Fusarium oxysporum strains as biocontrol agents against Fusarium wilt: effects on soil microbial biomass and activity

Before planning the large-scale use of nonpathogenic strains of Fusarium oxysporum as biocontrol agents of Fusarium wilt, their behaviour and potential impact on soil ecosystems should be carefully studied as part of risk assessment. The aim of this work was to evaluate the effects of antagonistic F. oxysporum strains, genetically manipulated (T26/6) or not (233/1), on soil microbial biomass and activity. The effects were evaluated, in North-western Italy, in two soils from different sites at Albenga, one natural and the other previously solarized, and in a third soil obtained from a 10-year-old poplar stand (Popolus sp.), near Carignano. There were no detectable effects on ATP, fluorescein diacetate hydrolysis, and biomass P that could be attributed to the introduction of the antagonists. A transient increase of carbon dioxide evolution and biomass C was observed in response to the added inoculum. Although the results showed only some transient alterations, further studies are required to evaluate effects on specific microorganism populations.

Distribuição do elemento transponível impala em isolados de fusarium oxysporum patogênicos e não-patogênicos ao feijoeiro

A variabilidade genética de 20 isolados de Fusarium oxysporum, nove não-patogênicos e 11 patogênicos ao feijoeiro (Phaseouls vulgaris), foi determinada com base na distribuição do elemento transponível impala. A presença de impala das subfamílias D e E foi determinada por experimentos de PCR, empregando oligonucleotídeos específicos para cada subfamília. Foi observada a presença de representantes das duas subfamílias na maioria dos isolados, sugerindo, portanto, que impala é um antigo componente do genoma de F. oxysporum f. sp. phaseoli. A hibridização do DNA total de cada isolado, clivado com a enzima EcoRI, com um fragmento do elemento impala da subfamíla E, mostrou uma variação nos padrões de bandas dos isolados não-patogênicos, indicando a possível atividade desses elementos. No entanto, no caso dos isolados patogênicos, foram observados padrões de bandas mais homogêneas e alguns isolados apresentaram o mesmo perfil de bandas, indicando que se trata de cópias de impala que, possivelmente, não são mais capazes de sofrer transposição. Estas cópias inativas são excelentes marcadores genéticos. Um dos isolados patogênicos, Fus4, não apresentou cópias endógenas de impala, o que torna esse isolado um candidato para experimentos de mutagênese insercional usando o vetor pNI160, que possui o elemento impala ativo interrompendo o gene niaD, que codifica a enzima nitrato redutase.

Enhancement of germination of spores of Verticillium dahliae and Fusarium oxysporum esp vasinfectum in vascular fluid from cotton plants infected with the root-knot nematode

Root-knot nematode [RKN] (Meloidogyne incognita) can increase the severity of Verticillium (V dahliae) and Fusarium (F oxysporum f.sp. vasinfectum) wilt diseases in cotton (Gossypium hirsutum). This study was conducted to determine some of the physiological responses caused by nematode invasion that might decrease resistance to vascular wilt diseases. The effect of RKN was investigated on spore germination and protein, carbohydrate and peroxidase content in the xylem fluids extracted from nematode-infected plants. Two cotton cultivars were used with different levels of resistance to both of the wilt pathogens. Spore germination was greater in the xylem fluids from nematode-infected plants than from nematode-free plants. The effect on spore germination was greater in the Fusarium-resistant cultivar (51%). Analysis of these fluids showed a decrease in total protein and carbohydrate levels for both wilt-resistant cultivars, and an increase in peroxidase concentration. Fluids from nematode-free plants of the Verticillium-resistant cultivar contained 46% more peroxidase than the Fusarium-resistant cultivar. The results provide further evidence that the effect of RKN on vascular wilt resistance is systemic and not only local. Changes in metabolites in the xylem pass from the root to the stem, accelerating disease development.

Desenvolvimento de substrato supressivo à murcha do crisântemo causada por Fusarium oxysporum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A foliar rating system for comparing the resistance of banana cultivars grown as tissue-cultured plantlets in the laboratory to Fusarium wilt

A foliar rating system was developed to assess the progress of Fusarium wilt ( Panama disease) caused by Fusarium oxysporum f. sp. cubense in seven banana cultivars differing in their resistance to race 1 of the pathogen. Plantlets were transplanted into unamended soil naturally infested with the pathogen, soil amended with urea and soil amended with aged chicken manure. A corm invasion score was also developed to assess the accuracy of the foliar symptom score as an indicator of cultivar resistance. On the basis of foliar symptom scores alone, the response of five of the seven cultivars in the chicken manure treatment corresponded to their known field response. However, the response of the other two cultivars, both susceptible to the pathogen in the field, fell into two categories. One had a high foliar symptom score and a correspondingly high corm invasion score, whereas the other had a low foliar symptom score and a high corm invasion score. Breeders need to be aware of the two categories of susceptible response, if inferior breeding material is to be rejected early on in a breeding program.

Effect of organic amendments and solarisation on Fusarium wilt in susceptible banana plantlets, transplanted into naturally infested soil

Despite extensive research since pathogenicity was first established in 1919, no cultural or chemical control strategy has proven effective against Fusarium wilt of bananas. The efficacy of cultural control is attributed to the suppression of pathogen activity. Yet, amending naturally infested soil with aged chicken manure has been shown to enhance disease severity, without any change in the activity of the pathogen Fusarium oxysporum f. sp. cubense (Foc) in the soil. In this study, the effect of amending soil with composted sawdust, and of solarising soil, was compared with the effect of amending soil with chicken manure. Bioassays comparing the activity of Foc in the soil with the extent of invasion of banana pseudostem tissue by Foc were used to investigate why strategies targetting pathogen survival have not proven successful in controlling this disease. The enhancement of Foc invasion of the banana plantlets was reproduced with the addition of chicken manure to the naturally infested soil. However, changes in the activity of Foc in the soil were not associated with changes in the frequency of invasion of the plantlets. Invasion of banana pseudostems in the sawdust and solarisation treatments was not significantly different from invasion in the respective control treatments, despite a reduction in the activity of Foc in the sawdust-amended soil and an enhancement in the solarised soil. Moreover, the increase in Foc activity in the solarised soil recorded during the bioassays occurred despite the effectiveness of solarisation in reducing the survival of Foc in pre-colonised banana root tips buried in the soil. Changes in the frequency of invasion were associated with changes in the availability of mineral nitrogen, particularly ammonium N. These results suggest that the physiological response of banana cultivars to ammonium N may be associated with their susceptibility to Fusarium wilt. Accordingly, cultural strategies for controlling Panama disease will only be effective if they enhance the ability of the host to resist invasion.

Mechanism of extracellular silver nanoparticles synthesis by Stereum hirsutum and Fusarium oxysporum

The increasing interest for greener and biological methods of synthesis has led to the development of non-toxic and comparatively more bioactive nanoparticles. Unlike physical and chemical methods of nanoparticle synthesis, microbial synthesis in general and mycosynthesis in particular is cost-effective and environment-friendly. However, different aspects, such as the rate of synthesis, monodispersity and downstream processing, need to be improved. Many fungal-based mechanisms have been proposed for the formation of silver nanoparticles (AgNPs), mainly those involving the presence of nitrate reductase, which has been detected in filtered fungus cell used for AgNPs production. There is a general acceptance that nitrate reductase is the main responsible for the reduction of Ag ions for the formation of AgNPs. However, this generally accepted mechanism for fungal AgNPs production is not totally understood. In order to elucidate the molecules participating in the mechanistic formation of metal nanoparticles, the current study is focused on the enzymes and other organic compounds involved in the biosynthesis of AgNPs. The use of each free fungal mycelium of both Stereum hirsutum and Fusarium oxysporum will be assessed. In order to identify defective mutants on the nitrate reductase structural gene niaD, fungal cultures of S.hirsutum and F.oxysporum will be selected by chlorate resistance. In addition, in order to verify if each compound identified as key-molecule influenced on the production of nanoparticles, an in vitro assay using different nitrogen sources will be developed. Lately, fungal extracellular enzymes will be measured and an in vitro assay will be done. Finally, The nanoparticle formation and its characterization will be evaluated by UV-visible spectroscopy, electron microscopy (TEM), X-ray diffraction analysis (XRD), Fourier transforms infrared spectroscopy (FTIR), and LC-MS/MS.

Potencial fungitóxico do óleo essencial de Piper hispidinervum (pimenta longa) sobre os fungos fitopatogênicos Bipolaris sorokiniana, Fusarium oxysporum e Colletotrichum gloeosporioides

O objetivo deste trabalho foi avaliar a atividade fungicida in vitro do óleo essencial das folhas de Piper hispidinervum sobre Bipolaris sorokiniana, Fusarium oxysporum e Colletotrichum gloeosporioides. Para os ensaios biológicos, empregou-se o teste bioanalítico in vitro utilizando as concentrações de 100, 200, 500, 1000, 1500 e 2000 µg.mL-1 do óleo essencial. Estas foram incorporadas no meio de cultura BDA (batata-dextrose-ágar) para avaliação do crescimento ou inibição micelial. O delineamento estatístico utilizado foi o inteiramente casualizado, com quatro repetições. Na concentração de 200 µg.mL-1, observou-se uma inibição total do fitopatógeno Bipolaris sorokiniana enquanto que, para o Fusarium oxysporum e o Colletotrichum gloeosporioides esta ocorreu na concentração de 1000 µg.mL-1.

Contribuição ao estudo da influência dos fatores físicos do solo, sôbre a incidência da murcha do algodoeiro, causada por Fusarium oxysporum f. vasinfectum (Atk.) Snyder & Hansen

A finalidade do trabalho, foi obter dados para o melhor conhecimento dos fatôres físicos do solo, que influenciam a incidência de murcha do algodoeiro, causada por Fusarium oxysporum f. vasinfectum (Atk.) Snyder & Hansen. Foi estudada a influência do tamanho das partículas de areia e porcentagem da mesma no solo. O primeiro ensaio mostrou que, a porcentagem de areia tinha um efeito sôbre a incidência de murcha na variedade IAC - 12. A maior incidência de murcha, foi notada nos tratamentos que possuiam maior porcentagem de areia. No segundo ensaio, foi notado o efeito do tamanho das partículas de areia sôbre a incidência de murcha na variedade IAC - 12. A incidência foi mais intensa nos substratos com Areias Fina e Média. O terceiro ensaio mostrou que a resistência da variedade RM4, ao Fusarium, aparentemente não foi afetada pela porcentagem de areia ou pelo tamanho das partículas.

Influência da porcentagem de areia no solo e Meloidogyne incognita (Kofoid e White) Chitwood sobre a incidência de murcha de algodoeiro causada por Fusarium oxysporum f. vasinfectum (Atk) Snyder e Hansen

O presente trabalho teve por finalidade estudar o efeito das diferentes porcentagens de areia, em substratos artificiais sobre a severidade de murcha do algodoeiro. Para êste fim foram utilizados substratos artificiais com quantidades variáveis de areia. Como inóculo foram utilizados suspensões de Meloidogyne, Fusarium e Fusarium mais Meloidogyne. Os três inóculos foram testados em duas; variedades de algodão. Os resultados obtidos com a variedade RM2 no 1.° ensaio mostraram uma maior incidência de murcha nos substratos, com maior porcentagem de areia, isto é, 90% e 60%. Quanto ao inóculo utilizado, a maior incidência da doença ocorreu nos tratamentos que receberam inoculação conjunta de Fusarium mais Meloidogyne. No segundo ensaio, utilizando-se a variedade RM4 não foram obtidos dados que mostrassem diferenças significativas entre substratos. Isto provavelmente se deve a um aumento no potencial de inóculo e uma melhor distribuição do mesmo. No entanto, houve uma diferença significativa para os tipos de inóculo utilizados, sendo que a maior severidade de doença ocorreu quando o inóculo era constituído de fungo e de nematóide, seguindo-se o tratamento cujo inóculo era constituido só de fungo No ensaio feito com variedade RM4 foi obtida significância para a interação tipos de inóculo versus substratos. A interação mostrou que o inóculo constituído só de Fusarium comportou-se diferentemente nos diferentes, tipos de substratos, utilizados. Tendo a maior severidade da doença ocorrido no substrato contendo a maior porcentagem de areia. No entanto, não houve diferenças significativas para a severidade da doença causada pelo inóculo constituido do fungo mais nematóides nos diferentes substratos, o que mostra efeito do inóculo de Meloidogyne sôbre o inóculo em potencial de Fusarium. Os diferentes tipos de inóculo comportaram-se de modo diferentes nos substratos de terra roxa, e nos substratos contendo 60% e 90% de areia, sendo que a maior coloração dos vasos foi obtida nos tratamentos que receberam os inóculos de Fusarium e Fusarium mais Meloidogyne.

Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .

Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection.

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.