Vegetative compatibility and heterokaryon stability in Fusarium oxysporum f.sp. radicis-lycopersici from Italy

Fusarium crown and root rot, caused by Fusarium oxysporum f.sp. radicis-lycopersici ( Forl ), is one of the most destructive soilborne diseases of tomato in Italy. Chlorate-resistant, nitrate-nonutilizing ( nit ) mutants were used to determine vegetative compatibility among 191 isolates of Forl collected in five geographic regions (Calabria, Emilia-Romagna, Liguria, Sardinia, Sicily) in Italy. The isolates were assigned to five vegetative compatibility groups (VCGs): 65 isolates to VCG 0090; 99 to VCG 0091; 23 to VCG 0092; two to VCG 0093; and two to VCG 0096. The population structure of Forl in Italy is similar to that reported for Israel, and differs from that found in North Atlantic European countries, where VCG 0094 is predominant. The stability of prototrophic heterokaryons originating from hyphal anastomosis between compatible complementary nit mutants was assessed through conidial analysis and mycelial mass transfer. Most monoconidial cultures (84%) recovered from 117 prototrophic heterokaryons were nit mutants, indicating that heterokaryons generally do not proliferate well through conidiation; most of the 177 prototrophic heterokaryons examined were unstable, and only 9% sustained prototrophic growth through the tenth mycelial transfer upon subculturing. The prototrophic growth is proposed to be maintained through restoration of the heterokaryotic state by continual anastomosis between adjacent homokaryotic hyphae. Since heterokaryosis is a prerequisite for parasexual recombination, we speculate that this mechanism is unlikely to play a major role in generating the VCG diversity found among Forl or other strains of F. oxysporum.     

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.     

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.     

Population Genetic Analysis Corroborates Dispersal of Fusarium oxysporum f. sp. radicis-lycopersici from Florida to Europe

ABSTRACT Fusarium oxysporum isolates from tomato plants displaying crown and root rot symptoms were collected in central and southern Florida and analyzed using vegetative compatibility grouping (VCG) and nuclear restriction fragment length polymorphism (RFLP) data. VCG 0094 of F. oxysporum f. sp. radicis-lycopersici, previously known only from northwestern Europe, was predominant among 387 isolates assessed. In addition, two newly described VCGs (0098 and 0099) were detected at low frequencies. Floridian VCG 0094 isolates displayed a continuum of compatibilities, which is in contrast to the three distinct subgroups previously identified among European VCG 0094 isolates. RFLP haplotypes were constructed using one repetitive and three low-copy probes. Population subdivision of VCG 0094 from various Floridian counties and from northwestern Europe (Belgium, the Netherlands, and the United Kingdom) was evaluated by analysis of molecular variance. A "natural" population structure was revealed, differentiating populations from the east and west coasts of Florida. In addition, isolates from Europe were statistically indistinguishable from the Palm Beach County, FL, population. Furthermore, gene diversity among Palm Beach County VCG 0094 isolates was more than five times greater than among European isolates. Results from both VCG and RFLP analyses strongly support the inference that the European VCG 0094 constitutes a founder population that resulted from intercontinental migration of a few isolates from Palm Beach County, FL.     

Disease Development Following Infection of Tomato and Basil Foliage by Airborne Conidia of the Soilborne Pathogens Fusarium oxysporum f. sp. radicis-lycopersici and F. oxysporum f. sp. basilici

ABSTRACT Fusarium oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato, and F. oxysporum f. sp. basilici, the causal agent of Fusarium wilt in basil, are soilborne pathogens capable of producing conspicuous masses of macroconidia along the stem. The role of the airborne propagules in the epidemics of the disease in tomato plants was studied. In the field, airborne propagules of F. oxysporum f. sp. radicis-lycopersici were trapped with a selective medium and their prevalence was determined. Plants grown in both covered and uncovered pots, detached from the field soil, and exposed to natural aerial inoculum developed typical symptoms (82 to 87% diseased plants). The distribution of inoculum in the growth medium in the pots also indicated the occurrence of foliage infection. In greenhouse, foliage and root inoculations were carried out with both tomato and basil and their respective pathogens. Temperature and duration of high relative humidity affected rate of colonization of tomato, but not of basil, by the respective pathogens. Disease incidence in foliage-inoculated plants reached 75 to 100%. In these plants, downward movement of the pathogens from the foliage to the crown and roots was observed. Wounding enhanced pathogen invasion and establishment in the foliage-inoculated plants. The sporulation of the two pathogens on stems, aerial dissemination, and foliage infection raise the need for foliage protection in addition to soil disinfestation, in the framework of an integrated disease management program.     

Population structure of Fusarium oxysporum f. sp. radicis-lycopersici in Florida inferred from vegetative compatibility groups and microsatellites

Fusarium crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL) is a serious soilborne disease reducing tomato yields in Florida, a leading state in fresh market tomato production in the United States. One hundred and twenty five isolates of FORL were collected from the three main tomato-growing counties in Florida between 2006 and 2008. Vegetative compatibility groups (VCGs) and 10 microsatellite loci were used to infer the population structure of FORL. Up to 69.8 % of the isolates could be assigned to one of three VCGs, 0094, 0098 or 0099, with frequencies of 38.6, 24.4, and 6.8 % respectively. A medium level of population differentiation (Φst?=?0.159) was detected among the three counties, and three optimal clusters (populations) were supported by discriminant analysis of principal components. In addition, each population had some individuals that likely migrated from other populations. Migration probably played an important role in shaping the population structure of FORL since repeated VCGs and multilocus genotypes were observed in the three counties. Considerable migrants (> 1.33 migrants per generation) were also detected between the three counties, resulting in an increase in the effective population size and genetic diversity of F. oxysporum f. sp. radicis-lycopersici. Although migration is an important evolutionary force, mutation and parasexual recombination could not be completely ruled out as contributing causes to the genetic diversity of FORL. Management strategies that limit the movement of F. oxysporum f. sp. radicis-lycopersici are necessary to reduce the evolutionary potential of the pathogen.     

Dispersal of Formulations of Fusarium oxysporum f. sp. erythroxyli and F. oxysporum f. sp. melonis by Ants

ABSTRACT A natural epidemic of Fusarium wilt on coca (Erythroxylum coca) in Peru prompted the suggestion of possibly using the pathogen Fusarium oxysporum f. sp. erythroxyli as a mycoherbicide against this narcotic plant. During field trials conducted in Kauai, HI, to test the pathogenicity of the coca wilt pathogen, ants were observed removing formulations from test plots. While removal of formulations by ants was considered detrimental with respect to conducting field tests, ant removal was considered potentially beneficial in disseminating the mycoherbicide. Thus, research was initiated to assess the ability of formulation additives to alter the preference of ants for the formulated mycoherbicide. In Hawaii, preference of indigenous ants for removing formulations was tested using three different food bases (rice, rice plus canola oil, and wheat flour [gluten]). Similar tests were conducted at Beltsville, MD, using F. oxysporum f. sp. melonis, in which the formulation based on wheat flour was replaced by a formulation based on canola meal. Formulations based on wheat were preferred by ants in both locations; up to 90% of the wheat plus rice flour granules (C-6) and the wheat gluten plus kaolin granules (pesta) were removed within 24 h, while only 20% of those containing rice without oils were taken. However, when either canola, sunflower (Maryland only), or olive oil was added to the rice formulation, up to 90% of the granules were taken. The formulation based on canola meal was less attractive to ants, as only 65% of the granules were removed within a period of 24 h. Ants showed no preference with respect to presence or absence of fungal biomass. To alter the attractiveness of the C-6 formulation to ants, C-6 was amended with three natural products. Canna and tansy leaves were added to C-6 at a ratio of 1:5 (wt/wt), while chili powder was added at 1:25 or 1:2.5 (wt/wt). Canna, tansy, and the higher rate of chili powder significantly reduced the number of C-6 granules removed by ants. Canna and tansy leaves affected neither germination nor sporulation of the mycoherbicide, while the high concentration of chili powder reduced viability of propagules in the formulation. More F. oxysporum f. sp. erythroxyli-type colonies were recovered from inside ant nests (9 cm depth) than from nest surfaces, indicating that ants may distribute the mycoherbicide in the soil profile. Ants passively carried propagules of F. oxysporum f. sp. erythroxyli outside their bodies, as well as either very closely adhering to the outside or within their bodies.     

Genetic diversity in Fusarium oxysporum f.sp. dianthi and Fusarium redolens f.sp. dianthi

Pathogenic isolates were selected representing all known vegetative compatibility groups (VCGs) and races of Fusarium oxysporum sensu lato from Dianthus spp. On basis of differences in the internal transcribed spacer region of the ribosomal DNA, six VCGs were classified as F. oxysporum f.sp. dianthi and four as F. redolens f.sp. dianthi. All VCGs of F. oxysporum f.sp. dianthi were characterized by unique restriction fragment length polymorphisms (RFLPs), unique overall esterase profiles, and unique virulence spectra, supporting a clonal lineage concept. Two VCGs of F. oxysporum f.sp. dianthi nevertheless comprised more than one race, but races within the same VCG shared the same distinct overall virulence spectrum. VCGs belonging to F. redolens f.sp. dianthi also had unique RFLPs and unique virulence spectra, but had grossly identical esterase profiles. Three new races (9, 10 and 11) are described for F. oxysporum f.sp. dianthi, and four for F. redolens f.sp. dianthi. Two races previously considered lost were recovered; race 7 was identified as a member of VCG 0021 of F. oxysporum f.sp. dianthi while race 3 was identified as a distinct VCG and race of F. redolens f.sp. dianthi. A summary of races and VCGs in F. oxysporum f.sp. dianthi and F. redolens f.sp. dianthi is presented.     

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.     

Biological control of Fusarium oxysporum f. sp. lycopersici

Fungi known to produce lytic enzymes were used in an attempt to control wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici (FOL). Some of the fungal species (Penicillium oxalicum, Penicillium purpurogenum and Aspergillus nidulans) damaged hyphae of FOL in vitro and reduced the numbers of microconidia in the soil. Treatments with fungi did not result in a reduction in either chlamydospores of FOL in soil or populations of FOL in the rhizosphere of tomato. P. oxalicum was the most effective agent of biocontrol, and it reduced disease severity in both non-autoclaved (20% decrease) and sterile soil. In sterile soil, P. oxalicum reduced disease with different levels of severity (27% decrease at high levels and 50% decrease at low levels). Disease control by A. nidulans and P purpurogenum was only achieved when disease severity was low in sterile soil (55% and 45%, respectively).     

Vegetative compatibility grouping in Fusarium oxysporum f.sp. radicis-lycopersici from the UK, the Netherlands, Belgium and France

The population structure of Fusarium oxysporum f.sp. radicis-lycopersici ( F.o.r.l .), the causal agent of crown and root rot disease in tomato, was studied using the vegetative compatibility grouping approach. Four vegetative compatibility groups (VCGs) were identified among 37 isolates from the UK, the Netherlands, Belgium and France. Three of these VCGs (0090, 0091, 0094) had already been described, whereas VCG 0097 was new. VCG 0094 was dominant in the UK, the Netherlands and Belgium, but not in France. The opposite was true for the cosmopolitan VCG 0091, while the cosmopolitan VCG 0090 was only found in France. Based on hyphal interactions, VCG 0094 was divided into three subgroups, each comprising isolates from at least two countries. One isolate of VCG 0094 did not belong to any of these subgroups, suggesting further variability in this VCG. Isolate FORL-19R from France, previously assigned to VCG 0090 I, was reassigned to VCG 0090 III, a new subgroup of VCG 0090 found in Israel. FORL-19R and additional members of its subgroup manifest cross-VCG compatibility between VCG 0090 and VCG 0092. Along with previous studies, the multiple VCGs and subgroups found among F.o.r.l . in western Europe demonstrate a high level of genetic diversity in this pathogen.     

农杆菌介导的西瓜枯萎病菌遗传转化

为获得带GFP标记的西瓜枯萎病菌转化株,用于后期观察病原菌侵染过程,采用农杆菌介导的方法,对西瓜枯萎病菌1号生理小种进行了遗传转化。结果表明:共培养时间为36h,枯萎病菌孢子和农杆菌AGL1比例为1∶1时该菌株的遗传转化效率最高,可以达到117.33个转化子/107个孢子。转化株的孢子、菌丝体及萌发的孢子均能发出稳定而强的绿色荧光。转化株的致病力检测显示其致病力与转化前的野生菌株致病力无明显差异。结果表明本研究获得的带GFP标记的西瓜枯萎病菌转化株可用于观察病菌在西瓜根系的侵染过程。     

Sensitivity of Fusarium oxysporum f. sp. cubense to phosphonate

Phosphonate (0.1 mM) significantly reduced growth of Fusarium oxysporum f. sp. cubense (Foc) race 4 grown at an optimal phosphate concentration of 0.3 mM in vitro. At higher phosphate concentrations, closer to physiological conditions within the plant, the sensitivity of Foc race 4 to phosphonate was greatly reduced, with 25 mM phosphonate required to reduce growth by 50% at 1 mM phosphate. Two isolates of Fusarium oxysporum f. sp. dianthi and another race of Foc, race 1, were shown to be similar to Foc race 4 in their sensitivity to phosphonate, while another species of Fusarium, F. avenaceum , was more sensitive to phosphonate in vitro.     

Genetic diversity in Fusarium oxysporum f. sp. melonis*

Fusarium oxysporum f. sp. melonis is an important vascular wilt pathogen of melon. Races 1, 2 and 1–2 of this fungus have been identified in Portugal by pathogenicity tests with appropriate hosts. The aim of this research was to examine the relationships between different races of F. o. melonis of Portuguese and French origin through analysis of random amplified polymorphic DNAs (RAPDs). DNA fingerprint profiles were developed for all the accessions. Each isolate showed 5–10 DNA bands with each of the 16 primers employed. A total of 126 bands was obtained. The size of amplified DNA fragments generated with these primers ranged from 0.5 to 3.2 kb. A phenogram based on the Jaccard coefficient of similarity was computed by the unweighed pair group method using arithmetic averages (UPGMA). It was found that Portuguese race 2 is very similar to French race 1, while French race 2 is the most dissimilar being clearly separated from all other races. The genetic diversity of these isolates is also being studied for vegetative compatibility by using the nit mutant system.