Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0-10.0). When studied by SDS-PAGE in the presence of beta-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30-60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Extracellular proteinases from the phytopathogenic fungus Fusarium culmorum

The growth of Fusarium culmorum fungus on a medium containing thermostable proteins from potato tubers was accompanied by the production of proteinases, exhibiting activity over a broad pH range (from 6.0–10.0). When studied by SDS-PAGE in the presence of β-mercaptoethanol, extracellular proteinases were represented by at least five species with a molecular weight of 30–60 kDa. Inhibitor analysis and studies of enzyme activities with synthetic substrates demonstrated that the culture liquid of Fusarium culmorum contained serine proteinases of various classes. The amount of subtilisin-like proteinases was the highest. A near-complete inhibition of the enzymes was caused by proteinaceous proteinase inhibitors from potato tubers. These data suggest that proteinases of the phytopathogen Fusarium culmorum serve as a metabolic target for natural inhibitors of potato proteinases.     

Formation of fusarenone X, nivalenol, zearalenone, alpha-trans-zearalenol, beta-trans-zearalenol, and fusarin C by Fusarium crookwellense.

Fusarium crookwellense KF748 (NRRL A-28100) (isolated from dry rotted potato tubers in Central Poland) produced six mycotoxins on both rice and corn substrates at 25 degrees C. The metabolites detected were zearalenone, alpha-trans-zearalenol, beta-trans-zearalenol, fusarin C, and the trichothecenes fusarenone X and nivalenol. This is the first report of formation of alpha-trans-zearalenol, beta-trans-zearalenol, fusarenone X, and nivalenol by F. crookwellense.     

Hydrolytic Activities of Fusarium solani and Fusarium solani f. sp. eumartii Associated with the Infection Process of Potato Tubers

The development of dry rot caused by Fusarium solani f. sp. eumartii was evaluated in susceptible (Huinkul) and resistant (Spunta) potato cultivars. Fungal proteolytic and polygalacturanase activities were measured at different days postinoculation either with the pathogenic F. solani f. sp. eumartii, isolate 3122 or with the non‐pathogenic F. solani, isolate 1042. After inoculation with the pathogenic fungus, proteolytic and polygalaturonase activities were higher in the susceptible than in the resistant cultivar. In addition, we found a correlation between the levels of proteolytic activity detected in the intercellular washing fluids with the size of the lesion area caused by F. solani f. sp. eumartii in Huinkul tubers. The action of the proteolytic activity over cell wall proteins of both potato cultivars was assayed. An extracellular potato protein with homology to proteinase inhibitors of the Kunitz family was identified as a substrate of the proteolytic activity in the susceptible cultivar. A microscopic study revealed differences between the potato genotypes in the rate of response to infection by F. solani f. sp. eumartii. In addition, the cell wall alteration caused by F. solani f. sp. eumartii in cortical cells of susceptible tubers was evaluated. The data with respect to the correlation between the course of cyto‐ and biochemical events of the two host–pathogen interactions were discussed.     

Formation of fusarenone X, nivalenol, zearalenone, alpha-trans-zearalenol, beta-trans-zearalenol, and fusarin C by Fusarium crookwellense

Fusarium crookwellense KF748 (NRRL A-28100) (isolated from dry rotted potato tubers in Central Poland) produced six mycotoxins on both rice and corn substrates at 25 degrees C. The metabolites detected were zearalenone, alpha-trans-zearalenol, beta-trans-zearalenol, fusarin C, and the trichothecenes fusarenone X and nivalenol. This is the first report of formation of alpha-trans-zearalenol, beta-trans-zearalenol, fusarenone X, and nivalenol by F. crookwellense.     

Analysis of intercellular washing fluids of potato tubers and detection of increased proteolytic activity upon fungal infection

We present the first procedure for extracting intercellular fluids of potato ( Solanum tuberosum L. cv. Spunta) tubers. Intercellular washing fluids were isolated from healthy and Fusarium ‐infected potato tissue. The electrophoretic pattern using SDS‐PAGE indicated differences between the fluids from the two tissues. A significant extracellular proteolytic activity was accumulated during the infection with Fusarium solani f. sp. eumartii . A major proteolytic band with an apparent molecular mass of 70 kDa and another of approximately 30 kDa were detected after separation of intercellular fluids by casein gel electrophoresis. Proteolytic activity was principally inhibited by diisopropylfluorophosphate, which is indicative of the involvement of serine protease(s). In vitro degradation assay indicated that specific potato proteins from healthy tubers were hydrolyzed by fluid proteases from infected tubers. The biological role of such activity in potato‐ Fusarium interaction is still unknown. Our results suggest that the intercellular serine protease has a fungal origin.     

A comparison of Fusarium avenaceum and Fusarium caeruleum as causes of wastage in stored potato tubers

Fusarium avenaceum is reported for the first time as a cause of rotting of potato tubers in Britain. The progress of rotting in tubers infected with F. avenaceum has been compared with dry rot due to F. caeruleum in the laboratory, clamp and potato store. Of the four varieties, Majestic, King Edward, Doon Star and Arran Pilot, tested for susceptibility, King Edward was the most susceptible to F. avenaceum and Doon Star to F. caeruleum.
Optimum temperatures for growth on potato-dextrose agar were 20-25 C. for F. avenaceum and 20 C. for F. caeruleum ; maximum temperatures were > 30 and 30 C. respectively. For infection of wounded potato tubers, cardinal temperatures for F. avenaceum were similar to those on agar, but for F. caeruleum the optimum was 15 C. and the maximum 25 C. The optimum temperature for rotting tended, with both species, to be higher in the more susceptible potato varieties. At low temperatures F. caeruleum caused quicker rotting than did F. avenaceum , even though its rate of growth on agar was scarcely more than half that of the latter.
High humidity favoured rotting especially by F. avenaceum; F. caeruleum was more tolerant of relatively low humidity. Both species caused quicker rotting in the clamp than in store, even though there was no appreciable difference in mean temperature between the clamp and the store. This was attributed to the higher atmospheric humidity in the clamp.     

向日葵花盘水提物对硫色镰刀菌及其细胞壁降解酶活性抑制特性

通过抑菌及细胞壁降解酶活性试验,研究向日葵花盘(sunflower disc,SFD)水提物对引起马铃薯干腐病主要病原菌——硫色镰刀菌(Fusarium sulphureum)生长及其侵染不同马铃薯品种时分泌的多聚半乳糖醛酸酶(polygalacturonase,PG)、果胶甲基半乳糖醛酸酶(polymethyl-ga...     

Fusarochromanone production by Fusarium isolates.

Sixty two Fusarium isolates representing nine species from many parts of the world were screened for fusarochromanone production. A simplified method for the detection of fusarochromanone in culture filtrates or grain cultures was used. Under UV irradiation (364 nm) the chloroform phase from fusarochromanone-positive culture extracts fluoresced a characteristic bright blue color. Results were confirmed by thin-layer-chromatography comparison with pure fusarochromanone standards. Detection was possible in cultures as young as 1 week old. Biosynthesis of fusarochromanone was rare in Fusarium spp. and was only detected in three isolates of Fusarium equiseti, namely R-4482 (barley [Federal Republic of Germany]), R-6137 (barley [Alaska]), and R-8508 (potato [Denmark]), among all the isolates tested from various geographic sources.     

A Cell Wall-degrading {alpha}-1,3-arabinofuranosidase Produced by the Potato Dry Rot Pathogen Fusarium caeruleum (Lib.) Sacc.

Fusarium caeruleum produced an arabanase which showed maximalactivity at pH 3·5 and which liberated arabinose andits oligomers from -l,3-arabinofuranan and isolated potato tuberparenchyma cell walls. This enzyme was most active in dry rotlesions produced in susceptible potato tubers by a fast-growingisolate of F. caeruleum. This arabanase is not of primary importancein causing tissue maceration, protoplast death or electrolyteloss but it may play a secondary role in these processes.     

Studies in the Physiology of Parasitism: XXI. The Production and Properties of Pectic Enzymes secreted by Fusarium moniliforme Sheldon

Fusarium moniliforme secreted macerating enzymes in liquid mediaonly when these contained certain natural extracts, pectic substances,or galacturonic acid. Apple extract was unsuitable for enzymesecretion and also inhibited enzyme secretion in synthetic mediaotherwise suitable. Protopectinase activity of solutions was highest in the pH range8·0–9·0, was rapidly lost at temperaturesabove 50–60° C., and was reduced by concentrationsof phosphate higher than 0·02 M. The enzyme was partiallypurified by precipitation in 60 per cent. acetone at pH 6·0. Protopectinase solutions also contained an enzyme which reducedthe viscosity of solutions of various pectic substances. Theproperties of this enzyme were, in general, similar to thoseof protopectinase. When activity of enzyme solutions was measured by the liberationof reducing groups, pectate solutions were more rapidly degradedthan were solutions of a high methoxyl pectin, particularlyin the early stages of the reaction. Paper chromatography ofthe products formed showed that pectate and pectin were degradedin different ways. Although the pathogen readily secreted protopectinase in potatoextract, potato tubers were not readily parasitized. In contrast,Fusarium avenaceum which readily attacked tubers, secreted littleprotopectinase in potato extract.     

The effect of date of haulm destruction and harvest on the development of dry rot caused by Fusarium solani var. coeruleum on potato tubers

Over 3 yr, the development of dry rot, caused by Fusarium solani var. coeruleum, and the efficacy of treating potato tubers with imazalil was examined in relation to the date of haulm destruction and harvest, and the interval between haulm destruction using diquat dibromide and harvest. The effect of these factors on skin set was also assessed. Planting inoculated seed tubers bearing small rots produced more dry rot on the daughter tubers than planting naturally contaminated seed tubers in 1992. The incidence of dry rot was higher on daughter tubers wounded by a standardised method than on those passed over a reciprocating riddle (riddling). The relationship between the two assessment methods was significantly (P<0.05) correlated in 2 out of 3 yr. Overall, the interval between haulm destruction and harvest had less effect on the incidence of dry rot on daughter tubers after riddling than the date of harvest. In 2 out of 3 yr, the incidence of dry rot on riddled tubers was least on those harvested in August and was much greater on September‐harvested tubers. On tubers harvested in October, the pattern was variable, with the incidence declining in 1 yr but increasing in the other. In the third year, the development of dry rot was similar on August‐ and September‐harvested tubers and was least on those harvested in October. Dipping tubers in imazalil gave significant reductions in dry rot although the amount of the reduction was variable and not affected by time of treatment or the amount of fungicide deposited within the range 6.7 to 19.4 mg imazalil kg^?1. Skin set at harvest, as measured by skin strength or the amount of scuff damage, increased the later the tubers were harvested but did not appear to be affected by the interval between haulm destruction and harvest.     

Production of volatile sesquiterpenes by Fusarium sambucinum strains with different abilities to synthesize trichothecenes.

Twenty-five strains of Fusarium sambucinum grown on wheat kernels were examined for trichothecene production and the synthesis of volatile sesquiterpenes. The volatiles were purged with air and collected on Tenax traps. Adsorbed compounds were eluted from the traps and injected into a gas chromatograph coupled with a mass spectrometer. Ten strains isolated from potato tubers produced high amounts of diacetoxyscirpenol and its derivatives. These strains were characterized by the production of high amounts of diverse sesquiterpenes. In 10 cultures, 19 compounds were detected, of which 6 were predominant and composed as much as 82% of the volatile sesquiterpene fraction (e.g., beta-farnesene, beta-chamigrene, beta-bisabolene, alpha-farnesene, trichodiene, and an unidentified compound). Fifteen strains isolated from various sources that did not produce trichothecenes produced much less volatile sesquiterpenes, with less chemical diversity. No more than six compounds were present in cultures. Two of these compounds were present in the toxigenic strains isolated from potatoes (beta-farnesene and acoradiene), but four were unique to the strains not producing trichothecenes (longifolene, isocaryophyllene, delta-elemene, and an unidentified one). The pattern of volatile sesquiterpenes was characteristic and distinctive for both toxic and nontoxic strains.     

Fungal Assciations: I. Synergistic Relation between Rhizoctonia solani Kuhn and Fusarium solani Snyder and Hansen in causing a Potato Tuber Rot

Rhizoctonia solani, Fusarium solani, and Phoma foveata werechosen for the study of disease caused by these fungi in differentcombinations in potato tubers. An initial Rhizoctonia infection,when followed by a Fusarium infection, gave an extensive rottingwith external pimple-like formations in some cases. This typeof rotting could not be brought about by individual infectionswith either of the two fungi, or jointly by them when Fusariumwas inoculated first. Microscopic observations of infected matureand young potato tubers showed that Rhizoctonia grew intracellularlywhen infected alone, whereas it grew inter- as well as intra-cellularlyin the successive double infection. Fusarium formed more haustorium-likestructures when inoculated alone that when it followed Rhizoctonia.The length of these structures in the double infection was greaterin mature than in young tubers. Atmospheric humidity affectedthe amount of rotting, the shape and colour of the rot, andthe morphology of the fungus in the tissue.     

Fusarochromanone production by Fusarium isolates

Sixty two Fusarium isolates representing nine species from many parts of the world were screened for fusarochromanone production. A simplified method for the detection of fusarochromanone in culture filtrates or grain cultures was used. Under UV irradiation (364 nm) the chloroform phase from fusarochromanone-positive culture extracts fluoresced a characteristic bright blue color. Results were confirmed by thin-layer-chromatography comparison with pure fusarochromanone standards. Detection was possible in cultures as young as 1 week old. Biosynthesis of fusarochromanone was rare in Fusarium spp. and was only detected in three isolates of Fusarium equiseti, namely R-4482 (barley [Federal Republic of Germany]), R-6137 (barley [Alaska]), and R-8508 (potato [Denmark]), among all the isolates tested from various geographic sources.     

The Influence of Glycoalkaloids and Impact Damage on Resistance to Fusarium solani var. coeruleum and Phoma exigua var. foveata in Potato Tubers

Potato glycoalkaloids were determined in tubers of a wide range of genotypes in order to study their influence on resistance to storage rots caused by Fusarium solani var. coeruleum and Phoma exigua var. foveata. No relationships were found. This indicates that breeders selection for low glycoalkaloid potato clones is not in conflict with the demands for high levels of resistance to these pathogens. It was also observed that genotypes susceptible to skin-breaking types of, damage were more easily infected than genotypes susceptible to other types of damage.     

Effect of chitosan for the control of potato diseases caused by Fusarium species

The antifungal activity of chitosan against Fusarium spp. was investigated based on in vitro and in vivo assays, and its possible modes of action were also explored. Chitosan applied at 4.0 g/L of acetic acid-distilled water solution significantly decreased the mycelial growth of Fusarium oxysporum, Fusarium sambucinum and Fusarium graminearum by 88.4%, 89.0% and 89.8%, respectively. Tuber treatment by chitosan (4.0 g/L) of acetic acid-distilled water solution, prior to inoculation, reduced dry rot severity induced by F. oxysporum and F. sambucinum by 60.0% and 48.2%, respectively. When tested as plant treatment, potato plants inoculated with Fusarium species, exhibited 33.5%–45.3% less wilting severity as compared to the control. This abiotic treatment improved the phenolic compounds activities and defence-related enzymes such as peroxidase and polyphenoloxidase in potato tubers inoculated with Fusarium spp. Results clearly demonstrated that chitosan could be explored as an alternative agent to chemical fungicides for the control of tuber dry rot and Fusarium wilt through induction of the plant defence system.     

Control of Fusarium solani rot of potato tubers with fungicides

Tecnazene (up to 33 ppm) and dichloran (up to 500 ppm) had little effect on germination of spores or growth of Fusarium solani isolated from and causing a rot of potato tubers; they also did not decrease rotting when applied to wounds later inoculated with the pathogen. Benomyl and thiabendazole (up to 500 ppm) also had little effect on spore germination but did greatly decrease growth at 5 ppm. A pronounced pink coloration developed in cultures growing slowly in the presence of benomyl; a similar though less striking effect appeared in agar cultures containing thiabendazole. Benomyl suspended in water or diluted with Fuller's earth gave good control of rotting when applied to wounds inoculated later with F. solani. Still better control was obtained with thiabendazole; dusts containing 1% a.i. substantially decreased rots and those containing 10 % a.i. gave almost complete control when applied to wounds shortly before inoculation. Thiabendazole was also very effective when used 24 h after inoculation and a fair measure of control was obtained when it was applied 24 h later. Benomyl and thiabendazole placed on apparently intact surfaces of tubers caused tissue 5 mm deep to become toxic to F. solani 10 days later, and, unexpectedly, this tissue prevented spore germination. Fuller's earth alone substantially decreased rotting. The results obtained suggest that dusts containing thiabendazole have some promise for the control of Fusarium rots of potato tubers, especially of early crops.     

Isolation and characterization of a potato cDNA corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene differentially activated by stress

1-Aminocyclopropane-1-carboxylate (ACC) oxidase enzyme catalyses the final step in ethylene biosynthesis, converting 1-aminocyclopropane-1-carboxylic acid to ethylene. A cDNA clone encoding an ACC oxidase, ST-ACO3, was isolated from potato (Solanum tuberosum L.) by differential screening of a Fusarium eumartii infected-tuber cDNA library. The deduced amino acid sequence exhibited similarity to other ACC oxidase proteins from several plants species. Northern blot analysis revealed that the ST-ACO3 mRNA level increased in potato tubers upon inoculation with F. eumartii, as well as after treatment with salicylic acid and indole-3-acetic acid, suggesting a cross-talk between different signalling pathways involved in the defence response of potato tubers against F. eumartii attack.     

Dryopteris crassirhizoma Dryocrassin ABBA for Postharvest Control of the Potato Dry Rot Pathogen Fusarium solani var. coeruleum

Dryopteris crassirhizoma dryocrassin ABBA treatment was tested for its effectiveness in controlling Fusarium solani var. coeruleum growth in vitro and for prevention of postharvest dry rot of potato tubers and slices. Dryocrassin ABBA strongly inhibited mycelial growth, resulting in reductions in both mycelium dry weight and per cent spore germination of F. solani var. coeruleum at concentrations of 2.0, 0.5 and 0.1 mg/ml. Scanning electron microscopy (SEM) observations showed that treatment induced abnormal, tightly twisted morphological changes in hyphae. Moreover, in vivo experiments demonstrated that dryocrassin ABBA treatment at 2 mg/ml effectively controlled dry rot of potato tubers inoculated with mycelial discs of F. solani var. coeruleum. After dryocrassin ABBA treatment, the content of soluble proteins, peroxidase (POD) and superoxide dismutase (SOD) activity increased, and malondialdehyde (MDA) content remained the stable situation. In addition, the expression level of plant lipid‐transfer proteins (LTPs) genes – StLTPa1, StLTPa7, StLTPb1 and StLTPb3 – was upregulated. These results collectively demonstrate that dryocrassin ABBA can inhibit growth of Fusarium pathogens to induce disease resistance. On the other side, dryocrassin ABBA maybe induce potato defence responses.