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The United States is the third largest producer of cotton and the largest exporter of cotton globally. Fusarium wilt, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum ( Fov ), was estimated to cause a $21 million cotton yield loss in 2022. Historically, Alabama was an important producer of cotton in the Southeastern United States and was the first state in which Fusarium wilt on cotton was described. To assess the genetic diversity of Fov field isolates in Alabama, 118 field isolates were collected from six counties across the state from 2014 to 2016. Phylogenetic analysis using TEF1 and RPB2 placed the Fov field isolates into 18 haplotypes. Upon profiling the Tfo1 transposon insertion in the NAT gene, it was determined that no race 4 isolates were recovered in Alabama. Representatives of all field isolate haplotypes caused disease on Upland cotton variety Rowden in a hydroponic test tube assay. Two haplotype A isolates were the most aggressive isolates recovered, and haplotype A isolate TF1 was more aggressive than the race 4 isolate 89-1A on Upland cotton and had similar symptom severity on Pima cotton. Karyotype profiling indicted an abundance of small chromosomes characteristic of karyotypes that include accessory chromosomes, with considerable variability between isolates. Collectively, our study indicates that Fov isolates from Alabama are genetically diverse, which may have been promoted by its persistence in cotton fields., Competing Interests: The author(s) declare no conflict of interest.

Fusarium root rot is usually classified as an extremely destructive soilborne disease. From 2020 to 2021, Fusarium root rot was observed in production areas and seriously affected the yield and quality of Scutellaria baicalensis in Shanxi Province, China. Based on morphological characteristics and combined analysis of the internal transcribed spacer region of ribosomal DNA and translation elongation factor 1-alpha sequences, 68 Fusarium isolates obtained in this work were identified as F. oxysporum (52.94%), F. acuminatum (20.59%), F. solani (16.17%), F. proliferatum (5.88%), F. incarnatum (2.94%), and F. brachygibbosum (1.47%). In the pathogenicity tests, all Fusarium isolates could infect S. baicalensis roots, presenting different pathogenic ability. Among these isolates, F. oxysporum was found to have the highest virulence on S. baicalensis roots, followed by F. acuminatum, F. solani, F. proliferatum, F. brachygibbosum , and F. incarnatum . According to fungicide sensitivity tests, Fusarium isolates were more sensitive to fludioxonil and difenoconazole, followed by carbendazim, thiophanate-methyl, and hymexazol. In brief, this is the first report of Fusarium species ( F. oxysporum, F. acuminatum, F. solani, F. proliferatum, F. incarnatum , and F. brachygibbosum ) as causal agents of root rot of S. baicalensis in Shanxi Province, China. The fungicide sensitivity results will be helpful for formulating management strategies of S. baicalensis root rot., Competing Interests: The author(s) declare no conflict of interest.

Coffee fruit rot (CFR) is a well-known disease worldwide, mainly caused by Colletotrichum spp., the most important species being C. kahawae subsp. kahawae . In Puerto Rico, Colletotrichum spp. were identified as pathogens of coffee fruits. The coffee berry borer (CBB) was shown to be a dispersal agent of these fungi, and interaction of Fusarium with Colletotrichum affecting coffee fruits was suggested. In this study, we demonstrated that Fusarium spp. also cause CFR in Puerto Rico. Fusarium spp. are part of the CBB mycobiota, and this insect is responsible for spreading the pathogens in coffee fields. We identified nine Fusarium spp. ( F. nirenbergiae, F. bostrycoides, F. crassum, F. hengyangense, F. solani-melongenae, F. pseudocircinatum, F. meridionale, F. concolor , and F. lateritium ) belonging to six Fusarium species complexes isolated from CBBs and from rotten coffee fruits. Pathogenicity tests showed that F. bostrycoides, F. lateritium, F. nirenbergiae, F. solani-melongenae , and F. pseudocircinatum were pathogens causing CFR on green coffee fruits. F. bostrycoides was the predominant species isolated from the CBB mycobiota and coffee fruits with symptoms of CFR, suggesting a close relationship between F. bostrycoides and the CBB. To our knowledge, this is the first report of F. bostrycoides, F. solani-melongenae, F. pseudocircinatum , and F. nirenbergiae causing CFR worldwide and the first report of F. lateritium causing CFR in Puerto Rico. Understanding the CFR disease complex and how the CBB contributes to dispersing different Fusarium spp. on coffee farms is important to implement disease management practices in Puerto Rico and in other coffee-producing countries., Competing Interests: The author(s) declare no conflict of interest.

The most common soilborne diseases affecting the strawberry industry in California include Verticillium wilt due to Verticillium dahliae , charcoal root rot due to Macrophomina phaseolina, and Fusarium wilt due to Fusarium oxysporum f. sp. fragariae . Detection of these pathogens in soil is an important facet of disease management and fumigation recommendations. Whereas the soil populations of both M. phaseolina and V. dahliae can be readily quantified with quantitative PCR (qPCR) assays using DNA extractions with 500 mg of soil, the single-cell nature of the F. oxysporum chlamydospore does not provide enough pathogen DNA from 500-mg extractions to be reliably quantified. Here, we describe an improved DNA extraction protocol from 10 to 15 g of soil that allows for the quantification of F. oxysporum f. sp. fragariae populations below 10 CFU/g. The relationship between results from the TaqMan qPCR assay and pathogen population density in soil was determined by using this extraction method in pathogen-free soils artificially infested with a hygromycin-resistant strain of F. oxysporum f. sp. fragariae to facilitate accurate colony counts when plated on a selective medium. Although the protocol was developed for F. oxysporum f. sp. fragariae , it is applicable for detection and quantification of other soilborne pathogens., Competing Interests: The author(s) declare no conflict of interest.

Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were collected from two locations. Compared with the wild type, bmr plants have reduced lignin content, altered cell wall composition, and different levels of phenolic intermediates. Multilocus maximum-likelihood analysis identified two Fusarium thapsinum operational taxonomic units (OTU). One was identified at greater frequency in grain and leaves of bmr and wild-type plants but was infrequently detected in air. Nine F. graminearum OTU were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright's F statistic (F ST ) indicated that Fusarium air populations differentiated between locations during crop anthesis but did not differ during vegetative growth, grain development, and maturity. F ST also indicated that Fusarium populations from wild-type grain were differentiated from those in bmr6 or bmr12 grain at one location but, at the second location, populations from wild-type and bmr6 grain were more similar. Thus, impairing monolignol biosynthesis substantially effected Fusarium populations but environment had a strong influence.

Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.

Fusarium oxysporum species complex (FOSC) isolates were obtained from celery with symptoms of Fusarium yellows between 1993 and 2013 primarily in California. Virulence tests and a two-gene dataset from 174 isolates indicated that virulent isolates collected before 2013 were a highly clonal population of F. oxysporum f. sp. apii race 2. In 2013, new highly virulent clonal isolates, designated race 4, were discovered in production fields in Camarillo, California. Long-read Illumina data were used to analyze 16 isolates: six race 2, one of each from races 1, 3, and 4, and seven genetically diverse FOSC that were isolated from symptomatic celery but are nonpathogenic on this host. Analyses of a 10-gene dataset comprising 38 kb indicated that F. oxysporum f. sp. apii is polyphyletic; race 2 is nested within clade 3, whereas the evolutionary origins of races 1, 3, and 4 are within clade 2. Based on 6,898 single nucleotide polymorphisms from the core FOSC genome, race 3 and the new highly virulent race 4 are highly similar with Nei's Da = 0.0019, suggesting that F. oxysporum f. sp. apii race 4 evolved from race 3. Next generation sequences were used to develop PCR primers that allow rapid diagnosis of races 2 and 4 in planta.

Fusarium spp. are ranked among the top 10 most economically and scientifically important plant-pathogenic fungi in the world and are associated with plant diseases that include fruit decay of a number of crops. Fusarium isolates infecting bell pepper in Trinidad were identified based on sequence comparisons of the translation elongation factor gene (EF-1a) with sequences of Fusarium incarnatum-equiseti species complex (FIESC) verified in the FUSARIUM-ID database. Eighty-two isolates were identified as belonging to one of four phylogenetic species within the subclades FIESC-1, FIESC-15, FIESC-16, and FIESC-26, with the majority of isolates belonging to FIESC-15. A comparison of the level of DNA polymorphism and phylogenetic inference for sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2) and EF-1a sequences for Trinidad and FUSARIUM-ID type species was carried out. The ITS sequences were less informative, had lower haplotype diversity and restricted haplotype distribution, and resulted in poor resolution and taxa placement in the consensus maximum-likelihood tree. EF-1a sequences enabled strongly supported phylogenetic inference with highly resolved branching patterns of the 30 phylogenetic species within the FIESC and placement of representative Trinidad isolates. Therefore, global phylogeny was inferred from EF-1a sequences representing 11 countries, and separation into distinct Incarnatum and Equiseti clades was again evident. In total, 42 haplotypes were identified: 12 were shared and the remaining were unique haplotypes. The most diverse haplotype was represented by sequences from China, Indonesia, Malaysia, and Trinidad and consisted exclusively of F. incarnatum isolates. Spain had the highest haplotype diversity, perhaps because both F. equiseti and F. incarnatum sequences were represented; followed by the United States, which contributed both F. equiseti and F. incarnatum sequences to the data set; then by countries representing Southeast Asia (China, Indonesia, Malaysia, Thailand, and Philippines) and Trinidad; both of these regions were represented by only F. incarnatum sequences. Trinidad shared two haplotypes with China and one haplotype with the United States for only F. incarnatum isolates. The findings of this study are important for devising disease management strategies and for understanding the phylogenetic relationships among members of the FIESC.

The Fusarium oxysporum species complex (FOSC) is a widely distributed group of fungi that includes both pathogenic and nonpathogenic isolates. In a previous study, isolates within the FOSC collected primarily from soybean were assessed for the presence of 12 fungal effector genes. Although none of the assayed genes was significantly associated with wilt symptoms on soybean, the secreted in xylem 6 (Six6) gene was present only in three isolates, which all produced high levels of vascular wilt on soybean. In the current study, a collection of F. oxysporum isolates from soybean roots and F. oxysporum f. sp. phaseoli isolates from common bean was screened for the presence of the Six6 gene. Interestingly, all isolates for which the Six6 amplicon was generated caused wilt symptoms on soybean, and two-thirds of the isolates showed high levels of aggressiveness, indicating a positive association between the presence of the effector gene Six6 and induction of wilt symptoms. The expression profile of the Six6 gene analyzed by quantitative reverse-transcription polymerase chain reaction revealed an enhanced expression for the isolates that caused more severe wilt symptoms on soybean, as established by the greenhouse assay. These findings suggest the suitability of the Six6 gene as a possible locus for pathogenicity-based molecular diagnostics across the various formae speciales.

Cover cropping is a prevalent conservation practice that offers substantial benefits to soil and water quality. However, winter cereal cover crops preceding corn may diminish beneficial rotation effects because two grass species are grown in succession. Here, we show that rye cover crops host pathogens capable of causing corn seedling disease. We isolated Fusarium graminearum, F. oxysporum, Pythium sylvaticum, and P. torulosum from roots of rye and demonstrate their pathogenicity on corn seedlings. Over 2 years, we quantified the densities of these organisms in rye roots from several field experiments and at various intervals of time after rye cover crops were terminated. Pathogen load in rye roots differed among fields and among years for particular fields. Each of the four pathogen species increased in density over time on roots of herbicide-terminated rye in at least one field site, suggesting the broad potential for rye cover crops to elevate corn seedling pathogen densities. The radicles of corn seedlings planted following a rye cover crop had higher pathogen densities compared with seedlings following a winter fallow. Management practices that limit seedling disease may be required to allow corn yields to respond positively to improvements in soil quality brought about by cover cropping.

The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

Root diseases have long been prevalent in Australian grain-growing regions, and most management decisions to reduce the risk of yield loss need to be implemented before the crop is sown. The levels of pathogens that cause the major root diseases can be measured using DNA-based services such as PreDicta B. Although these pathogens are often studied individually, in the field they often occur as mixed populations and their combined effect on crop production is likely to vary across diverse cropping environments. A 3-year survey was conducted covering most cropping regions in Western Australia, utilizing PreDicta B to determine soilborne pathogen levels and visual assessments to score root health and incidence of individual crop root diseases caused by the major root pathogens, including Rhizoctonia solani (anastomosis group [AG]-8), Gaeumannomyces graminis var. tritici (take-all), Fusarium pseudograminearum, and Pratylenchus spp. (root-lesion nematodes) on wheat roots for 115, 50, and 94 fields during 2010, 2011, and 2012, respectively. A predictive model was developed for root health utilizing autumn and summer rainfall and soil temperature parameters. The model showed that pathogen DNA explained 16, 5, and 2% of the variation in root health whereas environmental parameters explained 22, 11, and 1% of the variation in 2010, 2011, and 2012, respectively. Results showed that R. solani AG-8 soil pathogen DNA, environmental soil temperature, and rainfall parameters explained most of the variation in the root health. This research shows that interactions between environment and pathogen levels before seeding can be utilized in predictive models to improve assessment of risk from root diseases to assist growers to plan more profitable cropping programs.

Fusarium oxysporum f. sp. cepae causes Fusarium basal rot in onion (common onion) and Fusarium wilt in Welsh onion. Although these diseases have been detected in various areas in Japan, knowledge about the genetic and pathogenic variability of F. oxysporum f. sp. cepae is very limited. In this study, F. oxysporum f. sp. cepae was isolated from onion and Welsh onion grown in 12 locations in Japan, and a total of 55 F. oxysporum f. sp. cepae isolates (27 from onion and 28 from Welsh onion) were characterized based on their rDNA intergenic spacer (IGS) and translation elongation factor-1α (EF-1α) nucleotide sequences, vegetative compatibility groups (VCGs), and the presence of the SIX (secreted in xylem) homologs. Phylogenetic analysis of IGS sequences showed that these isolates were grouped into eight clades (A to H), and 20 onion isolates belonging to clade H were monophyletic and assigned to the same VCG. All the IGS-clade H isolates possessed homologs of SIX3, SIX5, and SIX7. The SIX3 homolog was located on a 4 Mb-sized chromosome in the IGS-clade H isolates. Pathogenicity tests using onion seedlings showed that all the isolates with high virulence were in the IGS-clade H. These results suggest that F. oxysporum f. sp. cepae isolates belonging to the IGS-clade H are genetically and pathogenically different from those belonging to the other IGS clades.

The genus Fusarium has a wide host range and causes many different forms of plant disease. These include seed rot and seedling blight diseases of cultivated plants. The diseases caused by Fusarium on wild plants are less well-known. In this study, we examined disease development caused by Fusarium sp. n on nondormant seeds of the important rangeland weed Bromus tectorum as part of broader studies of the phenomenon of stand failure or "die-off" in this annual grass. We previously isolated an undescribed species in the F. tricinctum species complex from die-off soils and showed that it is pathogenic on seeds. It can cause high mortality of nondormant B. tectorum seeds, especially under conditions of water stress, but rarely attacks dormant seeds. In this study, we used scanning electron microscopy (SEM) to investigate the mode of attack used by this pathogen. Nondormant B. tectorum seeds (i.e., florets containing caryopses) were inoculated with isolate Skull C1 macroconidia. Seeds were then exposed to water stress conditions (-1.5 MPa) for 7 days and then transferred to free water. Time lapse SEM photographs of healthy versus infected seeds revealed that hyphae under water stress conditions grew toward and culminated their attack at the abscission layer of the floret attachment scar. A prominent infection cushion, apparent macroscopically as a white tuft of mycelium at the radicle end of the seed, developed within 48 h after inoculation. Seeds that lacked an infection cushion completed germination upon transfer to free water, whereas seeds with an infection cushion were almost always killed. In addition, hyphae on seeds that did not initiate germination lacked directional growth and did not develop the infection cushion. This strongly suggests that the fungal attack is triggered by seed exudates released through the floret attachment scar at the initiation of germination. Images of cross sections of infected seeds showed that the fungal hyphae first penetrated the caryposis wall, then entered the embryo, and later ramified throughout the endosperm, completely destroying the seed.

Sugar beet (Beta vulgaris) Fusarium yellows is caused by Fusarium oxysporum f. sp. betae and can lead to significant reductions in root yield, sucrose percentage, juice purity, and storability. F. oxysporum f. sp. betae can be highly variable and many F. oxysporum strains isolated from symptomatic sugar beet are nonpathogenic. Identifying pathogenicity factors and their diversity in the F. oxysporum f. sp. betae population could further understanding of how this pathogen causes disease and potentially provide molecular markers to rapidly identify pathogenic isolates. This study used several previously described fungal effector genes (Fmk1, Fow1, Pda1, PelA, PelD, Pep1, Prt1, Rho1, Sge1, Six1, Six6, Snf1, and Ste12) as genetic markers, in a population of 26 pathogenic and nonpathogenic isolates of F. oxysporum originally isolated from symptomatic sugar beet. Of the genes investigated, six were present in all F. oxysporum isolates from sugar beet (Fmk1, Fow1, PelA, Rho1, Snf1, and Ste12), and seven were found to be dispersed within the population (Pda1, PelD, Pep1, Prt1, Sge1, Six1, and Six6). Of these, Fmk1, Fow1, PelA, Rho1, Sge1, Snf1, and Ste12 were significant in relating clade designations and PelD, and Prt1 were significant for correlating with pathogenicity in F. oxysporum f. sp. betae.

Fusarium crown and root rot (FCRR) of asparagus has a complex etiology with several soilborne Fusarium spp. as causal agents. Ninety-three Fusarium isolates, obtained from plant and soil samples collected from commercial asparagus fields in southwestern Ontario with a history of FCRR, were identified as Fusarium oxysporum (65.5%), F. proliferatum (18.3%), F. solani (6.4%), F. acuminatum (6.4%), and F. redolens (3.2%) based on morphological or cultural characteristics and polymerase chain reaction (PCR) analysis with species-specific primers. The intersimple-sequence repeat PCR analysis of the field isolates revealed considerable variability among the isolates belonging to different Fusarium spp. In the in vitro pathogenicity screening tests, 50% of the field isolates were pathogenic to asparagus, and 22% of the isolates caused the most severe symptoms on asparagus. The management of FCRR with soil organic amendments of pelleted poultry manure (PPM), olive residue compost, and fish emulsion was evaluated in a greenhouse using three asparagus cultivars of different susceptibility in soils infested with two of the pathogenic isolates (F. oxysporum Fo-1.5 and F. solani Fs-1.12). Lower FCRR symptom severity and higher plant weights were observed for most treatments on 'Jersey Giant' and 'Grande' but not on 'Mary Washington'. On all three cultivars, 1% PPM consistently reduced FCRR severity by 42 to 96% and increased plant weights by 77 to 152% compared with the Fusarium control treatment. Populations of Fusarium and total bacteria were enumerated after 1, 3, 7, and 14 days of soil amendment. In amended soils, the population of Fusarium spp. gradually decreased while the population of total culturable bacteria increased. These results indicate that soil organic amendments, especially PPM, can decrease disease severity and promote plant growth, possibly by decreasing pathogen population and enhancing bacterial activity in the soil.

At least 20 epidemics of Fusarium head blight (FHB) of wheat have been registered in the last 50 years in Argentina, with variable intensity. Damage induced by the disease is further aggravated by the presence of mycotoxins in affected grains that may cause health problems to humans and animals. The trichothecene chemotype was analyzed for 112 isolates of Fusarium graminearum from Argentina by polymerase chain reaction and two field trials were conducted to study the aggressiveness of a subsample of 14 representative isolates and to analyze deoxynivalenol (DON) production in planta and in vitro. All isolates belonged to the 15-acetyl-DON chemotype. Significant differences were observed in both the symptom severity induced in wheat spikes and the in vivo DON production, and a close correlation was found between these two variables. However, in vitro toxigenic potential was not correlated with the capacity of F. graminearum isolates to produce DON under natural conditions. The progress of infection in the rachis of inoculated wheat spikes was analyzed and the pathogen presence verified in both symptomatic and symptomless spikes. Even isolates with a limited capacity to induce symptoms were able to colonize the vascular tissue and to produce considerable amounts of DON in planta.

A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on 'Charentais-T' (susceptible) and 'Nad-1' (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host-pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

Fusarium spp. are commonly isolated from soybean roots but the pathogenic activity of most species is poorly documented. Aggressiveness and yield impact of nine species of Fusarium were determined on soybean in greenhouse (50 isolates) and field microplot (19 isolates) experiments. Root rot severity and shoot and root dry weights were compared at growth stages V3 or R1. Root systems were scanned and digital image analysis was conducted; yield was measured in microplots. Disease severity and root morphology impacts varied among and within species. Fusarium graminearum was highly aggressive (root rot severity >90%), followed by F. proliferatum and F. virguliforme. Significant variation in damping-off (20 to 75%) and root rot severity (<20 to >60%) was observed among F. oxysporum isolates. In artificially-infested microplots, root rot severity was low (<25%) and mean yield was not significantly reduced. However, there were significant linear relationships between yield and root symptoms for some isolates. Root morphological characteristics were more consistent indicators of yield loss than root rot severity. This study provides the first characterization of aggressiveness and yield impact of Fusarium root rot species on soybean at different plant stages and introduces root image analysis to assess the impact of root pathogens on soybean.

Fusarium circinatum is thought to have been moved around the world with pine planting stock consisting, most probably, of infected seed. In this effort, we investigate the genetic structure of F. circinatum in Spain and globally. In total, 223 isolates were studied from five regions in northern Spain and eight countries. Eight microsatellite markers revealed 66 multilocus genotypes (MLGs). Minimum spanning network analysis of MLGs by region within Spain as well as globally, discriminant analysis of principal components, and analysis of molecular variance revealed that Spanish populations are significantly differentiated and structured into two distinct groups, each one including one of the dominant genotypes observed. This result suggests that two independent introductions occurred into Spain that subsequently underwent clonal divergence and admixture. This result is further supported by the linkage disequilibrium and clonality observed for F. circinatum populations in northern Spain. The maintenance of differentiation between the clusters could result from the lack of or rare sexual reproduction in Spain. Possible introduction pathways from other countries and subsequent routes of dispersion of F. circinatum in Spain are discussed.

Fusarium oxysporum f. sp. cepae, which causes basal rot of onion, consists of seven vegetative compatibility groups (VCGs 0420 to 0426) and several single-member VCGs (SMVs). F. oxysporum f. sp. cepae populations in South Africa and Colorado each consist of one main VCG (namely, VCG 0425 and 0421, respectively). The aim of this study was to develop sequence-characterized amplified region (SCAR) markers for the identification of VCGs 0425 and 0421, using 79 previously characterized F. oxysporum isolates. A second aim was to investigate the prevalence of VCG 0425 among 88 uncharacterized South African onion F. oxysporum isolates using (i) the developed SCAR markers and (ii) inter-retrotransposon (IR)- and random amplified polymorphic DNA (RAPD) fingerprinting. Only two RAPD primers provided informative fingerprints for VCG 0425 isolates but these could not be developed into SCAR markers, although they provided diagnostic fragments for differentiation of VCG 0425 from VCG 0421. IR fingerprinting data were used to develop a multiplex IR-SCAR polymerase chain reaction method for the identification of VCG 0421, VCG 0425, and SMV 4 isolates as a group. Molecular identification of the uncharacterized collection of 88 F. oxysporum isolates (65 F. oxysporum f. sp. cepae and 23 F. oxysporum isolates nonpathogenic to onion) confirmed that VCG 0425 is the main VCG in South Africa, with all but 3 of the 65 F. oxysporum f. sp. cepae isolates having the molecular characteristics of this VCG. Genotyping and VCG testing showed that two of the three aforementioned isolates were new SMVs (SMV 6 and SMV 7), whereas the third (previously known as SMV 3) now belongs to VGC 0247.

Fusarium head blight (FHB), caused by Fusarium graminearum sensu stricto (s.s.), causes tremendous annual yield losses in wheat worldwide. Variation of aggressiveness of isolates from individual field populations in terms of FHB infection and deoxynivalenol (DON) concentration in the host are important population parameters reflecting parasitic ability. Our main objective was to estimate the variation of both traits within three populations of F. graminearum s.s., each consisting of 30 single-spore isolates collected from small wheat fields in Germany, and to compare it with 11 isolates of a collection (F. graminearum collection) from four countries. The same isolates were characterized using 19 single-sequence repeat markers. All isolates were spray inoculated on a moderately resistant spring wheat cultivar at two field locations over 2 years (i.e., in four environments). The genotypic proportion of phenotypic variance (σ(2)(G)) within populations was significant (P < 0.01) for both traits, and the σ(2)(G) × environment interaction was even more important for mean FHB severity. Ranges in mean FHB severity and DON concentration in the host were only slightly smaller for the field populations than for the F. graminearum collection. Both traits were significantly (P < 0.05) correlated within and across populations. A further partitioning of σ(2)(G) revealed 72% of σ(2)(G) within and 28% of σ(2)(G) across populations for both traits. Molecular variance of the three populations was similarly distributed (73.6% within versus 26.4% between populations). In view of this high within-field variation for traits of parasitic ability and selection, neutral molecular markers, multiple resistance genes of different origin should be employed in wheat breeding programs to obtain a long-term stable FHB resistance.

Based on a 2-year field trial at two locations in Lower Saxony (Germany), 395 Fusarium isolates belonging to 13 species were collected from more than 3,000 sugar beet roots that were apparently healthy at harvest. In a comparative screen, subsamples were analyzed for Fusarium infection directly after harvest and after different storage conditions. Depending on the storage duration, a different species composition was observed. F. redolens was predominant in freshly harvested beets, while F. culmorum, F. cerealis, and F. graminearum comprised 50.0% (2006) and 84.8% (2007) of the Fusarium mycoflora of sugar beets subjected to long-term pile storage. Randomly selected isolates of all species detected were tested for pathogenicity to sugar beet, but only isolates of F. graminearum and F. sambucinum caused severe root symptoms. Overall, 34 isolates of all species detected were characterized for their mycotoxin profile in rice culture to determine potentially produced toxins for future analysis of sugar beet. A total of 26 Fusarium mycotoxins were detected by liquid chromatography-tandem mass spectrometry, including trichothecenes, zearalenone, and especially high amounts of beauvericin, enniatins, and moniliformin. Further work is required to analyze the natural occurrence of these mycotoxins in sugar beet.

Fusarium lateritium is a globally distributed plant pathogen. It was recently reported as the causal agent of nut gray necrosis (NGN) on hazelnut. Isolate characterization within F. lateritium was undertaken to investigate how morphological and molecular diversity was associated with host and geographic origin. Morphological studies combined with inter-simple-sequence repeat (ISSR) analysis, and phylogenetic analyses using translation elongation factor 1α (TEF-1α), β-tubulin genes, and nuclear ribosomal DNA internal transcribed spacer (ITS) sequences were conducted to resolve relationships among 32 F. lateritium isolates from NGN-affected hazelnut fruit, and 14 from other substrates or 8 from other hosts than hazelnut. Colonies of F. lateritium from hazelnut showed dark grayish-olive differing from the orange-yellow color of all other isolates from other hosts. Generally, isolates from NGN-affected fruit failed to produce sporodochia on carnation leaf agar. The influence of host and substrate on the genetic structure of F. lateritium was supported by ISSR and analyzed with principal coordinates analysis. A relationship between hazelnut and genetic variation was inferred. Phylogenetic analysis of ITS provided limited resolution while TEF-1α and β-tubulin analyses allowed a clear separation between the European and non-European F. lateritium isolates retrieved from GenBank, regardless of host. Though morphological traits of F. lateritium isolates from hazelnut were generally uniform in defining a typical morphogroup, they were not yet phylogenetically defined. In contrast, the typology related to slimy deep orange cultures, due to spore mass, grouped clearly separated from the other F. lateritium isolates and revealed a congruence between morphology and phylogeny.

Fusarium oxysporum f. sp. phaseoli strains isolated from runner bean plants showing Fusarium wilt symptoms were characterized. The analysis of the genetic diversity of these strains and the comparison with strains formerly isolated from diseased common bean plants grown in the same region of Spain indicated a close genetic similarity among them. Pathogenicity assays carried out on runner bean plants showed virulence differences that allowed the classification of these strains into three groups: super virulent, highly virulent, and weakly virulent. However, all the analyzed strains behaved as highly virulent when inoculated on common bean plants, indicating that virulence is specific of the host-pathogen interaction. We also analyzed the number of copies and expression of the gene encoding the transcription factor ftf1, which has been shown to be specific of virulent F. oxysporum strains and highly up-regulated during plant infection. In planta real-time quantitative polymerase chain reaction expression analysis showed that expression of ftf1 was correlated with the degree of virulence. The comparative analysis of the polymorphic copies of ftf1 detected in the strains here characterized and those detected in the genome sequence of F. oxysporum f. sp. lycopersici strain 4287 indicates that some of the copies are likely nonfunctional.

The primary objective of this study was to characterize Fusarium spp. associated with the economically devastating mango malformation disease (MMD) in Mexico. In all, 142 Fusarium strains were isolated from symptomatic mango inflorescences and vegetative tissues in eight geographically diverse Mexican states from 2002 through 2007. Initially, all the Mexican isolates were screened for genetic diversity using appolymerase chain reaction and random amplified polymorphic DNA markers and were grouped into seven distinct genotypes. Based on results of these analyses, evolutionary relationships and species limits of the genetically diverse MMD-associated Fusarium spp. were investigated using multilocus DNA sequence data and phylogenetic species recognition. Maximum parsimony analyses of a five-locus data set comprising 5.8 kb of aligned DNA sequence data indicated that at least nine phylogenetically distinct Fusarium spp. within the Gibberella fujikuroi species complex are associated with MMD, including one species within the African clade (Fusarium pseudocircinatum), two species within the Asian clade (F. mangiferae and F. proliferatum), and at least six species within the American clade (F. sterilihyphosum and five undescribed Fusarium spp.). Molecular phylogenetic analyses indicate that a novel genealogically exclusive lineage within the American clade was the predominant MMD associate in Mexico. This new Fusarium sp. caused MMD and could be distinguished from all other known species morphologically by the production of mostly sterile, coiled hyphae which are typically associated with sporodochial conidiophores together with unbranched or sparsely branched aerial conidiophores. Koch's postulates were completed for isolates of the new species on nucellar seedlings of mango cv. Ataulfo. This pathogen is formally described herein as F. mexicanum.

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer-probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

Fusarium asiaticum and F. graminearum are the primary causal agents of Fusarium head blight (FHB) of wheat in China. In this study, sensitivities of 159 F. asiaticum and F. graminearum isolates to a benzimidazole fungicide carbendazim (MBC) and to sterol demethylation inhibitors (DMIs) tebuconazole and prochloraz were determined. Among the 159 isolates, 9 were resistant to MBC and designated as MBC-R isolates. Three showed resistance to tebuconazole and prochloraz and designated as DMI-R isolates. There was no cross-resistance between MBC and DMI. Genetic analysis by microsatellite-primed polymerase chain reaction (PCR) showed that MBC-R or DMI-R isolates had different genotypes, which indicated that they originated from different wild-type parents. Analysis of two 14alpha-demethylase (cyp51) homologous genes (cyp51A and cyp51B) showed that the F. asiaticum isolates could be distinguished from F. graminearum isolates based on the sequence of cyp51A. Analysis of deduced amino acid sequence of cyp51A and cyp51B suggested that no mutations were associated with DMI resistance. Real-time PCR analysis showed that the DMI resistance was not related to the expression of cyp51A and cyp51B in F. asiaticum and F. graminearum, but expressions of both genes were induced greatly by the tebuconazole. Results of this study indicated that cyp51A would be an informative marker for analysis of population structure of F. asiaticum and F. graminearum, and the existence of homologous cyp51 genes in F. asiaticum and F. graminearum could provide new insights into DMI resistance in phytopathogenic fungi.

Fusarium head blight (FHB) of wheat and other cereals, caused mainly by Fusarium graminearum, is one of the most economically important diseases worldwide, especially in the United States and China. The benzimidazole fungicides, particularly carbendazim (MBC), have been consistently used during the period of wheat heading and flowering in areas with warm and moist weather to control FHB in China for over 30 years. The effectiveness of MBC, however, has been threatened by the emergence of resistant pathogen populations in the field. JS399-19 (experimental number; a.i. 2-cyano-3-amino-3-phenylancryic acetate) is a novel cyanoacrylate fungicide discovered and patented by the Jiangsu Branch of National Pesticide Research & Development South Center of China. To evaluate the potential risk of resistance development in MBC-resistant F. graminearum isolates to this new fungicide JS399-19, five isolates each of MBC-resistant or -sensitive, which were classified into three different sensitivity phenotypes, such as sensitive (S), moderately resistant (MR), and highly resistant (HR) to MBC, were selected to induce JS399-19-resistant mutants by selecting resistance on potato sucrose agar (PSA) plates amended with JS399-19 at 10 microg/ml. In this way, a total of 24 JS399-19-resistant mutants were obtained from all tested MBC-resistant or -sensitive isolates. All 50 single-spore progenies of each of the resistant mutants could grow normally on PSA plates amended with JS399-19 at 10 microg/ml, indicating stability of resistance to this fungicide. Also, all of the resistant mutants maintained their resistance to JS399-19 and/or MBC through eight transfers on PSA plates for 40 days and when stored on PSA slants at 4 degrees C for 60 days. The mycelial growth and conidial production capacity were decreased in 52.4% of the resistant mutants, indicating that a fitness cost was associated with JS399-19-resistant phenotypes of F. graminearum isolates. However, most of the mutants resistant to both MBC and JS399-19 exhibited high sexual reproduction capacity and pathogenicity as their parental isolates. Nevertheless, the majority of these mutants possessed fitness levels comparable to their parents. The results on the efficacy of the two fungicides for controlling FHB incited by the fungicide-resistant mutants were generally consistent with those of the in vitro sensitivity tests. JS399-19 was effective in controlling FHB caused by MBC-resistant isolates under field conditions, while it was not effective in controlling FHB caused by isolates resistant to JS399-19 or those that were resistant to both MBC and JS399-19. Moreover, the efficacy of the mixture of MBC and JS399-19 was also significantly lower in controlling FHB caused by the isolates resistant to both MBC and JS399-19 than the efficacy against the disease caused by the sensitive isolates, the MBC-resistant isolates, or the JS399-19-resistant isolates. The results suggest that JS399-19 possessed a high risk in development of resistance in MBC-resistant and -sensitive F. graminearum isolates, and this double resistance to both of these fungicides could presumable emerge and create a major problem since both these fungicides are extensively used in China. Therefore, careful use of JS399-19 should be followed to delay resistance development in natural populations of F. graminearum, avoid unexpected control failures, and sustain the usefulness of MBC and the new product JS399-19.

We used a real time polymerase chain reaction-based assay and visual disease assessment to evaluate the efficacies of Js399-19, tebuconazole, a mixture of tebuconazole and thiram, azoxystrobin, carbendazim, and thiram on the development of Fusarium head blight (FHB) and deoxynivalenol (DON) contamination and on the yield of winter wheat (cv. Nannong no. 9918) after artificial inoculation under field conditions with Fusarium graminearum. The incidence of infected spikelets (IIS), amount of F. graminearum DNA (Tri5 DNA), total DON (containing DON, 3-acetyl-deoxynivalenol, and 15-acetyl-deoxynivalenol) concentration, and 1,000-grain weight (TGW) were quantified in 2006 and 2007. A strong positive correlation was found between IIS or Log10Tri5 DNA and total DON concentration in the harvested grain. The Js399-19, tebuconazole, and the mixture of tebuconazole and thiram significantly reduced IIS of FHB, amount of Tri5 DNA, and total DON within the grain and increased TGW. Although azoxystrobin, carbendazim, and thiram can increase TGW, they had no effect on the occurrence of F. graminearum compared with those of the untreated controls. Surprisingly, azoxystrobin and carbendazim significantly increased the total DON content in the harvested grain because they might have stimulated the amount of total DON production per Tri5 DNA. The fungicides Js399-19, tebuconazole, and the mixture of tebuconazole and thiram were the most effective in controlling FHB and reducing DON contamination of the wheat.

The recent observance of Fusarium fujikuroi, the causal agent of Bakanae disease of rice, in California provides a unique opportunity to assess the population diversity of an introduced pathogen in a new environment. We collected 172 isolates of this pathogen between 2000 and 2003 from California rice and two from water grass (Echinochloa spp.). Pathogenicity of F. fujikuroi was demonstrated on early water grass (E. oryzoides) and barnyard grass (E. crus-galli) indicating that weed control should be part of Bakanae management programs. Both mating types and six unique amplified fragment length polymorphism haplotypes corresponding to six identified vegetative compatibility groups were detected. The two most frequently isolated haplotypes encompassed 94% of the collected isolates, suggesting that clonal reproduction dominates. Coefficients of similarity between the unique haplotypes ranged from 0.94 to 0.98, and indicate that there is very little genotypic variation in the F. fujikuroi population in California. The near fixation of the MAT-1 idiomorph (observed ratio 170 MAT-1:4 MAT-2), is consistent with a hypothesis of predominant or exclusive asexual reproduction. The low level of introduced genotypic diversity, in conjunction with the asexual reproductive strategy of this population will slow evolutionary processes, including adaptation to the California environment.

We report on a large gene bank of Fusarium isolates established by a broad survey conducted in 2005 in which infected barley ears were collected in 23 counties of seven provinces and two municipalities along the Yangtze River in China. In total, 1,894 single spore isolates were obtained. The isolates were characterized at the species level by a newly developed and robust set of diagnostic primers based on single nucleotide polymorphisms (SNPs) among members of the F. graminearum clade. In addition, we determined their chemotype using previously described polymerase chain reaction (PCR) primers. The results showed that in all regions F. asiaticum was the predominant species causing Fusarium head blight (FHB) on barley in China (N = 1,706), while in the upper valleys of the Yangtze River also F. graminearum sensu stricto, F. meridionale, and F. proliferatum were found. Major differences in the chemotypes were found in the F. asiaticum populations, from very high to exclusive nivalenol (NIV) chemotypes in the mountainous upper valleys of the Yangtze River to predominantly deoxynivalenol (DON) chemotypes in the middle and lower valleys. In contrast to the F. asiaticum isolates from three counties in Sichuan province, which were largely NIV producers (278 of 291), F. graminearum isolates from these sampling sites were for the vast majority (27 of 28) DON producers, indicating that despite sharing the same habitat, these sympatric species apparently have unique mycotoxin chemotypes.

Crown rot, caused by the fungus Fusarium pseudograminearum (teleomorph Gibberella coronicola) is a major disease of wheat in the Australian grain belt. However, there is little information available on the population structure of this pathogen. We measured genetic diversity as assessed with amplified fragment length polymorphism (AFLP) analysis within and between populations of F. pseudograminearum from northeastern, south central, and southwestern regions of the Australian grain belt. Amongst the 217 isolates, 176 haplotypes were identified and grouped into two main clusters. One cluster contained isolates from populations in northeastern Australia, and the other cluster contained isolates from populations in south central and southwestern Australia. The southern populations were distinguished from the northeastern populations by higher levels of population differentiation (Gst) between them and genetic identity amongst the regional populations. We hypothesize that the F. pseudograminearum populations from northeastern and southern Australia are independent, which could result from different founding events or from geographic isolation and the accumulation of genetic differences due to genetic drift and/or selection.

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.