Accumulation of phytoalexins in susceptible and resistant near-isogenic lines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici

The time course of accumulation of two phytoalexins, the terpenoid rishitin and the polyacetylene cis-tetradeca-6-ene-1,3-diyne-5,8-diol, was determined in near-isogenic susceptible and resistant tomato lines inoculated with either Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici.Cultivars containing the Ve gene for verticillium wilt resistance accumulated phytoalexins at a rate similar to that in susceptible plants following stem inoculation with V. albo-atrum. Higher amounts of phytoalexins were isolated from susceptible than from resistant plants at 11 days after inoculation. Inoculum concentrations of 10⁵, 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ had no differential effect on phytoalexin accumulation at 3 days after inoculation. Also, no differences were observed between fungal growth in susceptible and resistant cultivars during that period.A cultivar containing the I-1 gene for fusarium wilt resistance contained more rishitin than did susceptible plants at 2 and 3 days after inoculation with 10⁷ conidia of F. oxysporum f.sp. lycopersici ml⁻¹, but at 7 and 11 days after inoculation more rishitin had accumulated in the susceptible plants.No difference was observed between the rate of accumulation of phytoalexin in stem segments from resistant and susceptible plants inoculated by vacuum-infiltration.To estimate the concentration of phytoalexins in the xylem fluid, sap was expressed from vascular tissue and amounts of phytoalexins were determined in the sap and in the expressed tissue. Less than 5% of the phytoalexins present in stem segments was recovered from the sap, indicating that their concentration in the xylem fluid may be relatively low.The role of phytoalexins in resistance to verticillium and fusarium wilt is discussed.     

Distribution of the FoToml gene encoding tomatinase in formae speciales of Fusarium oxysporum and identification of a novel tomatinase from F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato

The antifungal glycoalkaloid -tomatine accumulates in tomato plants and may protect plants from fungal infection. Fusarium oxysporum f. sp. lycopersici, the causal agent of vascular wilt of tomato, produces a tomatinase (FoToml) that degrades -tomatine to the nontoxic compounds tetrasaccharide lycotetraose and tomatidine. Induction of tomatinases and the distribution of FoToml homologs were examined among 30 strains belonging to 16 formae speciales of F. oxysporum. Tomatinase activity was found in 27 strains belonging to 15 formae speciales, but FoToml homologs (>98% sequence identity) were detected in only six strains belonging to four formae speciales. To identify tomatinases other than FoToml, -tomatine-inducible proteins of another tomato pathogen F. oxysporum f. sp. radicis-lycopersici were analyzed by two-dimensional gel electrophoresis. A protein with a molecular mass of 64kDa accumulated in the -tomatine-induced culture filtrates, and the protein had tomatinase activity, degrading -tomatine to lycotetraose and tomatidine.     

Induced resistance in tomato plants against Fusarium wilt invoked by Fusarium oxysporum f.sp. dianthi

Tomato plants, susceptible toFusarium oxysporum f. sp.lycopersici, were inoculated by immersing the roots in a conidial suspension ofF. oxysporum f. sp.lycopersici race 1,F. oxysporum f. sp.dianthi race 2 or a mixture of both fungi. Plants inoculated withF. oxysporum f. sp.lycopersici showed disease symptoms after 2 weeks, whereas plants inoculated withF. oxysporum f. sp.dianthi or a mixture of both fungi remained symptomless for over 7 weeks, the duration of the experiment. In another experiment root systems of plants were split and each half was separately inoculated. One half was firstly inoculated withF. oxysporum f. sp.dianthi or treated with water, followed after a week by a second inoculation of the other half withF. oxysporum f. sp.lycopersici or by a water treatment. The disease symptoms in the half firstly inoculated withF. oxysporum f. sp.dianthi were significantly delayed, compared to plants of which that half had been treated with water. BecauseF. oxysporum f. sp.dianthi reduced disease symptoms caused byF. oxysporum f. sp.lycopersici without any direct interaction with this pathogen, it is concluded thatF. oxysporum f. sp.dianthi is able to induce resistance againstF. oxysporum f. sp.lycopersici in tomato plants.     

番茄枯萎病菌和颈腐根腐病菌KASP-SNP检测技术的建立

为快速、准确地对番茄枯萎病菌Fusarium oxysporum f. sp. lycopersici(FOL)和番茄颈腐根腐病菌F. oxysporum f. sp. radicis-lycopersici(FORL)进行检测,基于尖孢镰刀菌F. oxysporum多聚半乳糖醛酸外切酶基因pgx4的单核苷酸多态性（single nucleotide polymorphism,SNP）位点,设计FORL、FOL生理小种1(FOL-R1)、2(FOL-R2)和3(FOL-R3)的竞争性等位基因特异性PCR-SNP(kompetitive allele specific PCR-SNP,KASP-SNP)引物,建立番茄颈腐根腐病菌和番茄枯萎病菌KASP-SNP检测技术,并通过与常规PCR比对及ITS与pgx4序列分析对该检测技术的可靠性进行验证。结果显示,在FORL、FOL-R1、FOL-R2和FOL-R3中存在35个变异SNP位点,设计出18对KASP-SNP引物,筛选出FORL＿KASP、FOLrace1＿KASP、FOLrace2＿KASP和FOLrace3＿KASP共4对分型清晰的...     

Cross protection provides evidence for race-specific avirulence factors inFusarium oxysporum

Simultaneous inoculation with races 1 and 2 of the vascular wilt pathogenFusarium oxysporumf.sp.lycopersiciprovided a high level of protection against race 2 in three tomato cultivars carrying resistance geneI, which confers resistance to race 1 but not race 2. However, simultaneous inoculation did not provide any protection in cultivars lacking this gene. Protection resulted in reduction and delay of wilt symptoms. Similarly, avirulent races ofF. oxysporumf.sp.melonisprotected muskmelon plants against virulent races of the sameforma specialis.A ratio 10:1 between spore concentrations of inducer and challenger organism gave the highest cross protection, but ratio 0.1:1 still provided significant disease reduction. Cross protection was also obtained when inoculation with the inducer organism was performed 6 or 12 h before inoculation with the challenger organism. Autoclaved spores of the inducer did not have any protective effect, indicating that living propagules were required to initiate protection. The results suggest the presence of a gene-for-gene interaction betweenF. oxysporumf.sp.lycopersici-tomato andF. oxysporumf.sp.melonis-muskmelon, in which cross protection against a virulent race is mediated by recognition of a specific elicitor from the avirulent race by the plant resistance gene product and by subsequent induction of the plant defense reaction.     

Cloning of the pathogenicity-related gene <Emphasis Type="Italic">FPD1</Emphasis> in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB110097     

Effect of mixed inoculations with Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. dianthi on the phenols content of tomato plants

Forms ofFusarium oxysporum specific on hosts other than tomato induce in this plant greater initial increases of the phenols content than the pathogenic f. sp.lycopersici. Mixed inoculations of f. sp.lycopersici and f. sp.dianthi are on the contrary no more effective in inducing the phenol accumulation 24 h after the infection than f. sp.lycopersici alone. This observation suggests that the pathogen can suppress the phenolic response that is typical of the incompatible combinations.Samenvatting Vormen vanFusarium oxysporum welke pathogeen zijn voor andere planten dan de tomaat induceren in deze plant aanvankelijk een grotere toename van het fenolgehalte dan de pathogene f. sp.lycopersici. Inoculaties met een gemengd inoculum van de f. sp.lycopersici en f. sp.dianthi hebben daarentegen geen groter effect op de toename van het fenolgehalte 24 uur na infectie dan de inoculaties met f. sp.lycopersici alleen. Verondersteld wordt dat het pathogeen de toename van het fenolgehalte, dat typerend is voor de incompatibele combinatie, kan onderdrukken.     

PCR-based differentiation of Fusarium oxysporum ff. sp. lycopersici and radicis-lycopersici and races of F. oxysporum f. sp. lycopersici

The pathogenic type (form and race) of Fusarium oxysporum, which generates wilt symptoms on tomato, was rapidly identified with a polymerase chain reaction (PCR)-based technique. We compared the partial nucleotide sequences of endo polygalacturonase (pg1) and exo polygalacturonase (pgx4) genes from isolates of F. oxysporum ff. sp. lycopersici (FOL) and radicis-lycopersici (FORL) from Japan and designed specific primer sets (uni, sp13, sp23, and sprl) based on the nucleotide differences that appeared among the pathogenic types. PCR with the uni primer set amplified a 670∼672-bp fragment from all isolates of FOL and FORL. With the sp13 primer set, an amplicon of 445 bp was obtained only from isolates of FOL race 1 and 3. With the sp23 primer set, a 518-bp fragment was obtained from isolates of FOL race 2 and 3. The sprl primer set yielded a 947-bp fragment from isolates of FORL, but not from FOL. A combination of amplifications with these primer sets effectively differentiated the pathogenic types of F. oxysporum in tomato.     

The elegans fusaria causing wilt disease of carnation. II. Distinction of vegetative compatibility groups

The vegetative compatibility patterns among isolates ofElegans fusaria causing wilt disease of carnation were investigated. Nitrate non-utilizing mutants were generated from 16 isolates labelledF. redolens, nine of which came from carnation, and from 33 isolates labelledF. oxysporum, 19 of which came from carnation. Pairings of the mutants revealed five vegetative compatibility groups among the isolates from carnation, corresponding withF. oxysporum f.sp.dianthi race 1 (VCG1), race 2 (VCG2) and race 4 (VCG3),F. redolens f.sp.dianthi (VCG4) andF. redolens isolates from foot rot-diseased carnations (VCG5). Besides three isolates typical ofF. redolens, VCG4 comprised a now slightly deviating subculture of the type isolate ofF. redolens f.sp.dianthi of which the cultural characteristics correspond toF. oxysporum instead ofF. redolens. This observation may be taken to support previous conclusions that the distinction between both taxa is not justified. Otherwise, the compatibility patterns did not provide decisive evidence to accept or reject conspecificity of both taxa. Isolates from carnation did not form heterokaryons with other formae speciales ofF. oxysporum.Samenvatting De vegetatieve compatibiliteitspatronen bij isolaten vanElegans-fusaria die verwelkingsziekte bij anjer veroorzaken werden onderzocht. Van 16 isolaten vanF. redolens, waarvan negen afkomstig van anjers, en van 33 isolaten vanF. oxysporum, waarvan 19 afkomstig van anjers, werden mutanten gegenereerd die zonder een organische stikstofbron geen luchtmycelium meer konden vormen. Paringen tussen mutanten van isolaten afkomstig van anjers brachten een vijftal vegetatieve compatibiliteitsgroepen aan het licht, die overeenkwamen metF. oxysporum f.sp.dianthi fysio 1 (VCG 1), fysio 2 (VCG 2) en fysio 4 (VCG3),F. redolens f.sp.dianthi (VCG4) enF. redolens isolaten afkomstig van aan voetrot lijdende anjers (VCG5). Naast drie voorF. redolens karakteristieke isolaten omvatte VCG4 ook een afwijkende subculture van het type-isolaat vanF. redolens f.sp.dianthi, die in cultuureigenschappen overeen kwam metF. oxysporum in plaats vanF. redolens. Deze waarneming geeft enige steun aan eerdere conclusies dat het onderscheid tussen beide taxa niet gerechtvaardigd is. Daarbuiten gaven de compatibiliteitspatronen geen uitsluitsel over de mogelijke conspecificiteit van beide taxa. Isolaten afkomstig van anjers vormden geen heterokaryons met andere formae speciales vanF. oxysporum.     

Biofumigation with Brassica plants and its effect on the inoculum potential of Fusarium yellows of Brassica crops

The use of Brassica crops as green manure in the so-called biofumigation treatment has been successfully exploited for the management of soilborne pathogens and is gaining interest particularly in the case of less intensive agricultural systems. A study was undertaken to investigate possible negative side-effects of biofumigation in order to prevent possible damage caused by wilt pathogens able to attack both plants used for biofumigation as well as agricultural crops. To do so, firstly the response of different Brassicas, including some used in biofumigation, to the formae speciales of Fusarium oxysporum known for being pathogenic on Brassica crops was evaluated. Secondly, the effect of green manure treatments on yield, quality of crops, and inoculum densities, infection and survival of Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. raphani was evaluated. In the second part of the work, four Brassica crops, selected for their response (susceptibility or resistance) to F. oxysporum f. sp. conglutinans and to F. oxysporum f. sp. raphani were evaluated in order to determine their response to the two pathogens during subsequent crops grown in soil where plants were incorporated as green manure into the soil at the end of each cycle. Moreover, the dynamics of the populations of F. oxysporum f. sp. conglutinans and F. oxysporum f. sp. raphani in the soil after several biofumigation cycles was studied. Many of the Brassica crops used for biofumigation tested were susceptible to F. oxysporum f. sp. conglutinans and or to F. oxysporum f. sp. raphani. Green manure treatment, carried out by growing nine cycles of biocidal plants, with a short crop cycle of 30–35 days, did not reduce Fusarium wilts on susceptible Brassica hosts. The population of the pathogen was partially increased as a result of the incorporation of tissues of the susceptible plants. When Brassica crops grown were resistant to the two F. oxysporum pathogens used for soil infestation, green manure simulation did inhibit both pathogens, thus confirming its biocidal activity. The results obtained under our experimental conditions show that biofumigation treatment is not applicable for soil disinfestation on crops susceptible to the same formae speciales of F. oxysporum affecting Brassica species used for biofumigation. Brassica crops resistant to Fusarium yellows should be grown where biofumigation is applied. Moreover, alternation of crops used for biofumigation should be encouraged.     

Defense strategies of pea embryo axes with different levels of sucrose to Fusarium oxysporum and Ascochyta pisi

The aim of this study was to compare the defense responses of embryo axes of Pisum sativum L. cv. Kwestor with different sucrose levels to pathogenic fungi, i.e. systemic acting Fusarium oxysporum f. sp. pisi and locally acting Ascochyta pisi. Embryo axes were cultured on Heller medium for 96 h. Four variants were compared: these included inoculated embryo axes cultured with or without 60 mM sucrose (+Si and −Si) and non-inoculated embryo axes cultured with or without 60 mM sucrose (+Sn and −Sn). After inoculation of the pea embryo axes with pathogenic fungi a generally higher concentration of free radicals was detected by electron paramagnetic resonance (EPR), in comparison to non-inoculated embryo axes. The inoculation with F. oxysporum caused stronger generation of free radicals in −Si than in +Si embryo axes. A different response was observed after inoculation with A. pisi; starting from 48 h, the concentration of free radicals in +Si axes was found to be 1.5 times higher than in −Si embryo axes. The values of spectroscopic splitting coefficients for these radicals suggest that they are semiquinone radicals. The EPR method also revealed Mn²⁺ ion accumulation after 24 h of culture. Over time, high levels of these ions were recorded in +Si embryo axes inoculated with F. oxysporum, while in +Si embryo axes inoculated with A. pisi they decreased. Up to 48 h after inoculation with the pathogenic fungi, Mn²⁺ ion levels were higher in +Si embryo axes than in +Sn axes. The activity of superoxide dismutase (SOD, EC 1.15.1.1) increased in +Si embryo axes up to 72 h after inoculation with pathogenic fungi; however, it was generally lower than in +Sn axes. Catalase activity (CAT, EC 1.11.1.6) increased up to 72 h after inoculation with F. oxysporum and the values were higher than in the non-inoculated tissue. Especially high activity of this enzyme was noted in −Si embryo axes after inoculation with either F. oxysporum or A. pisi. Peroxidase activity (POX, EC 1.11.1.7) towards pyrogallol in embryo axes increased during culture; however, it was lower or similar to that in non-inoculated embryo axes. SOD, CAT and POX zymograms showed that the synthesis of new isoforms was induced after inoculation with pathogenic fungi. Peroxidase isozymes detected by the reaction with diaminobenzidine in native PAGE were intensely stained in +Si embryo axes after inoculation with pathogenic fungi. Respiratory activity of the inoculated tissues was considerably higher than in non-inoculated tissues. The respiration rate was generally much higher in +Si than in −Si embryo axes. Growth of −Si embryo axes was more significantly retarded as a consequence of inoculation than that of +Si embryo axes.These results indicate that, depending on the manner of influence of a pathogenic fungus, both similar and differing defensive strategies may be initiated and a raised sugar levels in pea tissues limit the development of F. oxysporum and A. pisi.     

Development of sequence tagged site markers to identify races of Fusarium oxysporum f. sp. lactucae

By random amplified polymorphic DNA (RAPD) analysis of the representative isolates of each race of Fusarium oxysporum f. sp. lactucae, RAPD fragments of 0.6, 1.6, and 2.9kb were obtained. The 0.6-kb RAPD fragment was common to the representative isolates of all three races. Amplification of the 1.6- and 2.9-kb fragments were unique to the isolates of races 1 and 2, respectively. Sequence tagged site (STS) marker FLA0001, FLA0101, and FLA0201 were generated from the 0.6-, 1.6-, and 2.9-kb RAPD fragments, respectively. Polymerase chain reaction (PCR) analysis showed that FLA0001 was common to all 49 isolates of F. oxysporum f. sp. lactucae. FLA0101 was specifically generated from all 23 isolates of race 1 but not from races 2 or 3. FLA0201 was specifically amplified from all 12 isolates of race 2 but not from races 1 or 3. In two isolates of F. oxysporum f. sp. lactucum, PCR amplified FLA0001 and FLA0101 but not FLA0201. On the other hand, these STS markers were not detected from isolates of five other formae speciales. Because these STS markers were not generated from isolates of other plant pathogenic fungi, bacteria, or plant materials examined in this study, PCR analysis combined with the three STS markers should be a useful means for rapid identification of races of F. oxysporum f. sp. lactucae.     

Genetic diversity of Fusarium oxysporum and related species pathogenic on tomato in Algeria and other Mediterranean countries

In order to characterize the pathogen(s) responsible for the outbreak of fusarium diseases in Algeria, 48 Fusarium spp. isolates were collected from diseased tomato in Algeria and compared with 58 isolates of Fusarium oxysporum originating from seven other Mediterranean countries and 24 reference strains. Partial sequences of the translation elongation factor EF‐1α gene enabled identification of 27 isolates as F. oxysporum, 18 as F. commune and three as F. redolens among the Algerian isolates. Pathogenicity tests confirmed that all isolates were pathogenic on tomato, with disease incidence greater at 28°C than at 24°C. All isolates were characterized using intergenic spacer (IGS) DNA typing, vegetative compatibility group (VCG) and PCR detection of the SIX1 (secreted in xylem 1) gene specific to F. oxysporum f. sp. lycopersici (FOL). No DNA polymorphisms were detected in the isolates of F. redolens or F. commune. In contrast, the 27 Algerian isolates of F. oxysporum were shown to comprise nine IGS types and 13 VCGs, including several potentially new VCGs. As none of the isolates was scored as SIX1+, the 27 isolates could be assigned to F. oxysporum f. sp. radicis‐lycopersici (FORL). Isolates from Tunisia were also highly diverse but genetically distinct from the Algerian isolates. Several Tunisian isolates were identified as FOL by a PCR that detected the presence of SIX1. The results show that isolates from European countries were less diverse than those from Tunisia. Given the difference between Algerian populations and populations in other Mediterranean countries, newly emergent pathogenic forms could have evolved from local non‐pathogenic populations in Algeria.     

Sequence variation in the putative effector gene SIX8 facilitates molecular differentiation of Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc), causal agent of fusarium wilt of banana, is among the most destructive pathogens of banana and plantain. The development of a molecular diagnostic capable of reliably distinguishing between the various races of the pathogen is of key importance to disease management. However, attempts to distinguish isolates using the standard molecular loci typically used for fungal phylogenetics have been complicated by a poor correlation between phylogeny and pathogenicity. Among the available alternative loci are several putative effector genes, known as SIX genes, which have been successfully used to differentiate the three races of F. oxysporum f. sp. lycopersici. In this study, an international collection of Foc isolates was screened for the presence of the putative effector SIX8. Using a PCR and sequencing approach, variation in Foc‐SIX8 was identified which allowed race 4 to be differentiated from race 1 and 2 isolates, and tropical and subtropical race 4 isolates to be distinguished from one another.     

Effect of ammonium/nitrate ratio in nutrient solution on control of Fusarium wilt of tomato by Trichoderma asperellum T34

The behaviour of Fusarium oxysporum f.sp. lycopersici (Fol) and the effectiveness of the microbial control agent Trichoderma asperellum strain T34 were examined in hydroponically grown tomato plants under five ammonium/nitrate ratios. The results showed that disease severity was reduced by the action of T34 under increasing concentrations of ammonia. Furthermore, rhizosphere F. oxysporum populations decreased with T34 application. The presence of T34 augmented leaf nitrogen concentration in treatments infested with Fol. In addition, T34 application reduced iron concentration in tomato leaves at high ammonium/nitrate ratios and reduced the severity of Fusarium wilt at high iron and nitrogen leaf concentrations.     

Germination of Fusarium oxysporum in root exudates from tomato plants challenged with different Fusarium oxysporum strains

The response of microconidia from pathogenic and non-pathogenic Fusarium oxysporum to root exudates from tomato plants inoculated with different pathogenic and non-pathogenic F. oxysporum strains was studied. Root exudates from non-inoculated tomatoes highly stimulated the microconidial germination of the two tomato pathogens, F. oxysporum f.sp. lycopersici strain Fol 007 and F. oxysporum f.sp. radicis-lycopersici strain Forl 101587. In root exudates from tomato plants challenged with the pathogen Fol 007 the microconidial germination of Fol 007 was increased, whereas in root exudates from plants challenged with Forl 101587 the microconidial germination of Fol 007 was reduced. Root exudates of tomato plants challenged with the non-pathogenic unspecific F. oxysporum strain Fo 135 and the biocontrol strain Fo 47 clearly reduced microconidial germination of the pathogenic strain Forl 101587. Moreover, the microconidial germination rate of the biocontrol strain Fo 47 was increased in the presence of root exudates of tomato plants challenged with the tomato wilt pathogen Fol 007. These results indicate that pathogenic and non-pathogenic F. oxysporum strains alter the root exudation of tomato plants differently and consequently the fungal propagation of pathogenic and non-pathogenic F. oxysporum strains in the rhizosphere is affected differently.     

A new race of Fusarium oxysporum f. sp. lactucae of lettuce

Fusarium oxysporum f. sp. lactucae, the causal agent of fusarium wilt of lettuce (Lactuca sativa), occurs in most countries in which lettuce is grown and causes serious economic losses. Three races (1, 2 and 3) of the pathogen have previously been identified on the basis of their ability to cause disease on differential lettuce cultivars, as well as by means of molecular tools developed to characterize different races of this pathogen. Only race 1 has been detected in Europe so far. In this study, two isolates of F. oxysporum, obtained from lettuce plants grown in the Netherlands showing symptoms of wilt, have been characterized by combining the study of pathogenicity with differential cultivars of lettuce and molecular assays to determine whether the isolates are different from the known races of F. oxysporum f. sp. lactucae. This study reports the presence of F. oxysporum f. sp. lactucae for the first time in the Netherlands. The causal pathogen has been identified, using the IRAP‐SCAR technique, as a new race of F. oxysporum f. sp. lactucae. Specific primers have been designed to identify this new race.     

Fusarium oxysporum f. sp. lycopersici retardation through induction of defensive response in tomato plants using a liquid formulation of Pseudomonas fluorescens (Pf1)

A liquid based Pseudomonas fluorescens (Pf1) bioformulation was found to contribute the restriction of Fusarium oxysporum f. sp. lycopersici in tomato roots by inducing defence enzymes. Induction of defence enzymes such as phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenoloxidase (PPO), catalase, β-1,3 glucanase and super oxide dismutase (SOD), was studied in tomato plants pretreated with liquid as well as a talc based formulation of Pf1 challenged with F. oxysporum f. sp. lycopersici in glasshouse vegetable production systems. There were increased activities of PAL, PO, PPO, catalase and β-1 3-glucanases in tomato plants treated with a combined application of seedling dip?+?soil application?+?foliar spray of liquid and talc formulation of Pf1 when compared to pathogen inoculated and untreated healthy controls. The activities of the above enzymes started to increase at 3rd day, reached maximum levels on 8-9th day and thereafter declined gradually. Similarly, native polyacralamide gel electrophoresis (PAGE) analysis revealed that one to six isoforms of the defence enzymes each with a higher intensity were expressed in these treatments, whereas fewer isoforms with less intensity were noticed in inoculated controls. These results suggest that the retardation of the invasion of F. oxysporum f. sp. lycopersici in tomato roots resulting from treatment with the liquid formulation of Pf1 was due to enhancement of activities of enzymes involved in the phenylpropanoid pathway. These results suggest that induced systemic resistance occurred in the treated tomato plants.     

Molecular characterisation of vegetative compatibility groups in Fusarium oxysporum f. sp. radicis-lycopersici and f. sp. lycopersici by random amplification of polymorphic DNA and microsatellite-primed PCR

Random amplification of polymorphic DNA (RAPD-PCR) analysis was conducted on 48 isolates of Fusarium oxysporum f. sp. radicis-lycopersici (F.o.r.l.) from different geographic regions, representing all known vegetative compatibility groups (VCGs) except VCG 0097 and VCG 0099 and on eight isolates of F.oxysporum f. sp. lycopersici (F.o.l.), representing VCGs 0030, 0031, 0032 and 0033. Upon UPGMA (unweighted pair-group method with arithmetic averages) analysis of 86 RAPD-PCR markers generated by 16 informative primers and 44 markers obtained with eight microsatellite primers, a close relatedness was evident for F.o.r.l. isolates in VCGs 0090, 0092, 0096, and, to a lesser extent, for those in VCG 0093. Representatives of VCG 0091 formed a distinct group, while F.o.r.l. isolates in VCGs 0094 and 0098 were not distinguishable by the tested markers, most of which were also shared by F.o.l. isolates belonging to VCGs 0031 and 0033. F.o.l. isolates in VCGs 0030 and 0032 shared most of the molecular markers. The correlation between RAPD-PCR and microsatellite genetic distance was highly significant (R² = 0.77; P by Mantel test < 0.001). The molecular variability observed in both formae speciales is discussed in relation to the development of F.o.r.l.- and F.o.l.-specific diagnostic tools.     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.