Formae Speciales Research Articles

Pathogenic, Morphological, and Phylogenetic Characterization of Fusarium solani f. sp. cucurbitae Isolates From Cucurbits in Almería Province, Spain.

Fusarium solani f. sp. cucurbitae (syn. Neocosmosporum cucurbitae) is one of the most devastating soilborne pathogens affecting the production of cucurbits worldwide. Since its first detection in Almería Province in Spain in the spring of 2007, it has become one of the main soilborne pathogens affecting zucchini production. It has also been reported on melon, watermelon, and squash rootstocks in Spain, representing a high risk of dissemination in the area. The objectives of this study were to investigate the incidence and distribution of this disease in southeastern Spain and characterize isolates collected over 5 years. These strains were characterized on the basis of greenhouse aggressiveness assays on a range of cucurbit hosts, morphological characteristics, and elongation factor 1-α and RNA polymerase II second largest subunit phylogenies. All pathogenic isolates were highly aggressive on zucchini plants, causing a high mortality rate a few weeks after inoculation. The rest of the cucurbit hosts showed differential susceptibility to the pathogen, with cucumber being the least susceptible. Plants belonging to other families remained asymptomatic. Morphological characterization revealed the formation of verticilate monophialides and chlamydospores forming long chains, characteristics not described for this forma specialis. Phylogenetic studies of both the individual loci and combined datasets revealed that all pathogenic isolates clustered together with strong monophyletic support, nested within clade 3 in the F. solani species complex.

Isolation of Bacillus subtilis strain SEM-2 from silkworm excrement and characterisation of its antagonistic effect against Fusarium spp.

Fusarium wilt is a devastating soil-borne disease mainly caused by highly host-specific formae speciales of Fusarium spp. Antagonistic microorganisms play a very important role in Fusarium wilt control. Isolation of potential biocontrol strains has become increasingly important. Bacterial strain SEM-2 was isolated from the high-temperature stage of silkworm excrement composting. SEM-2 exhibited a considerable antagonistic effect against Fusarium graminearum mycelial growth and spore germination. The results of pot experiments suggested that SEM-2 has a better inhibitory effect on the early stage of disease occurrence. The green fluorescent protein labelled SEM-2 coated on the surface of tomato seeds colonised the roots of tomato plants in 15 days. Genome sequencing identified SEM-2 as a new strain of Bacillus subtilis, and genome annotation and analysis determined gene clusters related to the biosynthesis of antimicrobials, such as bacillaene, fengycin, bacillibactin, subtilosin A, surfactin, and bacilysin. Interestingly, liquid chromatography - quadrupole time-of-flight mass spectrometry revealed that metabolites in pathways associated with the synthesis of secondary metabolites and antibiotics were highly differentially expressed. These findings may help to explain the mode of action of B. subtilis SEM-2 against Fusarium spp.

Distinctive Gene Expression Profiles and Effectors Consistent With Host Specificity in Two Formae Speciales of Marssonina brunnea.

The knowledge on the host specificity of a pathogen underlying an interaction is becoming an urgent necessity for global warming. In this study, the gene expression profiles and the roles of effectors in host specificity were integrally characterized with two formae speciales, multigermtubi and monogermtubi, of a hemibiotrophic pathogen Marssonina brunnea when they were infecting respective susceptible poplar hosts. With a functional genome comparison referring to a de novo transcriptome of M. brunnea and Pathogen–Host Interaction database functional annotations, the multigermtubi strain showed abundant and significant differentially expressed unigenes (DEGs) (more than 40%) in colonizing the initial invasion stage and in the necrotrophic stage. The monogermtubi strain induced less than 10% of DEGs in the initial invasion stage but which abruptly increased to more than 80% DEGs in the necrotrophic stage. Both strains induced the least DEGs in the biotrophic stage compared to the initial invasion and necrotrophic stages. The orthologs of the effector genes Ecp6, PemG1, XEG1, ACE1, and Mg3LysM were exclusively induced by one of the two formae speciales depending on the infection stages. Some unigenes homologous to carbohydrate lytic enzyme genes, especially pectate lyases, were notably induced with multigermtubi forma specialis infection but not expressed in the monogermtubi forma specialis at an earlier infection stage. The extraordinary differences in the functional genome level between the two formae speciales of M. brunnea could be fundamental to exploring their host specificity determinant and evolution. This study also firstly provided the fungal transcriptome of the monogermtubi forma specialis for M. brunnea.

Genome Sequence of Fusarium oxysporum f. sp. matthiolae, a Brassicaceae Pathogen.

The filamentous fungus Fusarium oxysporum is a soilborne pathogen of many cultivated species and an opportunistic pathogen of humans. F. oxysporum f. sp. matthiolae is one of three formae speciales that are pathogenic to crucifers, including Arabidopsis thaliana, a premier model for plant molecular biology and genetics. Here, we report a genome assembly of F. oxysporum f. sp. matthiolae strain PHW726, generated using a combination of PacBio and Illumina sequencing technologies. The genome assembly presented here should facilitate in-depth investigation of F. oxysporum-Arabidopsis interactions and shed light on the genetics of fungal pathogenesis and plant immunity.

Characterization of Pathogenic and Nonpathogenic Fusarium oxysporum Isolates Associated with Commercial Tomato Crops in the Andean Region of Colombia.

In Colombia, tomato production under protected conditions represents an important economic contribution to the agricultural sector. Fusarium wilt diseases, caused by pathogenic formae speciales of the soil-borne fungus Fusarium oxysporum Schltdl., cause significant yield losses in tomatoes throughout the world. Investigation of the F. oxysporum–tomato pathosystem in Colombia is required to develop appropriate alternative disease management. In this study, 120 fungal isolates were obtained from four different departments in the Central Andean Region in Colombia from tomato crops with symptoms of wilt disease. A molecular characterization of the fungal isolates was performed using the SIX1, SIX3, and SIX4 effector genes of Fusarium oxysporum f. sp. lycopersici W.C. Snyder & H.N. Hansen (Fol). Additionally, we developed a new specific marker to distinguish between Fusarium oxysporum f. sp. radicis-lycopersici Jarvis & Shoemaker (Forl) and Fol isolates. Furthermore, a phylogenetic analysis using the Translation Elongation Factor 1-alpha (EF1a) gene was performed with the collected isolates. Two isolates (named Fol59 and Fol-UDC10) were identified as Fol race 2, four isolates were identified as Forl, six isolates were identified as F. solani, and most of the isolates were grouped within the F. oxysporum species complex. The phylogenetic tree of EF1a showed that most of the isolates could potentially correspond to nonpathogenic strains of F. oxysporum. Additional pathogenicity assays carried out with Fol59 and Fol-UDC10 confirmed that both isolates were highly virulent strains. This study represents a contribution to the understanding of the local interaction between tomatoes and F. oxysporum in Colombia.

Genome-wide annotation, comparison and functional genomics of carbohydrate-active enzymes in legumes infecting Fusarium oxysporum formae speciales

ABSTRACTFusarium wilt caused by soil borne ascomycetes fungi Fusarium oxysporum which has host-specific forms known as formae speciales (ff. spp.), apparently requires plant cell wall degrading enzymes (PCWDE) for successful invasion. In this study, 12 F. oxysporum ff. spp. were taken for genome-wide annotation and comparative analysis of CAZymes, with an assessment of secretory PCWDE and orthologues identification in the three legumes infecting ff. spp. Further, transcriptomic analysis in two legumes infecting ff. spp. using publically available data was also done. The comparative studies showed Glycoside hydrolase (GH) families to be abundant and Principle Component Analysis (PCA) formed two distinct clusters of ff. spp. based on the CAZymes modules and families. Nearly half of the CAZymes in the legumes infecting ff. spp. coded for signal peptides. The orthologue clusters of secretory CAZymes common in all the three legume infecting ff. spp. mostly belonged to families of AA9, GH28, CE5 and PL1 and the expression analysis revealed the abundant PCWDE were differentially expressed in these legumes infecting ff. spp. Therefore, this study gave an insight into the distribution of CAZymes especially extracellular PCWDE in legumes infecting ff. spp. with further shedding light onto some of the key PCWDE families through differential expression analysis.

Murcha de fusarium e podridão radicular em grão-de-bico: taxonomia, sintomatologia e etiologia

Chickpea ( Cicer arietinum ) wilt and root rot are the most relevant diseases in several production areas. There are some lineage of Fusarium oxysporum f. sp. ciceris , from the F . solani and F . redolens complex associated with the wilt symptom. The external disease symptoms are similar; however, both species show different colonization. Traditionally, for many years, the taxonomy of the Fusarium genus has been based on the evaluation of morphological markers, being an irrelevant technique to identify species or phylogenetic lineage. Nucleotide sequencing techniques in species from the genus Fusarium have broke down species into several different phylogenetic lineages, which are currently called the species complex. Thus, it is believed that many pathogens associated with chickpea that are currently denominated as formae speciales may be complex of phylogenetic lineage. Fusarium species of different Fusarium complex have the ability to induce wilt in different hosts, while other species or phylogenetic lineage, have the ability to induce rot root.

Chemical composition and biological activity of the essential oil of the fruits Pimenta dioica against formae speciales of fungus Fusarium oxysporum

We determined the chemical composition and explored the hypothesis that the essential oil of the fruits of Pimenta dioica inhibits the mycelial development of fungi Fusarium oxysporum f. sp. lycopersici, F. oxysporum f. sp. passiflorae, F. subglutinans f. sp. ananas , F. oxysporum f. sp. vasinfectum e F. oxysporum f. sp. Cubense. To do this, we extracted the oil by hydrodistillation, identified its components by gas chromatography coupled to mass spectrometry (GC-MS) and determined the fungal activity against five special forms of Fusarium species. The results showed that the oil had 76.88% of eugenol and inhibited the mycelial development of fungi up to 97.78% in an average of 7.2 days. Therefore, oil is a potential natural fungicide.

Basal Rot of Narcissus: Understanding Pathogenicity in Fusarium oxysporum f. sp. narcissi.

Fusarium oxysporum is a globally distributed soilborne fungal pathogen causing root rots, bulb rots, crown rots and vascular wilts on a range of horticultural plants. Pathogenic F. oxysporum isolates are highly host specific and are classified as formae speciales. Narcissus is an important ornamental crop and both the quality and yield of flowers and bulbs can be severely affected by a basal rot caused by F. oxysporum f. sp. narcissi (FON); 154 Fusarium isolates were obtained from different locations and Narcissus cultivars in the United Kingdom, representing a valuable resource. A subset of 30 F. oxysporum isolates were all found to be pathogenic and were therefore identified as FON. Molecular characterisation of isolates through sequencing of three housekeeping genes, suggested a monophyletic origin with little divergence. PCR detection of 14 Secreted in Xylem (SIX) genes, previously shown to be associated with pathogenicity in other F. oxysporum f. spp., revealed different complements of SIX7, SIX9, SIX10, SIX12 and SIX13 within FON isolates which may suggest a race structure. SIX gene sequences were unique to FON and SIX10 was present in all isolates, allowing for molecular identification of FON for the first time. The genome of a highly pathogenic isolate was sequenced and lineage specific (LS) regions identified which harboured putative effectors including the SIX genes. Real-time RT-PCR, showed that SIX genes and selected putative effectors were expressed in planta with many significantly upregulated during infection. This is the first study to characterise molecular variation in FON and provide an analysis of the FON genome. Identification of expressed genes potentially associated with virulence provides the basis for future functional studies and new targets for molecular diagnostics.

Identifikasi Infraspesifik Fusarium oxysporum asal Subtrat Nonpisang dan Kemampuan Pindah Inangnya ke Tanaman Pisang

Infra-specific Identification of Fusarium oxysporum from Nonbanana Substrates and Its Ability to Move Hosts to Banana PlantsFusarium oxysporum has various life style, i.e. saprobe, endophyte and pathogen. Plant pathogenic F. oxysporum are divided into many forma specialis (f. sp.) depending on the host, for instance F. oxysporum f. sp. cubense (Foc), a causal agent of Panama disease of banana. The study aimed to determine the infraspecific identity of F. oxysporum from non-banana host and evaluate its ability to jump banana plants. Infraspecific identity was determined through a molecular approach using 3 specific primers to recognize TR4 race (TR4 F/R, TR4 F/R1, and FocSc-1/ FocSc-2), while the host’s transfer ability was tested on 2 banana cultivars namely cv. Ambon and Tanduk. Thirteen strain studied i.e IPBCC 88,012, IPBCC 07,528, IPBCC 07,561, IPBCC 08,562, IPBCC 08,568, IPBCC 10.674, IPBCC 14.1236, IPBCC 14.1237, IPBCC 14.1238 and IPBCC 14.1239 were TR4 Foc; IPBCC 07,338 and IPBCC 14.1242 are race Foc 4. The pathogenicity test of Foc IPBCC 88,012, 07,328, 08,561, 10,674 and 14.1236 derived from cucumbers, soil, agarwood sapwood, insect nests and quinine tree endophytes showed that these strains were able to move to banana plant. This shows that Foc may not host-specific and the infaspecific term forma specialis is therefore debatable.

Orthologous receptor kinases quantitatively affect the host status of barley to leaf rust fungi

Global food security depends on cereal crops with durable disease resistance. Most cereals are colonized by rust fungi, which are pathogens of major significance for global agriculture1. Cereal rusts display a high degree of host specificity and one rust species or forma specialis generally colonizes only one cereal host2. Exploiting the non-host status and transferring non-host resistance genes between cereal crop species has been proposed as a strategy for durable rust resistance breeding. The molecular determinants that define the host status to rusts, however, are largely unknown. Here, we show that orthologous genes at the Rphq2 locus for quantitative leaf rust resistance from cultivated barley3 and Rph22 from wild bulbous barley4 affect the host status to leaf rusts. Both genes encode lectin receptor-like kinases. We transformed Rphq2 and Rph22 into an experimental barley line that has been bred for susceptibility to non-adapted leaf rusts, which allowed us to quantify resistance responses against various leaf rust species. Rphq2 conferred a much stronger resistance to the leaf rust of wild bulbous barley than to the leaf rust adapted to cultivated barley, while for Rph22 the reverse was observed. We hypothesize that adapted leaf rust species mitigate perception by cognate host receptors by lowering ligand recognition. Our results provide an example of orthologous genes that connect the quantitative host with non-host resistance to cereal rusts. Such genes provide a basis to exploit non-host resistance in molecular breeding.

Expression and role of CYP505A1 in pathogenicity of Fusarium oxysporum f. sp. lactucae

BackgroundCytochrome P450 enzymes (CYPs) are monooxygenases present in every domain of life. In fungi CYPs are involved in virulence. Fusarium wilt of lettuce, caused by F. oxysporum f. sp. lactucae, is the most serious disease of lettuce. F. oxysporum f.sp. lactucae MSA35 is an antagonistic fungus. Pathogenic formae specialis of F. oxysporum possess a CYP belonging to the new family CYP505. This enzyme hydroxylates saturated fatty acids that play a role in plant defence. MethodsMolecular tools were adopted to search for cyp505 gene in MSA35 genome. cyp505 gene expression analysis in pathogenic and antagonistic Fusarium was performed. The enzyme was expressed in its recombinant form and used for catalytic reactions with fatty acids, the products of which were characterized by mass spectrometry analysis. ResultsA novel MSA35 self-sufficient CYP505 is differentially expressed in antagonistic and pathogenic F. oxysporum. Its expression is induced by the host plant lettuce in both pathogenesis and antagonism during the early phase of the interaction, while it is silenced during the late phase only in antagonistic Fusarium. Mass-spectrometry investigations proved that CYP505A1 mono-hydroxylates lauric, palmitic and stearic acids. ConclusionsThe ability of CYP505A1 to oxidize fatty acids present in the cortical cell membranes together with its differential expression in its Fusarium antagonistic form point out to the possibility that this enzyme is associated with Fusarium pathogenicity in lettuce. General significanceThe CYP505 clan is present in pathogenic fungal phyla, making CYP505A1 enzyme a putative candidate as a new target for the development of novel antifungal molecules.

Population structure and linkage disequilibrium in a large collection of Fusarium oxysporum strains analysed through iPBS markers

AbstractIn the current study, 160 pathogenic strains of Fusarium oxysporum collected from tomato, eggplant and pepper were studied. Eighteen inter‐primer binding site (iPBS)‐retrotransposon primers were used, and these primers generated 205 scorable polymorphic bands. The number of polymorphic bands per primer varied between 9 and 19, with a mean of 11 bands per primer. The highest polymorphism information content (PIC) value was determined as 0.27, and the lowest was 0.05. The unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram including a heat map revealed that the 160 pathogenic strains of F. oxysporum were divided into two main clusters. The first cluster mainly included F. oxysporum f. sp. capsici (FOC) and F. oxysporum f. sp. melongenae (FOMG) isolates. The second cluster mainly comprised F. oxysporum f. sp. lycopersici (FOL) and F. oxysporum f. sp. radicis lycopersici (FORL) isolates. The highest percentage of loci in significant linkage disequilibrium (LD) was detected for FOL, whereas the lowest level of LD was found for FOC, and 95.2%, 99.4%, 99.1% and 97.4% of the relative kinship estimates were less than 0.4 for FOL, FOMG, FORL and FOC, respectively. LD differences were detected among formae speciales, and LD was higher in FOL as compare to FOC species. The findings of this study confirm that iPBS‐retrotransposon markers are highly polymorphic at the intraspecific level in Fusarium spp.

Pathogenicity and phylogenetic analysis of Fusarium oxysporum f. sp. capsici isolates from pepper in Turkey

The pathogenic Fusarium oxysporum species complex (FOSC) causes significant yield losses in the family Solanaceae including on tomato, eggplant and pepper in the Aegean and Mediterranean regions of Turkey. Fusarium wilt of pepper, caused by Fusarium oxysporum f. sp. capsici (Foc), has emerged recently in Turkey, where its significance has not been investigated thoroughly. In the present study, disease prevalence was determined with surveys conducted in 67 pepper-growing greenhouses and 56 pepper fields in the Aegean and Mediterranean regions in 2015 and 2016. In both years, about 32% of the greenhouses and 55% of the fields were found to be infested with the wilting disease. It was observed in a pathogenicity experiment that 64 isolates were pathogenic to pepper. In cross-inoculation experiments, these isolates did not cause wilting symptoms on tomato and eggplant and therefore were designated as Foc. Phylogenetic analyses were carried out with TEF-1α and actA DNA regions of a selected subset of 40 Foc isolates, along with non-host formae of F. oxysporum and outgroup Fusarium species. Sequence data for the TEF-1α and actA DNA regions displayed limited variation among Foc isolates and failed to distinguish reliably several formae speciales of F. oxysporum. The phylogenetic tree successfully separated the outgroup isolates. All Foc isolates were placed in the same branch, suggesting a single monophyletic group in the dendrogram. This is the first comprehensive study for pathogenic and phylogenetic analysis of Foc isolates from peppers in Turkey.

An Improved Phenotyping Protocol for Panama Disease in Banana.

Fusarium oxysporum (Fo) belongs to a group of soil-borne hyphomycetes that are taxonomically collated in the Fusarium oxysporum Species Complex (FOSC). Hitherto, those infecting bananas were placed in the forma specialis cubense (Foc). Recently, however, these genetically different Foc lineages were recognized as new Fusarium spp. placed in the Fusarium of Banana Complex (FOBC). A member of this complex F. odoratissimum II-5 that uniquely comprises the so-called Tropical Race 4 (TR4), is a major problem sweeping through production zones of Cavendish banana in several regions of the world. Because of this, there is an urgent need for a phenotyping method that allows the screening for resistance to TR4 of large numbers of banana genotypes. Most Fusarium species produce three types of spores: macroconidia, microconidia and the persistent chlamydospores that can contaminate soils for many years. Inoculum production has been an important bottleneck for efficient phenotyping due to the low or variable number of conidia and the elaborate laboratory procedures requiring specific infrastructure. Here, we report a rapid, simple and high-yielding spore production method for nine F. oxysporum formae speciales as well as the biocontrol species Fo47 and Fo618-12. For Fusarium spp. causing Fusarium wilt or Panama disease of banana, we used the protocol for four species comprising the recognized physiological races, including Tropical Race 4 (TR4). We subsequently tested the produced inoculum in comparative inoculation trials on banana plants to evaluate their efficiency. All assays resulted in typical symptoms within 10 weeks; significant differences in final disease ratings were observed, depending on inoculum concentration. Pouring inoculum directly onto banana plants showed the most consistent and reproducible results, as expressed in external wilting, internal discoloration and determined by real-time PCR assays on entire rhizomes. Moreover, this method allows the inoculation of 250 plants per hour by one individual thereby facilitating the phenotyping of large mutant and breeding populations.

A morphological comparison of Arceuthobium abietinum and A. campylopodum (Viscaceae) and nomenclatural changes for A. abietinum

Fir dwarf mistletoe (Arceuthobium abietinum, Viscaceae) parasitizes true firs (Abies spp.) in the Pacific Northwest through California, the Southwest, and into northern Mexico. At present, fir dwarf mistletoe consists of two special forms (formae speciales, f. sp.) and one subspecies that are morphologically similar, but exhibit a high degree of host specificity. However, A. abietinum has been treated as a subspecies of A. campylopodum or conspecific with it in some taxonomic treatments. Therefore, we undertook this study to compare the morphologies of these dwarf mistletoes; collecting morphological data from nearly 100 A. abietinum and 60 A. campylopodum populations collected across most of their geographic ranges and analyzing these data using univariate and multivariate statistical tests. Our results demonstrated that the special forms and subspecies of A. abietinum are morphologically distinct from A. campylopodum, thereby, supporting the continued recognition of A. abietinum as a separate species. Furthermore, our analysis found significant differences for several of the characters we examined between the special forms of A. abietinum. Therefore, we recombined the special forms as subspecies: A. abietinum subsp. abietinum (formerly f. sp. concoloris) and A. abietinum subsp. magnificae (formerly f. sp. magnificae). The previously described A. abietinum subsp. wiensii was morphologically distinct from subsp. abietinum and subsp. magnificae. The distinctiveness of these taxa was supported by their host affinities as well.

Rumble in the Effector Jungle: Candidate Effector Proteins in Interactions of Plants with Powdery Mildew and Rust Fungi

Rust and powdery mildew fungi are widespread obligate biotrophic phytopathogens. They colonize a broad range monocotyledonous and dicotyledonous host plant species and in the case of crop plants can cause severe yield losses. While powdery mildews (Ascomycota) grow mainly epiphytically and infect the host epidermis, rust fungi (Basidiomycota) typically enter host tissues through stomata and spread within the intercellular spaces. Both fungal taxa have unusually large genomes that are rich in repetitive elements (mostly derived from retrotransposons) and experienced a convergent loss of genes usually present in free-living fungi compared to their respective relatives. Genomes of rust and powdery mildew fungi encode many candidates for secreted effector proteins thought to aid the suppression of defense and cell death or to mediate nutrient acquisition. Although the precise biochemical activity of most effector proteins remains obscure, candidate host targets have been identified for several of them. In addition, some effectors are perceived by matching plant immune receptors and thus serve as avirulence determinants in plant-fungus interactions. This review article summarizes the current knowledge of rust and powdery mildew effector proteins and raises and discusses urgent questions regarding future research. Abbreviations: AVR: avirulence protein; BiFC: bimolecular fluorescence complementation; CSEP: candidate secreted effector protein; ETI: effector-triggered immunity; f. sp.: forma specialis; ff. spp.: formae speciales; MS: mass spectrometry; RIP: repeat-induced point mutation; R protein: resistance protein; sRNA: small RNA; TE: transposable element; Y2H: yeast-2-hybrid.

Complete mitochondrial genome sequence of lettuce pathogenic fungus, Fusarium oxysporum f. sp. lactucae 16-086

Fusarium oxysporum f. sp. lactucae 016-086 is a plant pathogenic filamentous fungus isolated from wilted lettuce in Korea. We reported complete mitochondrial genome sequence of F. oxysporum f. sp. lactucae 016-086. Total length of this mitogenome is 41,826 bp and it encoded 42 genes (14 protein-coding genes, 2 rRNAs, and 26 tRNAs). Nucleotide sequence of coding region takes over 30.6%, and overall GC content is 32.5%. Phylogenetic tree of Fusarium mitochondrial genomes presented distinct clades along with nine formae speciales. This mitogenome will contribute distinguishing formae speciales of F. oxysoporum claearly with additional mitogenomes sequenced in the near future.

Molecular characterization by PCR-ITS technique of Fusarium oxysporum isolated from tomato in Baghdad city

The present study aimed to estimate the pathogenesis of Fusarium oxysporum isolates and identify them by molecular technique Polymerase Chain Reaction-Internal Transcript Spacer (PCR-ITS). A total of thirty Fusarium oxysporum isolates were isolated from infected tomato plants and diagnosed depending on 'morphological characteristics. The pathogenicity test was performed for thirty Fusarium oxysporum isolates. So it was procedure
 steps that Genomic DNA was extracted from seven F. oxysporum isolates according to pathogenicity test by using'' ZR Fungal/Bacterial DNA'' MiniPrep™ kit. Seven F. oxysporum isolates showed high pathogenicity. The concentration and purity of the DNA extracted from the seven F. oxysporum isolates was (150 – 201) ng/µl and (1.4 -1.9) respectively. ITS gene was amplified by using PCR-ITS The sequencing results of amplified product of ITS gene from Fusarium oxysporum isolates indicated that all the seventh sequenced isolates where from one Formae speciales of Fusarium oxysporum as Fusarium oxysporum f. sp. Lycopersici. These results have been devoted to study genetic variability among the species involved in study. Molecular markers are highly important'' to focus on F. oxysporum isolates below'' the species level therefore, the DNA array containing "genus, species'' and forma specialis specific" detector'' for the detection and identification ''of F. oxysporum. Also, Cluster analysis and phylogenetic tree depending on genetic distance revealed the genetic relationship between the thirteen F. oxysporum isolates and confirmed sequencing analysis.

Three formae speciales of Puccinia striiformis were identified as heteroecious rusts based on completion of sexual cycle on Berberis spp. under artificial inoculation

Puccinia striiformis Westend. is an obligate biotrophic parasite that could infect wheat and grasses to cause stripe rusts. P. striiformis, a species of rust fungi, is divided into several formae speciales based on host specialization, including P. striiformis f. sp. tritici, P. striiformis f. sp. hordei, P. striiformis f. sp. elymi, P. striiformis f. sp. agropyri, and P. striiformis f. sp. secalis. Among the five forms of P. striiformis, sexual stage was confirmed only for the wheat form of the rust, P. striiformis f. sp. tritici, but not known for the rest four forms. In the present study, we investigated the host specialization of three forms of P. striiformis including P. striiformis f. sp. agropyri, P. striiformis f. sp. elymi, and P. striiformis f. sp. hordei, compared the morphology of their urediniospores and teliospores, and analyzed the molecular phylogenic tree of the three forms of P. striiformis and other related species in the genus Puccinia. Each of the three forms was able to infect barberry to complete pycnial and aecial stages under artificial inoculation. Further inoculations of grass, barley cv. Guoluo and wheat cv. Mingxian 169, highly susceptible to P. striiformis f. sp. tritici, using the resultant aeciospores showed that the aeciospores could successfully infect their respective hosts and produce uredinia with high infection types, with the exception on wheat cv. Mingxian 169 where only necrosis was observed. The results suggested that Berberis spp. were alternate hosts for the three formae speciales of P. striiformis, and confirmed that the three rusts were macrocyclic and heteroecious rust fungi completing the whole life cycle on primary hosts and alternate hosts. Our study provides a basis for studying pathogenic variation of the rust fungi through sexual hybridization between formae speciales of P. striiformis.