Races and virulence of Fusarium oxysporum f.sp. cubense on local banana cultivars in Kenya

Virulence of 31 Kenyan isolates of Fusarium oxysporum obtained from bananas showing symptoms of Panama disease was tested against the differential banana cvs Bluggoe, Gros Michel, Dwarf Cavendish, and two other local cvs Muraru and Wang'ae. Seventeen isolates were assigned to either race 1 or race 2 of F. oxysporum f.sp. cubense (FOC). Race 4 was not apparent in this sample of 31 isolates from Kenya as none were pathogenic to cv. Cavendish, and no wilted Cavendish have been observed in field surveys in Kenya. Races could not be assigned to 12 isolates as they were virulent on more than one differential cultivar, and two were apparently not pathogenic. All isolates assigned to races 1 and 2 belonged to the VCG bridging complex 0124/5/8/20, but some other isolates belonging to this VCG complex could not be assigned to race. All five isolates assigned to VCG 01212 could not be assigned to known races. Considerable variability thus exists within FOC isolates within this region. Local cultivars of banana showed differential resistance to the pathogen. The interaction of cultivars and isolates on the level of disease was significant. Overall, cv. Wang'ae was the most susceptible to most of the isolates tested, regardless of their race, and could therefore be used as a reference cultivar in pathogenicity tests of isolates of FOC in the East African region. Of the cultivars tested that are widely grown on smallholder farms in Kenya, Muraru was the least susceptible.     

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.     

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.     

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.     

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.     

Genetic Diversity and Pathogenicity of Fusarium oxysporum f.sp. melonis Races from Different Areas of Italy

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis (FOM) is a devastating disease of melon worldwide. Pathogenicity tests performed with F. oxysporum isolates obtained from Italian melon‐growing areas allowed to identify thirty‐four FOM isolates and the presence of all four races. The aims of this work were to examine genetic relatedness among FOM isolates by race determination and to perform phylogenetic analyses of identified FOM races including also other formae speciales of F. oxysporum of cucurbits. Results showed that FOM race 1,2 was the most numerous with a total of eighteen isolates, while six and nine isolates were identified as race 0 and 1, respectively, and just one isolate was assigned to race 2. Phylogenetic analysis was performed by random amplified polymorphic DNA (RAPD) profiling and by translation elongation factor‐1α (TEF‐1α) sequencing. The analysis of RAPD profiles separated FOM races into two distinct clades. Clade 1, which included races 0, 1 and 1,2, was further divided into ‘subclade a’ which grouped almost all race 1,2 isolates, and into ‘subclade b’ which included race 0 and 1 isolates. Clade 2 comprised only race 2 isolates. The phylogenetic analysis based on TEF‐1α separated FOM from the other formae speciales of F. oxysporum. Also with TEF‐1α analysis, FOM races 0, 1 and 1,2 isolates grouped in one single clade clearly separated from FOM race 2 isolates which grouped closer to F. oxysporum f.sp. cucumerinum. RAPD technique was more effective than TEF‐1α in differentiating FOM race 1,2 isolates from those belonging to the closely related races 0 and 1. Both phylogenetic analyses supported the close relationship between the three different FOM races which might imply the derivation from one another and the different origin of FOM race 2.     

Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Comparison of Fusarium oxysporum and Fusarium oxysporum var. redolens by Analyzing the Isozyme and Serological Patterns

Extracts from Fusarium oxysporum (F.o.) and F. oxysporum var. redolens (F.o.r.) isolates were compared by means of electrophoresis and crossed immunoelectrophoresis. The polymorphism of five isozyme systems allowed a distinction between F.o. and F.o.r. isolates. The isozyme patterns of three other isozyme systems did not allow this distinction between F.o. and F.o.r. to be made. Both fungi appeared almost identical serologically. Relative amounts of their corresponding proteins differed but the qualitative patterns of the proteins were nearly the same with the anti-F.o.r. serum, only one specific antigen was detected in the extracts from F.o.r., isolates. Although the results obtained indicate a strong similarity between F.o. and F.o.r., they are not sufficient for an unequivocal statement that the fungi belong to the same species.     

Fungus gnats vector Fusarium oxysporum f.sp. radicislycopersici1

Fungus gnat adults transported Fusarium oxysporum f.sp. radicis-lycopersici from Petri dish culture and infected host plants to the roots and hypocotyls of healthy tomato and bean plants. The source of the fungus did not affect the ability of fungus gnats to transport the fungus to healthy hosts. The presence of fungus gnat larvae in media in which young tomato plants were grown did not increase the incidence of plant infection by the pathogen. Fungus gnat adults appear to aid in the dissemination of F. oxysporum f.sp. radicis-lycopersici.     

Quantitative and Microscopic Assessment of Compatible and Incompatible Interactions between Chickpea Cultivars and Fusarium oxysporum f. sp. ciceris Races

Background

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris, a main threat to global chickpea production, is managed mainly by resistant cultivars whose efficiency is curtailed by Fusarium oxysporum f. sp. ciceris races.

Methodology

We characterized compatible and incompatible interactions by assessing the spatial-temporal pattern of infection and colonization of chickpea cvs. P-2245, JG-62 and WR-315 by Fusarium oxysporum f. sp. ciceris races 0 and 5 labeled with ZsGreen fluorescent protein using confocal laser scanning microscopy.

Findings

The two races colonized the host root surface in both interactions with preferential colonization of the root apex and subapical root zone. In compatible interactions, the pathogen grew intercellularly in the root cortex, reached the xylem, and progressed upwards in the stem xylem, being the rate and intensity of stem colonization directly related with the degree of compatibility among Fusarium oxysporum f. sp. ciceris races and chickpea cultivars. In incompatible interactions, race 0 invaded and colonized ‘JG-62’ xylem vessels of root and stem but in ‘WR-315’, it remained in the intercellular spaces of the root cortex failing to reach the xylem, whereas race 5 progressed up to the hypocotyl. However, all incompatible interactions were asymptomatic.

Conclusions

The differential patterns of colonization of chickpea cultivars by Fusarium oxysporum f. sp. ciceris races may be related to the operation of multiple resistance mechanisms.     

西瓜枯萎病菌的快速检测与鉴定

通过西瓜枯萎病菌与其他专化型枯萎病菌及瓜类几种重要病原菌的比较基因组分析,获得了西瓜枯萎病菌的基因组特异序列。在此基础上,设计出特异引物,筛选可扩增出西瓜枯萎病菌特异性DNA条带的引物。将特异性引物和尖孢镰刀菌专化型的通用引物W106R/W106S结合,建立双重PCR检测体系。该双重PCR检测体系可以在一次PCR反应中快速、准确的检测出西瓜枯萎病菌,为通过分子方法快速鉴定西瓜枯萎病菌提供技术支持。     

Two granular formulations of Fusarium oxysporum f.sp. orthoceras to mitigate sunflower broomrape Orobanche cumana

Fusarium oxysporum Schlecht. f.sp. orthoceras (Appel & Wollenw.) Bilai, a potential biocontrol agent against Orobanche cumana Wallr.,was formulated into two granular forms, wheatflour kaolin (`Pesta') granules and sodium alginatepellets. The formulations were compared in terms ofeffectiveness for mitigating O. cumanaparasitism in sunflower and shelf-life forstorage. `Pesta' granules reduced the emergence of O. cumana shoots by 64% while sodium alginatepellets did not reduce the emergence rate but increased thepercentage of diseased O. cumana plants.Calculated efficacy of the application was better for`Pesta' granules. Viability of the formulatedmaterial tested in the laboratory was higher in sodium alginatepellets than in the `Pesta' formulation.However, a loss of virulence after six months of storage wasalso observed in sodium alginate pellets in agreenhouse experiment.     

Lectins Specificity to Pathogenesis of Fusarium oxysporum f. sp. vasinfectum

Lectins of cotton were isolated from either resistant or susceptible seed cultivars. In agar-gel double diffusion tests, positive reactions took place between lectins of cotton cultivars and the antisera of their corresponding wilt Fusaria. The number of precipitin bands correlated with the degree of susceptibility of the tested cultivars. On the other hand, no visible reaction was detected when these antisera were subjected to react with either the resistant host or nonhost lectins.     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

Temperature-induced alterations in phospholipids of Fusarium oxysporum f. sp. lycopersici.

Ten phospholipids were identified in hyphal membrane preparations of Fusarium oxysporum f. sp. lycopersici when the cells were grown to the late log phase at 15, 25, and 37 degrees C, respectively. The major phospholipids present were phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which together made up about 70% of the total membrane phospholipids. The degree of unsaturation in the acyl group of the phospholipids was inversely related to growth temperature. The polar head group composition was also affected by growth temperature. Cells grown at 15 and 25 degrees C contained the same relative proportions of PC and PE, but when the growth temperature was raised to 37 degrees C, the ratio of PC to PE was doubled. A methylating system capable of converting PE to PC was demonstrated in vitro.     

应用PCR-RFLP和巢式PCR检测黄瓜尖镰孢菌

以3株黄瓜尖镰孢菌(Fusarium oxysporum f.sp.cucumarinum)、23株镰孢菌属(Fusariumspp.)真菌和分离自土壤的20株真菌、6株细菌和7株放线菌为材料,采用化学裂解法提取总DNA,进行PCR-RFLP和巢式PCR检测,试验证明PCR-RFLP程序不能完全区分Fusarium属内不同种,而巢式PCR对黄瓜尖镰孢菌具有特异性.运用优化的PCR-RFLP和巢式PCR检测程序对染病黄瓜组织进行了检测,结果表明,两种方法均可在接种发病早期(未显症时)检测出黄瓜枯萎病菌,PCR-RFLP在感病品种接种后3d即可检测到病原菌,而巢式PCR在接种后5d才能检测到病原菌.     

Antifungal activity of alkyl and heterocyclic aza-derivatives of gossypol as well as their complexes with NaClO4 against Fusarium oxysporum f. sp. lupini

Eight alkyl and six heterocyclic aza-derivatives of gossypol (2–15) have been synthesized using gossypol (1) extracted from Gossypium Herbaceum cottonseeds. The ability of gossypol aza-derivatives to form complexes with NaClO₄ has been investigated by electrospray ionisation (ESI) mass spectra recorded in the positive and negative ion detection modes. The gossypol aza-derivatives have been characterized by FT-IR, ¹H and ¹³C NMR spectroscopic methods and subsequently tested for their antifungal properties against Fusarium oxysporum. Four alkyl aza-derivatives (2–5), present in the enamine–enamine tautomeric form, have shown activity comparable or higher than that of gossypol against this fungus. To improve the antifungal activity the complexes of the most active compounds 2–5 with NaClO₄ were prepared. Complexes of 2 and 5 with NaClO₄ have shown antifungal activity higher than that of the uncomplexed compounds.     

Inoculum sources of Fusarium oxysporum f.sp. asparagi in asparagus production

Fusarium oxysporum f.sp. asparagi (Foa) incites crown and root rot of asparagus which causes early decline of asparagus plantings. The aim of the present study was to identify the main inoculum sources of the pathogen in the Netherlands. As has been reported for foreign seed lots, Dutch seed lots can be infested with Foa at low levels. We found that seed infestation occurs mainly during the seed harvesting process through infested soil adhering to fallen berries. Soil samples from 59 fields without a history of asparagus growing and differing in their distance from asparagus plantings were tested for infestation with Foa, using a bioassay with asparagus as a bait plant. A high correlation was found between the incidence of infestation and proximity to asparagus fields; Foa was found in 69% of the samples from fresh fields in an asparagus production centre, and in only 6% of the samples from fields at a distance of 1 km and more from asparagus fields and outside a production centre. To evaluate planting material as an inoculum source of Foa, 49 lots of one-year-old crowns from 23 nurseries were collected and rated for disease symptoms. Infestation was found to be common with only two lots free of symptomatic plants. Most of the lots had more than 75% of symptomatic plants. Although most of the plants were infested, they showed only slight root rot symptoms. The procedure for production of Foa-free planting material is discussed. Persistence and infestation of asparagus root residues in former asparagus fields was assessed by retrieving the residues from eight former asparagus fields with an asparagus-free period of one to 25 years, and three fields with a standing asparagus crop. Even after an asparagus-free period of 25 yr asparagus root residues were retrieved from soil, although at low levels. Mean population densities of Fusarium spp. declined from 2 times 10⁶ to 1 times 10⁵ colony forming units g^_1 air-dry root tissue during the first 10 years and were still > 10⁴ c.f.u. g“¹ air-dry root tissue 20 to 25 yr after asparagus produced was stopped. The population was dominated by F. oxysporum. Eighty-three of the 112 isolates (74%) of F. oxysporum belonged to the forma specialis asparagi. The proportion of Foa in the population did not decrease in time. It was concluded that persistence of Foa in asparagus root residues is a major reason for its long-term survival.