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.     

Pathogenic groups identified among Plasmopara halstedii isolates belonging to several races

Pathogenic groups among 50 Plasmopara halstedii (sunflower downy mildew) isolates belonging to races 100, 300, 304, 314, 710, 704 and 714 were identified. Based on the reaction for the P. halstedii isolates to sunflower differential line D3, these isolates were divided into two groups as more virulent isolates of the 7xx races and the less virulent isolates of 100 and 3xx races. Index of aggressiveness (sporulation density/latent period) was calculated for each isolate and the presence of significant differences between isolates of 100 and 3xx races (more aggressive) and isolates of 7xx races (less aggressive) was revealed. Consequently, it seems that P. halstedii isolates may be divided into two pathogenic groups as more virulent and less aggressive isolates of 7xx races and less virulent and more aggressive isolates of the 100 and 3xx races.     

Isozyme Analysis and Soluble Mycelial Protein Pattern in Iranian Isolates of Several formae speciales of Fusarium oxysporum

A total of 13 representative isolates of Fusarium oxysporum f. sp. melonis (FOM) from Iran, USA and France, eight isolates of seven formae speciales from Iran and one isolate of F. oxysporum f. sp. niveum from the USA were compared based on isozyme analysis and soluble mycelial protein pattern. Isozyme analyses of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD) and xanthine dehydrogenase (XDH) revealed polymorphism among the F. oxysporum isolates in which 22 electrophoretic phenotypes (EP) were determined. At least 10 putative loci for these six enzymes were detected and they were all polymorphic. Maximum genetic diversity was observed in CAT, EST and XDH loci. Using UPGMA, the 22 isolates were separated into three main groups with one of the groups divided into two subgroups. Group I included isolates belonging to five formae speciales from Iran, whereas group II that included FOM isolates from both Iran and the USA was divided into two subgroups each containing the vast majority of the respective isolates from either country. Group III constituted FOM isolates from France and one pathogenic isolate on pepper from Iran. FOM isolates representing five different geographical regions from Iran belonged to two different races of 1 and 1,2Y and one vegetative compatibility group (VCG)0134 and thus were genetically homologous. Isozyme polymorphism in these isolates was highly correlated with VCG and geographical origins and to a lesser extent with races. Variations in soluble protein profile in FOM isolates were correlated with genetic distances determined in isozyme analysis. This study suggests that isozyme analysis could be a useful tool for identifying genetic diversity not only in FOM but also several formae speciales of F. oxysporum.     

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.     

Race Profiling and Molecular Diversity Analysis of Fusarium oxysporum f.sp. ciceris Causing Wilt in Chickpea

Seventy isolates of Fusarium oxysporum f.sp. ciceris (Foc) causing chickpea wilt representing 13 states and four crop cultivation zones of India were analysed for their virulence and genetic diversity. The isolates of the pathogen showed high variability in causing wilt incidence on a new set of differential cultivars of chickpea, namely C104, JG74, CPS1, BG212, WR315, KWR108, GPF2, DCP92‐3, Chaffa and JG62. New differential cultivars for each race were identified, and based on differential responses, the isolates were characterized into eight races of the pathogen. The same set of isolates was used for molecular characterization with four different molecular markers, namely random amplified polymorphic DNA, universal rice primers, simple sequence repeats and intersimple sequence repeats. All the four sets of markers gave 100% polymorphism. Unweighted paired group method with arithmetic average analysis grouped the isolates into eight categories at genetic similarities ranging from 37 to 40%. The molecular groups partially corresponded to the states of origin/chickpea‐growing region of the isolates as well as races of the pathogen characterized in this study. The majority of southern, northern and central Indian populations representing specific races of the pathogen were grouped separately into distinct clusters along with some other isolates, indicating the existence of variability in population predominated by a single race of the pathogen. The present race profiling for the Indian population of the pathogen and its distribution pattern is entirely new. The knowledge generated in this study could be utilized in resistance breeding programme. The existence of more than one race, predominated by a single one, in a chickpea cultivation zone as supported by the present molecular findings is also a new information.     

Genetic Variability of Fusarium oxysporum f.sp. ciceris Population Affecting Chickpea in the Sudan

Fusarium wilt caused by Fusarium oxysporum f.sp. ciceris (Foc) is the most important soilborne disease of chickpea in the Sudan and many other countries. A total of 76 Foc isolates from six different chickpea‐growing states in the Sudan have been collected in this study to investigate the genetic diversity of Sudanese Foc isolates. Additional 14 Foc isolates from Syria and Lebanon were included in this study. All isolates were characterized using four random amplified polymorphic DNA (RAPD), three simple sequence repeats (SSR), five sequence‐characterized amplified region (SCAR) primers and three specific Foc genome primers. Based on the similarity coefficient, the results indicated two major clusters included seven subclusters. The isolates from the Sudan were grouped as identified as races 0, 2 and unknown races. The isolates from Syria and Lebanon were grouped together as they identified as races 1B/C and 6, respectively. This study identified a new race Foc (race 0) in the Sudan. The results of this study will be useful for breeders to design effective resistance breeding program in chickpea in the Sudan.     

The application of high-throughput AFLP's in assessing genetic diversity in Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc) is responsible for fusarium wilt of bananas. The pathogen consists of several variants that are divided into three races and 21 vegetative compatibility groups (VCGs). Several DNA-based techniques have previously been used to analyse the worldwide population of Foc, sometimes yielding results that were not always consistent. In this study, the high-resolution genotyping method of AFLP is introduced as a potentially effective molecular tool to investigate diversity in Foc at a genome-wide level. The population selected for this study included Foc isolates representing different VCGs and races, isolates of F. oxysporum f. sp. dianthi, a putatively non-pathogenic biological control strain F. oxysporum (Fo47), and F. circinatum. High-throughput AFLP analysis was attained using five different infrared dye-labelled primer combinations using a two-dye model 4200s LI-COR automated DNA analyser. An average of approx. 100 polymorphic loci were scored for each primer pair using the SAGA^MX automated AFLP analysis software. Data generated from five primer pair combinations were combined and subjected to distance analysis, which included the use of neighbour-joining and a bootstrap of 1000 replicates. A tree inferred from AFLP distance analysis revealed the polyphyletic nature of the Foc isolates, and seven genotypic groups could be identified. The results indicate that AFLP is a powerful tool to perform detailed analysis of genetic diversity in the banana pathogen Foc.     

Analysis of whole cellular fatty acids and anastomosis relationships of binucleate Rhizoctonia spp. associated with Ceratobasidium cornigerum

Previous research has demonstrated that whole cellular fatty acids analysis is a useful tool for identifying and establishing taxonomic relationships between anastomosis groups (AGs) and related Rhizoctonia isolates. In this experiment, the composition of fatty acid of 28 isolates of teleomorph genus Ceratobasidium cornigerum, consisting of binucleate Rhizoctonia, AG-A, AG-B(o), AG-C, AG-P, and AG-Q, was evaluated using gas chromatography. Eleven fatty acids identified, i.e., myristic, pentadecanoic, palmitic, 2-hydroxypalmitic, palmitoleic, heptadecanoic, 9-heptadecenoic, stearic, oleic, linoleic, and linolenic acids, were present in isolates of AG-A, AG-B(o), AG-C, AG-P, and AG-Q. The major fatty acids, palmitic, oleic, and linoleic acids, were common in all isolates, constituting 87.1% to 94.7% of the whole cellular fatty acids identified. Isolates within the same AG were closely clustered, whereas isolates from different AGs were clearly and distinctly clustered based on average linkage cluster analysis of whole cellular fatty acids. Principal-component analysis generated from all fatty acids also confirmed the divergent separation of the 5 AGs of binucleate Rhizoctonia.     

Fatty acid composition as a taxonomic characteristic for Microcystis and other coccoid cyanobacteria (blue-green alga) isolates

The taxonomic importance of fatty acid composition at genus and sub-genus level was evaluated by analysing the fatty acid composition of fourteen different Microcystis isolates and seven additional members of the order Chroococcales. Fatty acid composition proved to be consistent within isolates. Isolates were clustered into two major groups, namely A and B. Group B contained all the Microcystis isolates and was further divided into subgroups of varying similarity indicating the existence of different taxa. The Microcystis isolates were characterised by a high content of polyunsaturated fatty acids (27–44%) and a low content of palmitoleate. The test organisms were arranged in a scheme indicating their possible phylogenetic relationship based on fatty acid composition and other phenotypic characteristics. According to our data the toxic strains, represented by different isolates, of Microcystis appear as a distinct group. Furthermore two dubious species namely Microcystis incerta and a Synechocystis sp. could clearly be reasigned to different genera. The results demonstrated that fatty acid composition is an effective taxonomic tool in clarifying taxonomical problems of Microcystis isolates. Department of Microbiology, University of the Orange Free State     

Morphological and pathogenic variability of Indian isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt

Wilt of chickpea (Cicer arietinum) caused by Fusarium oxysporum f. sp. ciceris is prevalent in almost all chickpea growing areas of the world and its incidence varied from 14.1 to 32.0% in the different states of India surveyed. The isolates were highly variable in their colony growth pattern, size of colony and pigmentations. The size of microconidia varied from 5.1–12.8 × 2.5–5.0 μm, whereas macroconidia ranged from 16.5–37.9 × 4.0–5.9 μm with 1–5 septations. One hundred and twelve isolates were grouped into 12 categories on the basis of their radial growth, size of macroconidia and growth pattern. Majority of the isolates were highly pathogenic causing more than 50% wilt in chickpea cultivar JG 62. Virulence analysis of 56 representative isolates on a set of 18 cultivars of chickpea, including 10 international differentials, grouped them into three categories. Chickpea cultivar KWR 108 differentiated all isolates of Punjab, Haryana and Delhi states and a few isolates of Rajasthan from others by showing resistant reactions and were placed in the first group. The rest of the isolates of Rajasthan state showed susceptible reactions on KWR 108 placed in a second group. Cultivar CPS 1 distinguished the isolates of Jharkhand state and placed them into a third group. An international set of cultivars recommended for race differentiation were not able to distinguish all the isolates into known races of the pathogen, therefore cultivar KWR 108 should be included in the existing differential set of cultivars.     

Virulence Analysis and Race Classification of Xanthomonas oryzae pv. oryzae in China

Two hundred and eighty‐five isolates of Xanthomonas oryzae pv. oryzae were randomly collected from 22 rice‐growing provinces in China. Ninety‐one representative isolates were chosen to assess the differential characteristics of 24 near‐isogenic rice lines containing a single resistance gene or two to four genes. Most isolates were avirulent on pyramided lines, except IRBB51, and hence, the pyramided lines cannot be used as differentials for the virulence analysis of X. oryzae pv. oryzae in China. The 13 rice lines with a single gene were used further to establish a system of races classification of X. oryzae pv. oryzae in China. IR24 and IRBB10 were susceptible to the isolates with several exceptions, whereas IRBB5, IRBB7 and IRBB21 were resistant. Based on the interactions between the isolates of X. oryzae pv. oryzae and the 13 near‐isogenic rice lines, six single‐gene rice cultivars (IRBB5, IRBB13, IRBB3, IRBB14, IRBB2 and IR24) were chosen as differentials, and the 285 tested isolates were classified into nine races. The reaction patterns of the nine races in order were: RRRRRR, RRRRRS, RRRRSS, RRRSSS, RRSSSS, RSRRRS, RSSRRS, RSSSSS and SSSSSS. The race frequencies were 10.18%, 10.53%, 4.91%, 10.18%, 24.21%, 5.96%, 11.23%, 22.46% and 0.35% respectively. The virulence of representative strains of eight Philippine races on 13 rice lines with a single gene was determined and compared with the Chinese races. The frequency distributions of X. oryzae pv. oryzae races were primarily described for the different regions in China.     

Race Distribution, Vegetative Compatibility and Pathogenicity of Fusarium oxysporum f. sp. melonis Isolates in Greece

Races and vegetative compatibility groups (VCGs) in Greek isolates of Fusarium oxysporum f. sp. melonis(Fom) were characterized. Three races (0, 2 and 1–2) among 12 isolates tested and two VCGs among 19 isolates tested, were identified. Race 1–2 was the most common and race 1 was not detected. One widespread VCG corresponded to a VCG previously reported from Israel (coded 0138), and included seven isolates of races 0 and 1–2. The other VCG, which was unclassified, included four isolates of races 0, 2 and 1–2. The latter VCG was detected only in a specific melon‐growing location of Evros. The remaining eight isolates tested for VCG did not show positive reactions with other isolates, with each other or with the testers of VCGs 0135 or 0138, although they produced complementary mutants. Using two inoculation methods, the local cv. ‘Golden Head’ was found susceptible to all known Fom races, and especially to race 1–2. These results show the presence of more than one VCG and the widespread distribution of the race 1–2, in Greece.     

Differentiation of Physiologic Races of the Rice Blast Fungus, Pyricularia oryzae Cav. by PAGE-electrophoresis

Eighty-three isolates of the rice blast fungus (Pyricularia oryzae) were tested with respect to genetic diversity and the possibility of race differentiation by electrophoresis. The fungus was genetically very heterogeneous. The isolates were differentiated into 6 races by pathogenicity on race differential varieties. There was little correlation between pathogenicity and zymogram types of one particular enzyme such as esterase, phosphatase or catalase. The isolates were divided into 14 groups by the combination of the zymogram types of the three enzymes. The isolates in the same group showed similar pathogenicity. A new method is proposed which differentiates the blast fungus races by the combination of zymogram type of enzymes. The details, are discussed.     

Utilization of a polyphasic approach in the taxonomic reassessment of antibiotic- and enzyme-producing <Emphasis Type="Italic">Bacillus</Emphasis> spp. isolated from the Philippines

Summary The taxonomy of 58 locally isolated antibiotic- and enzyme-producing Bacillus isolates deposited at the Philippine National Collection of Microorganisms (PNCM)-BIOTECH was reassessed in this study using a polyphasic approach since they had been only partially identified prior to deposition in the culture collection. The isolates had 41.1–69% G + C, and possessed the characteristic diaminopimelic acid (DAP) and fatty acid methyl ester (FAMEs) properties of Bacillus species. Molecular analysis using specific PCR primers differentiated the isolates into two major groups, the Bacillus cereus group and the Bacillus subtilis group. To further differentiate these isolates, they were subjected to 39 phenotypic tests. Using the dichotomous key constructed for Bacillus, 45 isolates maintained their original identities, five were named at the species level, and 12 were re-identified and renamed. These results showed that the classical phenotypic tests allowed the reclassification of the isolates, while modern techniques of chemotaxonomy and the molecular approach led only to genus and cluster classification and confirmation.     

Relationship of Pseudomonas syringae pv. tabaci races to the rhizosphere of Wisconsin-grown tobacco

Isolates of Pseudomonas syringae pv. tabaci, including 21 strains of the wildfire pathogen and 2 strains of the angular leafspot pathogen, were isolated from 143 rhizosphere and soil samples collected from 11 tobacco fields in Wisconsin. These pathogens were isolated by inoculating rhizosphere and soil washings into tobacco leaves and isolating the bacteria from wildfire or angular leafspot lesions that developed on the leaves. The wildfire isolates were from the rhizospheres of tobacco and Panicum capillare and from soil. While the majority of these were from wildfire-diseased fields, one isolate was from a field without disease symptoms; both angular leafspot isolates were from fields without angular leafspot symptoms. The majority of wildfire isolates were race 1, but three were race 0, and one was a new race. In three fields multiple races of wildfire were found. Both angular leafspot isolates were race 1. Two wildfire and one angular leafspot isolates were from fields where the cultivars were resistant to the races isolated.     

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.     

Sexual reproduction contributes to the evolution of resistance-breaking isolates of the spinach pathogen Peronospora effusa

Peronospora effusa causes downy mildew, the economically most important disease of cultivated spinach worldwide. To date, 19 P. effusa races have been denominated based on their capacity to break spinach resistances, but their genetic diversity and the evolutionary processes that contribute to race emergence are unknown. Here, we performed the first systematic analysis of P. effusa races showing that those emerge by both asexual and sexual reproduction. Specifically, we studied the diversity of 26 P. effusa isolates from 16 denominated races based on mitochondrial and nuclear comparative genomics. Mitochondrial genomes based on long-read sequencing coupled with diversity assessment based on short-read sequencing uncovered two mitochondrial haplogroups, each with distinct genome organization. Nuclear genome-wide comparisons of the 26 isolates revealed that 10 isolates from six races could clearly be divided into three asexually evolving groups, in concordance with their mitochondrial phylogeny. The remaining isolates showed signals of reticulated evolution and discordance between nuclear and mitochondrial phylogenies, suggesting that these evolved through sexual reproduction. Increased understanding of this pathogen's reproductive modes will provide the framework for future studies into the molecular mechanisms underlying race emergence and into the P. effusa-spinach interaction, thus assisting in sustainable production of spinach through knowledge-driven resistance breeding.     

Characterisation,differentiation and biochemical diversity of Fusarium solani and Fusarium proliferatum based on cellular fatty acid profiles

The biochemical relationships between Fusarium solani and Fusarium proliferatum isolates were investigated using fatty acid analysis. Cellular fatty acid composition showed that palmitic acid, stearic acid, oleic acid and linoleic acid were the most abundant fatty acids in these species and accounted for 93.88 and 94.02% of the fatty acid profiles in F. solani and F. proliferatum, respectively. The most predominant fatty acids were linoleic acid (37.44%) in F. solani and oleic acid (39.81%) in F. proliferatum. The fatty acid compositions of F. solani and F. proliferatum were significantly different (p?<?0.05) for most of the individual fatty acids. This study demonstrated that fatty acid profiles may be useful to characterise and differentiate F. solani and F. proliferatum isolates at the species level. Using fatty acid analysis, biochemical diversity was observed among isolates of these species. The dendrogramme revealed that F. solani and F. proliferatum formed two distinct clusters with a distance of 7.2. Isolates of each species were clustered with each other, having a Euclidean distance of 6 and 6.6 for F. solani and F. proliferatum, respectively.     

Acclimation of Plasmopara halstedii (sunflower downy mildew) in relationship with trade-off between virulence and aggressiveness in a local pathogen population

The acclimation in relationship with virulence cost was analysed for seven Plasmopara halstedii (sunflower downy mildew) isolates including five progeny isolates of several races descending from two parental isolates of races 100 and 710. Aggressiveness criteria were analysed in one sunflower inbred line showing a high level of quantitative resistance. Isolates of races 100 and 3xx were characterised with shorter latent period and higher sporulation density than isolates of races 7xx. All isolates showed high percentage infection values and caused a large reduction in seedling size except for one isolate involved in dwarfing. The seven isolates were divided, according to their virulence and aggressiveness, into two main groups as more aggressive isolates of the 100 and 3xx races which do not overcome the sunflower differential host D3, and less aggressive isolates of 7xx races which can overcome D3. Consequently, the 100 and 3xx avirulent races had a virulence cost measured by differences in aggressiveness (from 45.5 to 76.3%) compared to 7xx virulent races carrying unnecessary virulence gene.     

Virulence and Vegetative Compatibility of Dutch and Italian Isolates of Fusarium oxysporum f. sp. lilii

The virulence and vegetative compatibility of eight Dutch and four Italian isolates of Fusarium oxysporum obtained from lily were compared. The virulence was tested by determination of the specific interaction between the Fusarium isolates and eight lily cultivars. A specific interaction was not found, so the existence of races was not demonstrated. Six of the twelve isolates turned out to be non-pathogenic for lily. The pathogenic isolates fell in four vegetative compatibility groups. No vegetative compatibility was found between isolates of F. oxysporum f. sp. lilii and those of f. sp. gladioli.