Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), is one of the most destructive emerging diseases affecting global wheat production. Although it is endemic to South America and has significantly affected Brazil, Paraguay, and Bolivia, it had not been reported in Uruguay until 2023. During that season, climatic conditions similar to those associated with Brazilian MoT outbreaks, particularly in northwestern Uruguay, prompted intensive monitoring for this disease. In October 2023, 17 wheat spike samples with blast-like symptoms were collected from 11 fields; nine were positive for M. oryzae based on morphology. Four monosporic isolates were recovered, and three were subjected to molecular and genomic characterization. PCR confirmed their identity as MoT, and pathogenicity tests demonstrated their ability to cause disease in both leaves and spikes of susceptible wheat genotypes. Phylogenetic comparisons among 57 Triticum, Lolium, and Eleusine M. oryzae strains, including strains from the B71 branch responsible for outbreaks in Bangladesh and Zambia, indicated that the Uruguayan strains are closely related to some Brazilian strains and are genetically distinct from the B71 branch. Specifically, PyrUy10.1 and PyrUy14.1 shared 99.9% of their SNPs, whereas PyrUy11.1 showed only 73% similarity to these strains, suggesting more than one introduction of the pathogen into Uruguay and the presence of distinct lineages. These findings confirm the presence of MoT in Uruguay and suggest that its introduction likely occurred through airborne dispersal due to its geographic proximity to Brazil. This study highlights the need for continuous surveillance and monitoring in Uruguayan regions with conducive environmental conditions for wheat blast, as the detection of multiple genetic lineages suggests the presence of strains with distinct adaptations. This genetic diversity poses a significant challenge for effective wheat blast management in Uruguay and may have important implications for national wheat production as the pathogen continues to evolve locally.

Abstract The genus Erwinia comprises a diverse group of bacteria associated with plants, insects, and the environment, including several economically important phytopathogens. The genus has been revised taxonomically many times, yet a thorough and genome-wide assessment of its evolutionary relationships and genomic diversity has been lacking. In this research, we carried out an extensive phylogenomic and comparative genomic analyses of the genus Erwinia using 104 genomes including historically important strains. Genome-wide analyses integrating average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH), core-genome phylogenomics, pan-genome analysis, and comparative genomics resolved evolutionary relationships across the genus and identified multiple taxonomic inconsistencies. The pan-genome analysis revealed a relatively small core genome alongside an extensive accessory genome, underscoring the substantial genomic plasticity and ongoing diversification within the genus. The comparative analyses further showed pronounced lineage-specific variation in secretion systems, exopolysaccharide biosynthetic loci, flagellar gene clusters, genomic islands, prophages, and iron acquisition systems, suggesting that virulence-associated determinants have evolved through differential gene gain, loss, and conservation across distinct lineages, thereby facilitating host and ecological niche adaptation. This lineage-specific variation indicates that pathogenicity in the genus is not driven by a single conserved set of virulence determinants but instead reflects distinct combinations of virulence-associated genes. These findings refine the genomic framework of the genus Erwinia , provide evidence for taxonomic revision of several lineages, and improve our understanding of the evolutionary relationships, genomic diversification, and lineage-specific adaptations associated with host interactions and ecological specialization. Impact Statement This study provides the first comprehensive genome-wide phylogenomic framework for the genus Erwinia , integrating taxonomy, pan-genome diversity, virulence-associated determinants, and mobile genetic elements across all 18 currently recognized species. Analyses resolve evolutionary relationships, uncover multiple taxonomic inconsistencies, identify previously unrecognized species-level lineages, including a putative novel Erwinia species PL328 isolated from Cornus florida (dogwood), and reveal lineage-specific genomic features. These findings establish a valuable genomic foundation for future studies of Erwinia evolution, taxonomy, and plant-microbe interactions. Data Summary Genomes sequenced in this study were submitted to the NCBI database under the accession numbers: JCBCPT000000000

Xanthomonas axonopodis pv. vasculorum (Xav), the causative agent of sugarcane gumming disease, represents a significant threat to global sugarcane production due to its systemic and destructive nature. A field-deployable tool specific to Xav is required for rapid infection detection and timely disease management. This resulted in a loop-mediated isothermal amplification (LAMP) assay targeting the gene region, unique to Xav strains, as a rapid and precise diagnostic assay. The selection of a target gene region was informed by comprehensive in silico genomes analyses of Xav and other closely related Xanthomonas species. The target gene region's specificity was validation against the NCBI GenBank database and internally sequenced genomes. Primers for both endpoint PCR and LAMP assays were designed using this unique gene. The LAMP assay underwent extensive testing against inclusivity and exclusivity panels. Use of exclusivity panel, comprising 81 strains from related species, other bacterial genera, and host genomes, demonstrated the assay's specificity with no false positives. The assay exhibited a detection limit of 1 pg, and its effectiveness was unimpeded by crude host lysate (sugarcane). Further validation through multi-device and multi-operator testing underscored the assay's 100% reproducibility and robustness. Application to infected plant samples resulted in the detection of all infected specimens without any false positives or negatives. This novel LAMP assay is accurate and reliable tool for Xav detection, with promising applications in routine diagnostics, biosecurity measures, microbial forensics, and epidemiological research.

Corn stunt is one of the most significant corn diseases in the Neotropics, leading to severe plant stunting and substantial yield losses. Although four pathogens have been found either singly or in combination in infected plants in the Americas, corn stunt spiroplasma (Spiroplasma kunkelii) has been the most predominant pathogen associated with the disease in the United States, due to its widespread distribution in the Rio Grande Valley region and persistent occurrence in California and Florida. During the 2024 growing season, reports of chlorosis, leaf reddening, and stunting in corn fields in Southern, Great Plains, Central Corn Belt, and Northeastern states raised concern regarding the possibility of a more widespread distribution of corn stunt spiroplasma in the United States. Symptomatic corn leaf samples were collected in commercial and experimental field sites across the United States. Detection and identification of S. kunkelii were performed using a polymerase chain reaction assay targeting a section of the spiralin gene, followed by amplicon sequencing. This study provides the first report of the pathogen S. kunkelii associated with corn stunt symptoms distributed across 6 counties in Oklahoma, 14 counties in Kansas, 2 counties in Missouri and Arkansas, 4 counties in New York, and 1 county in each of Nebraska, South Dakota, Wisconsin, Minnesota, Indiana, and Alabama. All states with submitted samples had at least one confirmed case of S. kunkelii.

Potatoes, among the most economically significant crops worldwide, are susceptible to various plant pathogens that significantly impact their propagation, production, storage, and distribution. Soft rot disease, caused primarily by Dickeya and Pectobacterium , results in substantial economic losses to the agricultural industry annually. In this study, we developed a rapid, reliable, and field-deployable loop-mediated isothermal amplification (LAMP) assay for detecting D. dadantii , a common soft rot causing bacteria. The D. dadantii -specific LAMP primers were designed targeting a highly conserved genomic region within D. dadantii , the TetR/AcrR family transcriptional regulator CDS and its flanking sequences. This assay was thoroughly validated with the members of inclusivity (nine strains of D. dadantii ) and exclusivity panels (85 strains, including all Dickeya species, related taxa, and host DNA), detecting no false positives or negatives. The limit of detection (LOD) was established by performing assays with 10-fold serially diluted pure gDNA of D. dadantii and gDNA spiked with host crude extract; the assay detected the target pathogen down to 1 pg (188 copies) without being adversely affected by the host crude extract. The developed LAMP assay specifically detected the target pathogen in infected plant materials. Additional multi-operator blind and multi-instrument tests were conducted to assess the assay’s robustness and applicability, consistently yielding accurate results without false positives or negatives. These findings demonstrate the assay’s potential utility for biosecurity, routine diagnostics, and epidemiological studies.### Competing Interest StatementThe authors have declared no competing interest.

AbstractXanthomonas axonopodispv.vasculorum(Xav), the causative agent of sugarcane gumming disease, represents a significant threat to global sugarcane production due to its systemic and destructive nature. Despite the economic implications, a field-deployable, Xav-specific diagnostic tool has not been developed. This resulted in a loop-mediated isothermal amplification (LAMP) assay targeting thepelLgene, unique to Xav strains, as a rapid and precise diagnostic assay. The selection of thepelLgene was informed by comprehensivein silicoanalyses of Xav genomes and relatedXanthomonasspecies and other close relatives. Validation against the NCBI GenBank database and internally sequenced genomes confirmed the gene’s exclusivity to Xav. Subsequent primers for both endpoint PCR and LAMP assays were designed using thepelLgene region. The LAMP assay underwent extensive testing against inclusivity and exclusivity panels. Use of exclusivity panel, comprising 81 strains from related species, other bacterial genera, and host genomes, demonstrated the assay’s specificity with no false positives. The assay exhibited a detection limit of 1 pg, and its effectiveness was unimpeded by crude host lysate (sugarcane). Further validation through multi-device and multi-operator testing underscored the assay’s 100% reproducibility and robustness. Application to infected plant samples resulted in the detection of all infected specimens without any false positives or negatives. This novel LAMP assay is accurate and reliable tool for Xav detection, with promising applications in routine diagnostics, biosecurity measures, microbial forensics, and epidemiological research.

Xanthomonas axonopodis pv. vasculorum (Xav), the causative agent of sugarcane gumming disease, represents a significant threat to global sugarcane production due to its systemic and destructive nature. Despite the economic implications, a field-deployable, Xav-specific diagnostic tool has not been developed. This resulted in a loop-mediated isothermal amplification (LAMP) assay targeting the pelL gene, unique to Xav strains, as a rapid and precise diagnostic assay. The selection of the pelL gene was informed by comprehensive in silico analyses of Xav genomes and related Xanthomonas species and other close relatives. Validation against the NCBI GenBank database and internally sequenced genomes confirmed the gene’s exclusivity to Xav. Subsequent primers for both endpoint PCR and LAMP assays were designed using the pelL gene region. The LAMP assay underwent extensive testing against inclusivity and exclusivity panels. Use of exclusivity panel, comprising 81 strains from related species, other bacterial genera, and host genomes, demonstrated the assay’s specificity with no false positives. The assay exhibited a detection limit of 1 pg, and its effectiveness was unimpeded by crude host lysate (sugarcane). Further validation through multi-device and multi-operator testing underscored the assay’s 100% reproducibility and robustness. Application to infected plant samples resulted in the detection of all infected specimens without any false positives or negatives. This novel LAMP assay is accurate and reliable tool for Xav detection, with promising applications in routine diagnostics, biosecurity measures, microbial forensics, and epidemiological research. ### Competing Interest Statement The authors have declared no competing interest.

The ability to minimize negative economic, environmental, or productivity impacts to plant systems caused by recurrent and emerging pathogens and pests depends on the early detection and accurate identification of those pests and pathogens. New pest and pathogen populations and genotypes are being detected and reported monthly in peer-reviewed journals across the globe. Accurate and timely identification of pests and pathogens is dependent on the capability and capacity of the diagnostic laboratory. Among the important attributes for a diagnostic laboratory are the technologies and methods used and the number and types of samples that the laboratory can process. Because pathogen or pest identification informs response, accurate identification is also dependent on the reliability of the assay employed and the correct interpretation of assay results. Confidence in the test results generated, and ultimately in the identification rendered, is dependent on the design and rigor of the validation process for the assay(s) used. Well-designed and -executed validation requires access to an array of resources including positive controls, reference strains to build inclusivity and exclusivity panels, reference nucleic acid sequence databases, and fully vetted standard operating procedures. A communications ecosystem to facilitate the sharing of such resources, as well as to provide access to taxon and technology expertise, will greatly accelerate the development, validation, and reliability of trusted diagnostic tests to support plant biosecurity specifically and plant health in general. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Plant biosecurity depends heavily on early detection of biological pest and disease threats and accurate diagnosis of causal agents. The information from these activities is disseminated and communicated with decision makers to promote effective mitigation. In the United States, most land-grant university, state, and private diagnostic and expert pest identifier labs are successfully networked via the U.S. Department of Agriculture Animal Plant Health Inspection Service, Plant Protection and Quarantine (USDA APHIS PPQ; https://www.aphis.usda.gov/ ), the National Plant Diagnostic Network (NPDN; https://www.npdn.org ), and the National Clean Plant Network (NCPN; https://www.nationalcleanplantnetwork.org/ ). These organizations provide access to training, shared protocols, and standardized communications. For example, the NPDN has secure communications tools that network leadership built into its IT infrastructure and deploys a formal communications process and protocol for regulatory and high-risk samples. Additionally, the diagnostic networks adopt protocols from regulatory organizations such as the USDA-APHIS, European and Mediterranean Plant Protection Organization (EPPO), North American Plant Protection Organization (NAPPO), and other international and national plant protection organizations (NPPOs). However, plant health diagnosticians also need access to validated protocols for endemic and nonregulated organisms. Although there is strong networking of diagnostic laboratories, plant disease diagnostic assay development and validation are siloed, representing a critical gap in our biosecurity infrastructure, especially for new or emerging pathogens. Most assays are developed in independent research labs and then chosen independently by the end-use laboratory. Assay developers, those who provide reference materials, labs that can provide ring testing for validation, and assay end users could be connected and integrated for more streamlined operations. Terminology and validation protocols need to be standardized within the United States and harmonized with our trade partners to ensure understanding. The ideal would be availability of standard, taxon-specific validation protocols, essential reference materials, and appropriate control materials for use to quickly develop and deploy assays in emergencies, as well as for day-to-day testing. This perspective article provides a summary of principles of assay validation, fitness-for-purpose concepts, and the need for continuous evaluation of assay performances. We describe the existing capacity and resources needed to efficiently develop, validate, and use diagnostic tests, as well as the inefficiencies and resource shortfalls currently faced. We propose development of a Diagnostic Assay Validation Network (DAVN) system to coordinate resources at the national level and harmonize with our partners at the international level. Here, we outline a project to set up the DAVN with the objectives of standardizing terminology and statistics and facilitating location of reference materials used in diagnostic assay development and validation via a portal of tools designed by and for the plant disease research and extension community. Although many of the concepts are also relevant to insects and other pests, the focus of this article is primarily plant pathogens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Dickeya fangzhongdai , a bacterial pathogen of taro ( Colocasia esculenta ), onion ( Allium sp.), and several species in the orchid family ( Orchidaceae ) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai . Multiple genomes of D. fangzhongdai , and other species of Dickeya , Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18–20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis ( Cn ), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter - and Cn -specific primers/probes, respectively. The detection limit for Cn -specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter -specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae ), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae . Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri . Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri ) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Wheat blast is an explosive new fungal disease of wheat caused by an Magnaporthe oryzae (synonym of Pyricularia oryzae) host-adapted subpopulation, the M. oryzae Triticum pathotype (MoT). MoT has been found in South America, South Asia, and Africa, but not in the United States. Wheat blast caused by the MoT fungus was first reported in Brazil in 1985 and subsequently spread to Bolivia, Paraguay, and Argentina in the 1990s and 2000s. The disease first appeared in Bangladesh in 2016 and in Zambia in 2017. The MoT fungus is seedborne, and the most likely route for movement across oceans was though grain trade. Wheat head (spike) blast is the predominant form of the disease in the field, although foliar and stem blast also occurs. The disease has proven hard to control when weather conditions are conducive, often resulting in devastating yield and quality losses. The only currently effective resistance, contained in the 2NvS translocation from the wild wheat relative Aegilops ventricosa, confers partial resistance that is variable depending on the genetic background of the specific wheat variety. Fungicides are not fully effective in controlling wheat head blast if warm, humid weather occurs during the heading stage. A major disease management strategy in areas where the disease occurs involves timing the wheat planting date so that heading does not coincide with warm rainy weather. A climate suitability model for the United States indicates that all of U.S. soft red winter wheat and about half of the hard red winter wheat are at risk.

Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.

The National Plant Diagnostic Network (NPDN), comprising diagnostic professionals from more than 70 pathology, entomology, and nematology laboratories, safeguards U.S. plant systems through accurate diagnosis and effective communications with clients, partners, and stakeholders. As a USDA-NIFA extension program built on the land-grant university system, the network has dual responsibilities to extension clientele such as farmers and the green industry, as well as state and federal regulatory agencies. Following strategic planning in 2019, the network emerged with a concise plan and strong committees of network participants to enhance and sustain service to NPDN clientele and partners, even through significant disruptions like the 2020 coronavirus pandemic. The commitment to building diagnostic capacity and expertise across the country allows these plant clinics to assist during a response to detections of high-consequence plant pathogens by clearing healthy plants for commerce while identifying potential positives for regulators to quarantine and/or eradicate, similar to the test and trace efforts for human diseases such as COVID-19. In this review, we describe the network's recent activities to protect U.S. plant agriculture and natural ecosystems and its plans to improve and expand capacity for national plant biosecurity.

Rathayibacter toxicus is a toxigenic bacterial pathogen of several grass species and is responsible for massive livestock deaths in Australia and South Africa. Due to concern for animal health and livestock industries, it was designated a U.S. Select Agent. A rapid, accurate, and sensitive in-field detection method was designed to assist biosecurity surveillance surveys and to support export certification of annual ryegrass hay and seed. Complete genomes from all known R. toxicus populations were explored, unique diagnostic sequences identified, and target-specific primers and a probe for recombinase polymerase amplification (RPA) and endpoint PCR were designed. The RPA reaction ran at 37 °C and a lateral flow device (LFD) was used to visualize the amplified products. To enhance reliability and accuracy, primers and probes were also designed to detect portions of host ITS regions. RPA assay specificity and sensitivity were compared to endpoint PCR using appropriate inclusivity and exclusivity panels. The RPA assay sensitivity (10 fg) was 10 times more sensitive than endpoint PCR with and without a host DNA background. In comparative tests, the RPA assay was unaffected by plant-derived amplification inhibitors, unlike the LAMP and end-point PCR assays. In-field validation of the RPA assay at multiple sites in South Australia confirmed the efficiency, specificity, and applicability of the RPA assay. The RPA assay will support disease management and evidence-based in-field biosecurity decisions.

Rathayibacter toxicus is a Gram-positive, nematode-vectored bacterium that infects several grass species in the family Poaceae. Unique in its genus, R. toxicus has the smallest genome, possesses a complete CRISPR-Cas system, a vancomycin-resistance cassette, produces tunicamycin, a corynetoxin responsible for livestock deaths in Australia, and is designated a Select Agent in the United States. In-depth, genome-wide analyses performed in this study support the previously designated five genetic populations, with a core genome comprising approximately 80% of the genome for all populations. Results varied as a function of the type of analysis and when using different bioinformatics tools for the same analysis; e.g., some programs failed to identify specific genomic regions that were actually present. The software variance highlights the need to verify bioinformatics results by additional methods; e.g., PCR, mapping genes to genomes, use of multiple algorithms). These analyses suggest the following relationships among populations: RT-IV ↔ RT-I ↔ RT-II ↔ RT-III ↔ RT-V, with RT-IV and RT-V being the most unrelated. This is the most comprehensive analysis of R. toxicus that included populations RT-I and RT-V. Future studies require underrepresented populations and more recent isolates from varied hosts and geographic locations.

Fusarium proliferatum and F. verticillioides are mycotoxin-producing, seedborne pathogens of maize. They are often asymptomatic in seed, eluding symptom-based detection. Experiments were conducted in nonsterile soil to determine whether interspecific competition influenced establishment in maize plants of an introduced isolate of F. proliferatum or F. verticillioides. Hygromycin-resistant, green fluorescent protein (GFP) transformed (GFP-tagged) F. proliferatum (F. proliferatum-green) and hygromycin-resistant, monomeric red fluorescent protein (mRFP) transformed (mRFP-tagged) F. verticillioides (F. verticillioides-red) strains were developed to provide molecular markers to track fungal establishment. Heat-killed Fusarium-free maize seed, colonized with F. proliferatum-green or F. verticillioides-red by immersion in a spore suspension for 16 h, served as the source of inoculum. The ability of F. proliferatum-green and F. verticillioides-red to colonize viable maize plants already colonized by the other species was determined. Maize plants were retrieved from soil after 14 days and DNA was extracted from three consecutive root segments and three consecutive stem segments. A TaqMan multiplex real-time quantitative PCR protocol was developed to identify and quantify F. proliferatum-green and F. verticillioides-red from each plant segment from each treatment; the experiment was repeated three times. This experiment confirmed that F. proliferatum-green and F. verticillioides-red effectively colonized roots and stems of the maize plant already colonized with the other species. Prior colonization of maize tissues by F. verticillioides-red (P = 0.6749) and other seedborne microorganisms (P = 0.1910) reduced but did not prevent subsequent colonization by F. proliferatum-green. Similarly, prior colonization of maize tissues by F. proliferatum-green (P = 0.7032) and other seedborne microorganisms (P = 0.1447) reduced but did not prevent subsequent colonization by F. verticillioides-red.