Defense Proteins from Vigna Unguiculata Seed Exudates: Characterization and Inhibitory Activity Against Fusarium Oxysporum

Plants exude a variety of substances through their roots, germinating seeds and aerial parts. Some of these released compounds seem to have an inhibitory effect against pathogens. The aim of this work was to investigate and identify antifungal proteins present in exudates from imbibed cowpea seeds (Vigna unguiculata (L.) Walp). The obtained exudation was analyzed in regard to specific protein activities by enzymatic or immunological assays for plant defense proteins, from 4 h to 48 h of seed imbibition. Our results show that cowpea seeds exudates present several defense related proteins characterized as β-1,3-glucanases, cystatins, vicilins and lipid transfer proteins (LTPs), as well as a storage vacuole membrane α-TIP protein, since the very first hours of imbibition. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus Fusarium oxysporum f. sp. phaseoli. Our results suggest that seed exudates should promote seed protection from soil pathogens.     

Effect of kind and method of fungicidal treatment of bean seed on infections by the VA-mycorrhizal fungus Glomus macrocarpum and by the pathogenic fungus Fusarium solani

To test the effect of seed treatment with fungicides on the development of mycorrhizal fungi, bean seeds were treated with fungicide dry or vehicled in the organic solvents, ethanol or dichloromethane and then planted in soil inoculated with the mycorrhizal fungus Glomus macrocarpum and/or the plant pathogenic fungus Fusarium solani. Measurements were made at 4 day intervals, to evaluate the location and extent of colonization of either Glomus macrocarpum or Fusarium solani in the root system. Most combinations of fungicide-solvent had little effect on the extent of colonization by each fungus individually. However, when both fungi were inoculated together, symptoms of F. solani were seen only in the tips of roots which indicate that the mycorrhizal fungus was able to limit the occurrence of the pathogenic fungus.     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.     

Fusarium nygamai, a potential bioherbicide for Striga hermonthica control in sorghum

Glasshouse trials were performed to investigate the control of the parasitic weed Striga hermonthica by Fusarium nygamai and the performance of the host plant sorghum (Sorghum bicolor) using different inoculum substrates and inoculum amounts of the fungus. Optimal constant and alternating temperatures for the growth of the fungus were 25°C and 30/20°C, respectively. Striga incidence was decreased up to 100% when the fungus was incorporated into the soil preplanting. Emerged Striga plants at different stages of growth up to the flowering stage were killed by the fungus when the fungus was applied postemergent. In root-chamber trials none of the Striga seeds germinated when 10 ml inoculum suspension of 8 × 10⁶ spores/ml of F. nygamai was applied on seeds of the parasitic weed sprinkled on the surface of filter paper. F. nygamai has potential as a bioherbicide for Striga control. Further studies regarding its performance under field conditions and its safety to the environment and humans should be assessed.     

Fusarium asiaticum: an Emerging Pathogen Jeopardizing Postharvest Asparagus Spears

Asparagus spears are usually vulnerable to pathogenic micro‐organisms. In this study, 217 pathogens were isolated from symptomatic asparagus, and one highly virulent fungus (designated EXAP‐08) isolated from the rotted asparagus spears in cold storage was characterized in detail. Koch's postulates were checked through pathogenicity tests, indicating that EXAP‐08 infection could cause reproducible rot symptoms similar to those observed on naturally infected asparagus spears, and the pathogenicity of EXAP‐08 was also relatively higher than other Fusarium pathogens, especially at 4°C. Through morphological and molecular identification, EXAP‐08 was characterized as Fusarium asiaticum. This identification was further confirmed by phylogenetic analysis with the Histone gene H3 of EXAP‐08 and other Fusarium species. EXAP‐08 also belongs to 3A‐DON (3‐acetyl‐4‐deoxynivalenol) chemo‐type, and the mycotoxin was detected during the infection of plant, implying the potential risks of mycotoxin contamination in fresh crops infected by this pathogen. Thus, this emerging pathogen threatening edible safety of asparagus spears should deserve particular quarantine inspection in the future.     

Induction of PR proteins and resistance by the biocontrol agent Clonostachys rosea in wheat plants infected with Fusarium culmorum

Clonostachys rosea (CR) is a common worldwide saprophyte with destructive effect against several plant pathogenic fungi showing antagonistic features against a wide variety of pathogens. We recently isolated a strain of C. rosea, named CR47, from wheat crown infected with Fusarium culmorum (FC); this strain proved to be effective against Fusarium seed borne diseases of cereals under field condition. In this paper the function of C. rosea applied as seed treatment on wheat seedling growth was investigated. In addition, we investigated the expression pattern of peroxidases and chitinases as well as PR4 proteins following both CR treatments of seeds and FC infection and also in the three-component system pathogen–antagonist–wheat. Several chitinase isoforms were induced by CR-treatment both in coleoptiles and roots, whereas some peroxidase isoforms were induced only in the presence of both antagonist and pathogen. In the latter case, it seems that CR-treatment by itself promotes plant growth and reduces the peroxidase expression, while enhances some chitinase isoforms probably involved in cell wall disruption. Moreover, both the antagonist and the pathogen studied induced PR4 protein expression, which probably exerts its role on the invading microorganisms by a translation-inhibitory process that could be ascribed to their ribonuclease activity.     

Influence of substrate on the interaction ofGlomus intraradices andFusarium oxysporum f.sp.radicis-lycopersici on tomatoes

Summary The influence of five substrates on the interaction betweenGlomus intraradices andFusarium oxysporum f.sp.radicis-lycopersici and its effect on tomato plants development was investigated. The presence ofG. intraradices decreased root necrosis in all substrates and affected the Fusarium population with different intensity depending on the substrate used. Substrates were found to influence disease development, Fusarium population in the substrate, root colonization by the endomycorrhizal fungus and growth of the host plant. In addition to providing good experimental conditions, the use of calcined montmorillonite clay also facilitated washing, recuperation, necrosis evaluation and staining of roots. Its use is proposed as a standard medium for experimental work on the interactions between endomycorrhizal fungi, root pathogens and host plants.Contribution no J 981 of the Saint-Jean Research Station and no 268 of the Sainte-Roy Research Station     

Impact of the exodermis on infection of roots byFusarium culmorum

Patterns of infection withFusarium culmorum (W G Smith) Saccardo were observed in seedling roots of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), maize (Zea mays L.) and asparagus (Asparagus officinalis L). Apical regions of the main roots were not infected. Since penetration into the root occurred several days after inoculation and the roots were growing during the experiment, these regions had apparently not been in existence long enough to be infected. In older regions of barley, wheat and asparagus, hyphae entered through the tips of lateral roots. In barley and wheat, which had not developed any suberin lamellae in their subepidermal layer, infection occurred randomly over the remainder of the root. In maize, the fungus penetrated the epidermis at many sites but did not breach the exodermis in which all cells possessed both Casparian bands and suberin lamellae. Maize roots, therefore, sustained only minimal infections. In asparagus, the fungus grew through the short (passage) cells but never the long cells of the exodermis. In doing so, it penetrated cells possessing Casparian bands but lacking suberin lamellae. The results support the hypothesis that suberin lamellae provide effective barriers to the growth ofF. culmorum hyphae.     

Interactions of Fusarium moniliforme,its metabolites and bacteria with corn

Fusarium moniliforme Sheldon is an economically important pathogen of corn (Zea mays L.) which causes stalk, root and ear rot. Several mycotoxins have also been isolated, identified and implicated in both animal and human toxicoses. The fungus can be disseminated in symptomless corn seed and can also survive in crop residues in the soil. Asymptomatic infection may be related to different corn cultivars, fungal strains, and environmental factors. Symptomatic expression of pathogenicity may vary, but usually the result of such infections is death of the plant. The greatest concern is the asymptomatic infection, since it is in this form that fungal toxins may surreptitiously enter animal and human food chains. F. moniliforme produces both fusaric acid, which is phytotoxic to corn and interferes with seed germination, and plant growth regulators that may affect pathogenicity of the fungus or be associated with the production of mycotoxins. Other metabolites, including fusarin C, moniliformin, and the fumonisins, may or may not be phytotoxic, but are associated with animal and human toxicoses. The control of F. moniliforme in corn is therefore quite important. One potential means to accomplish this reduction is biocontrol by the application of antagonistic rhizobacteria to corn kernels at planting. To be effective the bacteria must be able to colonize the corn root system and be able to prevent root infection by successful competing with F. moniliforme which may be accomplished by siderophore and or antibiotic activity.     

Identification of defence proteins from the seed exudates of Cicer arietinum L. and its effect on the growth of Fusarium oxysporum f.sp. ciceri

Germinating seeds tend to release a variety of proteins into their surrounding surfaces; some of which have an inhibitory action against plant pathogens. The aim of this study was to investigate and identify defence proteins present in the exudates from water-imbibed and chitosan-imbibed (0.1% w/v) seeds of chickpea (Cicer arietinum L). Chickpea seeds imbibed in chitosan released a higher amount of proteins in the exudate when compared to the seeds imbibed in water. The obtained exudates were analysed in regard to specific protein activities by enzymatic assays and SDS-PAGE analysis. Results showed that the exude obtained from chickpea seeds imbibed in chitosan solution exhibited a new isoform of chitinase, chitosanase and protease inhibitors. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus, Fusarium oxysporum f.sp. ciceri. Our results suggest that seed exudates protect seeds during their germination from soil pathogens.     

Seed predation,pathogen infection and life-history traits in Brassica rapa

Herbivory and disease can shape the evolution of plant populations, but their joint effects are rarely investigated. Families of plants of Brassica rapa (Brassicaceae) were grown from seeds collected in two naturalized populations in an experimental garden. We examined leaf infection by the fungus Alternaria, seed predation by a gall midge (Cecidomyiidae) and plant life-history traits. Plants from one population had heavier seeds, were more likely to flower, had less fungal infection, had more seed predation and were more fecund. Fungal infection score and seed predation rate increased with plant size, but large plants still had the greatest number of undamaged fruits. Spatial heterogeneity in the experimental garden was significant; seed predation rate and fecundity varied among blocks. An apparent tradeoff existed between susceptibility to disease and seed predation: plants with the highest fungal infection score had the lowest seed predation rate. Alternaria infection varied between populations, but the disease had no effect on fecundity. Seed predation did reduce fecundity. Damaged fruits had 31.4% fewer intact seeds. However, evidence for additive genetic variation in resistance to seed predation was weak. Therefore, neither disease nor seed predation was likely to be a strong agent of genetically based fecundity selection.     

Trichoderma spp. tolerance to Brassica carinata seed meal for a combined use in biofumigation

Biofumigation by Brassicaceae green manure or seed meal incorporation into soil is an ecological alternative to chemical fumigation against soil-borne pathogens, based on the release of glucosinolate-derived compounds. This study aimed at investigating the tolerance of the beneficial fungus Trichoderma to these compounds in view to combined utilization with Brassica carinata seed meal (BCSM). Forty isolates of Trichoderma spp. were tested in vitro for tolerance to toxic volatiles released by BCSM and in direct contact with the meal. They were found to be generally less sensitive than the assayed pathogens (Pythium ultimum, Rhizoctonia solani, Fusarium oxysporum), even if a fungistatic effect was observed at the highest dose (10 μmole of sinigrin). Most of them also were able to grow on BCSM and over the pathogens tested. A preliminary experiment of integrating BCSM with Trichoderma in soil was carried out under controlled conditions with the patho-system P. ultimum—sugar beet. BCSM incorporation increased pathogen population, but reduced disease incidence, probably due to indirect mechanisms. The greatest effect was achieved when BCSM was applied in combination with Trichoderma, regardless of meal ability to release isothiocyanate. These findings suggest that disease control can be improved by this integrated approach. This study also highlighted that a reduction of allyl-isothiocyanate concentration in soil could occur due to the activity of some Trichoderma isolates. This effect could protect resident or introduced Trichoderma isolates from depressing effects due to the biocidal compounds, but, on the other hand, could reduce the efficacy of biofumigation against target pathogens.     

Isolation, Identification, and Activity of Mycoherbicidal Pathogens from Juvenile Broomrape Plants

Although there are reports of isolation of mycoherbicidal pathogens attacking the widespread broomrapes (Orobanche spp.) that parasitize legumes and vegetables, none is in use or available. This is despite there being no good method of controlling broomrapes in most crops other than by preplant fumigation with methyl bromide. Two highly parasitic fungi, Fusarium arthrosporioides strain E4a (CNCM I-164) and F. oxysporum strain E1d (CNCM I-1622), were isolated from nearly 100 organisms found on diseased, juvenile, emerging Orobanche flower stalks. A near-axenic polyethylene envelope system for culturing broomrape on tomato roots was used to ascertain pathogenicity of these strains. Both organisms fulfilled Koch's postulates for being primary pathogens. Their DNAs were analyzed and fingerprinted by restriction fragment length polymorphism and random amplified polymorphic DNA, showing that they are indeed different from each other and from many other Fusarium spp. and other formae speciales of F. oxysporum including a strain that attacks O. cumana on sunflowers. Both strains infect O. aegyptiaca, O. cernua, and O. ramosa, but not O. cumana. They did not infect any of the vegetable and legume crops tested and thus seem specific to Orobanche. Tomato plant roots dipped into a fungal spore and mycelial suspension and planted in broomrape-infested soil were protected for 6 weeks, as were tomato transplants in pot experiments. About 90% control was also achieved by posttransplant soil drench with fungal suspensions in pot experiments. These pathogens may be effective as seed, transplant, or soil-drench treatments of high-value vegetable and other crops.     

带叶兜兰种子原地共生萌发及有效菌根真菌的分离与鉴定

为获得带叶兜兰(Paphiopedilum hirsutissimum)种子萌发的共生真菌,采用原地共生萌发技术获得了2株自然萌发的小幼苗,并分离和筛选出了有效的种子萌发共生菌——瘤菌根菌(Epulorhiza sp.)。为验证分离菌株对带叶兜兰种子萌发的有效性,将Phs34号菌株与带叶兜兰种子在灭菌后的原生境基质上进行室内共生萌发试验,结果表明,经过6周的培养,对照组没有观察到种子的萌发;接菌的种子胚明显膨大,突破种皮,形成原球茎,平均萌发率为(58.35±3.41)%。这表明分离得到的瘤菌根菌能促进带叶兜兰的种子萌发。     

Fungus gnats vector Fusarium oxysporum f.sp. radicislycopersici1

Fungus gnat adults transported Fusarium oxysporum f.sp. radicis-lycopersici from Petri dish culture and infected host plants to the roots and hypocotyls of healthy tomato and bean plants. The source of the fungus did not affect the ability of fungus gnats to transport the fungus to healthy hosts. The presence of fungus gnat larvae in media in which young tomato plants were grown did not increase the incidence of plant infection by the pathogen. Fungus gnat adults appear to aid in the dissemination of F. oxysporum f.sp. radicis-lycopersici.     

The putative gymnosperm plant defensin polypeptide (SPI1) accumulates after seed germination,is not readily released,and the SPI1 levels are reduced in Pythium dimorphum-infected spruce roots

The putative plant defensin SPI1 cDNA from the conifer Norway spruce (Picea abies) is the only known plant defensin-like sequence from a gymnosperm. The predicted translational product SPI1 was not detected in the embryo or other parts of the seed by means of antibodies, but it accumulated in the root cortex after germination. In roots of seedlings infected with the root pathogenic oomycete Pythium dimorphum and the blue stain fungus Ceratocystis polonica, variable levels of SPI1 was detected during the first day as a response to the infection, however a significant increase was seen as an initial response to the root-rot fungus Heterobasidion annosum. After the first day of infection, the amount of SPI1 polypeptide was dramatically reduced in response to either of the pathogens, but not in response to the ectomycorrhizal fungus Laccaria bicolor. During the same time of infection, extensive damage to cortical root cells resulted from the infecting pathogens, but not from the mycorrhiza. These results indicate that pathogens may reduce the level of SPI1 by suppressing its expression, but may also reduce the SPI1 level by invading and disrupting the root cortical cells or by a combination of these mechanisms.     

Onychomycosis Caused by Fusarium Solani and Fusarium Oxysporum In São Paulo,Brazil

Fusarium species are common soil saprophytes and plant pathogens that have been frequently reported as etiologic agents of opportunistic infections in humans. We report eight cases of onychomycosis caused by Fusarium solani (4) and Fusarium oxysporum (4) in São Paulo, Brazil. These species were isolated from toenails in all cases. The infections were initially considered to be caused by dermatophytes. The clinical appearance of the affected toenails was leukonychia or distal subungual hyperkeratosis with yellowish brown coloration. The eight cases reported here suggest that Fusarium spp. should be taken into consideration in the differential diagnosis of tinea unguium.     

Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva

Podophyllotoxin, a well-known naturally occurring aryltetralin lignan occurs in few plant species that is used as a precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. The availiability of this lignan is becoming increasingly limited because of the scarce occurance of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. This paper reports first time the production of podophyllotoxin by an endophytic fungus Fusarium oxysporum isolated from the medicinal plant Juniperus recurva. Further confirmation and quantification of podophyllotoxin was performed by HPLC, LC-MS, and LC-MS/MS.     

First Record of Fusarium Wilt of Tobacco in Greece Imported as Seedborne Inoculum

Leaf yellowing and brown discoloration was observed in tobacco plants cv. Burley TN97 in tobacco fields of central Greece in 2002. Fusarium oxysporum f. sp. nicotianae was isolated from symptomatic plants and Koch's postulates were fulfilled. The pathogenicity of the isolated fungus was examined on five tobacco cultivars (Burley TN97, BurleyB21, VirginiaBE9, Virginia Niki and Anatolika KE26/2). The pathogen was present in tobacco seed batches imported in 2000 and 2001, which indicates that the infected seed is most probably the primary source of the disease in Greece. As Fusarium oxysporum f. sp. vasinfectum can also cause vascular wilt in tobacco, the hypothesis that the isolated F. oxysporum strain belongs to f. sp. vasinfectum was excluded by a pathogenicity test to cotton cv. Acala SJ‐2. This is the first report of F. oxysporum f. sp. nicotianae in Greece and the second in the European Union, although the seedborne nature of the pathogen has not been previously reported in Europe.     

Barley hordothionin accumulates in transgenic oat seeds and purified protein retains anti-fungal properties in vitro

Summary Transgenic anti-fungal gene expression in heterologous species provides a means to test resistance protein combinations across species barriers. This is the first report of transgenic anti-fungal seed storage protein accumulation in oat seed. An anti-fungal barley (Hordeum vulgare L.) hordothionin (Hthl) gene was genetically engineered into oat (Avena sativa L.) to determine the effect of hordothionin on pathogen resistance. The transgene was expressed in both leaf and seed tissue, with transgenic protein accumulation occurring only in the seed. Transgenic oat line HTH-Av5 expressed c. 94 μg HTH/g seed, 19% of native barley seed levels. The anti-fungal activities of HTH fractions from barley cv. Morex and oat (transgenic and control) were tested in an in vitro growth assay against an important small grain pathogen. Fusarium graminearum. The partially purified HTH fractions from control oat seeds did not inhibit fungal growth, while HPLC-purified HTH positive control, as well as partially purified barley and transgenic oat HTH inhibited growth similarly over a range of concentrations. These results indicate hordothionin can be expressed in a heterologous cereal species and still maintain its anti-fungal properties. Future studies with HTH targeted to additional tissues are planned to test for increased fungal resistance. The University of Wisconsin and the USDA neither guarantee nor warrant the standard of the products named herein, and the use of the name by University of Wisconsin or USDA implies no approval of the product to the exclusion of others that may also be suitable.