Lysis of mycelium ofFusarium oxysporum f. sp.udum in soil amended with organic matters

Summary Autoclaved or natural field soil amended with 0.1 to 5.0 per cent (W/W) of margosa cake, rice husk and sawdust with or without supplemental nitrogen were tested for lytic activity and bacterial numbers. Generally, non-amended autoclaved soil caused little or no lysis of mycelium ofF. oxysporum f. sp.udum; non-amended natural soil caused more lysis. Amendment of soil with margosa cake, rice husk or saw-dust with or without supplemental nitrogen greatly enhanced its lytic effect on the fungus. The degree of lysis depended on the dosage of amendment used and the stage of its decomposition in the soil. The extent of lysis increased as the bacterial population increased. Amongst bacteria,Bacillus subtilis was very common in most lytic zones.     

Variability in Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea

The variability in cultural characteristics and the virulence among three isolates of Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea was studied under laboratory conditions. The three isolates (Foc-1, Foc-2 and Foc-3) did not show any significant difference in their mycelial dry weight production at any temperature regimes, pH level or the growth media tested. The radial growth on PDA also did not differ significantly in the three isolates. However, some quantitative differences were noted in colony characters and septations in macroconidia of these isolates. The isolate Foc-1 exhibited dull white, thin and flat hairy growth, spreading out like thread, Foc-2 showed a white fluffy colony with irregular aerial margin, while Foc-3 exhibited a pinkish white, slightly fluffy colony with regular margin. Conidia also differed with regard to septation. Three to six septa were present in Foc-2, while there were 2–3 in isolates Foc-1 and Foc-2. These isolates differed significantly with regard to their virulence on test varieties. Isolate Foc-1 was more virulent that Foc-2 or Foc-3 and produced abundant spores.     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.     

Interaction of Fusarium oxysporum f. sp. ciceri and Meloidogyne javanica on Cicer arietinum

Interaction of Meloidogyne javanica and Fusarium oxysporum f. sp. ciceri was studied on Fusarium wilt-susceptible (JG 62 and K 850) and resistant (JG 74 and Avrodhi) chickpea cultivars. In greenhouse experiments, inoculation of M. javanica juveniles prior to F. oxysporum f. sp. ciceri caused greater wilt incidence in susceptible cultivars and induced vascular discoloration in roots of resistant cultivars. Nematode reproduction was greatest (P = 0.05) at 25 °C. Number of galls and percentage of root area galled increased when the temperature was increased from 15 °C to 25 °C. Wilt incidence was greater at 20 °C than at 25 °C. Chlorosis of leaves and vascular discoloration of plants did not occur at 15 °C. The nematode enhanced the wilt incidence in wilt-susceptible cultivars only at 25 °C. Interaction between the two pathogens on shoot and root weights was significant only at 20 °C, and F. o. ciceri suppressed the nematode density at this temperature. Wilt incidence was greater in clayey (48% clay) than in loamy sand (85% sand) soils. The nematode caused greater plant damage on loamy sand than on clayey soil. Fusarium wilt resistance in Avrodhi and JG 74 was stable in the presence of M. javanica across temperatures and soil types.     

Potential of microsatellites to distinguish four races of Fusarium oxysporum f. sp. ciceri prevalent in India

Fusarium oxysporum f. sp. ciceri, the causal agent of chickpea wilt, is an important fungal pathogen in India. Thirteen oligonucleotide probes complementary to microsatellite loci, in combination with 11 restriction enzymes, were used to assess the potential of such markers to study genetic variability in four Indian races of the pathogen. Hybridisation patterns, which were dependent upon both the restriction enzyme and oligonucleotide probe used, revealed the presence of different repeat motifs in the F. oxysporum f. sp. ciceri genome. Among the restriction enzymes used, hexa-cutting enzymes were more informative than tetra- and penta-cutting enzymes, whereas tetranucleotide and trinucleotide repeats yielded better hybridisation patterns than dinucleotide repeats. Dependent upon the levels of polymorphism detected, we have identified (AGT)₅, (ATC)₅ and (GATA)₄ as the best fingerprinting probes for the F. oxysporum f. sp. ciceri races. The distribution of microsatellite repeats in the genome revealed races 1 and 4 to be closely related at a similarity index value of 76.6%, as compared to race 2 at a similarity value of 67.3%; race 3 was very distinct at a similarity value of 26.7%. Our study demonstrates the potential of oligonucleotide probes for fingerprinting and studying variability in the F. oxysporum f. sp. ciceri races and represents a step towards the identification of potential race diagnostic markers. Received: 12 March 2000 / Accepted: 14 April 2000     

Chromatographic Characterization and Phytotoxic Activity of Fusarium oxysporum f. sp. albedinis and Saprophytic Strain Toxins

The bayoud disease, vascular fusariosis of date palm tree (Phoenix dactylifera L.), is caused by the pathogenic fungus Fusarium oxysporum f. sp. albedinis. The characteristic symptoms of the bayoud disease were elicited on detached leaves of F. oxysporum f. sp. albedinis‐susceptible cultivars of date palm trees, which were treated either with the F_II (F. oxysporum f. sp. albedinis) fraction purified from the organic extracts of a F. oxysporum f. sp. albedinis liquid culture, or with a solution of fusaric acid. Enniatins, which are secreted by several Fusarium species, were tested at different concentrations and were not capable of inducing symptoms on such detached leaves. The F_II (F. oxysporum f. sp. albedinis) fraction was unable to induce necrosis of potato slices, which indicates that it does not contain significant amounts of enniatins. The high‐performance liquid chromatography (HPLC) profiles of the F_II (F. oxysporum f. sp. albedinis) fraction showed toxic peaks different from fusaric acid. A fraction, named F_II (AZ₄), was obtained from culture filtrates of a saprophytic Fusarium strain maintained in the same cultural conditions as for the F. oxysporum f. sp. albedinis. The HPLC profile of the F_II (AZ₄) fraction did not show the characteristic phytotoxic peaks present in the F_II (F. oxysporum f. sp. albedinis) fraction. This finding well agrees with the fact that the F_II (AZ₄) fraction is not toxic to detached date palm leaves. Moreover, the HPLC profiles of F_II fractions obtained from other special forms of F. oxysporum are different the F_II (F. oxysporum f. sp. albedinis) profile. The phytotoxic compounds purified from the F_II (F. oxysporum f. sp. albedinis) fraction are probably new molecules that may help in understanding the pathogenesis of bayoud disease.     

Selection of Chickpea-Rhizosphere-Competent Pseudomonas fluorescens NBRI1303 Antagonistic to Fusarium oxysporum f. sp. ciceri,Rhizoctonia bataticola and Pythium sp.

A procedure that consumes less screening time was developed for screening chickpea rhizosphere-competent bacteria for suppression of the chickpea pathogenic fungi Fusarium oxysporum f. sp. ciceri, Rhizoctonia bataticola and Pythium sp. Of the 478 bacteria obtained by random selection of the predominant, morphologically distinct colonies, 386 strains that effectively colonize chickpea roots could be divided broadly into three different groups. The first group consisted of 44 good chickpea rhizosphere colonizers with 10⁷ to 10⁸ colony-forming units (CFU)/g root; the second group consisted of 253 medium chickpea rhizosphere colonizers with 10⁴ to 10⁶ CFU/g root; and the third group consisted of 89 poor chickpea rhizosphere colonizers with 10⁰ (nondetectable) to 10³ CFU/g root. Forty-four Rif^r strains from the first group of good chickpea rhizosphere colonizers were further screened for their in vitro biocontrol activity against F. oxysporum f. sp. ciceri, R. bataticola, and Pythium sp. One bacterial strain was selected for further work because of its unique ability to inhibit all three fungi and its good chickpea rhizosphere colonization ability. This is the first report of a single biocontrol bacterium active against three most devastating pathogenic fungi of chickpea. In a greenhouse test, chickpea seed bacterization with P. fluorescens NBRI1303 increased the germination of seedlings by 25%, reduced the number of diseased plants by 45%, compared with nonbacterized controls. Increases in seedling dry weight, shoot length, and root length ranged from 16% to 18%. Significant growth increases in shoot length, dry weight, and grain yield, averaging 11.59%, 17.58%, and 22.61% respectively above untreated controls, were attained in field trials in Agra and Jhansi. A rifampicin-resistant mutant P. fluorescens NBRI1303R of the P. fluorescens NBRI1303, used to monitor chickpea root colonization, confirmed the rapid and aggressive colonization by the bacterium, making it a potential biocontrol agent against chickpea phytopathogenic fungi. The results, demonstrating an increase in the efficiency of screening and detection of plant beneficial strains, should greatly benefit future studies. Received: 23 December 1996 / Accepted: 28 January 1997     

Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f. sp. ciceri in chickpea

Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. A marked increase in shoot and root length was observed in P. fluorescens treated plants. The isolates of P. fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium. oxysporum f.sp. ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control. Varied degree of protection against Fusarium wilt was recorded with chemical inducers. The reduction in disease was more pronounced when chemical inducers were applied with P. fluorescens. Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers. A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested. Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application. All the isolates of P. fluorescens produced SA in synthetic medium and in root tissues. HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates. 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog. After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.     

Salicylic acid and salicylic acid sensitive and insensitive catalases in different genotypes of chickpea against Fusarium oxysporum f. sp. ciceri

Differential expression of catalase isozymes in different genotypes of chickpea resistant genotypes- A1, JG-315, JG-11, WR-315, R₁-315, Vijaya, ICCV-15017, GBS-964, GBM-10, and susceptible genotypes- JG-62, MNK, ICCV-08321, ICCV-08311, KW-104, ICCV-08123, ICC-4951, ICC-11322, ICC-08116 for wilt disease caused by Fusarium oxysporum. f. sp. ciceri (Foc) was analyzed. Salicylic acid (SA) and H₂O₂ concentrations were determined in control as well as in plants infected with F. ciceri and found that the high and low levels of salicylic acid and H₂O₂ in resistant and susceptible genotypes of chickpea respectively. Catalase isozyme activities were detected in the gel and found that no induction of new catalases was observed in all the resistant genotypes and their some of the native catalase isozymes were inhibited; whereas, induction of multiple catalase isozymes was observed in all the screened susceptible genotypes and their activities were not inhibited upon Foc or SA treatments. The above results support the possible role of these isozymes as a marker to identify which genotype of chickpea is expressing systemic acquired resistance.     

Saprophytic growth of inoculated Frankia sp. in soil microcosms

The potential of two Frankia strains to grow saprophytically was studied in nonsterile soil microcosms with ground leaf litter of Alnus glutinosa as the sole carbon and nitrogen sources. Strains Ag45/Mut15 and ArI3, which represent two taxonomic subgroups within the Alnus host infection group were inoculated alone, or together to investigate potential competition. Their growth was analyzed by in situ and dot-blot hybridization. A significant increase in cell numbers and filament length was observed during the first 6 weeks after inoculation for strain Ag45/Mut15, both alone and in mixed culture with strain ArI3, followed by a decrease until the end of the study after 12 weeks. The number of filaments remained unchanged. In contrast, the cell numbers and filament length of strain ArI3 were reduced significantly during the first 2 weeks and were undetectable for the remainder of the study. These results were comparable with those obtained in sterile mineral medium amended with leaf litter of A. glutinosa, although reductions in cell numbers and filament length were less pronounced than in soil microcosms. In concomitant control studies without leaf litter amendments for both experimental setups, filaments of both strains could only be detected immediately after inoculation. These results were matched in all experimental setups by concomitant shifts in the rRNA content of both strains, i.e., an immediate decline in the rRNA content for strain ArI3 after inoculation, and an increase in the rRNA content, followed by a late decline during incubation for strain Ag45/Mut15. These results demonstrated that Frankia strain Ag45/Mut15 could grow saprophytically in soil with complex carbon and nitrogen sources such as leaf litter, while the growth of strain ArI3 was not supported.     

Mineral Uptake in Fusarium oxysporum f. sp. vasinfectum

A method for growing Fusarium oxysporum, a mycelial fungus, and a technique for its use in mineral uptake studies have been described. Some general characteristics of the uptake process were determined. The fungus, grown for 54 hours, was found to take up as much K as 15 to 20 meq/100 g dry weight in 2 to 4 hours from a solution of 5 meq/l KCl. Approximately 3 to 5 meq of this uptake was readily removed by a CaCl₂ rinse. The uptake was only slightly sensitive to pH over the range of 4 to 9. Below pH 4 uptake dropped rapidly. The age of the culture appeared to be the dominant factor in determining the rate of uptake. In contrast to other fungi, the presence of glucose during uptake was detrimental to K uptake. Conditions unfavorable for metabolic activity as low temperature, anaerobiosis, or the presence of DNP markedly reduced the uptake rate. Although the fungus took up Na from single salt solutions nearly as well as K, the latter ion was much preferred in mixtures of the two ions. The organism showed no significant metabolic uptake of Ca or Cl. During uptake from KCl solutions, the organic acid content increased. The increase, chiefly in succinic acid and to a lesser extent in acetic and citric acids, amounted to about half the K uptake. The remainder of the K taken up was correlated with a roughly equivalent efflux of cellular Mg.     

The effect of soil temperature on survival ofFusarium oxysporum f.melonis (Leach and Currence) Snyder and Hansen

Summary Most of the washed conidia ofFusarium oxysporum f.melonis introduced into either autoclaved or naturally infested soils disappeared after 30 days incubation at temperatures between 5 and 30°C, but more rapid decline in population took place at the higher temperatures and in naturally infested soil. The population of f.melonis after 14 months incubation was greatest at 15 and lowest at 5°C in both autoclaved and nonautoclaved soils. The survival units were chlamydospores associated with the previously invaded organic particles. This association is considered to be an important factor in the organism's survival in soil.     

Dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici

The genomes of many filamentous fungi consist of a ‘core’ part containing conserved genes essential for normal development as well as conditionally dispensable (CD) or lineage‐specific (LS) chromosomes. In the plant‐pathogenic fungus Fusarium oxysporum f. sp. lycopersici, one LS chromosome harbours effector genes that contribute to pathogenicity. We employed flow cytometry to select for events of spontaneous (partial) loss of either the two smallest LS chromosomes or two different core chromosomes. We determined the rate of spontaneous loss of the ‘effector’ LS chromosome in vitro at around 1 in 35 000 spores. In addition, a viable strain was obtained lacking chromosome 12, which is considered to be a part of the core genome. We also isolated strains carrying approximately 1‐Mb deletions in the LS chromosomes and in the dispensable core chromosome. The large core chromosome 1 was never observed to sustain deletions over 200 kb. Whole‐genome sequencing revealed that some of the sites at which the deletions occurred were the same in several independent strains obtained for the two chromosomes tested, indicating the existence of deletion hotspots. For the core chromosome, this deletion hotspot was the site of insertion of the marker used to select for loss events. Loss of the core chromosome did not affect pathogenicity, whereas loss of the effector chromosome led to a complete loss of pathogenicity.     

Race Differentiation in Fusarium oxysporum f.sp. chrysanthemi

The pathogenicity of different isolates of Fusarium oxysporum obtained from plants of Gerbera (Gerbera jamesonii), Chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens) and African daisy (Osteospermum sp.), all in the family Asteraceae, was tested on different cultivars of these hosts, to assess their pathogenicity. The reactions were compared with those of isolates of F. oxysporum f. sp. chrysanthemi and of f.sp. tracheiphilum obtained from the American Type Culture Collection. We found that isolates of F. oxysporum f. sp. chrysanthemi can be distinguished as three physiological races on the basis of their pathogenicity to the panel of differential cultivars. Sequencing of the intergenic spacer (IGS) region of ribosomal DNA (rDNA) and phylogenetic analysis showed that the Fusarium races fell into three phylogenetic groups, which coincided with those observed in pathogenicity tests. Analysis of the IGS sequences revealed a high degree of similarity among strains from Italy and Spain from different host species, suggesting that recent outbreaks in these ornamentals were probably caused by introduction of infected nursery material from a common origin.     

Production of Intraspecific Hybrids of Fusarium oxysporum f. sp. radicis-lycoperisici and Fusarium oxysporum f. sp. lycopersici by Protoplast Fusions

The fusion of protoplasts from the cycloheximide-resistant mutant FOL(C) of Fusarium oxysporum f. sp. lycopersici (FOL) and the mycostatin-resistant mutant FORL(M) of F. oxysporum f. sp. radicis-lycopersici (FORL), produced hybrids which expressed significant differences from the parents in their pathogenicity and growth and in the electrophoretic separation patterns of their proteins, enzymes and isoenzymes. The results suggest a transformed genetic basis for these altered expressions and the feasibility of using protoplast fusion technology for examining the biology of pathogenicity genes and for elucidating the disease and virulence potential for new races from within hybridisable taxa of Fusarium spp. Such information would be useful for the design and development of long-term control systems for Fusarium diseases, particularly in breeding programs for disease resistance in crops.     

A highly efficient Agrobacterium mediated transformation system for chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri using DsRed-Express to follow root colonisation

The soil-borne fungus Fusarium oxysporum f. sp. ciceri (Foc) causes vascular wilt of chickpea (Cicer arietinum L.), resulting in substantial yield losses worldwide. Agrobacterium tumefaciens mediated transformation (ATMT) has served as a resourceful tool for plant-pathogen interaction studies and offers a number of advantages over conventional transformation systems. Here, we developed a highly efficient A. tumefaciens mediated transformation system for Foc. In addition, a binary vector for constitutive expression of red fluorescent protein (DsRed-Express) was used to study developmental stages and host-pathogen interactions. Southern hybridisation was performed to confirm the transformation event and the presence of T-DNA in selected hygromycin resistant transformants. Most of the transformants showed single copy integrations at random positions. Microscopic studies revealed significant levels of fluorescent protein, both in conidia and mycelia. Confocal microscopy of chickpea roots infected with the transformed Foc showed rapid colonisation. These studies will allow us to develop strategies to determine the mechanisms of Foc-chickpea interaction in greater detail and to apply functional genomics for the characterisation of involved genes at the molecular level either by insertional mutagenesis or gene knock-out.     

Survival and inoculum potential of conidia and chlamydospores of Fusarium oxysporum f.sp. lini in soil

The kinetics of survival and inoculum potential of Fusarium oxysporum f.sp. lini were studied in soil. Two types of inoculum were compared: microconidia freshly harvested from a laboratory-grown culture and microchlamydospores produced in sterilized soil. Introduced at the same inoculum densities into a natural soil, the two types of inoculum showed similar behavior; the inoculum densities changed little with time, at least during 100 days. However, the two types of inoculum did differ in disease potential. A higher percentage of microchlamydospores than microconidia germinated in the rhizosphere of flax seedlings, and the heterotrophic fluorescein diacetate hydrolysing activity of the microchlamydospores was 100 times higher than that of microconidia. Moreover, the microchlamydospores produced more disease on flax than the microconidia even at a much lower inoculum density.     

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.