of the mother plant root system up until plant crop harvest when weather conditions continue to favour infection.