Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Field assessment of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> based mycoherbicide for control of <Emphasis Type="Italic">Striga hermonthica</Emphasis> in Nigeria

Fusarium oxysporum (isolate PSM 197) based mycoherbicide was evaluated for its efficacy under field conditions in trials conducted during 1999--2001 cropping seasons in the Nigerian savanna. In the 1999 cropping season, spot application of 5--10 g of mycoherbicide was found to give effective control of Striga hermonthica. Results of on-farm trials at Barhim and Dutsen-Ma areas showed the application of the mycoherbicide to significantly (p= 0.05) increase stand count at both 3 weeks and at harvest, reduced Striga shoot count and increased crop yield in both improved and local sorghum varieties, as compared with the same varieties not treated with the mycoherbicide. Results establish the efficacy of F. oxysporum as a mycoherbicide and the need for further development of the mycoherbicide into formulated granules or seed treatment for use in control of S. hermonthica under field conditions.     

Molecular mapping of<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis> f. sp.<Emphasis Type="Italic"> ciceris</Emphasis> race 3 resistance gene in chickpea

Sequence-tagged microsatellite site (STMS) and sequence-tagged site (STS) markers linked closely to Fusarium oxysporum f. sp. ciceris race 3 resistance gene in chickpea were identified, and linkage between three wilt resistance genes was elucidated. The resistance to race 3 in chickpea germplasm accession WR-315 was inherited as a single gene, designated foc-3, in 100 F₇ recombinant inbred lines derived from the cross of WR-315 (resistant) × C-104 (susceptible). The foc-3 gene was mapped 0.6 cM from STMS markers TA96 and TA27 and STS marker CS27A. Another STMS marker, TA194, at 14.3 cM, flanked the gene on the other side. Linkage between foc-3 and two other chickpea wilt resistance genes, foc-1 (syn. h ₁ ) and foc-4, was established. foc-3 was mapped 9.8 cM from foc-1 and 8.7 cM from foc-4, whereas foc-1 and foc-4 are closely linked at 1.1 cM. The identification of closely linked markers to resistance genes will facilitate marker-assisted selection for introgression of the race 3 resistance gene to susceptible chickpea lines.Communicated by H.C. Becker     

Genetic relatedness of <Emphasis Type="Italic">Trichoderma</Emphasis> isolates antagonistic against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">dianthi</Emphasis> inflicting carnation wilt

Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and beta-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20-35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.     

ITS-RFLP fingerprinting and molecular marker for detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">ciceris</Emphasis>

Genetic diversity of 11 representative isolates of Fusarium oxysporum f.sp. ciceris causing chickpea wilt was determined through internal transcribed spacer (ITS) region of the ribosomal DNA-restriction fragment length polymorphism (ITS-RFLP). ITS1+5.8s+ITS2 regions of the isolates were amplified with a set of primers ITS1 and ITS4 and amplified products were digested with 4 restriction enzymes (AluI, MboI, RsaI, MseI). Six different kinds of ITS-RFLP patterns were obtained. The ITS region of these isolates was sequenced and deposited to NCBI GeneBank. The nucleotide sequence homology of ITS region grouped the isolates into 5 categories. Primers were designed with sequence information using Primer 3 software. F. oxysporum f.sp. ciceris specific markers (FOC F2 and FOC R2) based on ITS region were developed for the first time for detection of the pathogen. The markers produced an amplicon of 292 bp; they were validated against the isolates of the pathogen collected from different locations of India.     

Expression of a rice <Emphasis Type="Italic">GLP</Emphasis> in <Emphasis Type="Italic">Medicago truncatula</Emphasis> exerting pleiotropic effects on resistance against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> through enhancing FeSOD-like activity

To evaluate the effectiveness of a germin-like protein (GLP) in legumes against the serious soil-borne pathogen Fusarium oxysporum f. sp. lentis, an Oryza sativa root-expressed GLP (OsRGLP1) was expressed in the model legume Medicago truncatula using the recombinant vector pCOsRGLP1. The transgene was highly expressed in M. truncatula transformed lines as assessed by RT-qPCR. Consistent with the active status of the transgene there was an elevated accumulation of H₂O₂ in transformed progeny. Enzymatic characterization of T₁ transgenic progeny showed increased superoxide dismutase (SOD) activity. The additional SOD activity in transgenic lines was insensitive to potassium cyanide and sensitive to H₂O₂ indicating its resemblance to FeSOD. The effectiveness of the OsRGLP1 gene was tested by monitoring the root disease after infection of wild-type and transgenic lines. Wild-type plants were greatly affected by the pathogen infection showing a percent disease index value of 50 compared to 10–18 for the transgenic lines. The tolerance of the transgenic lines leads to recovery in fresh weight and pod production to an almost normal level. Analysis of defense-related genes downstream of hydrogen peroxide (H₂O₂) in transgenic plants showed induction of salicylic acid and jasmonate signaling pathways and increased expression of some pathogenesis-related-1 (PR-1) genes and a plant defensin gene. Overall, the findings suggest that OsRGLP1 provides protection against the fungal pathogen F. oxysporum that may involve the direct influence of H₂O₂ on signaling pathways leading to the activation of defense-related genes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Acacia mangium</Emphasis>

A protocol was developed for Agrobacterium-mediated genetic transformation of Acacia mangium using rejuvenated shoots as the explant. Axillary buds and shoot apices of adult trees were rejuvenated by culturing them on Murashige and Skoog (MS) medium, and stem segments of rejuvenated shoots were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI121. The selection for transgenic shoots was performed through five consecutive steps on MS medium supplemented with 1.0 mg/l thidiazuron, 0.25 mg/l indole-3-acetic acid and different concentrations of geneticin (G418; 12–30 mg/l) and timentin (T; 50–300 mg/l) in the following order: 12 mg/l G418 and 300 mg/l T for 30 days, 20 mg/l G418 and 200 mg/l T for 60 days, 30 mg/l G418 and 100 mg/l T for 30 days, 12 mg/l G418 and 50 mg/l T for 30 days, and finally 15 mg/l G418 and 5 mg/l gibberellic acid (GA₃) for 60 days. Thirty-four percent of the stem segments produced resistant multiple adventitious shoot buds, of which 30% expressed the β-glucuronidase gene. The shoot buds were subjected to repeated selection on MS medium supplemented with 2.0 mg/l 6-benzylaminopurine, 2.5 mg/l GA₃ and 20 mg/l G418. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 2.0 mg/l α-naphthaleneacetic acid, 0.1 mg/l kinetin and 20 mg/l G418. Genomic Southern blot hybridization confirmed the incorporation of the NPTII gene into the host genome.     

Physiological and biochemical responses of <Emphasis Type="Italic">in vitro Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">niveum</Emphasis> to benzoic acid

The allelopathic potential of an artificially applied allelochemical, benzoic acid, on in vitro Fusarium oxysporum f.sp. niveum (a soil-borne pathogen causing watermelon wilt) was evaluated. Benzoic acid strongly inhibited its growth, sporulation and conidia germination, whereas it stimulated virulence factors of this pathogen. The biomass was reduced by 83–96 % and the conidia germinating rate and conidia production rate were decreased by 100 % at a concentration of >200 mg/L. However, phytopathogenic enzyme activities and mycotoxin production were stimulated with an increase of 10.2–1250 % for enzyme activities and 610–2630 % for mycotoxin yield.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Allelopathic impact of artificially applied coumarin on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">niveum</Emphasis>

Watermelon production is threatened by fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON) in continuous cultivation system. Some elements, mainly allelochemicals, released from living roots or decayed plants might be associated with the disease. The purpose of this work was to evaluate the possible impact of coumarin, one kind of watermelon allelochemical, on FON. Furthermore, possible new mechanisms might be investigated during the ecological interactions of plant-microbe. Results showed that coumarin strongly inhibited growth of FON leading to a decrease in its biomass, dry weight of mycelia of FON in a liquid culture. The dry weight was decreased by 62.9% compared with control. The hyphal growth of FON on plates was stopped at high (>400 mg l⁻¹) concentrations of coumarin. At 320 mg l⁻¹, sporulation and enzyme activities of FON were also severely suppressed by coumarin. The yield of conidia, and the activities of proteinase, cellulase, and amylase were reduced by 98.9%, 79.7%, 29.8% and 15.9% respectively. However, conidial germination and mycotoxin (MT) production of FON were greatly stimulated, being increased by 55.7% and 14.9 fold at 320 mg l⁻¹ respectively. We conclude that coumarin acted as an allelochemical substance to inhibit growth and pathogenic enzyme activities of FON but to stimulate mycotoxin production and conidial germination. It was suggested that coumarin acted as a signal transduction element bridging plant and pathogen in the process of plant-microbe interactions.     

QTL-seq and marker development for resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> race 1 in cultivated watermelon

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the predominant diseases of watermelon. Resistance to Fon race 1 is conferred by a single major quantitative trait locus (QTL), Fo-1.1, but resolution of this region has been poor due to low marker density. In this study, a combination of whole genome resequencing of bulked segregants (QTL-seq analysis) followed by QTL mapping with kompetitive allele specific PCR (KASP) markers developed across Fo-1.1 successfully increased the resolution from 2.03 to 1.56 Mb and 315 kb, respectively. The linkage of the KASP markers to Fon race 1 resistance across a wide range of watermelon germplasm was validated in a set of elite watermelon cultivars. The linked markers described here provide a breeder-friendly toolkit immediately available for high-throughput genotyping in large-scale breeding programs for fine mapping and incorporation of Fon race 1 resistance in watermelon.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> cell elicitor enhances betalain content in the cell suspension culture of <Emphasis Type="Italic">Celosia cristata</Emphasis>

We started a cell suspension culture from magenta coloured calli of cockscomb to study the effect of biotic and abiotic elicitors on the biosynthesis of betalain pigments. The cultures were grown in a flask containing 30 ml MS media fortified with 13.5 μM 2,4-D and 0.44 μM BAP. These cultures were elicited during its log-phase of growth using fungal elicitors (prepared from mycelia of Fusarium oxysporum), yeast extract, copper sulphate and cobalt chloride. The elicitation reduced the cell count, cell viability and percent pigmented cell in the suspension culture. Similarly, it also resulted in reduced betalain content by all the elicitors except 0.125 × 10^?3% fungal elicitor. Rather, fungal elicitor at this concentration significantly enhanced the amaranthin, betanin, betalamic acid and betaxanthin content in the culture. Besides this, copper sulphate doubled the pigment contribution (ratio of particular pigment content to total pigment content) of betaxanthin at all the concentrations. Therefore, we conclude that fungal elicitor can further be investigated to enhance the content of betalain pigments in suspension culture at a larger scale.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

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.     

Detection of pea wilt pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis> using DNA-based markers

Identification of the fungus Fusarium oxysporum f. sp. pisi (Fop), the causal organism of wilt disease of pea, is a time consuming and arduous task. Diagnosis of Fop by traditional means requires more than 2 months and involves two steps, identification of species using morphological characters and formae specialis ‘pisi’ using pathogenicity assays. The ambiguous morphological differences between F. solani and F. oxysporum further complicate the diagnosis of F. oxysporum. A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) based method was developed to detect Fop from India. A PCR–RFLP marker, HPACAPS1₃₈₀, generated after restriction of 28S rDNA region with enzyme MvaI, detected accurately the Fop among several other fungi with detection sensitivity of 5 fg of Fop genomic DNA. In a mixture of Fop and pea DNA, the sensitivity was 500 pg of Fop DNA in 50 ng of pea DNA. The assay was further refined to detect the Fop from infected tissues and infested soil. The current assay can detect Fop from culture, plant tissues and soil in a considerably shorter period of time compared to traditional methods.