Highly Diverse Endophytic and Soil Fusarium oxysporum Populations Associated with Field-Grown Tomato Plants

The diversity and genetic differentiation of populations of Fusarium oxysporum associated with tomato fields, both endophytes obtained from tomato plants and isolates obtained from soil surrounding the sampled plants, were investigated. A total of 609 isolates of F. oxysporum were obtained, 295 isolates from a total of 32 asymptomatic tomato plants in two fields and 314 isolates from eight soil cores sampled from the area surrounding the plants. Included in this total were 112 isolates from the stems of all 32 plants, a niche that has not been previously included in F. oxysporum population genetics studies. Isolates were characterized using the DNA sequence of the translation elongation factor 1α gene. A diverse population of 26 sequence types was found, although two sequence types represented nearly two-thirds of the isolates studied. The sequence types were placed in different phylogenetic clades within F. oxysporum, and endophytic isolates were not monophyletic. Multiple sequence types were found in all plants, with an average of 4.2 per plant. The population compositions differed between the two fields but not between soil samples within each field. A certain degree of differentiation was observed between populations associated with different tomato cultivars, suggesting that the host genotype may affect the composition of plant-associated F. oxysporum populations. No clear patterns of genetic differentiation were observed between endophyte populations and soil populations, suggesting a lack of specialization of endophytic isolates.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Variations of Phenoloxidase Activities as a Consequence of Stresses that Induce Resistance to Fusarium Wilt of Tomato

The variations over 7–8 day of peroxidase (PO) and polyphenoloxidase (PPO) activity have been investigated in tomato plants the roots of which had been subjected to stresses (heat, chloroform and a non-pathogenic form of Fusarium oxysporum) that induce resistance to Fusarium oxysporum f. sp. lycopersici. All treatments induced increase of PO and PPO activity that reached a maximum 3 days after the treatments in leaves, 4 days in stem and roots and were higher in leaves than in other parts. Activity decreased to levels for the control plants after 8 days. Inoculation with Fusarium oxysporum f. sp. lycopersici further stimulated PO and PPO activity in all treated plants over that caused by the treatments alone. Again, activity of treated plants was lower than in controls 7 days after inoculation. It is concluded that 1. increased PO and PPO activity in tomato is a systemic response to cellular injury caused in the root by heat, chloroform and non-pathogenic Fusarium oxysporum, 2. these treatments do no prevent the pathogen from interacting with the plants and inducing further enzyme increase, 3. treated plants react more strongly to the challenge inoculation than untreated plants.     

Host-specific Fusarium oxysporum Causes Wilt of Jojoba

Jojoba [Simmondsia chinensis (Link) Schneider] plantations in Israel originated from vegetative propagation, planted during 1991–92, have shown symptoms of wilting and subsequent death. Verticillium dahliae was only rarely isolated from these plants and artificial inoculation showed only mild disease symptoms. Fusarium oxysporum caused severe chlorosis, desiccation, defoliation and wilt in leaves of jojoba plants, resulting in plant death. Recovery of the fungus from artificially inoculated stem cuttings and seedlings showed for the first time that F. oxysporum was the primary pathogen. Inoculated cuttings exhibited wilt within 3 weeks, while in seedlings wilt occurred 10–24 weeks after inoculation. Seedlings and cuttings of jojoba which were inoculated with other Fusarium isolates originating from different crops (F. oxysporum f. sp. vasinfectum from cotton, F. oxysporum f. sp. dianthi from carnation, F. oxysporum f. sp. lycopersici from tomato and F. oxysporum f. sp. basilicum from basil) did not develop symptoms. Moreover, cotton, tomato, melon and cucumber seedlings inoculated with several virulent F. oxysporum isolates from jojoba did not show any symptoms of wilt or defoliation. These results indicate a high degree of specificity of the Fusarium isolates from jojoba; therefore, it is suggested that this isolate be defined as F. oxysporum f. sp. simmondsia.     

Bio-Synthesis of Gold Nanoparticles by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> and Assessment of Their Conjugation Possibility with Two Types of β-Lactam Antibiotics without Any Additional Linkers

In the present study, biosynthesis of gold nanoparticles (GNPs) by Fusarium oxysporum was carried out and their conjugation possibility with two β-lactam antibiotics was evaluated. F. oxysporum was cultured and the fungal culture supernatant was subjected to the 1 mmol final concentration of chloroauric acid solution. The produced GNPs were analyzed using visible spectrophotometer, X-ray diffraction analysis (XRD) and transmission electron microscope (TEM). After the purification of GNPs, they were subjected to penicillin G and ceftriaxone without any additional linkers. Finally, the mixture was analyzed using visible spectrophotometer, Fourier transform infrared spectroscopy (FTIR) and TEM and subjected to antibacterial activity test using the well diffusion method. Results confirmed the presence of GNPs in the F. oxysporum culture supernatant after the addition of chloroauric acid. TEM results showed that GNPs were spherical and amorphous with sizes around 10–25 nm and XRD confirmed the presence of GNPs in the fungal culture supernatant. After the incubation of GNPs with the antibiotics, FTIR results demonstrated the successful linking of GNPs with the corresponded antibiotics and TEM images showed that GNPs sizes were bigger than the pure ones (around 50–100 nm). Finally the antibacterial activity test indicated that absolutely, the antibacterial properties of the GNPs-β-lactam antibiotics was lowered or not changed in contrast to the pure antibiotics. The present study showed that GNPs had high tendency of conjugation with antibiotics but unlike the previous researches, linking of the antibiotics to GNPs always cannot improve their antibacterial activity based on the antibiotics that were used. The high conjugation affinity of GNPs made them a good candidate as detoxification agent in diverse areas of medicine or environmental sciences.     

Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.     

Comparative physiological responses of the yeast halotolerance genes expressed in transgenic lines of tomato cv Rio Grande under saline conditions

We previously analyzed the transgenic lines of tomato cv Rio Grande over-expressing the yeast HAL I and HAL II genes for their response to salt stress under in vitro conditions. In this study, six homozygous tomato lines harbouring the yeast HAL I or HAL II genes with highest expression level were selected for exploring their physiological responses against different salt stresses in the field. These transgenic plants showed significant growth and improved water content in comparison with control under 100 and 150 mM salt stress conditions. The HAL I and HAL II lines showed better Ca²⁺ content than their control counterparts. Furthermore, the transgenic lines exhibited lower values of relative electrical conductivity and improved resistance against the fungal pathogens Fusarium oxysporum and Alternaria solani when tested by detached leaf and agar tube dilution assays. Physiological analyses carried out in this study suggest an involvement of multiple mechanisms in transgenic tomato plants harbouring yeast genes to confer biotic and abiotic tolerance under stress conditions.     

Synthetic chitinase gene driven by root-specific LjNRT2 and AtNRT2.1 promoters confers resistance to Fusarium oxysporum in transgenic tobacco and tomato

Fusarium wilt is a soil-borne disease causing substantial yield losses in various crops and vegetables. We have previously reported the synthetic chitinase (NIC) gene (1.2 kb), in which codon usage of fungus, replaced with that of plant, conferred resistance against Botrytis cinerea. In this study, the NIC or GUS gene was linked to two root-specific promoters, LjNRT2 or AtNRT2.1 (nitrate transporter 2), derived from Lotus japonica and Arabidopsis thaliana, respectively. Transgenic tobacco lines expressing LjNRT2-GUS and LjNRT2-NIC, and tomato lines expressing AtNRT2.1-NIC, were produced by Agrobacterium-mediated transformation. GUS histochemical staining was observed in vascular regions of the roots but was conspicuously absent in the leaves of transgenic plants. Western blot analysis showed the production of NIC proteins in the roots but not in the leaves of transgenic tobacco and tomato lines. These results indicate that LjNRT2 and AtNRT2.1 promoters expressed transgenes in a root-specific manner. When in vitro whole plant resistance assay against Fusarium oxysporum was conducted, transgenic plants showed increased levels of resistance compared to non-transgenic plants. Antifungal activities of the root extract against spore germination of F. oxysporum showed lower CFU (colony-forming unit) than those of the leaf extract. Root colonization assay against F. oxysporum showed much lower CFU values in the roots of transgenic plants than in those of non-transgenic plants. These results suggest that NIC gene triggered by the root-specific promoters successfully expressed only in the roots and conferred increased levels of resistance against the root pathogen, F. oxysporum.     

Further Investigation of the Specificity of Interactions Between Wild Lactuca spp. and Bremia lactucae Isolates from Lactuca serriola

Callus cultures derived from isogenic lines of the tomato cultivars Moneymaker and Craigella, resistant or susceptible to F. oxysporum f. sp. lycopersici, were inoculated with Fusarium oxysporum f. sp. lycopersici race 1. Fungal growth was restricted on callus derived from resistant plants, after inoculation with a conidial suspension, whereas callus derived from susceptible plants was totally overgrown by the fungus within 7 days. The concentration of the phytoalexin rishitin was significantly higher in the callus culture derived from a resistant tomato line compared with the callus culture from a susceptible line, 2 and 3 days after inoculation with mycelium. The results of the experiments were compared with experiments with whole plants. Rishitin production as well as growth of the fungus was comparable with responses in plant-fungus interaction. Therefore callus culture may be useful in studying the interaction between tomato plants and race 1 of F. oxysporum f. sp. lycopersici.     

Biotic and abiotic initiators for rishitin formation and accumulation in tomato

γ-Ray irradiation of pre-sowing seeds of tomato did not trigger the formation of the phytoalexin “rishitin” in either leaves or fruits of tomato plants through different growth seasons. Application of copper sulfate initiated rishitin formation in both leaves and fruits. Increasing of γ-ray dose was accompanied by decreasing rishitin accumulation in the presence of copper sulfate. Rishitin of tomato leaves was found to be reduced significantly, concomitant with increasing the disease incidence for late and early blight, andFusarium wilt disease, after applying γ-irradiation, in the case of biotic initiatorsPhytophthora infestans, Alternaria solani orFusarium oxysporum alone or together with the abiotic inducer copper sulfate. Shelf-extending γ-ray doses of 1.0, 1.5 and 2.0 kGy decreased rishitin amounts in tomato fruits treated with copper sulfate alone or infected withPhytophthora infestans. Also, the amount of formed rishitin was reduced by extending the storage period.     

The intercropping partner affects arbuscular mycorrhizal fungi and Fusarium oxysporum f. sp. lycopersici interactions in tomato

Arbuscular mycorrhizal fungi (AMF) and their bioprotective aspects are of great interest in the context of sustainable agriculture. Combining the benefits of AMF with the utilisation of plant species diversity shows great promise for the management of plant diseases in environmentally compatible agriculture. In the present study, AMF were tested against Fusarium oxysporum f. sp. lycopersici with tomato intercropped with either leek, cucumber, basil, fennel or tomato itself. Arbuscular mycorrhizal (AM) root colonisation of tomato was clearly affected by its intercropping partners. Tomato intercropped with leek showed even a 20 % higher AM colonisation rate than tomato intercropped with tomato. Positive effects of AMF expressed as an increase of tomato biomass compared to the untreated control treatment could be observed in root as well as in shoot weights. A compensation of negative effects of F. oxysporum f. sp. lycopersici on tomato biomass by AMF was observed in the tomato/leek combination. The intercropping partners leek, cucumber, basil and tomato had no effect on F. oxysporum f. sp. lycopersici disease incidence or disease severity indicating no allelopathic suppression; however, tomato co-cultivated with tomato clearly showed a negative effect on one plant/pot with regard to biomass and disease severity of F. oxysporum f. sp. lycopersici. Nonetheless, bioprotective effects of AMF resulting in the decrease of F. oxysporum f. sp. lycopersici disease severity were evident in treatments with AMF and F. oxysporum f. sp. lycopersici co-inoculation. However, these bioprotective effects depended on the intercropping partner since these effects were only observed in the tomato/leek and tomato/basil combination and for the better developed plant of tomato/tomato. In conclusion, the effects of the intercropping partner on AMF colonisation of tomato are of great interest for crop plant communities and for the influences on each other. The outcome of the bioprotective effects of AMF resulting in the decrease on F. oxysporum f. sp. lycopersici disease severity and/or compensation of plant biomass does not depend on the degree of AM colonisation but more on the intercropping partner.     

Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3?kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease.     

Reactive oxygen species (ROS) and antioxidative enzyme status in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> on priming with fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

In plants, ROS signaling and increase in activities of antioxidants are among defense responses. The present study describes the oxidative stress profiling in model host plant tomato (Solanum lycopersicum L.), during an invasion of the wilt pathogen Fusarium oxysporum f. sp. lycopersici with or without seed priming with Pseudomonas isolates M80, M96 and T109. Tomato seeds were primed with known Pseudomonas isolates M80, M96 and T109 and the forty-day- old plants were challenged with spores of F. oxysporum under greenhouse conditions. Leaf samples were collected at 0, 24, 48 72 and 96 h post fungal challenge and analysed for systemic level of oxidative stress parameters including total phenolics, proline, hydrogen peroxide, lipid peroxidation and enzymatic antioxidants. Disease incidence in the plants under greenhouse conditions was also calculated. Results revealed that priming with Pseudomonas isolates resulted in reduced oxidative stress in the host, during pathogen invasion. M80-priming showed highest antioxidative protection to the host plants during F. oxysporum invasion. The observed reduction in hydrogen peroxide and lipid peroxidation in primed plants was in agreement with the increased activities of the corresponding antioxidant enzymes. Greenhouse results showed that the highest wilt disease symptoms were with M80-priming followed by M96 and T109. The present study gives substantial evidences on the oxidative stress mitigation in response to Pseudomonas-priming on the model tomato-Fusarium interaction system.     

Impact of soil salinity on the plant-growth – promoting and biological control abilities of root associated bacteria

The effectiveness of plant growth – promoting bacteria is variable under different biotic and abiotic conditions. Abiotic factors may negatively affect the beneficial properties and efficiency of the introduced PGPR inoculants. The aim of this study was to evaluate the effect of plant growth – promoting rhizobacteria on plant growth and on the control of foot and root rot of tomatoes caused by Fusarium solani under different soil salinity conditions. Among the five tested strains, only Pseudomonas chlororaphis TSAU13, and Pseudomonas extremorientalis TSAU20 were able to stimulate plant growth and act as biological controls of foot and root rot disease of tomato. The soil salinity did not negatively affect the beneficial impacts of these strains, as they were able to colonize and survive on the roots of tomato plants under both saline and non-saline soil conditions. The improved plant height and fruit yield of tomato was also observed for plants inoculated with P. extremorientalis TSAU20. Our results indicated that, saline condition is not crucial factor in obtaining good performance with respect to the plant growth stimulating and biocontrol abilities of PGPR strains. The bacterial inoculant also enhanced antioxidant enzymes activities thereby preventing ROS induced oxidative damage in plants, and the proline concentrations in plant tissue that play an important role in plant stress tolerance.     

Suppressiveness of Vermicompost against Fusarium Wilt of Tomato

Vermicompost added to various container media significantly inhibited the infection of tomato plants by Fusarium oxysporum f. sp. lycopersici. The protective effect increased in proportion to the rate of application of vermicompost. Every type of container media amended with this substrate, used in the experiments, were suppressive to the pathogen. Vermicompost lost its activity after heating. Sterilized extracts of vermicompost added to potato dextrose agar stimulated the growth of F. oxysporum mycelium. The results indicate that chemical factors in this substrate had no direct inhibiting effect on the fungus. The total number of micro-organisms and populations of antagonistic bacteria and fungi were significantly higher in vermicompost than in the control peat substrate. A biotic nature is suggested for the suppressiveness of the vermicompost.     

A Tolerant Behavior in Salt-Sensitive Tomato Plants can be Mimicked by Chemical Stimuli

Lycopersicon esculentum plants exhibit increased salt stress tolerance following treatment with adipic acid monoethylester and 1,3-diaminepropane (DAAME), known as an inducer of resistance against biotic stress in tomato and pepper. For an efficient water and nutrient uptake, plants should adapt their water potential to compensate a decrease in water soil potential produced by salt stress. DAAME-treated plants showed a faster and stronger water potential reduction and an enhanced proline accumulation. Salinity-induced oxidative stress was also ameliorated by DAAME treatments. Oxidative membrane damage and ethylene emission were both reduced in DAAME-treated plants. This effect is probably a consequence of an increase of both non-enzymatic antioxidant activity as well as peroxidase activity. DAAME-mediated tolerance resulted in an unaltered photosynthetic rate and a stimulation of the decrease in transpiration under stress conditions without a cost in growth due to salt stress. The reduction in transpiration rate was concomitant with a reduction in phytotoxic Na⁺ and Cl⁻ accumulation under saline stress. Interestingly, the ABA deficient tomato mutant sitiens was insensitive to DAAME-induced tolerance following NaCl stress exposure. Additionally, DAAME treatments increased the ABA content of leaves, therefore, an intact ABA signalling pathway seems to be important to express DAAME-induced salt tolerance. Here, we show a possibility of enhance tomato stress tolerance by chemical induction of the major plant defences against salt stress. DAAME-induced tolerance against salt stress could be complementary to or share elements with induced resistance against biotic stress. This might be the reason for the observed wide spectrum of effectiveness of this compound.Key Words: adipic acid monoethyl ester, 1,3-diaminepropane, Lycopersicon esculentum, salt stress, oxidative stress, ethylene, chemical induced tolerance     

Molecular markers reflect differentiation of Fusarium oxysporum forma speciales on tomato and forma on eggplant

Selective pressure induces pathogens to change their method of infection and, sometimes, causes species to become infectious. Pathogenic fungi must differentiate different morphological and physiological properties during the process of host specialization in their life cycle. In the present study, we conducted a genetic investigation and compared similarities within a generation of Fusarium oxysporum forma speciales (f. sp.) infecting tomato and forma (f.) infecting eggplants using selected ISSR and RAPD markers, two horticultural commodities belonging to the same taxon of the Solanaceae. Interestingly, genetic data showed that fungi belonging to F. oxysporum f. sp. infecting tomato have a close genetic relationship with the fungi f. infecting eggplant. Furthermore, F. oxysporum f. sp. infecting tomato showed less genetic variation than F. oxysporum f. melongenae, suggesting that it could be developed more recently during host adaptation. On the other hand, the gene sequence of inter-simple sequence repeat (ISSR) markers resulting in high polymorphism showed matches with gene sequences encoding specific proteins related to pathogenicity of F. oxysporum species. These findings support the notion that selected ISSR markers can be used to follow host-associated divergence of F. oxysporum species infecting tomato and eggplant and that differentiation of their specific genes can also be related to pathogenicity and development as predictive studies before initiating detailed sequencing analysis.     

Genetic Diversity of Fusarium oxysporum Populations Isolated from Tomato Plants in Tunisia

Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. radicis‐lycopersici is a new devastative disease of tomato greenhouse crops in Tunisia. Nothing is known neither about the population of this pathogen in this region, nor about the population of F. oxysporum f. sp. lycopersici the causal agent of Fusarium wilt of tomato. In order to examine the genetic relatedness among the F. oxysporum isolates by intergenic spacer restriction fragment length polymorphism (IGS‐RFLP) analysis and to elucidate the origin of the formae specialesradicis‐lycopersici in Tunisia by looking for genetic similarity of Tunisians isolates with isolates from a foreign source, the genetic diversity among F. oxysporum f. sp. radicis‐lycopersici and F. oxysporum f. sp. lycopersici populations was investigated. A total of 62 isolates of F. oxysporum, obtained from symptomless tomato plants, were characterized using IGS typing and pathogenicity tests on tomato plants. All Fusarium isolates were highly pathogenic on tomato. Fusarium oxysporum f. sp. radicis‐lycopersici isolates were separated into five IGS types. From the 53 F. oxysporum f. sp. radicis‐lycopersici isolates, 34 isolates have the same IGS types (IGS type 25), and the remaining 19 isolates were distributed into four IGS types. However, the only nine isolates of F. oxysporum f. sp. lycopersici have six different IGS types. This difference of diversity between the two formae speciales suggests that F. oxysporum f. sp. radicis‐lycopersici isolates have a foreign origin and may have been accidentally introduced into Tunisia.