Detection of tomatinase from Fusarium oxysporum f. sp. lycopersici in infected tomato plants

The antifungal glycoalkaloid alpha-tomatine of the tomato plant (Lycopersicon esculentum) is proposed to protect the plant against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a vascular pathogen of tomato, produces a tomatinase enzyme which hydrolyses the glycoalkaloid into non-fungitoxic compounds. Detoxification of alpha-tomatine may be how this fungus avoids the plant glycoalkaloid barrier. As an initial step to evaluate this possibility we have studied the induction of tomatinase; (i) in fungal cultures containing extracts from leaf, stem or root of tomato plants; and (ii) in stem and root of tomato plants infected with the pathogen at different infection stages. The kinetics of tomatinase induction with leaf extract (0.6% dry weight) was similar to that observed with 20 micrograms ml-1 of alpha-tomatine. In the presence of stem extract, tomatinase activity was less than 50% of that induced with leaf extract, whereas in the presence of root extract tomatinase activity was very low. In the stem of infected tomato plants tomatinase activity was higher at the wilt stage than in previous infections stages and in root, tomatinase activity appeared with the first symptoms and was maintained until wilting. TLC analysis showed that the tomatinase induced in culture medium with plant extracts and in infected tomato plants had the same mode of action as the enzyme induced with pure alpha-tomatine, hydrolysing the glycoalkaloid into its non-fungitoxic forms, tomatidine and beta-lycotetraose. The antisera raised against purified tomatinase recognized in extracts of root and stem of infected tomato plants a protein of 50000 (45000 when proteins were deglycosylated), corresponding to the tomatinase enzyme. Therefore, it is concluded that F. oxysporum f. sp. lycopersici express tomatinase in vivo as a result of the infection of tomato plant.     

Inheritance and genetic mapping of resistance to Alternaria alternata f. sp. lycopersici in Lycopersicon pennellii

The fungal pathogen Alternaria alternata f. sp. lycopersici produces AAL-toxins that function as chemical determinants of the Alternaria stem canker disease in the tomato (Lycopersicon esculentum). In resistant cultivars, the disease is controlled by the Asc locus on chromosome 3. Our aim was to characterize novel sources of resistance to the fungus and of insensitivity to the host-selective AAL-toxins. To that end, the degree of sensitivity of wild tomato species to AAL-toxins was analyzed. Of all members of the genus Lycopersicon, only L. cheesmanii was revealed to be sensitive to AAL-toxins and susceptible to fungal infection. Besides moderately insensitive responses from some species, L. pennellii and L. peruvianum were shown to be highly insensitive to AAL-toxins as well as resistant to the pathogen. Genetic analyses showed that high insensitivity to AAL-toxins from L. pennellii is inherited in tomato as a single complete dominant locus. This is in contrast to the incomplete dominance of insensitivity to AAL-toxins of L. esculentum. Subsequent classical genetics, RFLP mapping and allelic testing indicated that high insensitivity to AAL-toxins from L. pennellii is conferred by a new allele of the Asc locus.     

Fot 1 insertions in the Fusarium oxysporum f. sp. albedinis genome provide diagnostic PCR targets for detection of the date palm pathogen

Populations of Fusarium oxysporum f. sp. albedinis, the causal agent of Bayoud disease of date palm, are derivatives of a single clonal lineage and exhibit very similar Fot 1 hybridization patterns. In order to develop a sensitive diagnostic tool for F. oxysporum f. sp. albedinis detection, we isolated several DNA clones containing a copy of the transposable element Fot 1 from a genomic library of the date palm pathogen. Regions flanking the insertion sites were sequenced, and these sequences were used to design PCR primers that amplify the DNA regions at several Fot 1 insertion sites. When tested on a large sample of Fusarium isolates, including 286 F. oxysporum f. sp. albedinis isolates, 17 other special forms, nonpathogenic F. oxysporum isolates from palm grove soils, and 8 other Fusarium species, the primer pair TL3-FOA28 allowed amplification of a 400-bp fragment found only in F. oxysporum f. sp. albedinis. Sequence analysis showed that one of the Fot 1 copies was truncated, lacking 182 bp at its 3' terminus. The primer pair BI03-FOA1 amplified a 204-bp fragment which overlapped the Fot 1 truncated copy and its 3' site of insertion in the F. oxysporum f. sp. albedinis genome and identified 95% of the isolates. The primer pairs BIO3-FOA1 and TL3-FOA28 used in PCR assays thus provide a useful diagnostic tool for F. oxysporum f. sp. albedinis isolates.     

Cloning and characterization of mycovirus double-stranded RNA from the plant pathogenic fungus, Fusarium solani f. sp. robiniae

A mycovirus (named FusoV) from the phytopathogenic fungus, Fusarium solani f. sp. robiniae SUF704, has two kinds of double-stranded (ds) RNA genomes, designated M1 and M2. The cDNAs were constructed from FusoV genomic dsRNAs. The sequences of M1 and M2 cDNAs comprised 1645 and 1445bp, respectively. Sequence analysis showed that each dsRNA had a single long open reading frame (ORF) on only one of the strands. M1 ORF encodes a 519-amino acid residue polypeptide with a predicted molecular mass of 60 kDa. RNA-dependent RNA polymerase-conserved motifs were identified in the predicted amino acid sequence, and the polymerase synthesized dsRNA in vitro. The M2 ORF encodes a polypeptide of 413 amino acid residues with a predicted molecular mass of 44 kDa. The predicted amino acid sequence contained the sequence corresponding to those found in the purified 44-kDa capsid protein of FusoV.     

DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora

Samples of ambient air collected with three different types of spore traps in a rural location were examined for the presence of Pneumocystis carinii by screening for P. carinii-specific DNA sequences by DNA amplification. Eleven spore trap samples were analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of P. carinii f. sp. carinii and P. carinii f. sp. hominis. The samples were collected over a 3-year period during the months of May to September, with a range of sampling times from 9 to 240 h. One air sample from an animal facility housing P. carinii-infected rats was also examined. P. carinii-specific amplification products were obtained from samples from each of the spore traps. The amplification products from eight air samples were cloned and sequenced. The majority of the recombinants from each of these samples had sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis, and a number of clones had single-base differences. These data suggest that sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis can be detected in samples of air collected in a rural location and that P. carinii may be a component of the air spora of rural Oxfordshire.     

Purification and properties of two fibrinolytic enzymes from Fusarium oxysporum N.R.C.1

OBJECTIVE: To find differences in effect on sperm motility of agents that increase intracellular cAMP: manganese ion, methyl-isobutyl-xanthine (MIX), 2-deoxyadenosine, glucose, and Mn-MIX and Mn-glucose. DESIGN: Nine men with asthenozoospermia vs. fertile donors. METHODS: Sperm was washed in Hepes-buffered saline, motility tested by laser-Doppler technique. RESULTS: Best activation was obtained with Mn and 2-deoxyadenosine; generally poor response to MIX or glucose. CONCLUSIONS: Usually, poor endogenous stimulation of adenylyl cyclase, and probably not limited energy supply, is the cause of impaired motility.     

Skin and nail infections due to Fusarium oxysporum in Tuscany, Italy

Nine cases of skin and nail infection due to Fusarium oxysporum, diagnosed in Tuscany in the period 1985-97, are described. Two manifested as interdigital intertrigo of the feet and seven as onychomycosis. All were diagnosed on the basis of repeated mycological examination, direct microscope observation and culture, as well as histological examination of biopsy specimens in two cases of intertrigo. Fragments of the fungal colonies were examined by scanning electron microscopy (SEM) for more detailed observation of fungal morphology. All patients had normal immune status and a history of the infection extending several years. Four of the patients with onychomycosis were treated with oral itraconazole, and clinical and mycological recovery was achieved in three cases. Two others were treated with cyclopyrox nail lacquer, successfully in one case. One patient with intertrigo was treated with oral itraconazole and one with oral terbinafine; both were also treated and with topical drugs, however clinical recovery was not confirmed by the mycological results.     

镰刀菌酸及其枯萎病菌培养液对棉花的毒性

[目的]了解镶刀菌酸及其枯萎病菌培养滤液对棉花感病品种及其抗性转化品系的致萎作用.[方法]用2种类型毒素制品浸泡棉花种子及幼苗,以无菌水作为CK,根据浸泡的时间不同测定种子萌发率及病情指数.[结果]25%培养滤液中镰刀菌酸的浓度为21.46μg/ml,但其处理种子萌发率却比30.00μg/ml的纯镰刀菌酸处理低,50%培养滤液镰刀菌酸的浓度为30.62μg/ml,其处理种子萌发率与50.00μg/ml的纯镰刀菌酸处理一样.病原菌培养滤液浓度不同、处理后时间不同,其病情指数均不同,随着处理后时间的延长和处理浓度的增加,病情指数也大都呈逐渐增加趋势.[结论]病原菌培养滤液对种子萌发的抑制作用要高于纯镰刀菌酸.抗性转化品系与原感病品系相比对病原菌培养滤液的抗性明显增强.     

Determination of copy number of rRNA genes in Pneumocystis carinii f. sp. hominis

Differential PCR was performed to determine the copy number of rRNA genes in Pneumocystis carinii f. sp. hominis. Two different reference genes, thymidylate synthase (TS) and beta-tubulin (BTU) genes, were used. Primers for the internal transcribed spacer (ITS) region of nuclear rRNA genes and either the TS or BTU gene were mixed together to perform PCR on seven different bronchoalveolar lavage specimens from patients with P. carinii pneumonia. The radioactivity derived from the incorporated radioactive nucleotides of each PCR product band was then used to calculate the copy number of the ITS relative to that of the TS or BTU gene. The copy number ratio between the ITS and the TS gene was determined to be 0.8, and that between the ITS and the BTU gene was also 0.8. These results suggest that the ITS has the same copy number as the TS or BTU gene. Since the copy number of the TS or BTU gene is presumed to be 1, the results also suggest that P. carinii f. sp. hominis has only one copy of the ITS and thus one copy of the nuclear rRNA genes. Therefore, two types of ITS sequences derived from a specimen would indicate that the patient is infected by two types of P. carinii f. sp. hominis.     

Identification of Pneumocystis carinii f. sp. hominis gene sequences in filtered air in hospital environments

To evaluate the risk of a nosocomial spread of Pneumocystis carinii f. sp. hominis (P. carinii hominis), air filter samples from rooms of P. carinii pneumonia (PCP) patients, adjacent corridors, and other hospital environments have been investigated for the presence of P. carinii hominis. Amplified DNA from air filters and sputum or bronchoalveolar lavage samples from the PCP patients have been genotyped with the P. carinii hominis genes of the mitochondrial large-subunit (mtLSU) rRNA and the internal transcribed spacers (ITS1 and ITS2) of the rRNA. Genotypes of the two loci were identified by direct sequencing, and for site 85 of the mtLSU locus, three allele-specific PCR assays were used. P. carinii hominis DNA was identified in the air of five of seven PCP patient rooms and in the air of two of four air filtrations from the ward corridors. The P. carinii hominis genotypes were the same in four of the five room air samples as those in the corresponding patients, suggesting a risk of person-to-person transmission of P. carinii hominis from PCP patients. Three of 16 air samples collected in infectious disease wards without the presence of PCP patients and one sample from a cardiology unit in a separate hospital building were also positive, which further strengthens the possibility of acquisition of P. carinii hominis from the environment.     

Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum

An extracellular beta-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0-6.0 and at 60 degrees C. It hydrolysed 1-->4-linked aryl-beta-glucosides and 1-->4-linked, 1-->3-linked and 1-->6-linked beta-glucosides. The apparent Km and kcat values for p-nitrophenyl beta-D-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 microM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, beta-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.     

海盐苦卤对玉米青枯病菌生长的影响

[目的]研究海盐苦卤对玉米青枯病菌生长的影响.[方法]采用菌丝生长速率法测定海盐苦卤对玉米青枯病菌的抑制率;采用叶片离体培养法测定海盐苦卤对玉米青枯病菌侵染玉米的抑制作用.[结果]随着海盐苦卤农度的降低,其对玉米青枯病菌的抑制规律性不强,表现为随着海盐苦卤浓度的降低而先降低后升高再降低,在浓度为0.005 0%时具有最佳抑制效果,抑制率为31.2%;0.050 0%海盐苦卤对玉米青枯病菌侵染玉米具有最佳的抑制作用,抑制率为44.3%.[结论]研究结果为海盐苦卤在植物病害防治方面的应用奠定了理论基础.     

Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei

Particle gun acceleration appears to be a possible way to transform mycelium cells of obligate plant parasites growing on host surfaces. GUS expression was obtained in E. graminis f.sp. hordei cells after bombardment with the GUS gene under the control of the E. graminis f.sp. hordei β-tubulin promoter. Three heterologous promoters, onefrom Aspergillus nidulans and two from Cochliobolus heterostrophus, gave very low or no expression of GUS.     

Typing of Pneumocystis carinii f. sp. hominis by single-strand conformation polymorphism of four genomic regions

To better investigate Pneumocystis carinii f. sp. hominis epidemiology, we have developed a molecular typing method. Because of the limited genetic variability of the P. carinii hominis genome, a multitarget approach was used. Four variable regions of the genome were amplified by PCR, polymorphism in each region was assessed by the single-strand conformation polymorphism (SSCP) technique, and the results for the four regions of each patient were combined. Bronchoalveolar lavage specimens collected from 11 patients were examined. Four patients were probably infected by a single strain, since their specimens yielded simple SSCP patterns (two bands corresponding to one allele). The combinations of these patterns were unique, suggesting that the strains which infected these patients were different. For the other seven patients, complex patterns were found (three or four bands corresponding to two alleles). The presence of more than one allele of a region in a patient is likely to be due to coinfection. Polymorphism was also assessed by sequencing, which revealed variations at nucleotide positions previously reported to vary. About half of the observed alleles had already been reported by laboratories in different countries. Multitarget typing of P. carinii hominis by PCR-SSCP should allow investigation of strain diversity and thus be useful for future epidemiological studies.     

Biosynthesis of bikaverin in Fusarium oxysporum. Use of 13C nuclear magnetic resonance with homonuclear 13C decoupling to locate adjacent 13C labels

Bikaverin obtained by supplementing cultures of Fusarium oxysporum with singly and doubly 13C labeled acetate was enriched by approximately 0.5 atom percent with the 13C isotope. At this low enrichment 13C NMR spectra of samples labeled from (1-13C)- and (2-13C) acetate did not show, unequivocally, the pattern of isotopic incorporation. Small sample size, poor solubility and difficulties in the assignment of resonances also restricted the amount of information thacetate. The difficulty was overcome by using 13C homonuclear single-frequency decoupling in conjunction with 1H heteronuclear decoupling to locate bonded 13C-13C pairs. The carbon skeleton of bikaverin was shown to be biosynthesized entirely by condensation of acetate units and the pattern of assembly was established.     

Detection of cross-reactive antigens shared by Fusarium oxysporum and Glycine max by indirect ELISA and their cellular location in root tissues

Pathogenicity test of Fusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium of F. oxysporum. Polyclonal antisera were raised against the mycelial suspension of F. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.     

N-terminal processing and amino acid sequence of two isoforms of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum

Cytochrome P450nor (P450nor), a nitric oxide reductase involved in the denitrifying system of the fungus Fusarium oxysporum, revealed molecular multiplicity. Two isoforms of P450nor, termed P450norA (norA) and P450norB (norB), were isolated. They had distinct isoelectric points of 5.1 (norA) and 4.9 (norB). However, their catalytic, spectroscopic, and immunological properties were almost identical. Partial amino acid sequences, involving 263 amino acid residues of norA and 278 residues of norB among 404 and 402 residues, respectively, were determined. Corresponding sequences in the isoforms were identical, and all of the determined partial sequences of norA or norB coincided with the sequence deduced from the CYP 55 gene or its cDNA. The amino acid sequence determination ruled out the possibility that there is a redox center in P450nor derived from amino acid residues, e.g., quinonoid cofactors. The only difference between norA and norB was in their N-termini. The N-terminus of norA was a threonine residue, whereas that of norB was an N-acetylated alanyl residue and norB was shorter by 2 residues than norA. The results suggested that norA and norB may be the products of the same gene, but translated from different initiation codons. The hypothetical precursor of norA would have a presequence containing targeting and sorting signals for transportation to the intermembrane space of mitochondria. This is consistent with the results of a Western-blot analysis which showed that norA was recovered only in particulate fractions, whereas norB was in the soluble fraction. It is therefore likely that the intracellular localizations as well as the N-termini of norA and norB are different, owing to the differences in the translational initiation codons and co/post-translational processings.