Oxysporum Isolates Research Articles

Whole genome analysis of the koa wilt pathogen (Fusarium oxysporum f. sp. koae) and the development of molecular tools for early detection and monitoring

BackgroundDevelopment and application of DNA-based methods to distinguish highly virulent isolates of Fusarium oxysporum f. sp. koae [Fo koae; cause of koa wilt disease on Acacia koa (koa)] will help disease management through early detection, enhanced monitoring, and improved disease resistance-breeding programs.ResultsThis study presents whole genome analyses of one highly virulent Fo koae isolate and one non-pathogenic F. oxysporum (Fo) isolate. These analyses allowed for the identification of putative lineage-specific DNA and predicted genes necessary for disease development on koa. Using putative chromosomes and predicted gene comparisons, Fo koae-exclusive, virulence genes were identified. The putative lineage-specific DNA included identified genes encoding products secreted in xylem (e. g., SIX1 and SIX6) that may be necessary for disease development on koa. Unique genes from Fo koae were used to develop pathogen-specific PCR primers. These diagnostic primers allowed target amplification in the characterized highly virulent Fo koae isolates but did not allow product amplification in low-virulence or non-pathogenic isolates of Fo. Thus, primers developed in this study will be useful for early detection and monitoring of highly virulent strains of Fo koae. Isolate verification is also important for disease resistance-breeding programs that require a diverse set of highly virulent Fo koae isolates for their disease-screening assays to develop disease-resistant koa.ConclusionsThese results provide the framework for understanding the pathogen genes necessary for koa wilt disease and the genetic variation of Fo koae populations across the Hawaiian Islands.

Influence of the combined action of <i>Fusarium</i> spp. fungi and temperature on some tomatoes growth traits

Relevance. Tomato root rot is a complex disease caused by a variety of soil-borne fungal pathogens, among which Fusarium oxysporum, F. solani and F. gibbosum occur with the greatest frequency in the conditions of the Republic of Moldova. The originality of the work consists in identifying the differentiated influence of pathogens of Fusarium root rot and temperature on the variability and heritability of the organs of growth and development of tomato plants.Material and methods. Two varieties and 3 lines of tomatoes with good economically valuable characteristics were used as material for the study. To test the response of plants to pathogens of fusarium root rot, we used culture filtrates of 6 – F. oxysporum isolates, 6 – F. solani, and 4 – F. gibbosum, prepared in a standard liquid Czapek medium. The plants were grown at the optimum temperature (23...24°C) and with alternating temperatures of 23...24°C and 10...11°C for two days each. Seed germination and root length were used as indicators of growth and development. The genetic parameters of the variability and heritability of the studied traits were studied.Results. An analysis of the reaction based on seeds germination and root growth showed its significant differentiation in the tomato genotype x type of fungus x isolate of the fungus x temperature system. A factor analysis of the interaction of the tomato genotype x Fusarium fungus revealed a significant role of the plant genotype in response to the pathogens. A significant effect of Fusarium fungi and temperature on the genetic and phenotypic variance of signs of growth and development of tomato plants, heritability in a broad sense, genotypic and phenotypic coefficient of variation, and genetic progress have been established. It is concluded that it is advisable to use the reaction of signs of growth and development of tomato to Fusarium pathogens in order to identify genotypes with good inheritance of the resistance trait.

Azole antifungal resistance in fungal isolates from wastewater treatment plant effluents.

Wastewater treatment plants (WWTPs) can be significant sources of antifungal resistant fungi, which can disseminate further in the environment by getting into rivers together with effluents discharged from WWTPs and pose a risk for human health. In this study, the presence of azole resistance was determined in fungal isolates from treated effluents of two WWTPs using the standard microdilution method from Clinical and Laboratory Standards Institute (CLSI). A total of 41 fungal isolates representing 23 fungal species and 16 fungal genera were obtained. Fungal genera related to the known human and/or plant pathogens such as Aspergillus, Fusarium, and Candida were detected. Among the observed species, the susceptibility of Aspergillus fumigatus and Fusarium oxysporum was tested against fluconazole (FCZ), ketoconazole (KTZ), itraconazole (ITZ), and voriconazole (VCZ). The isolate A. fumigatus was susceptible to KTZ, ITZ, and VCZ, while it showed resistance against FCZ. On the contrast, the isolate F. oxysporum showed resistance to KTZ, ITZ, and VCZ. Comparatively, VCZ showed highest activity against both A. fumigatus and F. oxysporum. Analysis of the gene Cyp51A for the A. fumigatus isolate showed no evidence of drug resistance that could be related to point mutations and/or tandem repeats in the gene. To the best of our knowledge, this is the first susceptibility test study on A. fumigatus and F. oxysporum isolates from the WWTPs of South Africa. In conclusion, this study indicated an urgent need for thorough investigation with larger group of fungal isolates from different regions of South Africa to broadly understand the role of WWTPs in the dissemination of azole antifungal drug resistance.

Morphological and cultural variability of Fusarium spp. causing wilt in chilli in Karnataka

Morphological and cultural variability was studied among the isolates of Fusarium spp. collected from fusarium wilt infested chilli fields of north Karnataka. The isolates cultured on potato dextrose agar medium showed considerable cultural variabilities in colony diameter, colony morphology, pigmentation and morphological variabilities in conidial size, septation and sporulation rate. Colony diameter among F. oxysporum isolates ranged from 63.93- 89.87 mm and produced white fluffy cottony growth with orangish pigmentation. The isolates produced macro conidia of size 11.5-24 × 2.2-5.5 µm with 3-4 septation, micro conidia of size 5-6 × 2.7-3 µm and chlamydospore of size 10.5-11.2 µm in diameter. Colony diameter among F. solani isolates ranged from 57.80-89.87 mm, and produced white-creamish cottony/fluffy/submersed growth with creamish, light orangish, purplish and pinkish pigmentation. The isolates produced macro-conidia of size 10-25.5×3-6.5 with 2-3 septation, micro-conidia of size 5-12.5×2.1-3.5 µm and chlamydospores of size 10-12 µm in diameter. The isolates also showed variation in sporulation rate.

The development of a multiplex PCR assay for the detection of Fusarium oxysporum f. sp. cubense lineage VI strains in East and Central Africa

Banana is a staple food and cash crop grown in East and Central Africa (ECA). The main banana varieties grown in ECA are the East African highland bananas (EAHB), although dessert/beer bananas such as Sukari Ndizi, Kayinja (Pisang Awak) and Gros Michel are also produced due to their high value at local markets. The Fusarium wilt fungus Fusarium oxysporum f. sp. cubense (Foc) causes disease of susceptible dessert/beer bananas, which significantly reduces yields. Banana Fusarium wilt is managed by excluding the pathogen from disease-free areas and by planting disease-resistant varieties in infested fields. Six phylogenetically closely-related vegetative compatibility groups (VCGs) of Foc, VCGs 0124, 0125, 0128, 01212, 01220 and 01222 are present in ECA, which all group together in Foc Lineage VI. Rapid and accurate detection of Foc Lineage VI strains is thus important to prevent its spread to disease-free areas. In this study, molecular markers specific to Foc Lineage VI were therefore developed. Primer sets were then combined in a multiplex PCR assay, and validated on a worldwide population of 623 known Foc isolates, other formae speciales and non-pathogenic Fusarium oxysporum isolates. The Foc Lineage VI multiplex PCR was used to identify Foc isolates collected in banana fields at five locations in Uganda and Tanzania. Foc Lineage VI DNA was detected at a concentration as low as 0.1 ng/μl, both in the absence and presence of banana DNA, and can therefore be used as an accurate diagnostic tool for Foc Lineage VI strains.

Isolation of Fusarium from vanilla plants grown in the Huasteca Potosina Mexico

La vainilla es una orquídea de importancia económica en México y el mundo. Entre las principales zonas de México donde se produce esta orquídea se encuentra la región Huasteca en el estado de San Luis Potosí. Recientemente se observó en plantas de vainilla cultivadas en esta región, la presencia de síntomas como clorosis, necrosis y marchitamiento general. Considerando que dichos síntomas son característicos de infecciones causadas por hongos patógenos, en este trabajo se planteó el objetivo de identificar los hongos asociados a dichas plantas. Para ello se colectaron hojas, tallos y frutos de plantas con síntomas; los cuales se lavaron, cortaron, y colocaron sobre medio PDA. Bajo estas condiciones se obtuvieron colonias fúngicas que fueron usadas para obtener aislados monospóricos, los cuales fueron identificados mediante microcultivos y mediante la amplificación y secuenciación de las regiones génicas conocidas como Espaciadores Transcritos Internos (ITS). Dichas secuencias fueron analizadas mediante BLAST en NCBI. Los hongos aislados mostraron características morfológicas del género <em>Fusarium</em>: micelio color violeta, coloración amarillo-marrón en el medio de cultivo, desarrollo de clamidosporas, macroconidios y microconidios. Las secuencias ITS mostraron una cobertura del 100% y una similitud del 99.5% con el aislado de <em>Fusarium oxysporum</em> O309 (MT032690.1), confirmando la presencia de <em>Fusarium</em> en las plantas analizadas. Interesantemente, este es el primer reporte del aislamiento e identificación de <em>Fusarium</em> en plantas de vainilla cultivadas en el estado de S.L.P.

Fusarium oxysporum isolates showed varied benzimidazole fungicides sensitivity under low temperature and heavy metal stress

Emergence of fungicide resistant phytopathogens under changing environmental condition is a serious problem in agriculture. Four Fusarium oxysporum isolates were isolated from four wilted crop plants (tomato, potato, pea and grass pea) and identified through conidial morphology and rDNA sequence analysis. Sensitivity of carbendazim and thiophanate methyl was tested through estimation of radial growth based ED50 values and conidia germination based EC50 values. The isolates were able to grow in presence of carbendazim (1.2 mg/L) with ED50 values ranging from 0.62 to 0.97 mg/L and EC50 values ranging from 0.10 to 1.13 mg/L. Moreover, they could also tolerate thiophanate methyl (upto 100 mg/L) with ED50 values ranging from 12.5 to 30.6 mg/L and EC50 values ranging from 23.0 to 43.0 mg/L. Thiophanate methyl was less effective to inhibit conidial germination under in vitro condition. The isolates were also showed pleiotropic inhibitory effect on mycelia growth to carbendazim (1 mg/L) and thiophanate methyl (10 mg/L) at low temperature. They could tolerate heavy metals such as arsenic, cadmium and lead (each at 100 mg/L concentration). Metal supplementation increased carbendazim sensitivity, but decreased thiophanate methyl sensitivity. Spectrophotometric analysis revealed that the suppressed activity of thiophanate methyl was due to interaction with the metals, but no such interactions occurred with carbendazim. Thus, under low temperature and heavy metal stress, carbendazim might be used judiciously for control of the pathogen.

Species-Specific Impact of Fusarium Infection on the Root and Shoot Characteristics of Asparagus.

Soil-borne pathogens can have considerable detrimental effects on asparagus (Asparagus officinalis) growth and production, notably caused by the Fusarium species F. oxysporum f.sp. asparagi, F. proliferatum and F. redolens. In this study, their species-specific impact regarding disease severity and root morphological traits was analysed. Additionally, various isolates were characterised based on in vitro physiological activities and on protein extracts using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The response of two asparagus cultivars to the different Fusarium species was evaluated by inoculating experiments. Differences in aggressiveness were observed between Fusarium species and their isolates on roots, while no clear disease symptoms became visible in ferns eight weeks after inoculation. F. redolens isolates Fred1 and Fred2 were the most aggressive strains followed by the moderate aggressive F. proliferatum and the less and almost non-aggressive F. oxysporum isolates, based on the severity of disease symptoms. Fungal DNA in stem bases and a significant induction of pathogenesis-related gene expression was detectable in both asparagus cultivars. A significant negative impact of the pathogens on the root characteristics total root length, volume, and surface area was detected for each isolate tested, with Fred1 causing the strongest effects. No significant differences between the tested asparagus cultivars were observed.

First detection of Fusarium oxysporum f. sp. radicis–lycopersici across major tomato–producing regions in Brazil

Fusarium oxysporum f. sp. lycopersici (FOL) races 1 and 2 were, up to recent years, the only pathogenic Fusaria associated with tomatoes (Solanum lycopersicum L.) in Brazil. However, recent outbreaks of F. oxysporum isolates with the ability to breakdown the I–2-mediated resistance prompted us to conduct nationwide surveys of the pathogens associated with vascular wilt of tomatoes. During these surveys, eight atypical F. oxysporum isolates (collected across five tomato–producing Brazilian states) displayed a peculiar ability of infecting accessions carrying the I–3 and I–7 genes (controlling resistance to FOL race 3 isolates). These observations suggested the involvement of either a new FOL race or of F. oxysporum f. sp. radicis-lycopersici (FORL) isolates. Koch’s postulates were fulfilled for these eight atypical isolates via root dipping inoculation (106 conidia mL− 1) of seedlings from two tomato cultivars ‘Ponderosa’ and ‘Dominador’. All eight isolates induced crown-rot symptoms only in ‘Ponderosa’, but not in ‘Dominador’ (which is resistant to FORL). To confirm the FORL identity, PCR assays were done using a set of race-specific and forma specialis-specific primers. A single amplicon of ≈ 947 bp was observed with the primer pair Sprl only with DNA template obtained from standard FORL isolates and from the eight atypical isolates, confirming the first detection of this forma specialis in Brazil. The geographical distribution of FORL isolates across major tomato–producing areas may require the incorporation of resistant factors into adapted cultivars.

MOLECULAR IDENTIFICATION OF FUNGI THE CAUSAL AGENT OF STRAWBERRY WILT DISEASE IN BALI

The development of strawberry farming in Bali experiencing some obstacles that cause a decline in production, such as wilting disease. The disease was reported caused by the fungi base on morphological recognition. There are two fungi were recognized caused the strawberry wilt disease in Bali, they are from genus Verticillium and Fusarium. More specific information about causal agent of wilt disease in strawberry especially in Bali is needed. The one accurate identification is done through the molecular approach by analyzing DNA that encode the ribosomal DNA (rDNA). The 18S rDNA, including the internal areas of transcribed spacers (ITS), ITS1 and ITS4 have been widely used in phylogenetic studies. The amplification results of this area produce bands in different sizes that can be used to identify fungal species. Based on that the identification of strawberry wilt disease using molecular analysis was conducted. The 542 bp of Internal Transcribed Spacer (ITS) DNA was successfully amplified using PCR with pairing primers ITS 1 (5-TCCGTAGGTGAACCTGCGG-3’), and ITS 4 (5’-TCCTCCGCTTATTGATATGC-3’). The sequences of three isolates were successfully obtained through sequencing. Homology levels were tested between sequences and showed that Candi Kuning sequence and Gobleg sequence had 95% similarity with sequence of Fusarium oxysporum NRRL 13307 (U34571) from America. While Pancasari sequence have 94% similarity with sequence of Fusarium oxysporum NRRL 13307 (U34571) from America. Candi Kuning, Gobleg, and Pancasari sequences had the same 86% with sequence of Fusarium oxysporum isolate C34-294 Brazil (KJ439088) and had 89% similarity with sequence Fusarium oxysporum f.sp. fragariae China (KT833080). Homology levels were tested between sequences and showed that Candi Kuning sequence and Gobleg sequence had 95% similarity with sequence of Fusarium oxysporum NRRL 13307 (U34571) from America. While Pancasari sequence have 94% similarity with sequence of Fusarium oxysporum NRRL 13307 (U34571) from America. Candi Kuning, Gobleg, and Pancasari sequences had the same 86% with sequence of Fusarium oxysporum isolate C34-294 Brazil (KJ439088) and had 89% similarity with sequence Fusarium oxysporum f.sp. fragariae China (KT833080). Based on phylogeny analysis of Pancasari, Gobleg and Candi Kuning isolates were obtained in one group with Fusarium oxysporum identified in America and Brazil, and also in one group with Fusarium oxysporum f. sp. fragariae that identified in China.

Colonization of human opportunistic Fusarium oxysporum (HOFo) isolates in tomato and cucumber tissues assessed by a specific molecular marker.

Fusarium oxysporum is a large complex cosmopolitan species composed of plant pathogens, human opportunistic pathogens, and nonpathogenic isolates. Many plant pathogenic strains are known based on host plant specificity and the large number of plant species attacked. F. oxysporum is an opportunistic pathogen in humans with a compromised immune system. The objectives of this study were: (1) to develop a specific marker to detect human opportunistic F. oxysporum (HOFo) isolates; (2) to determine whether or not HOFo isolates can colonize and cause disease symptoms in plants; and (3) to assess Taiwan isolates sensitivity to two agro-fungicides. The primer pair, Primer 5/ST33-R, specifically amplifying Taiwan and international reference HOFo isolates was developed and used to detect and assess the distribution of a Taiwan isolate in inoculated tomato plants and tomato and cucumber fruit. Taiwan HOFo isolate MCC2074 was shown to colonize tomato roots, hypocotyls, and cotyledons, but did not show any visible symptoms. Four days after surface inoculation of tomato and cucumber fruit with the same isolate, MCC2074 was detected in the pericarp and locular cavities of both tomato and cucumber fruit and in columella of tomato fruit. Three Taiwan HOFo isolates were found to be moderately sensitive to azoxystrobin and highly sensitive to difenconazole.

Identification of main pathogenic Fusarium species for biological control of poppy plants in Afghanistan

Afghanistan is one of the major countries which contributes to illegal poppy (Papaver somniferum L.) cultivation, globally. The objectives of the present study were to report the Fusarium species associated with P. somniferum and to investigate a potential biocontrol agent against P. somniferum. A total of 155 Fusarium isolates were obtained from four provinces in Afghanistan, including Helmand, Kandahar, Daikundi and Bamyan. The pathogenic Fusarium spp. isolates were characterized by morphological and molecular identification, using the translation elongation factor-1α (TEF-1α) gene. The results of isolation revealed the most predominant species as F. proliferatum (27%), followed by F. equiseti (26%), F. solani (13%), F. incarnatum (12%), F. acuminatum (7%), F. oxysporum (5%), F. verticillioides (5%), F. pseudograminearum (2%) and F. coeruleum (2%). With the exception of F. oxysporum and F. solani, other identified species are the first reported Fusarium species associated with poppy plants in Afghanistan. Furthermore, pathogenicity testing of F. oxysporum, F. proliferatum, F. acuminatum and F. verticillioides isolates were performed. Although disease symptoms were observed in all species; F. oxysporum infected poppy samples showed an early development of wilt symptoms and their disease severity was significantly higher than all other isolates. The results suggest that F. oxysporum isolates have the potential to be used as biological control agents against poppy plants in Afghanistan.

Soil management under tomato-wheat rotation increases the suppressive response against Fusarium wilt and tomato shoot growth by changing the microbial composition and chemical parameters

Crop rotation is a common practice aimed at enhancing both plant disease suppression and crop productivity under open field condition. The objective of the present study was to evaluate the effects of tomato-wheat rotation on Fusarium wilt suppression and shoot growth on tomato in comparison to tomato monoculture. The following factors were determined and integrated through a transdisciplinary approach: (i) soil chemical parameters; (ii) abundance, composition, richness and diversity of soil fungal and bacterial communities; (iii) soil biochemical and enzymatic indicators; (iv) abundance of Fusarium oxysporum isolates in rhizosphere under tomato cultivation and evaluation of the pathogenicity of the isolates; (v) suppressive response of the bulk soil samples against a selected pathogenic strain of Fusarium oxysporum f. sp. lycopersici on tomato; and (vi) tomato shoot growth. The integrated approach included soil amplicon sequencing by metabarcoding analyses carried out by Illumina MiSeq platform in association with the standard procedure for soil analyses. In addition, the pathogenicity of F. oxysporum from the tomato rhizosphere and the suppressive response of the soil bulk to Fusarium wilt were performed by bioassays under greenhouse condition. The results showed that, if durum wheat was rotated with cherry tomato for at least four consecutive years, it had the following beneficial effects: (i) increased suppressive response of the bulk soil samples against F. oxysporum f. sp. lycopersici, (ii) decreased abundance of the F. oxysporum isolates in tomato rhizosphere, (iii) decreased pathogenicity of the F. oxysporum isolates, and (iv) increased tomato shoot growth. The increased abundance of certain microbial groups belonging to potential biocontrol agents against fusaria-related wilt including Bacillales (Bacillus and Paenibacillus), Pseudomonadales (Pseudomonas), Streptomycetales (Streptomyces), Eurotiales (Aspergillus and Penicillium) and Mortierellales (Mortierella), as well as the highest tomato shoot growth, coincided with the end of the second wheat rotation cycle. There were significant Spearman cross-correlations and two-way ANOVA interactions among the microbiome variables with the (i) soil chemical parameters, (ii) abundance and pathogenicity of the F. oxysporum isolates in tomato rhizosphere, (iii) soil suppression to Fusarium wilt, and (iv) tomato shoot growth. The results indicate that a soil managed under tomato-wheat rotation displays increased suppressive response against Fusarium wilt and improved shoot growth in tomato plants by changing the microbial composition and chemical parameters of the soil.

Identification of Pathogenicity Groups and Pathogenic Molecular Characterization of Fusarium oxysporum f. sp. sesami in China.

Fusarium oxysporum f. sp. sesami is an extremely destructive pathogen, causing sesame Fusarium wilt disease worldwide. To clarify the pathogenicity and the genetic characters of F. oxysporum f. sp. sesami, we systematically investigated 69 F. oxysporum isolates collected from major sesame-growing areas in China. Among these isolates, 54 isolates were pathogenic and 15 were nonpathogenic according to pathogenicity testing on sesame seedlings. For the pathogenic isolates, three F. oxysporum f. sp. sesami pathogenicity groups were defined based on the three differential sesame hosts for the first time. A translation elongation factor 1α gene tree was constructed to determine the genetic diversity of the F. oxysporum isolates but could not separate F. oxysporum f. sp. sesami isolates from the nonpathogenic isolates and other F. oxysporum formae speciales. Ten secreted-in-xylem (SIX) genes (one family of effectors) were identified in F. oxysporum f. sp. sesami isolates by a search with the genome data, and were subsequently screened in the 69 F. oxysporum isolates. Compared with the SIX gene profiles in other F. oxysporum formae speciales, the presence and sequence variations of the SIX gene homologs directly correlated with the specific pathogenicity of F. oxysporum f. sp. sesami toward sesame. Furthermore, eight of these F. oxysporum f. sp. sesami SIX genes were significantly expressed in sesame plants as infection of the F. oxysporum f. sp. sesami isolate. These findings have important significance for understanding the pathogenic basis of F. oxysporum f. sp. sesami isolates, and will contribute to improve the diagnostics to effectively control Fusarium wilt disease in sesame.

Non-pathogenic Fusarium oxysporum as a biocontrol agent

Non-pathogenic Fusarium oxysporum are endophytes which can suppress disease development by pathogenic F. oxysporum isolates through plant growth promotion and local as well as systemic induced resistance. Competition for space and nutrients, production of secondary metabolites and induction of resistance are their different modes of action against pathogens. These have been reported to be compatible with biocontrol agents and selected fungicides. They were reported to be evolved from pathogenic isolates and transfer of specific genes encoding pathogenicity can convert them to pathogenic forms. There is the possibility of rapid emergence of new pathogenic lineages in distinct non-pathogenic strains by simple co-cultivation with pathogenic strains. Detailed studies are required to undermine the genes and the pathways involved in their antagonistic as well as plant growth promoting potential. This review paper attempts to describe various aspects of non-pathogenic F. oxysporum isolates and the possible threats in their usage as biocontrol agents.

ELISA-Based Development of Functional Antibodies for the Efficient Detection of Mycotoxigenic Fusarium (F. oxysporum and F. circinatum) and Diplodia (D. pinea) Species

Fusarium contains the most toxigenic and important fungal pathogens, producing a wide range of mycotoxins that include the more toxic secondary metabolites called fumonisins. This study analyzed the presence of common exo- and mycelium-soluble antigens, purified proteins using reducing SDS-PAGE and the expression of antibodies against different fungal pathogens. The results showed the presence of common antigens between pathogens and development of antibodies against antigens from F. circinatum, F. oxysporum and D. pinea. A strong reactivity against the antigens ranged between 0.510 to 0.868 in F. circinatum and F. oxysporum isolates. The reactivity of anti-D. pinea antibodies was relatively low, with the highest absorbance value of 0.650, accompanied by a minimal cross reactivity. Overall, these findings indicated that, antibodies against mycelium-soluble antigens were the most immuno-reactive and could be helpful for the development of a rapid Fusarium detection tool for use in various industries.

Phylogenetic relationship between Australian Fusarium oxysporum isolates and resolving the species complex using the multispecies coalescent model

BackgroundThe Fusarium oxysporum species complex (FOSC) is a ubiquitous group of fungal species readily isolated from agroecosystem and natural ecosystem soils which includes important plant and human pathogens. Genetic relatedness within the complex has been studied by sequencing either the genes or the barcoding gene regions within those genes. Phylogenetic analyses have demonstrated a great deal of diversity which is reflected in the differing number of clades identified: three, five and eight. Genetic limitation within the species in the complex has been studied through Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analyses with varying number of phylogenetic ‘species’ identified ranging from two to 21. Such differing views have continued to confuse users of these taxonomies.ResultsThe phylogenetic relationships between Australian F. oxysporum isolates from both natural and agricultural ecosystems were determined using three datasets: whole genome, nuclear genes, and mitochondrial genome sequences. The phylogenies were concordant except for three isolates. There were three concordant clades from all the phylogenies suggesting similar evolutionary history for mitochondrial genome and nuclear genes for the isolates in these three clades. Applying a multispecies coalescent (MSC) model on the eight single copy nuclear protein coding genes from the nuclear gene dataset concluded that the three concordant clades correspond to three phylogenetic species within the FOSC. There was 100% posterior probability support for the formation of three species within the FOSC. This is the first report of using the MSC model to estimate species within the F. oxysporum species complex. The findings from this study were compared with previously published phylogenetics and species delimitation studies.ConclusionPhylogenetic analyses using three different gene datasets from Australian F. oxysporum isolates have all supported the formation of three major clades which delineated into three species. Species 2 (Clade 3) may be called F. oxysporum as it contains the neotype for F. oxysporum.

Population Structure and Genetic Diversity of Fungi Causing Rice Seedling Blight in Northeast China Based on Microsatellite Markers.

Rice seedling blight, which is caused by diverse pathogenic microorganisms, occurs worldwide and is the most important seedling disease affecting rice production in Northeast China. To further characterize the population structure and genetic diversity of the fungi responsible for rice seedling blight in Northeast China, 225 fungal strains were isolated from diseased rice seedlings collected from various rice-producing areas. The isolated strains included Fusarium oxysporum (48.0%), F. verticillioides (11.6%), F. tricinctum (8.0%), F. redolens (6.7%), F. equiseti (6.2%), F. solani (6.2%), Rhizoctonia solani (6.7%), Alternaria alternata (4.0%), and Curvularia coatesiae (2.7%). F. oxysporum was the dominant fungal species causing rice seedling blight, with most isolates exhibiting moderate pathogenicity. Moreover, to our knowledge, this is the first study to identify A. alternata and C. coatesiae as causal agents of rice seedling blight in Northeast China. None of the F. oxysporum isolates were sensitive to 10 μg/ml of carbendazim, implying that carbendazim is ineffective for controlling rice seedling blight in Northeast China. The F. oxysporum isolates were divided into nine groups based on a simple sequence repeat analysis involving 14 primer pairs. In addition, an analysis of molecular variance revealed a significant correlation between the F. oxysporum population and geographical location, which had a significant effect on the differentiation of the dominant isolate population. The results of this study provide insights into the genetic diversity of F. oxysporum strains causing rice seedling blight and may be useful for selecting isolates to screen for disease-resistant rice varieties, evaluating fungicide efficacy, and developing effective disease management strategies.

The biological activity of subspecies Trichoderma harzianum against Fusarium oxysporum, the causative agent of fusarium wilt cucumber in vitro

The study of the effect of the strains of the fungus Trichoderma: Trichoderma atrobrunneum VKPM F-1434, Trichoderma harzianum 5/14, Trichoderma Lixii T4/14 on the number of micromycetes populations of the pathogenic fungi Fusarium oxysporum isolate B/14, Fusarium oxysporum isolate MOS509, Fysarium oxysporum isolate IMI58289 in vitro. It was found that the Trichoderma atrobrunneum fungus strain VKPM F-1434 showed the highest degree of inhibition on 10 days of cultivation with phytopathogenic microorganisms, which was 100 %. The study revealed that all Trichoderma species are capable of producing lytic enzymes. Trichoderma atrobrunneum strain VKPM F-1434 exhibits strong lipase and chitinase activity and average proteinase activity. In addition, Trichoderma atrobrunneum strain VKPM F-1434 has a growth-promoting ability, which was reflected in the germination of seeds of cucumber “German F1”. The maximum values of indicators of germination energy were noted – 98.4 % and germination – 100 %.

First Report of Fusarium oxysporum Causing Root Rot in Ophiopogon bodinieri in China

Ophiopogon bodinieri H. Lev. is an important ornamental groundcover widely used in urban gardens in southern China (Liu et al. 2011). In September 2017, a disease occurred on approximately 20% of O. bodinieri in 9 ha in Guangzhou, Guangdong province in China. Symptoms included etiolation in the leaves, wilt, root rot, and necrotic vascular systems. Three diseased plants were sampled for pathogen isolation. Portions (about 5 mm²) of symptomatic root tissues were dissected and surface disinfected (3% NaClO for 10 s and 70% ethanol for 30 s). Tissues were rinsed three times using sterile distilled water, dried on sterile filter paper, and transferred to Petri plates with potato dextrose agar (PDA) supplemented with streptomycin sulfate (150 µg/ml). Petri plates were incubated at 28°C for 5 days (Dita et al. 2010). Only one isolate was obtained from all the plates and was subcultured to new PDA plates. A single-spore isolate was obtained from a hyphal tip, and the culture characteristics and conidial morphology were studied on PDA and carnation leaf agar (CLA) (Neish 1983). The isolate grown on PDA formed abundant white-colored fungal colonies with radial mycelium in 5 days at 28°C. Microscopic observations from CLA medium revealed the curved macroconidia were usually three- to five-septate, with the size of 2.2 to 4.0 × 18.3 to 42.4 μm. Microconidia were kidney shaped with the size of 4.7 to 6.8 × 7.3 to 12.1 μm. Chlamydospores were single or in clusters, with the size of 9.1 to 11.0 μm in diameter. The elongation factor 1-alpha (EF1α) gene (accession no. MN026924, 686 bp), amplified and sequenced using primer pair EF-1/EF-2 (O’Donnell et al. 1998), showed 100% identification to a Fusarium oxysporum strain (accession no. KY508353.1) (Geiser et al. 2004). The molecular identification was confirmed via BLAST on the Fusarium ID and Fusarium MLST databases. Ten-week-old plants were used for pathogenicity tests. First, the plants were wounded by cutting off 1 cm of the roots. Then, 10 plants were inoculated by root dipping (30 min, 10⁴ spores/ml), and another 10 plants were treated with sterile water as a control. The plants were then repotted in potting mix and incubated at 28°C. The assay was conducted three times. After 15 days, the plants showed symptoms of leaf wilting, root rot, and necrosis in vascular tissues. After 40 days, all the inoculated plants were dead, whereas no symptoms were observed in the controls. Subsequently, the F. oxysporum isolate was successfully reisolated from the inoculated plants and was identified again by sequencing the EF1α. The pathogen was further identified by PCR amplification and sequencing the internal transcribed spacer (ITS) gene region using the primers ITS5/ITS4 and the 18s nuclear ribosomal small subunit (SSU) using the primers NS1/NS4 (Schoch et al. 2012). The isolate showed 100% and 99% identity to those of F. oxysporum (accession KF498869.1 for ITS and KU512835.1 for SSU). The sequences of ITS (accession no. MH752745.1), SSU (accession no. MH752591.1), and EF1α (accession no. MN026924) were deposited in GenBank. The first F. oxysporum causing disease on another Ophiopogon species (O. japonicus) was discovered in Florida in 1991 (Farr and Rossman 2019). To our knowledge, this is the first report of root rot disease of O. bodinieri caused by F. oxysporum in the world. This disease may pose a risk for urban landscapes in China.