Oxysporum Isolates Research Articles

Isolate-Cultivar Interactions, In Vitro Growth, and Fungicide Sensitivity of Fusarium oxysporum Isolates Causing Seedling Disease on Soybean.

Fusarium oxysporum is frequently associated with soybean root rot in the United States. Information about pathogenicity and other phenotypic characteristics of F. oxysporum populations is limited. The objective of the research described herein was to assess phenotypic characteristics of F. oxysporum isolates from soybean, including the interaction between isolates and soybean cultivars, fungal growth characteristics in culture, and sensitivity to fungicides commonly used as seed treatment products. The pathogenicity of 14 isolates was evaluated in rolled-towel and Petri-dish assays using 11 soybean cultivars. In the rolled-towel assay, seed were inoculated with a conidial suspension and disease severity was observed. In the Petri-dish assay, F. oxysporum isolates were grown on 2% water agar and seed were placed on the F. oxysporum colony to observe the symptoms that developed. Cultivars differed in susceptibility to F. oxysporum, and significant (P = 0.0140) isolate-cultivar interactions were observed. F. oxysporum isolates differed in radial growth on potato dextrose agar at 25°C. Pyraclostrobin and trifloxystrobin reduced conidial germination with average 50% effective concentration (EC50) of 0.15 and 0.20 µg active ingredient (a.i.)/ml, respectively. Ipconazole reduced fungal growth with average EC50 of 0.23 µg a.i./ml, whereas fludioxonil was ineffective. Our results illustrate soybean F. oxysporum isolate variability and the potential for their management through cultivar selection or seed treatment.

Discriminant analysis of volatile organic compounds of Fusarium oxysporum f. sp. cepae and Fusarium proliferatum isolates from onions as indicators of fungal growth

Basal rot is a common onion disease and is mainly caused by Fusarium oxysporum f. sp. cepae and Fusarium proliferatum. To study the possibility of using volatile organic compounds (VOCs) as biomarkers for these fungi, pathogenic isolates of F. oxysporum and F. proliferatum from onions were cultivated in onion medium and VOCs were measured by solid phase microextraction (SPME). Forty-two compounds were detected, and thirty of these compounds were highly related to fungal metabolic activity. Allyl mercaptan was specific to F. oxysporum isolate Fox006. Analysis of the VOCs showed significant differences between the two species and among different isolates within the same species. Sixteen of the VOCs showed were highly positively correlated with the fungal biomass estimated by real-time polymerase chain reaction (PCR). Ethanol, ethyl formate, ethyl acetate, 2-methyl-1-propanol, methyl thioacetate, n-propyl acetate and 3-methyl-1-butanol are volatile metabolites that were potential indicators of Fusarium growth on onions.

RNA seq analysis of a novel fish pathogenic fungus, Fusarium oxysporum: In quest of unveiling the virulence factors associated with fish infection

In the present study, a novel species of Fusarium oxysporum involved in the disease outbreak among freshwater murrel, Channa striatus was identified from natural lake in India and the pathogenesis was confirmed by infection studies. Using Illumina paired-end sequencing technology, we have sequenced, assembled and annotated the transcriptome of F. oxysporum isolate. A total of 53,397,192 high quality paired-end reads were assembled into 27,886 unique reads and gene ontology (GO) analysis revealed that the transcripts of F. oxysporum belonged to 1656 unique GO terms. An in-depth search for the genes with putative virulence functions revealed well-represented virulence-associated domains containing proteins such as cellulose binding proteins, proteases, kinases, kinase inhibitor and toxins. Sequence alignment revealed clear insights into the diversity and similarity of those virulence genes with plant pathogens. qRT-PCR analysis of five genes with putative virulence genes confirms the virulence roles of those genes in the pathogenesis of F. oxysporum in C. striatus. This study provides novel information regarding the transcriptome of fungal pathogen and delivers insights into the virulence genes associated with the pathogenesis of the F. oxysporum isolate which might be useful for the control of the fungal pathogenesis.

Fusarium Wilt of Sweet Potato Caused by Fusarium oxysporum f.sp. batatas in Egypt

Fusarium wilt in sweet potato was first observed in Middle Egyptgovernorates, i.e., Beni Sweif and Minia, between early April toSeptember 2016. Disease symptoms started as a stunted growth,yellowing and wilting of the leaves, browning and discolouration ofthe xylem vessels. Koch’s postulates were fulfilled and all Fusariumoxysporum isolates tested were able to infect sweet potato plantscausing typical wilt symptoms. Prevalence, incidence and severity ofsweet potato wilt (SPW) were varied with districts examined. Fungibelonging to five generae.g., Alternaria, Ceratocystis, Fusarium,Macrophomina and Rhizoctonia were found to be associated withwilted sweet potato plants. Fusarium spp recorded the highestfrequency (80.6%) and Fusarium oxysporum showed 51.5%.frequency. Sweet potato genotypes were varied in their response toinfection by Fusarium oxysporum isolate F1. Genotypes Menoufia 6and Menoufia 2 reacted as resistant, while Local A and Line 26 werehighly susceptible. Fusarium oxysporum f.sp. batatas isolate F1 wasinfective to convolvulaceous plants such as Cairo morning glory andfield bindweed but was not pathogenic to non convolvulaceous plants such as alfalfa, carrot, cotton, potato, sugar beet, sugarcane, turnip andwheat. Chlorophyll degradation was ralated to sweet potato genotypesreaction towards Fusarium oxysporum f.sp. batatas infection. Leastdegradation values were found in resistant genotypes and vice versaexhibited by susceptible genotypes. In contrast, phenols were enhanced when Fusarium oxysporum f.sp. batatas infected theresistant genotypes and decreased in the susceptible genotypes compared with uninfected.

Genetic diversity of Fusarium oxysporum f. sp. cumini isolates analyzed by vegetative compatibility, sequences analysis of the rDNA IGS region and iPBS retrotransposon markers

Fusarium oxysporum f. sp. cumini, the causal agent of cumin (Cuminum cyminum L.) wilt disease is a devastating fungal pathogen, limiting cumin production in Turkey. Genetic diversity among Fusarium wilt isolates of cumin was analyzed by vegetative compatibility grouping (VCG), DNA sequencing of the intergenic spacer region (IGS) of rDNA, and Inter-PBS (iPBS) amplification of LTR retrotransposons. The pathogen isolates were grouped into thirty-three VCGs by complementation tests using nitrate non utilizing mutants; most of these VCGs contained only a single isolate. Phylogenetic analysis of IGS region revealed two main clades among the pathogen isolates in the dendrogram with a bootstrap support of 99%. The unique VCGs had similar IGS sequences and grouped into the same clades with closely related other F. oxysporum formae speciales. iPBS analysis revealed high level of intraspecific diversity among the pathogen isolates and provided a similar discrimination that was consistent in the groupings based the partial IGS sequence. The results showed that F. oxysporum isolates from cumin has a polyphyletic origin, consisting of distinct clonal lineages. Also, the results of this study supported that iPBS marker system is a powerful tool for estimating intraspecific variation within the populations of F. oxysporum.

Detección de plantas asintomáticas de Solanum lycopersicum L. infectadas con Fusarium oxysporum usando espectroscopia de reflectancia VIS

Las plantas asintomáticas son reservorios de patógenos, ya que pueden permanecer infectadas la mayor parte de su ciclo de desarrollo, convirtiéndose en fuente de contaminación para el resto del cultivo. El objetivo de este estudio fue evaluar un método de detección y discriminación de dos cepas de Fusarium oxysporum en tomate usando espectroscopia. La enfermedad en las plantas de tomate inoculadas con la cepa aislada de gulupa (F05) fue mayor a la observada en la cepa aislada de tomate (F07), presentando valores de 60,0% (11 días) y 81,8% (22 días); la cepa F07 presentó incidencias de 30,0 y 64,3% en ambas mediciones. La planta infectada con la cepa F05 fue mejor discriminada en el periodo de incubación de la enfermedad en ambos periodos de tiempo en los Análisis de Componentes Principales (PCA) y Análisis Discriminantes Lineales (LDA) realizados con los controles en comparación con la cepa F07. Estos resultados sugieren que la espectroscopia de reflectancia VIS es un método sensible y confiable que puede ser adecuado para el diagnóstico temprano de enfermedades en plantas.

Molecular diversity in Fusarium oxysporum isolates from common bean fields in Brazil

The common bean (Phaseolus vulgaris L.) is widely cultivated in Brazil and is known as a very important crop for families in this country. Fusarium wilt severely harms common beans and has become a big issue for this crop. In order to assist the breeding programs that target resistance to this disease, the evaluation of genetic diversity of the pathogen and its molecular characterization are crucial. Thus, the present goal was to identify Fusarium isolates obtained from several places in Brazil using molecular tools; select molecular markers for these isolates; and analyze their diversity. All of isolates were molecularly identified as Fusarium oxysporum f. sp. phaseoli (Fop). By using seven selected SSR markers, the results of diversity obtained by the dendrogram and the Bayesian analysis formed four groups where a large diversity of this fungus was found within each state. However, the groups were more homogenous according to the collection source and the pathogenicity test. More specifically, group 2 was composed of the most virulent strains and originated from Minas Gerais State – UFV, and group 3 was mostly composed by isolates from Goias state. Group I was also more diverse in terms of location and virulence. The overall results indicated a positive correlation between Fusarium diversity and its virulence to common bean. Furthermore, the use of these markers was effective in molecular identification and in detecting polymorphism within F. oxysporum f. sp. phaseoli.

Relationship between pathogenicity, race and vegetative compatibility grouping among Algerian populations of Fusarium oxysporum f. sp. pisi causing pea wilt

Abstract Fusarium oxysporum f. sp. pisi (FOP) is a significant and destructive pathogen of field pea in Algeria. In the present study, 50 isolates of F. oxysporum f. sp. pisi, the causal agent of pea (Pisum sativum) wilt, collected from different parts of western Algeria and representing four races of the pathogen, were analyzed for virulence. The wilt incidence ranged from 6.66 to 88.33% on a highly susceptible cultivar (Little Marvel). Twenty-one isolates belonging to four races of FOP and one nonpathogenic F. oxysporum (FO) isolate were analyzed for vegetative compatibility in order to reveal the genetic structure of the population and to check the reliability of the method for the identification of physiological races of FOP. Obtained results showed that the FOP isolates could be classified into four main vegetative compatibility groups (VCGs) that corresponded to races l, 2A, 2B and 5. The race 6 isolate fell into the race 1 VCG. To our knowledge, this is the first such study in Algeria of its kind.

Genetic Diversity of Fusarium oxysporum f. sp. cubense in East and Central Africa.

Banana Fusarium wilt is a major production constraint globally and a significant threat to the livelihoods of millions of people in East and Central Africa (ECA). A proper understanding of the diversity and population dynamics of the causal agent, Fusarium oxysporum f. sp. cubense (Foc), could be useful for the development of sustainable disease management strategies for the pathogen. The current study investigated the diversity of Foc in ECA using vegetative compatibility group (VCG) analysis, PCR-RFLPs of the ribosomal DNA's intergenic spacer region, as well as phylogenetic analysis of the elongation factor-1α gene. Six VCGs (0124, 0125, 0128, 01212, 01220, and 01222), which all belong to one lineage (Foc lineage VI), were widely distributed throughout the region. VCGs 0128 and 01220 are reported for the first time in Burundi, the Democratic Republic of Congo (DRC), Rwanda, Tanzania, and Uganda, while VCG 01212 is reported in the DRC and Rwanda. Isolates that did not belong to any of the known VCGs were identified as Foc lineage VI members by phylogenetic analysis and may represent novel VCGs. CAV 2734, a banana pathogen collected in Rwanda, clustered with nonpathogenic F. oxysporum isolates in lineage VIII. Results from this study will contribute significantly toward the implementation of banana Fusarium wilt disease management practices in the region, such as the restricted movement of infected planting material and the selective planting of resistant banana varieties.

Root Rot Caused by Species of Fusarium on Brassica carinata in South Dakota

Brassica carinata is an emerging oilseed crop in the United States, and root diseases caused by Fusarium have the potential to cause yield losses in production. In this study, B. carinata plants were randomly sampled at vegetative and seed development plant stages from South Dakota State University experimental plots. Reddish-brown lesions were observed on roots of sampled plants from which F. acuminatum, F. oxysporum, F. solani, and F. sporotrichioides were recovered. The Fusarium species were identified based on morphology and phylogenetic analyses of the translation elongation factor 1-α gene region. Pathogenicity of the four Fusarium species was evaluated on five B. carinata accessions using a modified inoculum layer method in the greenhouse. At 21 days after inoculation, root rot severity caused by Fusarium on the B. carinata accessions was assessed on a rating scale of 0 to 4 and evaluated using relative treatment effects (RTEs). The F. oxysporum isolate caused significant differences in RTE (P = 0.01) among the B. carinata accessions. However, there were no significant differences in RTE among the B. carinata accessions in response to F. acuminatum (P = 0.82), F. solani (P = 0.76), and F. sporotrichioides (P = 0.47) isolates.

First Report of Fusarium oxysporum f. sp. niveum Race 3 Causing Wilt of Watermelon in Florida, U.S.A.

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is an important watermelon (Citrullus lanatus) disease in Florida and the southeast. This disease can cause complete yield loss when susceptible cultivars are grown (Egel and Martyn 2013). Disease symptoms include vascular discoloration, especially around crown and upper taproot and withering and wilting leaves followed by death of either a vine or the whole plant. A Fon isolate was collected from Madison County, Florida, in May 2015. It was recovered from infected stem slices that were surface sterilized for 1 min in 0.85% sodium hypochlorite and rinsed with sterile water. The slices were incubated on quarter strength potato dextrose agar (QPDA) until hyphae emerged from the vascular tissue. Conidia from these slices were streaked onto water agar. After 15 h, a germinated conidium was transferred to QPDA to obtain a monoconidial culture and labeled 150525. The isolate was amplified by PCR using EF-1α and IGS (O’Donnell et al. 2009) loci. BLAST analysis of the resulting EF locus sequence (accession no. MF784504) revealed >99% similarity with e-value of zero to F. oxysporum isolate ICMP:20827 (KX534005). Our IGS sequence (MF784503) had a 99% identity to the F. oxysporum accession no. KU128972. Although KU128972 belongs to f. sp. cyclaminis, previous studies had shown F. oxysporum f. sp. are polyphyletic and could cluster with different groups of the F. oxysporum species complex (O’Donnell et al. 2009). Race typing of isolate 150525 was completed using a root dip inoculation method with four differential watermelon cultivars Black Diamond, Charleson Gray, Calhoun Gray, and PI-296341-FR (Martyn and Netzer 1991; Zhou et al. 2010). PI-296341-FR is resistant to race 2 but susceptible to race 3. All other differentials are susceptible to both race 2 and 3. The roots of 2-week-old seedlings were dipped in a conidial suspension (1 × 10⁶ conidia/ml) for 30 s and then planted in transplant flats with potting media. Six seedlings of each differential were challenged with 150525 and a known race 2 identified at the University of Georgia. The flats were kept in a greenhouse with 26°C daytime and 22°C nighttime temperature, and a 14-h photoperiod. Control seedlings were inoculated with water. Seedlings were observed for disease symptoms every week for 1 month. Since there are reports of low-level segregation of PI-296341-FR line (Martyn and Netzer 1991), we conducted four race typing experiments. On all occasions, all differentials inoculated with 150525 were completely wilted with 100% incidence while control seedlings were asymptomatic. The verified race 2 isolate caused PI-296341-FR wilting only once in four experiments with 67% incidence. The pathogen was reisolated from symptomatic plants and confirmed as F. oxysporum using colony and spore morphology (Leslie and Summerell 2006). After confirmation of isolate 150525 as race 3, we tested an additional 26 Fon isolates collected in 2015 using the same techniques mentioned above. Of these, nine isolates from Madison, Levy, and Lee counties of Florida were confirmed as race 3. Currently, commercially available cultivars lack resistance to Fon race 3. Thus, producers in Florida will need to modify their disease management strategies to include delayed planting dates, fumigant and fungicide applications, and novel methods such as grafting for adequate disease control.

BIOLOGICAL AND MOLECULAR STUDIES ON FUSARIUM WILT DISEASE OF TOMATO

Tomato (Solanum lypersicum Mill.) is ranked number 1 among fruits and vegetables with 14% of the total production worldwide (Food & Agriculture Organization of the United Nations, FAO, 2003). Fusarium wilt disease is only responsible for 10 to 50% of these losses (Lukyanenko 1991). In Egypt, the losses due to tomato wilt disease reached up to 67% (Lasheen 2009). Different Fusarium oxysporum isolates were tested under greenhouse conditions to determine their virulence potential toward tomato plants. Severity and development of Fusarium wilt disease was recorded 4, 6 and 8 weeks after pathogen inoculation. All the tested isolates were pathogenic and varied significantly with regard to their virulence potential on tomato under the test conditions. The ability of a Fusarium isolate to infect particular plant species dependent on specific genes encoding host determining ‘virulence factors’ that distinguish virulent from avirulent strains. Eight fungal proteins were identified from xylem sap of infected plants, encompassing the small secreted proteins called SIX1, Six2, Six3, Six4, Six5, Six6, Six7 and SIX 8. The results showed that the tested isolates were varied regarding to their pathogenicity potential toward tomato plants under greenhouse conditions. On the other hand, the amplicons of SIX1, SIX5 and SIX 7 were detected with most hyper virulent isolates while no amplicons for any tested SIX genes were observed with the nonpathogenic isolate (Fo162) as well as with the most hypo virulent isolates. These results, suggested that SIX1, SIX5 and SIX6 may play a distinct role in virulence potential of these Fusarium oxysporum isolates toward tomato plants under Egyptian conditions .

Evaluation of Trichoderma Harzianum Biological Control Against Fusarium Oxysporum F. Sp. Melongenae

The present study was conducted to biocontrol in vitro and in vivo of Fusariumoxysporum that cause Fusarium wilt diseases for eggplant plants by using biologicalcontrol agent fungus Trichoderma harzianum. Fourteen isolates from F. oxysporumwere isolated and identified from two fields in Iraq. Pathogenicity test indicated thatall F. oxysporum isolates were pathogenic for eggplant but differed in its level ofpathogenicity. Four of the fourteen isolates from F. oxysporum were selecteddepending on their highest pathogenicity for eggplant plants, F. oxysporum fourisolates F5, F6, F13 and F14 achieved at pre emergence 83.3%, 83.3%, 86.7% and83.3% and at post emergence 90.0%, 90.0%, 83.3% and 76.7% respectively. Invitro, the antagonistic activity evaluation of T. harzianum against F. oxysporum fourisolates revealed that T. harzianum was highly significantly inhibited F. oxysporumfour isolates growth (F5, F6, F13, F14) which recorded the inhibition percentage87.00, 89.00, 89.00, and 96.33% respectively. Greenhouse (In vivo) results of thebiocontrol efficiency of T. harzianum against F. oxysporum showed significantreduction in eggplant Fusarium wilt incidence compared with pathogens controltreatments. The pathogen F. oxysporum treatments with T. harzianum (TF) showedthat all the treatments (TF) achieved high germination rate for eggplant seedscompared with control treatment, while the treatment TF13 was significantlysuperior than all other treatments in number of leaflets, stem height, shoot freshweight, shoot dry weight, root fresh weight, root dry weight and root size whichrecorded 8.67, 19.00cm, 20.33gm, 4.73gm, 12.00gm, 1.53gm, 17.67cm³respectively.

Comparative analysis of pathogenic and nonpathogenic Fusarium oxysporum populations associated with banana on a farm in Minas Gerais, Brazil

Fusarium wilt is one of the most devastating diseases on banana. The causal agent, Fusarium oxysporum f. sp. cubense (Foc) is genetically diverse and its origin and virulence are poorly understood. In this study, pathogenic Foc isolates and nonpathogenic F. oxysporum isolates from Minas Gerais in Brazil were compared using EF‐1α and IGS sequences. This allowed the examination of the origin and evolutionary potential of Foc in a country outside the region of origin of the banana plant. Two different sequence types were found among Foc isolates. One appeared to be of local origin because it was identical to the sequence type of the largest group of nonpathogenic isolates. To explore if the ‘local’ Foc isolates had acquired pathogenicity either independently through coevolution with the host, or through horizontal gene transfer (HGT) of pathogenicity genes from other, probably introduced, Foc isolates, the presence and sequence of putative SIX effector genes were analysed. Homologues of SIX1, SIX3 and SIX8 were found. SIX1 sequences were identical and exclusively found in all pathogenic isolates, while variable ratios of sequences of multicopy gene SIX8 were found among nonpathogenic and different pathogenic isolates. This observation supports the HGT hypothesis. Horizontal transfer of genes between isolates of F. oxysporum has important implications for the development of reliable diagnostic tools and effective control measures. Full genome sequencing is required to confirm HGT and to further unravel the virulence mechanisms of forma specialis cubense.

Development and evaluation of polyclonal antibodies for detection of Pythium aphanidermatum and Fusarium oxysporum in ginger

ABSTRACT Polyclonal antibodies against Pythium aphanidermatum and Fusarium oxysporum proteins were developed for the detection of rhizome rot in ginger using serological assays. Under optimal experimental conditions, the detection limit of P. aphanidermatum by indirect ELISA was 10 µg/ml with a linear working range from 5 to 100 µg/ml (R 2 = 0.994). In case of F. oxysporum, the linear working range was 5–100 µg/ml (R 2 = 0.991) and the limit of detection was 25 µg/ml. The developed antibodies showed the highest titer in ELISA at 1:2000 dilutions. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis identified proteins ranging from molecular weights 15–97 kDa and 14–116 kDa of P. aphanidermatum and F. oxysporum isolates, respectively. In Western blot analysis, the developed antisera gave positive reactions against the isolated antigens of the fungi. The antibodies revealed immune-reactive bands of molecular weights 59 and 65 kDa in P. aphanidermatum and 44 and 75 kDa in F. oxysporum. The results suggest that the developed antibodies could be successfully applied for the specific immunodetection of P. aphanidermatum and F. oxysporum at an early stage of rhizome rot disease.

The distribution and host range of the banana Fusarium wilt fungus, Fusarium oxysporum f. sp. cubense, in Asia.

Fusarium oxysporum formae specialis cubense (Foc) is a soil-borne fungus that causes Fusarium wilt, which is considered to be the most destructive disease of bananas. The fungus is believed to have evolved with its host in the Indo-Malayan region, and from there it was spread to other banana-growing areas with infected planting material. The diversity and distribution of Foc in Asia was investigated. A total of 594 F. oxysporum isolates collected in ten Asian countries were identified by vegetative compatibility groups (VCGs) analysis. To simplify the identification process, the isolates were first divided into DNA lineages using PCR-RFLP analysis. Six lineages and 14 VCGs, representing three Foc races, were identified in this study. The VCG complex 0124/5 was most common in the Indian subcontinent, Vietnam and Cambodia; whereas the VCG complex 01213/16 dominated in the rest of Asia. Sixty-nine F. oxysporum isolates in this study did not match any of the known VCG tester strains. In this study, Foc VCG diversity in Bangladesh, Cambodia and Sri Lanka was determined for the first time and VCGs 01221 and 01222 were first reported from Cambodia and Vietnam. New associations of Foc VCGs and banana cultivars were recorded in all the countries where the fungus was collected. Information obtained in this study could help Asian countries to develop and implement regulatory measures to prevent the incursion of Foc into areas where it does not yet occur. It could also facilitate the deployment of disease resistant banana varieties in infested areas.

Specific PCR detection of Fusarium oxysporum f. sp. raphani: a causal agent of Fusarium wilt on radish plants.

Because plant pathogenic Fusarium oxysporum has been classified into special forms based on its host specificity, identification of F. oxysporum usually requires a pathogenicity assay as well as knowledge of the morphological characteristics. For rapid and reliable diagnosis, this study provides PCR primer sets that specifically detect Fusarium oxysporum f. sp. raphani (For) which is a devastating pathogen of radish plants. Because one of the primer sets directly detected the DNA fragment of For isolates from infected plants, the specific PCR method demonstrated in this study will provide a foundation for integrated disease management practices in commodity crops.

First Report of Fusarium oxysporum Causing a Vascular Wilt of Statice (Limonium sinuatum) in the U.K.

Statice (Limonium sinuatum) is grown commercially in many countries as a cut-flower crop. Fungal and oomycete pathogens reported for this plant include Colletotrichum, Botrytis, Cercospora, Rhizoctonia, and Peronospora (Moorman 2016). In the UK, 80% of all statice production (2.5 ha, value $800,000) is through a specialist grower in Lincolnshire. In 2016, 30% of the 70,000 statice plants raised at this nursery were affected by a vascular wilt. Symptoms observed on approximately 1 m tall flowering plants (cv. Velvet Wings) consisted of wilted leaves and flower stalks, often progressing down one side of the stem, followed by leaf necrosis and plant death. Brown staining of vascular tissue was observed in dissected stems and, in advanced infections, mycelium was evident at the stem base. Isolations were carried out from 18 plants by excising 2 to 3 cm sections of infected stem, surface disinfecting in 5% sodium hypochlorite for 2 min, rinsing in sterile water and plating on potato dextrose agar (PDA) containing 20 µg/ml chlortetracycline. After incubation at 20°C for 4 days under natural light, fungal colonies were sub-cultured onto PDA and grown for a further 10 days. Cultures displayed morphology typical of Fusarium species, with a peach/pale pink color. Macroconidia were 3-septate, straight or slightly curved, 16.1 to 25.7 µm x 2.8 to 5.4 µm. Microconidia were abundant, elliptical or reniform, 6.1 to 12.4 µm x 1.6 to 4.9 µm. DNA was extracted from four isolates, and the translation elongation factor 1-α (EF1-α) gene amplified by PCR and sequenced using exTEF-F/ FUexTEF-R primers (Taylor et al. 2016). All isolates shared identical EF1-α sequences (GenBank accession number KX822794) and were confirmed as Fusarium oxysporum using BLAST analysis. The sequences were 99% identical to the EF1- α sequences of a wide range of F. oxysporum isolates with the greatest homology to F. oxysporum f. sp. freesia (KP964900, 99.7% over 989 bp). To confirm pathogenicity, statice (cv. Velvet Wings) plants produced by tissue culture were raised in pots. Once the first flower stalk reached approximately 30 cm, plants were removed, the bottom third of the root system excised and the remaining roots soaked in a conidial suspension (1 x 106 conidia/ml) for 5 min. As a negative control treatment, F. oxysporum isolate Fo47, a biocontrol strain known to be non-pathogenic on a range of hosts (Aime et al. 2013), was used to inoculate plants in the same way, while noninoculated control plants were soaked in sterile water. All statice (six plants / isolate or control treatment) were then replanted in compost and pots placed in a randomized design in a temperature controlled glasshouse (25°C day, 18°C night, 16 hour photoperiod). After 20 days, typical wilt symptoms were observed in all the plants inoculated with the four F. oxysporum isolates from statice, and after 40 days all the plants were dead (e-Xtra). All noninoculated control plants and those inoculated with F. oxysporum isolate Fo47 remained healthy. The pathogen was successfully re-isolated from infected stems with the fungal cultures exhibiting the same morphology as described previously. Furthermore, following DNA extraction, amplification and sequencing of the EF1-α gene of the re-isolated fungi, all sequences were 100% identical to those obtained for the original isolates, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of Fusarium wilt of statice. Further work is required to establish the host range of this potentially new forma specialis.

IDENTIFICATION OF FUSARIUM OXYSPORUM f. sp. OPUNTIARUM ON NEW HOSTS OF THE CACTACEAE AND EUPHORBIACEAE FAMILIES

Fusarium oxysporum has recently been detected in commercial nurseries in the Ligurian region (northern Italy) on new succulent plants belonging to the Cactaceae family (Astrophytum myriostigma, Cereus marginatus var. cristata, C. peruvianus monstruosus and C. peruvianus florida) and to the Euphorbiaceae family (Euphorbia mammillaris). The pathogen has been identified, for all the new hosts, from morphological characteristics observed in vitro. The identifications have been confirmed by means of ITS (Internal Transcribed Spacer) analysis and/or by Translation Elongation Factor 1α (TEF) analysis. The aim of this work was to identify the forma specialis of the F. oxysporum isolates obtained from new succulent plants. This has been investigated by means of phylogenetic analysis based on the TEF gene and intergenic spacer (IGS), carried out on single-spore isolates, together with pathogenicity assays. The results of this research led to include four new succulent hosts from Cactaceae as additional hosts to F. oxysporum f. sp. opuntiarum. This forma specialis has been identified for the first time on a new host (Euphorbia mammillaris) not belonging to the Cactaceae family.

Genetic diversity and phylogenetic analysis of Fusarium oxysporium strains isolated from the Cucurbitaceae hosts revealed by SRAPs

Cucurbitaceae wilt of different hosts could be caused by Fusarium oxysporum. To clear the genetic diversity and phylogenetic relationship of F. oxysporum isolates from different Cucurbitaceae hosts and regions, genomic DNA of ninety-five strains of F. oxysporum isolated from different hosts and different regions were amplified by sequence-related amplified polymorphism (SRAP) molecular markers. All strains could be uniquely distinguished with 238 polymorphism bands which generated 100% of polymorphic ratio with 19 selective primer pairs. On average, each primer pairs amplified 12.5 loci and 12.5 polymorphic loci. A total of 166 bands of F. oxysporum f. sp. momordicae (FOM) were amplified, among which 145 polymorphic bands generated 87.4% of polymorphic ratio. On average, each primer combination amplified 8.7 loci and 7.7 polymorphic loci. This indicated that genetic variation of F. oxysporum was extensive. The genetic similarity coefficient of F. oxysporum was 0.68-0.99, and the average indexes of Nei's genetic diversity and Shannon were 0.2390 and 0.3718, respectively. Ninety-five strains of F. oxysporum were divided into four formae speciales from bitter gourd, cucumber, watermelon and melon, when the genetic similarity coefficient was 0.74. In SRAP dendrogram all the FOM isolates were gathered into one phylogenetic branch with the genetic similarity coefficient ranging from 0.78 to 0.99. The average indexes of Nei's genetic diversity and Shannon indices were 0.1811 and 0.2750, respectively. This indicated that genetic diversity of FOM was abundant, and the classification of phylogenetic group was related to geographic origin to some extent.