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.     

Phylogenetic relationships of Fusarium oxysporum f. sp. melonis in Iran

Fusarium wilt of melon caused by Fusarium oxysporum f. sp. melonis is a destructive fungal disease in melon growing regions. Isolates of F. oxysporum obtained from six major melon producing provinces in Iran, from melons and other hosts, were characterized based on pathogenicity to melon, vegetative compatibility groups (VCGs) and nuclear ribosomal DNA intergenic spacer (IGS) sequencing. Thirty-four of 41 isolates from Iran in this study were identified as race 1,2 which belonged to either VCG 0134 or an unassigned VCG, which based on IGS sequencing grouped with the VCG 0135 tester isolate. The seven remaining isolates were identified as nonpathogenic to melon belonging to two undescribed VCGs. Based on sequence analyses of the IGS region of Iranian and foreign isolates, nine lineages were identified, each including one VCG. The separation of VCGs into distinct lineages based on IGS sequences is mostly consistent with Repetitive extragenic palindromic PCR (Rep-PCR) results. Exceptions are VCGs 0130 and 0131, which could be differentiated with IGS sequences, but not with Rep-PCR. Different races from the USA, France and Iran associated with VCG 0134 grouped into one IGS lineage but could be differentiated with Rep-PCR, suggesting that this VCG is more diverse than previously thought. Given the long history of melon cultivation in Iran and the Rep-PCR diversity of isolates belonging to this VCG, it could be speculated that VCG 0134 perhaps evolved in Iran.     

Physiological races and vegetative compatibility groups within Fusarium oxysporum f.sp. gladioli

The pathogenicity and vegetative compatibility of mainly Dutch isolates ofFusarium oxysporum collected from diseased gladioli and other Iridaceae were investigated. Based on their pathogenicity to two differential gladiolus cultivars, the isolates could tentatively be divided into two races. All self-compatible isolates ofFusarium oxysporum f.sp.gladioli belonged to one of three distinct vegetative compatibility groups, VCG 0340, 0341 or 0342, and were incompatible with isolates that were not pathogenic to gladiolus. Isolates of one of the two races were restricted to one VCG while isolates of the other race were present in all three VCGs.     

Occurrence of Fusarium oxysporum f. sp. melonis Race 1 in Japan

In 1994, Fusarium wilt of melon cultivars which are resistant to races 0 and 2 of Fusarium oxysporum f. sp. melonis was observed in southern area of the Lake Biwa region, Shiga prefecture. In commercial fields, mature plants of cv. Amus which were grafted onto cv. Enken Daigi 2, and of cv. FR Amus showed yellowing, wilting and finally death before harvesting of fruits. Diseased plants had vascular and root discolorations, and their stem sections yielded typical colonies of F. oxysporum. When the Shiga strains were tested for their pathogenicity to 12 species of cucurbits, they caused wilts only on melon. Using race differential cultivars of melon, the Shiga strains were classified as race 1 of F. oxysporum f. sp. melonis, which has not been reported in Japan. To further characterize their pathogenicity, the strains were used to inoculate 46 additional cultivars of melon, oriental melon and oriental pickling melon. All the race 1 strains were pathogenic to the cultivars tested, and their host range was apparently different from those of strains belonging to other races (races 0, 2 and 1,2y). DNA fingerprinting with a repetitive DNA sequence, FOLR3, differentiated race 1 strains from strains of races 0 and 2, but not from race 1,2y strains. Received 2 July 1999/ Accepted in revised form 30 September 1999     

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.     

Isolation of Pathogenicity Mutants of Fusarium oxysporum f. sp. melonis by Insertional Mutagenesis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to isolate mutants of Fusarium oxysporum f. sp. melonis impaired in pathogenicity. The race 2 strain Mel02010 was transformed with linearized pSH75, conferring resistance to hygromycin B, with or without the enzyme used to linearize the plasmid. Addition of restriction enzymes did not affect the transformation frequency. A total of 2929 REMI transformants were tested for pathogenicity to three melon cultivars, Amus, Ogon 9 and Ohi. The race 2 strains are pathogenic to Amus and Ogon 9, but not to Ohi. Of 43 transformants with reduced pathogenicity on susceptible melon cultivars, 12 mutants were examined in detail for pathogenicity, vegetative growth and integrative mode of pSH75. The levels of pathogenicity varied among these mutants. Two mutants (B48 and B137) almost completely lost pathogenicity to both susceptible cultivars, and the others had reduced pathogenicity. Mutants B48, B241, B886 and X36 were also impaired in vegetative growth. Mutant B809 was a biotin auxotroph. By DNA gel blot analysis, nine mutants were found to contain a single copy of the transformation vector. These mutants may thus be useful in isolating genes involved in pathogenicity. Received 22 December 2000/ Accepted in revised form 16 April 2001     

香蕉枯萎病菌生理分化研究摘要本研究

本研究对香蕉枯萎病菌菌株FOCAAA9(来自香蕉)和FOCABB1(来自粉蕉)进行培养试验和接种试验;在含粉蕉和香蕉组织浸提液的培养基上2个菌株的培养性状、菌丝生长速度、孢子形态、大小型孢子比率和产孢量显示出差异;接种结果FOCAAA9能侵染香蕉(MusaAAA)品种巴西蕉、红香蕉和台蕉引起枯萎病,而FOCABB1对3个香蕉品种无致病性。研究结果表明侵染香蕉和粉蕉的古巴尖镰孢[Fusariumoxysporumf.sp.cubense(E.F.Smith)Snyder]存在生理分化现象。     

Pathogenic and Genetic Variation in the Japanese Strains of Fusarium oxysporum f. sp. melonis

ABSTRACT Pathogenic variation among 41 Japanese strains of Fusarium oxysporum f. sp. melonis was analyzed by pathogenicity tests with muskmelon, oriental melon, and oriental pickling melon cultivars. Based on pathogenicity to muskmelon cvs. Amus and Ohi and oriental melon cv. Ogon 9, 41 strains were divided into 3 groups that corresponded completely to Risser's races 0, 2, and 1,2y. To further characterize pathogenic variation within the forma specialis and races, strains were assayed for pathogenicity to 42 additional muskmelon, oriental melon, and oriental pickling melon cultivars. All strains of race 1,2y were pathogenic to all cultivars tested. Strains of race 0 were divided into six variants based on differences in pathogenicity to three muskmelon cultivars; strains of race 2 also were classified into six variants based on differences in pathogenicity to two muskmelon cultivars and one oriental melon cultivar. Genetic variation among strains was analyzed by DNA fingerprinting with four repetitive DNA sequences: FOLR1 to FOLR4. Thirty-six fingerprint types were detected among forty-one strains by pooling results of fingerprinting with four probes. Cluster analysis showed distinct genetic groups correlated with races: the fingerprint types detected in each of races 2 and 1,2y were grouped into a single cluster, and two distinct genetic groups were found in race 0. However, pathogenic variation detected within races 0 and 2 could not be differentiated based on the nuclear markers examined.     

香蕉枯萎病菌生理分化研究

本研究对香蕉枯萎病菌菌株FOCAAA9（来自香蕉）和FOCABB1（来自粉蕉）进行培养试验和接种试验；在含粉蕉和香蕉组织漫提液的培养基上2个菌株的培养性状、菌丝生长速度、孢子形态、大小型孢子比率和产孢量显示出差异；接种结果FOCAAA9能侵染香蕉（Musa AAA）品种巴西蕉、红香蕉和台蕉引起枯萎病，而FOCABB1对3个香蕉品种无致病性。研究结果表明侵染香蕉和粉蕉的古巴尖镰孢[Fusarium oxysporum f．sp．cubeilse（E．F．Smith）Snyder]存在生理分化现象。     

Physiological races and vegetative compatibility groups of butterhead lettuce isolates of Fusarium oxysporum f. sp. lactucae in Japan

Races were identified among butterhead lettuce isolates of Fusarium oxysporum f. sp. lactucae collected from three geographical areas of Hokkaido, Shizuoka, and Fukuoka in Japan by inoculation tests using Fujinagas race differential cultivars of lettuce (i.e., Patriot, Costa Rica No. 4, and Banchu Red Fire). Eighteen isolates from Shizuoka and Fukuoka were designated race 3, with two unknown vegetative compatibility groups (VCGs) that differed from Ogisos VCG 1 and 2. These two new VCGs were obtained from both Shizuoka and Fukuoka. On the other hand, three isolates from Hokkaido were classified as race 1 and identified as VCG 1, which represents a VCG of crisphead isolates from Nagano.     

Abundant production of chlamydospores by Fusarium oxysporum f. sp. melonis in soil extract with glucose

Samenvatting In grondextract waaraan een kleine hoeveelheid glucose werd toegevoegd, vormtFusarium oxysporum f. sp.melonis overvloedig chlamydosporen. Gedurende een incubatieperiode van 30 dagen neemt het aantal chlamydosporen toe met stijgende glucoseconcentratie (10–1000 ppm). 98% van de gevormde chlamydosporen kiemde na 7 uren incubatie in vers medium of in steriele grond.     

Isolation of Nonpathogenic Mutants of Fusarium oxysporum f. sp. melonis for Biological Control of Fusarium Wilt in Cucurbits

ABSTRACT Two nonpathogenic mutant strains 4/4 and 15/15 of Fusarium oxysporum f. sp. melonis (race 1,2) were isolated by a continuous dipinoculation technique following UV mutagenesis of the virulent wild-type isolate FOM1.2. No disease symptoms or detrimental effects were observed following inoculation of muskmelon seedlings by strain 4/4. In contrast, strain 15/15 caused mortality of susceptible cultivars although to a lesser extent than the wild-type isolate. Strain 4/4 colonized a variety of muskmelon and watermelon cultivars. In muskmelon cv. Ein Dor, seedlings were dipped in a conidial suspension of strain 4/4 and planted in medium amended with the mutant to achieve 100% colonization of roots and between 30 to 70% of the lower stem tissues 7 days after planting. Similar percent colonization of watermelon seedlings by strain 4/4 was recorded. In cross-protection experiments with muskmelon cultivars, significant reduction in seedling mortality was observed between 4/4-colonized FOM1.2. challenged plants compared with that of wild-type challenged plants alone. Similarly, strain 4/4 was able to significantly reduce mortality of watermelon seedlings caused by F. oxysporum f. sp. niveum race 2. This novel approach of generating nonpathogenic mutants for biological control in Fusarium spp. and other fungal pathogens from virulent wild-type isolates may be beneficial for control, because the mutant strains, lacking only in pathogenicity, may compete more efficiently than other biocontrol organisms against the pathogen of origin.     

Polygenic Inheritance of Partial Resistance to Fusarium oxysporum f. sp. melonis Race 1.2 in Melon

ABSTRACT Fusarium oxysporum f. sp. melonis is responsible for Fusarium wilt of melon. Race 1.2 strains overcome two dominant resistance genes (Fom-1 and Fom-2) and are further divided into two types depending on the symptoms they cause, yellowing or wilting. Partial resistance to F. oxysporum f. sp. melonis race 1.2 was studied by using a recombinant inbred line (RIL) population that was developed by single seed descent from an F(1) hybrid between 'Isabelle', a partially resistant line, and a susceptible line, 'Védrantais'. Artificial inoculations were performed with a yellowing strain (TST) and a wilting strain (D'Oléon 8) and replicated in six locations. Disease reactions of the parental lines, controls, and RILs were scored using a 1-to-5 scale and by using the area under the disease progress curve (AUDPC). Phenotypic correlations were highly significant between the different locations and experiments. The heritability of the resistance was high, from 0.72 to 0.96, and 4 to 14 genetic factors were estimated to confer resistance to F. oxysporum f. sp. melonis race 1.2. Thirteen other strains were tested with an RILs subset. Some small strainspecific effects may be involved. These results contribute to a better understanding of the polygenic inheritance of the partial resistance to F. oxysporum f. sp. melonis race 1.2.     

Recovery of Mutants Impaired in Pathogenicity After Transposition of Impala in Fusarium oxysporum f. sp. melonis

ABSTRACT The ability of transposon impala to inactivate genes involved in pathogenicity was tested in Fusarium oxysporum f. sp. melonis. Somatic excision of an impala copy inserted in the nitrate reductase-encoding niaD gene was positively selected through a phenotypic assay based on the restoration of nitrate reductase activity. Independent excision events were analyzed molecularly and shown to carry reinsertedimpala in more than 70% of the cases. Mapping of reinserted impala elements on large NotI-restriction fragments showed that impala transposes randomly. By screening 746 revertants on plants, a high proportion (3.5%) of mutants impaired in their pathogenic potential was recovered. According to the kinetics of wilt symptom development, the strains that were impaired in pathogenicity were clustered in three classes: class 1 grouped two strains that never induced Fusarium wilt symptoms on the host plant; class 2 and class 3 grouped 15 and 9 revertants which caused symptoms more than 50 and 30 days after inoculation, respectively. The first results demonstrate the efficiency of transposition in generating mutants affected in pathogenicity, which are usually difficult to obtain by classical mutagenesis, and open the possibility to clone the altered genes with impala as a tag.     

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.     

Physiologic races ofFusarium oxysporum f.sp.melonis in the southeastern anatolia region of turkey and varietal reactions to races of the pathogen

Thirty-four isolates ofFusarium oxysporum f.sp.melonis (F.o.m.) obtained from 205 fields in melon-producing areas in the southeastern Anatolia Region of Turkey were identified on the basis of colony morphology and pathogenicity by the root dip method. In this region the mean prevalence of wilt disease was 88.1% and the mean incidence of disease was 47.5%. Physiologic races 0, 1, 2, and 1,2 of the pathogen were determined by their reactions on differential melon cultivars ‘Charentais T,’ ‘Isoblon’, ‘Isovac’ and ‘Margot’ in the greenhouse. Race 1,2, representating 58.8% (20/34) of all isolates, was widely distributed. Of the other pathogenic isolates, eight were identified as race 0, five as race 1, and one as race 2. This is the first report of physiologic races ofF.o.m. in Turkey. Of 44 melon cultivars tested in the greenhouse for resistance toF.o.m. races, 36 were found to be moderately resistant to race 0, 17 were susceptible to race 1,2, 34.1% were highly resistant to race 1, and 52.2% had moderate resistance to race 2. http://www.phytoparasitica.org posting July 16, 2002.     

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 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.     

Examination of the relationships between vegetative compatibility groups and races in Fusarium oxysporum f.sp. dianthi

The feasibility of identifying races of Fusarium oxysporum f.sp. dianthi by tests for vegetative compatibility type was investigated. Nitrate non-utilizing nitl and NitM mutants were generated from 51 isolates of F. oxysporum f.sp. dianthi , 18 isolates of f. oxysporum from Dianthus spp. not belonging to f.sp. dianthi and, for comparison, 11 isolates of F. proliferatum from Dianthus spp. Vegetative compatibility groups (VCGs) among the isolates were identified by pairing all nitl with all NitM mutants.
Vegetative compatibility was found between isolates of F. oxysporum f.sp. dianthi races 1 and 8 (VCG 0022), races 2, 5 and 6 (VCG 0021) and race 4 (VCG 0020), and wilt-causing isolates previously classified as F. redolens from D. caryophyllus (VCG 0023) and D. barbatus (VCG 0024), Three self-compatible wilt-causing isolates were vegetatively incompatible with all other isolates (VCGs 0025,0026 and 0027), Two VCGs were found among isolates of F. oxysporum from D. caryophyllus not belonging to f.sp. dianthi ; six non-pathogenic isolates were self-compatible but vegetatively incompatible with all other isolates. The foot-rot-associated isolates of F. proliferatum from D. caryophyllus constituted a separate VCG.
Virulence analyses revealed at least four new races among VCGs 0023 to 0027, New Isolates could be categorized as races as a result of VCG analysis and VCG classification correctly indicated that the race identities previously ascribed to two old isolates had been incorrect. Vegetative compatibility tests offer the prospect for rapid identification of races, although inoculation tests continue to be necessary to differentiate races that belong to a single VCG.