Antifungal activity of bacterium symbiotically associated with Steinernema abbasi towards Fusarium oxysporum

Fusarium oxysporum f.sp. lycopersici (Fol) is the causal agent of the Fusarium wilt disease of tomato. Soil fumigant (mainly methyl bromide) applications are in use for its control. With the increasing environmental awareness, biological control methods are under investigation for their effectiveness, including the use of antagonists. Pseudomonas oryzihabitans (=Flavimonas oryzihabitans), a symbiont of the entomopathogenic nematode Steinernema abbasi was investigated as an antagonism of a Fol isolate in two laboratory and two glasshouse experiments. Bacteria and cell-free filtrate antifungal activity were tested both in dual cultures and in broth culture. In pot experiments, suspensions of bacteria in five concentrations (106, 105, 104, 103 and 102 cells/ml) were tested for their ability to control the pathogen at 25±3°C. In all tests the bacterium significantly inhibited the growth of Fol mycelium in vitro. Similar results were obtained when the bacterium was also tested against Fusarium oxysporum f.sp. radicis lycopersici and against Rhizoctonia solani. Moreover, when it was introduced into the soil, it was able to suppress the Fusarium wilt of tomato.

Variabilidade genética de Fusarium spp. isolados de solos e de bananeiras com sintomas do Mal-do-Panamá

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

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.

Growth of Fusarium species as affected by temperature and osmotic potential (NaCl and KCl) interactions.

Myceliar growth of 90 Fusarium strains os F. acuminatum, F. chlamydosporum, F. culmorum, F. equiseti, F.verticillioides, F. oxysporum, F. proliferatum, F. solani an F. sambucinum isolated from fluvial channels and sea beds of the south-eastern coast of Spain was tested on potato-dextrose-agar adjusted to different matric potentials with either KCl or NaCl (from - 1.50 to - 144.54 bars).

Biogeografía de especies de Fusarium en el litoral mediterráneo de España

El trabajo presentado estudia la presencia de Fusarium oxysporum, F.solani(sensulato), F. equiseti y F.acuminatum en puntos del litoral de Almería, Alicante, Gerona e Islas Baleares (Menorca, Ibiza, Espalmador). Se analizaron tanto arenas de las playas (zonas intermareal y supramareal) como fondos marino situados a 27,9 y 7,2 metros de profundidad en Almería y a 10 m de profundidad en las Islas Baleares. Exceptuando el litoral de Gerona, en el resto de los enclaves se presentaron varias especies de Fusarium que se aislaron de las arenas de las playas, confirmando así resultados obtenidos con anterioridad. Lo más novedoso fue encontrar especies de Fusarium a diferentes profundidades marinas. En Almería F.oxysporum y F.equisti se aislaron a 27,9 y7,2 m profundidad. F. acuminatum se aisló de la muetra recogida a 27m de profundidad. En las islas Baleares, a10m de profundidad, se aislaron F. oxysporum, F. solani (sensulato), F.equiseti y F.acuminatum. El efecto antrópico, el comportamiento como "airborne" o los arrastres de aguas por las ramblas y torrentes podría explicar la presencia de estas especies en los hábitats mencionados. La permanencia de estas especies en los hábitats mencionados, especialmente en la zona intermareal de las playas y en los fondos marinos donde soportan elevadas presiones osmóticas por la alta salinidad del agua del mar Mediterráneo, permitirá estudios específicos sobre el comportamiento de estos hongos en medios muy salinos. Otros hongos aislados de arenas de playa y fondos marinos fueron: Acremonium, Alternaria, Aspergillus, Cladosporium, Dreschlera, Gliocladium Humicola, Penicillium, Phialophora, Rhizopus, Stemphylium, Trichoderma, Trichocladium y Ulocladium. Muchos de ellos fueron aislados del fondo marino, testimoniando así que estos hábitats no son exclusivos de Fusarium.

Especies de Fusarium aisladas de aguas de cauces fluviales y fondos marinos del litoral sureste de España

Este trabajo es continuación de una serie de estudios sobre la biogeografía de Fusarium que se están realizando desde hace 5 años en España. En él se presentan los resultados analíticos para el género Fusarium de muestras de aguas del cauce del río Andarax y de fondos del mar Mediterráneo en las provincias de Granada y Almería (Sureste de España). Se analizan un total de 18 muestras de agua del río Andarax. De ellas se aislaron 10 especies de Fusarium: F. anthophilum, F. acuminatum, F. chlamydosporum, F. culmorum, F. equiseti, F. verticillioides, F. oxysporum, F. proliferatum, F. solani y F. sambucinum. De las 23 muestras del mar Mediterráneo se aislaron 5 especies: F. equiseti,F. moliniforme, F. oxysporum, F. proliferatum y F. solani. Sobre el total de muestras analizadas, un 27,45% de las muestras de aguas del río y un 29,41% de muestras de procedencia marina presentaron como mínimo una especie de Fusarium a lo largo de casi 12 meses de muestreo. Considerando las muestras según sus orígenes se encuentra que en las de origen aguas del río un 77,77% presentaron alguna especie de Fusarium; en el caso de los fondos marinos un 45,45% de las muestras presentó alguna especie de Fusarium. La mayor presencia de especies en las aguas del río puede ser debida a los contenidos en el agua de partículas de suelo y materia orgánica, después de los arrastres producidos en las orillas por las lluvias. La presencia de especies encontradas en el mar puede ser consecuencia de las aguas de los cauces que desembocan en éste. Sin embargo, no pueden excluirse otras vías.

Evaluación del poder patógeno de especies de Fusarium aisladas de aguas de cauces fluviales y fondos marinos de España sobre cuatro especies vegetales.

En este artículo se estudia la patogenicidad de las especies de Fusarium aisladas de muestras de fondos marinos del Mediterráneo y de aguas del cauce del río Andarax en las provincias de Granada y Almería (Sureste de España) sobre plántulas de cebada, colirrábano, melón y tomate. La evaluación del poder patógeno se hizo para 41 aislados de 9 especies de Fusarium aisladas de agus de mar y de río: F. acuminatum, F. chlamydosporum, F.culmorum, F. equiseti, F. verticillioides, F. oxysporum, F. proliferatum, F. sambucinum y F. solani. Todos los aislados de las diferentes especies mostraron patogenicidad tanto en preemergencia como en postemergencia de plántulas. No fue posible distiguir a los aislados según su procedencia: aguas marinas o de río.

Species of Fusarium isolated from river and sea water of Southeastern Spain and pathogenicity on four plant species

Species of Fusarium were isolated from water samples collected from the Andarax River and coastal sea water of the Mediterranean in Granada and Almería provinces of southeastern Spain. In total, 18 water samples were analyzed from the Andarax River, and 10 species of Fusarium were isolated: Fusarium anthophilum, F. acuminatum, F. chlamydosporum, F. culmorum, F. equiseti, F. verticillioides, F. oxysporum, F. proliferatum, F. solani, and F. solani. When considering the samples by their origins, 77.8% of the river water samples yielded at least one species of Fusarium , with F. oxysporum comprising 72.2% of the total isolates. In the case of marine water, 45.5% of the samples yielded at least one species of Fusarium, with F. solani comprising 36.3% of the total isolates. The pathogenicity of 41 isolates representing nine of the species collected from river an sea water during the study ws evluated on barley, kohlrabe, melon, and tomato. Inoculation with F. acuminatum, F. chlamydosporum, F. culmorum, F. equiseti, F. verticillioides, F. oxysporum, F. proliferatum F. solani, and F. sambucinum resulted in pre-and post-emergence damping off. Pathogenicity of Fusarium isolates did not seem to be related to the origin of the isolates (sea water or fresh water). However, the presence of pathogenic species of Fusarium in river water flowing to the sea could indicate long-distance dispersal in natural water environments

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Apoptosis-related genes confer resistance to Fusarium wilt in transgenic ‘Lady Finger’ bananas

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, ‘Lady Finger’, were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3’ UTR, and independently transformed plant lines were regenerated for testing. Following a 12 week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 x Bcl-xL, 3 x Ced-9 and 2 x Bcl-2 3’ UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible ‘Lady Finger’ banana plants used as positive controls. Of these, one Bcl-2 3’ UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23 weeks post-inoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type ‘Lady Finger’ plants consistent with a necrotrophic phase in the lifecycle of this pathogen. This was further supported by the observed reduction of these effects in the roots of the resistant Bcl-2 3’ UTR transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

Generating transgenic banana (cv. Sukali Ndizi) resistant to Fusarium Wilt

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

Plant height affects Fusarium crown rot severity in wheat

Effects of plant height on Fusarium crown rot (FCR) disease severity were investigated using 12 pairs of near-isogenic lines (NILs) for six different reduced height (Rht) genes in wheat. The dwarf isolines all gave better FCR resistance when compared with their respective tall counterparts, although the Rht genes involved in these NILs are located on several different chromosomes. Treating plants with exogenous gibberellin increased FCR severity as well as seedling lengths in all of the isolines tested. Analysis of the expression of several defense genes with known correlation with resistance to FCR pathogens between the Rht isolines following FCR inoculation indicated that the better resistance of the dwarf isolines was not due to enhanced defense gene induction. These results suggested that the difference in FCR severity between the tall and dwarf isolines is likely due to their height difference per se or to some physiological and structural consequences of reduced height. Thus, caution should be taken when considering to exploit any FCR locus located near a height gene.

Survey of Fusarium species associated with crown rot of wheat and barley in eastern Australia

Fusarium species associated with crown rot were isolated and identified from 409 wheat, barley or durum wheat crops from the eastern Australian grain belt between 1996 and 1999. Fusarium pseudograminearum was almost the only species isolated from crops in Queensland and New South Wales. F. pseudograminearum was also the most common species in Victoria and South Australia, but F. culmorum was frequently isolated in these states. F. culmorum accounted for more than 70% of isolates from the Victorian high-rainfall (> 500 mm) region and the South-East region of South Australia. F. culmorum comprised 18% of isolates from the Victorian medium-rainfall (350-500 mm) region, and 7% of isolates from each of the Victorian low-rainfall region and the Mid-North region of South Australia. F. avenaceum, F. crookwellense and F. graminearum were isolated very infrequently. The proportion of F. culmorum among isolates of Fusarium from districts in Victoria and South Australia was strongly correlated with climatic conditions around the end of the growing season, especially with rainfall in November.

Towards the development of a Cavendish banana resistant to race 4 of fusarium wilt: gamma irradiation of micropropagated Dwarf Parfitt (Musa spp., AAA group, Cavendish subgroup)

'Dwarf parfitt', an extra-dwarf Cavendish cultivar with resistance to subtropical race 4 fusarium oxysporum f. sp. cubense 9Foc), was gamma irradiated at a dose of 20 Gy and putative mutants were recovered with improved agronomic characteristics. Further screening of putative mutants for improved yield and fruit size, as well as a degree of resistence to fusarium wilt, led to the selection of a line (DPM25) with improved productivity when grown on soils infested with subtropical race 4 Foc. DPM25 was equal to the industry standard, 'Williams', in every agronomic trait measured and it consistently showed a lower incidence of fusarium wilt. Further improvement of field resistance to race 4 Foc is needed in DPM25 and further cycles of mutation induction and selction is an option discussed.

Mycelial compatibility reactions of Australian Fusarium graminearum and F. pseudograminearum isolates compared with AFLP groupings

Using the mycelial reactions of 435 combinations of 14 Fusarium pseudograminearum and 15 F. graminearum isolates, it was demonstrated for the first time that mycelial reactions/barrage formation cannot be clearly used to distinguish F. graminearum and F. pseudograminearum. Mutually compatible isolates produced very different patterns of compatibility with other isolates. However, about 60% of pairings between F. graminearum and F. pseudograminearum isolates were compatible, indicating common ancestry. The Mantel tests used to determine any possible associations between mycelial compatibility reactions and AFLP genotypic diversity data revealed no association between the two systems in either species. In addition, no association was found between mycelial compatibility reactions and sexual reproduction in the two species. Implications of the higher frequency of mycelial compatibility reactions observed in F. pseudograminearum than in F. graminearum are discussed.